Liposome-Based Drug Delivery Systems 3662493187, 9783662493182
This volume describes the protocols for fabrication of liposomal drug delivery system, and consists of two parts. The fi
351 76 13MB
English Pages 489 [499] Year 2021
Series Preface
Volume Preface
Contents
About the Series Editor
About the Volume Editors
Contributors
Part I: General Protocols for Fabrication of Drug Liposomes
1 Liposomes in Drug Delivery: Status and Advances
1 Introduction
2 Features of Liposomes
3 Classification of Liposomes
3.1 Passive Targeting Liposomes
3.1.1 Conventional Liposomes
3.1.2 Long Circulating Liposomes
3.2 Active Targeting Liposomes
3.2.1 Ligand-Mediated Liposomes
3.2.2 Antibody-Mediated Liposomes
3.3 Physicochemical Targeting Liposomes
3.3.1 pH-Sensitive Liposomes
3.3.2 Temperature-Sensitive Liposomes
3.3.3 Enzyme-Sensitive Liposomes
3.3.4 Physical Adsorption-Mediated Liposomes
3.4 Multifunctional Liposomes
4 Preparation Materials and Methods of Liposomes
4.1 Preparation Materials
4.1.1 Phospholipids
4.1.2 Cholesterol
4.1.3 Other Materials
4.2 Preparation Methods
4.2.1 Film Dispersion Method
4.2.2 Reverse-Phase Evaporation Method
4.2.3 Chemical Gradient Methods
pH Gradient Method
Ammonium Sulfate Gradient Method
Calcium Acetate Gradient Method
4.2.4 Additional Methods
5 Application of Liposomes
6 Concluding Remarks
References
2 Preparation of Drug Liposomes by Thin-Film Hydration and Homogenization
1 Introduction
1.1 Thin-Film Hydration
1.2 Homogenization
1.2.1 Sonication
1.2.2 Extrusion
2 Materials
2.1 Preparation of DTX-Encapsulating Liposomes
2.2 Preparation of siRNA-Encapsulating Liposomes
3 Methods
3.1 Preparation of DTX-Encapsulating Liposomes
3.1.1 Thin Film Hydration-MLVs Formation
3.1.2 Homogenization (Sonication)-SUVs Formation
3.2 Preparation of siRNA-Incorporating Liposomes
3.2.1 Thin Film Hydration- MLVs Formation
3.2.2 Homogenization (Extrusion)-SUVs Formation
4 Notes
5 Conclusion
References
3 Preparation of Drug Liposomes by Reverse-Phase Evaporation
1 Introduction
2 Materials
2.1 Chemicals
3 Methods
3.1 Preparation of Liposomes by Reverse-Phase Evaporation
3.2 The Determination of Encapsulation Efficiency (%)
3.3 Observation of Particle Size and Electron Micrograph
3.4 Measurement of Surface Charge
3.5 Diffusional Exchange Measurements
4 Notes
5 Conclusion
References
4 Preparation and Characterization of Drug Liposomes by pH-Gradient Method
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Liposomes Loaded by Fluorescent Probes or Adriamycin
3.2 Fluorescence and Release Measurements
3.3 Data Analysis
4 Notes
References
5 Preparation and Characterization of Drug Liposomes by Ammonium Sulfate Gradient
1 Introduction
2 Materials and Apparatuses
2.1 Synthesis of Targeting Molecule (DSPE-PEG2000-PTDHIV-1 Conjugate)
2.2 Preparation of the Liposomes
2.3 Determination of Epirubicin and Celecoxib
2.4 Characterization of the Targeting Epirubicin Plus Celecoxib Liposomes
2.5 Cell Cultures
2.6 Evaluations on Breast Cancer Cells
2.7 Evaluations on Breast Cancer Spheroids
2.8 Evaluations on Breast Cancer Bearing Nude Mice
2.9 Statistics
3 Procedures and Methods
3.1 Synthesis of DSPE-PEG2000-PTDHIV-1 Conjugate
3.2 Preparation of the Liposomes
3.2.1 Preparation of Targeting Celecoxib Liposomes
3.2.2 Preparation of Targeting Epirubicin Plus Celecoxib Liposomes
3.2.3 Preparation of Epirubicin Liposomes
3.2.4 Preparation of Targeting Epirubicin Liposomes
3.2.5 Preparation of Fluorescence Labeling Liposomes
3.3 Determination of Epirubicin and Celecoxib by HPLC
3.3.1 Methodology of the HPLC Determination
3.3.2 EE Determination of Epirubicin and Celecoxib in the Liposomes
3.4 Characterization with DLS
3.5 Morphology Characterization with AFM and TEM
3.6 In Vitro Release of Epirubicin and Celecoxib
3.7 Cell Cultures
3.8 Evaluations on Breast Cancer Cells
3.8.1 Cytotoxic Effects on Breast Cancer Cells
3.8.2 Cellular Uptakes in Breast Cancer Cells
3.8.3 Targeting Effects on Breast Cancer Cells
3.9 Evaluations on Breast Cancer Cell Spheroids
3.9.1 Establishing Multicellular Cancer Cell Spheroids
3.9.2 Penetrating Effects in Breast Cancer Cell Spheroids
3.9.3 Destructing Effects on the Breast Cancer Spheroids
3.10 Evaluations on Breast Cancer-Bearing Nude Mice
3.10.1 Establishing Breast Cancer-Bearing Nude Mice Models
3.10.2 Anticancer Evaluations of the Targeting Liposomes on Nude Mice
3.10.3 In Vivo Imaging in Mice
4 Notes
References
6 Preparation of Drug Liposomes by EDTA Gradient Methods
1 Introduction
2 Materials
2.1 Materials for Preparation of Ammonium EDTA Solution
2.2 Materials for Preparation of Empty Liposomes
2.3 Materials for Preparation of EDTA Gradient Empty Liposomes
2.4 Materials for Preparation of Drug-Loaded EDTA Gradient Liposomes
2.5 Materials for Determination of Lipid Concentration Using a Phosphate Assay
3 Methods
3.1 Doxorubicin-Loaded EDTA Gradient Liposomes (Song et al. 2014)
3.1.1 Preparation of Ammonium EDTA Solution (See Note 1)
3.1.2 Preparation of Empty Liposomes (Modified Ethanol Injection Method) (See Note 3)
3.1.3 Preparation of EDTA Gradient Empty Liposomes (Dialyzed Method) (See Note 10)
3.1.4 Doxorubicin-Loaded EDTA Gradient Liposomes
3.2 Preparation of Topotecan-Loaded EDTA Gradient Liposomes (Yang et al. 2012)
3.2.1 Preparation of Ammonium EDTA Solution
3.2.2 Preparation of Empty Liposomes (Modified Ethanol Injection Method)
3.2.3 Preparation of EDTA Gradient Empty Liposomes (Anion and Cation Mixed Ion-Exchange Resin Mini-Column Method)
3.2.4 Preparation of Topotecan-Loaded EDTA Gradient Liposomes
3.3 Epirubicin-Loaded EDTA Gradient Liposomes (Yang et al. 2014)
3.3.1 Preparation of Ammonium EDTA Solution
3.3.2 Preparation of Empty Liposomes (Modified Ethanol Injection Method)
3.3.3 Preparation of EDTA Gradient Empty Liposomes (Sephadex G-50 Mini-Column Method)
3.3.4 Preparation of Epirubicin-Loaded EDTA Gradient Liposomes
3.4 Idarubicin-Loaded EDTA Gradient Liposomes (Gubernator et al. 2010)
3.4.1 Preparation of Diammonium EDTA Solution
3.4.2 Preparation of Empty Liposomes (Thin Lipid Film Method)
3.4.3 Preparation of EDTA Gradient Empty Liposomes (Sephadex G-50 Mini-Column Method)
3.4.4 Preparation of Idarubicin-Loaded EDTA Gradient Liposomes
3.5 Determination of Lipid Concentration Using a Phosphate Assay (Torchilin and Weissig 2003)
3.5.1 Preparation of Solutions
3.5.2 Determination of Lipid Concentration
4 Notes
5 Conclusion
References
7 Lipid-Coated Cisplatin Nanoparticles for Insoluble Drug Loading
1 Introduction
2 Materials
2.1 Materials
2.2 Synthesis of cis-[Pt(NH3)2(H2O)2](NO3)2 Precursor
2.3 Preparation of LPC NPs
2.4 Characterization of Particle Size and Zeta Potential
2.5 Transmission Electron Microscopy (TEM)
2.6 Characterization of Drug-Loading Capacity
2.7 In Vitro Release of Drug from NPs in Medium
2.8 In Vitro Release of Drug from NPs in Cells
3 Methods
3.1 Synthesis of Cis-[Pt(NH3)2(H2O)2](NO3)2 Precursor
3.2 Preparation of LPC NPs
3.3 Characterization of Particle Size and Zeta Potential
3.4 Transmission Electron Microscopy (TEM)
3.5 Characterization of Drug-Loading Capacity
3.6 In Vitro Release of Drug from NPs in Medium
3.7 In Vitro Release of Drug from NPs in Cells
4 Notes
5 Conclusion
References
8 Purification Method of Drug-Loaded Liposome
1 Introduction
2 Dialysis
3 Column Chromatographic Separation Method
4 Centrifugation
5 Microcentrifugation
6 Ion-Exchange Resin
7 Ultrafiltration
8 Protamine Aggregation Method
9 Others
10 Conclusion
References
9 Quality Evaluation of Drug-Loaded Liposomes
1 Introduction
2 Particle Size
3 Size Distribution
4 Morphology
5 Zeta Potential
6 Entrapment Efficiency
6.1 Gel Column Chromatography
6.2 Dialysis
6.3 Ultracentrifugation
6.4 Ultrafiltration
6.5 Protamine Aggregation Method
7 In Vitro Release
8 Osmotic Pressure
9 Sterilization
10 Oxidation Index
11 Lysophospholipid
12 Drug Contents
13 Leakage Rate
14 Stability
15 Pharmacokinetics and Pharmacology
16 Assessment of Efficacy
17 Safety and Toxicity
18 Conclusion
References
Part II: Functionalized Liposome-Based Drug Delivery Systems and Potential Application
10 Coupling Methods of Antibodies and Ligands for Liposomes
1 Introduction
1.1 Coupling Methods
1.2 Targeted Antibody-Drug Conjugates
1.3 Targeted Drug Liposomes
2 Materials
2.1 ADC Conjugates
2.1.1 Preparation of Conjugate P/PEG(5%)GH-DOX (Rameshwar and Jose 2012)
2.1.2 Preparation of mAb-taxoid Conjugates (Ojima and Geng 2002)
2.1.3 Preparation of cAC10-Valine-Citrulline-MMAE (Russell and Sanderson 2004)
2.1.4 Preparation of β-Galactosidase-Sensitive Antibody Drug Conjugates (Sergii and Chloe 2017)
2.2 Preparation of Mitochondrial Targeting Topotecan-Loaded Liposomes (Yu et al. 2012)
3 Methods
3.1 ADC Conjugates
3.1.1 Preparation of Conjugate P/PEG(5%)GH-DOX (Fig. 9, Notes 1, 2, 3)
Preparation of Conjugate P/PEG(5%)GH
Preparation of Conjugate P/PEG(5%)GH-DOX(5%)
3.1.2 Preparation of mAb-taxoid Conjugates (Fig. 10, Note 4)
Preparation of 3′-Dephenyl-3′-(2-methylprop-1-enyl)-10-(3-methyldisulfanylopropa- noyl-docetaxel)
Preparation of Antibody-Taxoid Conjugates
3.1.3 Preparation of cAC10-Valine-Citrulline-MMAE (Fig. 11, Notes 5, 6)
Preparation of Valine-Citrulline-MMAE
Preparation of cAC10-Valine-Citrulline-MMAE
3.1.4 Preparation of β-Galactosidase-Sensitive Antibody Drug Conjugates (Figs. 12 and 13, Notes 7, 8)
Preparation of Payloads
Preparation of ADCs
3.2 Preparation of Mitochondrial Targeting Topotecan-Loaded Liposomes (Yu et al. 2012)
4 Notes
5 Summary
References
11 Preparation and Evaluation of Folate Receptor Mediated Targeting Liposomes
1 Overview
2 Protocol
2.1 Materials
2.1.1 Synthesis of DPPE-PEG2000- Folate
2.1.2 Characterization of DPPE-PEG2000-Folate
2.1.3 Preparation of Doxorubicin Liposomes
2.1.4 Characterization of Folate-Targeted Doxorubicin Liposomes
2.1.5 Cellular Uptake Evaluation of Folate-Targeted Doxorubicin Liposomes
2.2 Rationale and Procedures
2.2.1 Synthesis of DPPE-PEG2000-Folate
2.2.2 Characterization of DPPE-PEG2000-Folate
2.2.3 Preparation of Folate-Targeted Doxorubicin Liposomes
Liposome Formation
Micellization of DPPE-PEG2000-Folate and Insert the Micelle into Preformed Liposomes
Folate-Targeted Doxorubicin Liposomes
2.2.4 Characterization of Folate-Targeted Doxorubicin Liposomes
2.2.5 Cellular Uptake Evaluation of Folate-Targeted Doxorubicin Liposomes
3 Notes
References
12 Preparation of Cell Penetrating Peptides-Mediated Targeting Drug Liposomes
1 Introduction
2 Materials
2.1 Materials
2.2 Synthesis of DSPE-PEG2000-R8
2.3 Preparation of Liposomes
2.4 Cell Culture, Cellular Uptake, and Cytotoxicity
3 Rationale and Procedures
3.1 Synthesis of DSPE-PEG2000-R8
3.2 Preparation of Liposomes
3.3 Determination of Particle Size and Zeta Potential
3.4 Morphological Examination of Liposomes
3.5 Stability of Liposomes in Serum
3.6 PTX Release Study
3.7 Cellular Uptake Study
3.8 Cytotoxicity Study In Vitro
4 Notes
5 Conclusion
References
13 Preparation of Multifunctional Paclitaxel Liposomes for Treatment of Brain Glioma
1 Introduction
2 Materials
2.1 Synthesis of Targeting Molecule Conjugates
2.1.1 MAN-TPGS1000 (See Note 1)
2.1.2 DQA-PEG2000-DSPE (See Note 2)
2.2 Preparation of Multifunctional Paclitaxel Liposomes
2.3 Measurement of Encapsulation Efficiency of Paclitaxel and Artemether
2.4 Measurement of Particle Size, Zeta Potential, and Polydispersity Index (PDI)
2.5 Morphology Characterization with Atomic Force Microscope (AFM)
2.6 In Vitro Release of Paclitaxel or Artemether
2.7 Cell Culture
2.8 Cytotoxic Effects on Brain Cancer Cells and Brain CSCs
2.9 Transport Across the BBB and Targeting of Brain Cancer Cells
2.10 In Vivo Imaging in Mice
2.11 Anticancer Efficacy in Rats
3 Methods
3.1 Synthesis of Targeting Molecule Conjugates
3.1.1 Synthesis of MAN-TPGS1000 Conjugate
3.1.2 Synthesis of DQA-PEG2000-DSPE Conjugate
3.2 Method for Determination of Paclitaxel and Artemether
3.2.1 Linearity of Paclitaxel
3.2.2 Linearity of Artemether
3.2.3 HPLC Method for Measurement of Paclitaxel or Artemether
3.3 Preparation of Multifunctional Paclitaxel Liposomes by Thin-Film Hydration
3.4 Measurement of Encapsulation Efficiency of Paclitaxel or Artemether
3.5 Measurement of Particle Size, Zeta Potential, and Polydispersity Index (PDI)
3.6 Morphology Characterization with AFM
3.7 In Vitro Release of Paclitaxel or Artemether
3.8 Cell Culture
3.9 Cytotoxic Effects on Brain Cancer Cells or Brain CSCs
3.9.1 Cytotoxic Effects on Brain Cancer Cells
3.9.2 Cytotoxic Effects on Brain CSCs
3.10 Transport Across the BBB and Targeting of Brain Cancer Cells
3.11 In Vivo Imaging in Mice
3.12 Anticancer Efficacy in Rats
4 Notes
5 Conclusion
References
14 Preparation and Evaluation of Integrin Receptor-Mediated Targeting Drug Liposomes
1 Introduction
2 Materials
2.1 Synthesis of RGD Peptide
2.2 Preparation of RGD Conjugated Polyethylene Glycol-Lipid (RGD-PEG-DSPE)
2.3 Characterization of RGD-PEG-DSPE
2.4 Preparation of RGD-Modified Liposome
2.4.1 Preparation of RGD-Modified Liposome Loaded with Doxorubicin (RGD-LS/DOX)
2.4.2 Preparation of RGD-Modified Liposome Loaded with pDP (RGD-LS/pDP)
2.5 Characterization of RGD-Modified Liposomes
2.6 Tumor Targeting Ability of RGD-Modified Liposomes
2.6.1 Cellular Uptake
Preparation of RGD-Modified Liposome Loaded with 5-Carboxyfluorescein (RGD-LS/FAM)
Uptake by Glioblastoma Cells
2.6.2 In Vivo Tumor Targeting Ability
Preparation of RGD-Modified Liposome Loaded with Near-Infrared Fluorescent Dyes (RGD-LS/Dir)
In Vivo Distribution
2.7 Antitumor Activity of RGD-Modified Liposomes
2.7.1 In Vitro Antitumor Activity
2.7.2 In Vivo Antiglioblastoma Efficacy
3 Methods
3.1 Synthesis of RGD Peptide
3.2 Preparation of RGD-PEG-DSPE
3.3 Characterization of RGD-PEG-DSPE
3.4 Preparation of RGD-Modified Liposomes
3.4.1 Preparation of RGD-LS/DOX
3.4.2 Preparation of RGD-LS/pDP
3.5 Characterization of RGD-Modified Liposomes
3.5.1 Physicochemical Characterization of RGD-Modified Liposomes
3.5.2 Characterization of Conjugated RGD on the Surface of Liposomes
3.5.3 DOX Encapsulation Efficacy
3.5.4 pDP Encapsulation Efficacy
3.6 Tumor Targeting Ability of RGD-Modified Liposomes
3.6.1 Cellular Uptake
Preparation of RGD-Modified Liposome Loaded with 5-Carboxyfluorescein (RGD-LS/FAM)
Uptake by Glioblastoma Cells
Cell Culture
Observation Using Confocal Laser Microscope
3.6.2 In Vivo Tumor Targeting Ability
Preparation of RGD-Modified Liposome Loaded with Near-Infrared Fluorescent Dyes (RGD-LS/Dir)
In Vivo Distribution
Establishment of Intracranial Glioma Model
3.7 Antitumor Activity of RGD-Modified Liposomes
3.7.1 In Vitro Antitumor Activity
3.7.2 In Vivo Antiglioblastoma Efficacy
4 Notes
5 Conclusion
References
15 Preparation of Functional Vincristine Liposomes for Treatment of Invasive Breast Cancer
1 Introduction
2 Materials
2.1 Synthesis of Targeting Molecule
2.2 Preparation of the Liposomes
2.3 Determination of Vincristine and Dasatinib
2.4 Characterization of the Liposomes
2.5 Cell Cultures
2.6 Evaluations on Invasive Breast Cancer Cells
2.7 Evaluations on Invasive Breast Cancer Spheroids
2.8 Evaluations on Breast Cancer-Bearing Nude Mice
3 Methods
3.1 Synthesis of Targeting Molecule
3.2 Preparation of the Liposomes
3.2.1 Preparation of Functional Vincristine Plus Dasatinib Liposomes
3.2.2 Preparation of Vincristine Plus Dasatinib Liposomes
3.2.3 Preparation of Vincristine Liposomes
3.2.4 Preparation of Dasatinib Liposomes
3.2.5 Preparation of Functional Liposomes
3.2.6 Preparation of Fluorescence Labeling Liposomes
3.3 Determination of Vincristine and Dasatinib
3.3.1 Linearity of Vincristine
3.3.2 Linearity of Dasatinib
3.3.3 Precision of Vincristine or Dasatinib
3.3.4 Detection Limits of Vincristine or Dasatinib
3.4 Characterization of the Liposomes
3.4.1 Encapsulation Efficiency Determination of Drug-Loaded Liposomes
3.4.2 Characterization with Dynamic Light Scatter
3.4.3 Morphology Characterization with AFM
3.4.4 In Vitro Release of Vincristine or Dasatinib
3.5 Cell Cultures
3.6 Evaluations on Invasive Breast Cancer Cells
3.6.1 Cellular Uptakes in Breast Cancer Cells
3.7 Evaluations on Invasive Breast Cancer Spheroids
3.7.1 Construction of Multicellular Cancer Spheroids
3.7.2 Penetrating Effect in Breast Cancer Spheroids
3.7.3 Destructing Effect on the Breast Cancer Spheroids
3.8 Evaluations on Breast Cancer-Bearing Nude Mice
3.8.1 Establishing Breast Cancer-Bearing Nude Mice Models
3.8.2 Anticancer Evaluations of the Functional Liposomes on Nude Mice
3.8.3 In Vivo Imaging in Mice
3.9 Statistics
4 Notes
5 Conclusion
References
16 Preparation and Characterization of DNA Liposomes Vaccine
1 Introduction
2 Materials
2.1 Preparation of Liposomes
2.2 Preparation of Plasmid DNA Liposome Vaccine
2.3 Encapsulation Efficiency of DNA Liposome Vaccine
2.4 Size Reduction of DNA Liposome Vaccine
3 Methods (Gregoriadis et al. 1999)
3.1 Preparation of MLV and SUV
3.1.1 Preparation of Phospholipid Membranes
3.1.2 Preparation of MLV
3.1.3 Preparation of SUV
3.2 Encapsulation of Plasmid DNA by Liposomes
3.2.1 Dehydration of SUV After Encapsulating Plasmid DNA
3.2.2 Rehydration of the Freeze-Dried Material
3.2.3 Depletion of Free DNA
3.3 Encapsulation Efficiency of DNA in the Liposomes
3.4 Reduction of DRV Liposomes Size
3.4.1 Reduction of DRV Liposomes Size by a Microfluidizer
3.4.2 Preparation of Small DNA Liposomes
3.4.3 Separation of Free DNA
3.4.4 Characterization of DNA Content and Properties of DRVs After Microfluidization
3.4.5 An Example of DNA Liposomes Vaccine
4 Notes
5 Conclusions
References
17 Preparation and Evaluation of Biomineral-Binding Antibiotic Liposomes
1 Introduction
2 Materials
2.1 Preparation of Biomineral-Binding Lipid ALN-TEG-Chol
2.2 Preparation and Characterization of Biomineral-Binding Liposomes (BBL)
2.3 Binding Kinetics of Biomineral-Binding Liposomes (BBL) on HA
2.4 In Vitro Oxacillin Release from Biomineral-Binding Liposomes (BBL)
2.5 In Vitro Inhibition of S. aureus Biofilm Growth Using Oxacillin-Loaded Biomineral-Binding Liposomes (BBL) Staphylococcus a...
3 Methods
3.1 Preparation of Biomineral-Binding Lipid ALN-TEG-Chol (Fig. 2, Note 1)
3.1.1 Preparation of Azido-Terminated Cholesterol (Azido-TEG-Chol, Compound 5) (Note 2)
3.1.2 Preparation of Acetylene-Terminated Alendronate (Acetylene ALN, Compound 7) (Note 3)
3.1.3 Preparation of Biomineral-Binding Alendronate-Cholesterol Lipid (ALN-TEG-Chol, Compound 8) Using Click Reaction (Note 4)
3.2 Preparation and Characterization of Biomineral-Binding Liposomes (BBL) and Non-Binding Liposome (NBL)
3.2.1 Preparation of Oxacillin-Loaded Liposomes by Extrusion Method (Gabizon et al. 2003; Note 5-8)
3.2.2 Preparation of Oxacillin-Loaded Liposomes by Sonication Method (Note 9, 10)
3.3 Binding Kinetics of Biomineral-Binding Liposomes (BBL) on HA
3.4 In Vitro Oxacillin Release from Biomineral-Binding Liposomes (BBL)
3.5 In Vitro Inhibition of S. aureus Biofilm Growth Using Oxacillin-Loaded Biomineral Binding Liposomes (BBL) (Note 11)
3.6 Statistical Analysis
4 Notes
5 Conclusion
References
18 Dual-Modified siRNA-Loaded Liposomes for Prostate Cancer Therapy
1 Introduction
2 Materials
2.1 Synthesis of DSPE-PEG2000-PRP
2.2 Synthesis of DSPE-PEG5000-Folate
2.3 Preparation and Characterization of siRNA-Loaded Liposomes
2.4 Gel Electrophoresis
2.5 Cell Culture
2.6 Cellular Uptake and Flow Cytometric Analysis
2.7 Cell Apoptosis Assay
2.8 Analytical Instruments
3 Methods
3.1 Synthesis of DSPE-PEG2000-PRP (See Note 2)
3.2 Synthesis of DSPE-PEG5000-Folate (See Note 5)
3.3 Preparation and Characterization of siRNA-Loaded Liposomes (See Note 8)
3.4 Gel Electrophoresis
3.5 Cell Culture
3.6 Cellular Uptake and Flow Cytometric Analysis
3.7 Cell Apoptosis Assay
4 Notes
5 Conclusion
References
19 Fabrication and Evaluation of Dual Peptides-Modified Liposomes Coencapsulating siRNA and Docetaxel
1 Introduction
2 Materials
2.1 Synthesis of DSPE-PEG2000-Angiopep
2.2 Synthesis of DSPE-PEG2000- tLyP-1
2.3 Preparation of Dual Peptides-Modified Liposomes Coencapsulating siRNA and Docetaxel
2.4 Characterization of Dual Peptides-Modified Liposomes Coencapsulating siRNA and Docetaxel
2.5 Gel Electrophoresis
2.6 Determination of Entrapment Efficiency of DTX
2.7 Antiproliferation Study
2.8 Analytical Instruments
3 Methods
3.1 Synthesis of DSPE-PEG2000-Angiopep (see Note 1)
3.2 Synthesis of DSPE-PEG2000-tLyP-1(see Note 1)
3.3 Preparation of Liposomes Coencapsulating siRNA and Docetaxel
3.4 Characterization of Liposomes
3.5 Gel Electrophoresis
3.6 Determination of Entrapment Efficiency of DTX
3.7 In Vitro Antiglioblastoma Efficacy
3.7.1 Cell Culture
3.7.2 Antiproliferation Study
4 Notes
5 Conclusion
References
20 Preparation and Evaluation of Rivastigmine Liposomes for Intranasal Delivery
1 Overview
2 Protocol
2.1 Materials
2.1.1 Preparation of Rivastigmine Liposomes
2.1.2 Synthesis of DSPE-PEG2000-CPP
2.1.3 Preparation of CPP-Modified Rivastigmine Liposomes
2.1.4 Characterization of Liposomes
2.1.5 Determination of Entrapment Efficiency of Rivastigmine
2.1.6 In Vitro Release of Liposomes
2.1.7 The BBB Model and Transport Across the BBB
2.1.8 Distribution of Rivastigmine Liposomes
2.1.9 Pharmacodynamic
2.1.10 Analytical Instruments
2.2 Methods
2.2.1 Preparation of Rivastigmine Liposomes
2.2.2 Synthesis of DSPE-PEG2000-CPP
2.2.3 Preparation of CPP-Modified Rivastigmine Liposomes
2.2.4 Characterization of Liposomes
2.2.5 Determination of Entrapment Efficiency of Rivastigmine
2.2.6 In Vitro Release of Liposomes
2.2.7 The BBB Model and Transport Across the BBB
2.2.8 Distribution of Rivastigmine Liposomes in the Brain
2.2.9 Pharmacodynamic Study of Liposomes
3 Discussion
References
21 Dequalinium-Mediated Mitochondria-Targeting Drug Liposomes for the Treatment of Drug-Resistant Lung Cancer
1 Introduction
2 Materials
2.1 Synthesis of DQA-PEG2000-DSPE Conjugate
2.2 Preparation of Targeting Liposomes
2.3 Characterization of Liposomes
2.4 Cell Culture
2.5 Cytotoxicity
2.6 Targeting Mechanism and Effect
2.6.1 Mitochondrial Co-localization
2.6.2 Drug Content in Mitochondria
2.6.3 Mitochondrial Depolarization
2.6.4 Release of Cytochrome C from Mitochondria
2.6.5 Caspase Activation
2.6.6 Effects on Pro-apoptotic Bax and Anti-apoptotic Mcl-1
2.6.7 ROS Assay
2.6.8 ATP Assay
2.7 Efficacy in Drug-Resistant Lung Cancer Xenografts
2.8 In Vivo Imaging Observation
3 Methods
3.1 Synthesis of DQA-PEG2000-DSPE Conjugate
3.2 Preparation of Liposomes
3.2.1 Preparation of Targeting Lonidamine Liposomes
3.2.2 Preparation of Lonidamine Liposomes
3.2.3 Preparation of Targeting Coumarin Liposomes (See Note 3)
3.2.4 Preparation of Coumarin Liposomes
3.2.5 Preparation of Targeting Epirubicin Liposomes
3.2.6 Preparation of Epirubicin Liposomes
3.3 Characterization of Liposomes
3.4 Cell Culture
3.5 Cytotoxicity
3.6 Targeting Mechanism and Effect
3.6.1 Mitochondrial Co-localization
3.6.2 Drug Content in Mitochondria
3.6.3 Mitochondrial Depolarization
Measure the Mitochondria Membrane Potential (DeltaPsim)
Confirm the Specificity of Induced Mitochondrial Depolarization by Targeting Lonidamine Liposomes or Targeting Epirubicin Lipo...
3.6.4 Release of Cytochrome C from the Mitochondria
3.6.5 Caspase Activation
3.6.6 Effects on Pro-apoptotic Bax and Anti-apoptotic Mcl-1
3.6.7 ROS Assay
3.6.8 ATP Assay
3.7 Efficacy in Drug-Resistant Lung Cancer Xenografts
3.8 In Vivo Imaging Observation
3.9 Statistical Analysis
4 Notes
5 Conclusion
References
22 Preparation of Anthracyclines Liposomes for Tumor-Targeting Drug Delivery
1 Introduction
2 Materials
2.1 Preparation of Liposomes by pH Gradient Method
2.2 Preparation of Liposomes Modified with MAN
2.3 Daunorubicin Liposomes Modified with MAN and TF
2.4 Determination of Encapsulation Efficiency, Drug Release, and Particle Size
2.5 Determination of Morphology
2.6 Cell Culture
2.7 BBB Model In Vitro
2.8 Transport Across the BBB and Competition Assay of MAN
2.9 C6 Glioma Cellular Uptake and Competition Assay of TF
2.10 Antiproliferative Activity Against C6 Glioma Cells
2.11 Dual-Targeting Effects In Vitro
2.12 Effect on the Avascular C6 Glioma Spheroids
2.13 Effects on the Survival of Brain Tumor-Bearing Animals
2.14 Measurement of Daunorubicin in Plasma
2.15 Measurement of Daunorubicin in Tissues
2.16 Pharmacokinetics and Biodistribution
3 Methods
3.1 Preparation and Characterization of the Liposomes
3.1.1 Blank Liposomes
3.1.2 Daunorubicin Liposomes (Note 1)
3.1.3 Daunorubicin Liposomes Modified with MAN (Note 2)
3.1.4 Daunorubicin Liposomes Modified with MAN and TF (Note 3)
3.1.5 Daunorubicin Liposomes Modified with TF (Note 3)
3.1.6 Encapsulation Efficiency (Note 4)
3.1.7 Drug Release (Note 5)
3.1.8 Particle Size (Note 6)
3.1.9 Determination of the Morphology Under TEM (Note 6)
3.1.10 Determination of the Morphology Under AFM (Note 6)
3.2 Cell Culture
3.3 BBB Model In Vitro (Note 7)
3.4 Transport Across the BBB and Competition Assay of MAN
3.5 C6 Glioma Cellular Uptake Assay of TF (Note 8)
3.6 Competition Assay of TF
3.7 Antiproliferative Activity Against C6 Glioma Cells (Note 9)
3.8 Dual-Targeting Effects In Vitro (Note 10)
3.9 Effect on the Avascular C6 Glioma Spheroids (Note 11)
3.10 Effects on the Survival of Brain Tumor-Bearing Animals (Note 12)
3.11 Measurement of Daunorubicin in Plasma (Note 13)
3.12 Measurement of Daunorubicin in Tissues (Note 14)
3.13 Pharmacokinetics and Biodistribution (Note 15)
3.14 Statistical Analysis
4 Notes
5 Conclusion
References
23 Preparation and Characterization of pH Sensitive Drug Liposomes
1 Introduction
2 Materials
2.1 Synthesis of Zwitterionic Oligopeptide Lipid (e.g., 1,5-Dioctadecyl-L-Glutamyl 2-Histidyl-Hexahydrobenzoic Acid, HHG2C18)
2.2 Preparation of pH-Sensitive Drug Liposomes by Thin-Film Dispersion Methods (HHG2C18-L)
2.3 Preparation of Conventional Drug Liposomes by Thin-Film Dispersion Methods (SPC-L)
2.4 Determination of Particle Size
2.5 Determination of Zeta Potential
2.6 Determination of the Buffering Capacity
2.7 Determination of the Degradation of Hexahydrobenzoic Amide from the HHG2C18-L
2.8 Determination of Zeta Potential Variation of the HHG2C18-L with the Degradation of the Amide
2.9 Cell Culture
2.10 Cellular Uptake of the HHG2C18-L Under Different pH Values
2.11 Qualitatively Evaluation of Endolysosomal Escape and Mitochondrial Targeting of the HHG2C18-L by Confocal Laser Scanning ...
3 Methods
3.1 Synthesis of Zwitterionic Oligopeptide Lipid (e.g., HHG2C18)
3.2 Preparation of pH-Sensitive Drug Liposomes by Thin-Film Dispersion Methods (HHG2C18-L)
3.3 Preparation of Conventional Drug Liposomes by Thin-Film Dispersion Methods (SPC-L)
3.4 Determination of Particle Size
3.5 Determination of Zeta Potential
3.6 Determination of the Buffering Capacity
3.7 Determination of the Degradation of Hexahydrobenzoic Amide from the HHG2C18-L
3.8 Determination of Zeta Potential Variation of the HHG2C18-L with the Degradation of the Amide
3.9 Cell Culture
3.10 Cellular Uptake of the HHG2C18-L Under Different pH Values
3.11 Qualitatively Evaluation of Endolysosomal Escape and Mitochondrial Targeting of the HHG2C18-L by CLSM
4 Notes
5 Conclusion
References
24 HER2-Specific PEGylated Immunoliposomes Prepared by Lyophilization/Rehydration Method
1 Introduction
2 Materials
2.1 Reagents
2.2 Preparation of Lyophilized PEGylated Immunoliposomes (LPIL)
2.3 Determination of Particle Size and Zeta Potential
2.4 Flow Cytometry
2.5 SiRNA Serum Stability
3 Methods
3.1 Preparation of LPIL
3.2 Encapsulation of siRNA into the Liposomes
3.3 Determination of Particle Size and Zeta Potential
3.4 Measurement of Transfection Efficiency
3.5 Measurement of Gene Silencing Efficiency
3.6 SiRNA Serum Stability
4 Notes
5 Conclusion
References
25 Preparation and Characterization of Drug Liposomes by Nigericin Ionophore
1 Introduction
2 Materials
2.1 Preparation of Ion Liposomes by Thin-Film Hydration
2.2 Formation of Ion Gradient
2.3 Loading of Vincristine or Topotecan and Measurement of Encapsulation Efficiency
2.4 Measurement of Particle Size, Zeta Potential, and Polydispersity Index (PDI)
2.5 Transmission Electron Microscopy (TEM)
2.6 In Vitro Release of Vincristine or Topotecan
2.7 Cell Culture
2.8 Cytotoxicity to MDA-MB231or MCF-7 Cells
2.9 In Vivo Inhibition of the Tumor Growth
2.10 Pharmacokinetics and Tissue Distribution
3 Methods
3.1 Preparation of Standard Curve of Vincristine
3.2 Preparation of Standard Curve of Topotecan
3.3 Linearity
3.4 HPLC Method for Measurement of Vincristine
3.5 HPLC Method for Measurement of Topotecan
3.6 Stability Testing of Vincristine or Topotecan
3.7 Precision of Vincristine or Topotecan
3.8 Experiment Design
3.9 Preparation of Vincristine Liposomes and Measurement of Encapsulation Efficiency
3.9.1 Preparation of K+ Liposomes by Thin-Film Hydration (Fig. 1, Note 1, 2, 3, 4, 5, and 6)
3.9.2 Formation of K+ Gradient (Note 7, 8, 9, and 10)
3.9.3 Loading of Vincristine and Measurement of Encapsulation Efficiency (Note 11 and 12)
3.10 Preparation of Topotecan Liposomes and Measurement of Encapsulation Efficiency (Note 1, 2, 3, 4, 5, and 6)
3.10.1 Preparation of Na+ Liposomes by Thin-Film Hydration (Fig. 2)
3.10.2 Formation of Na+ Gradient (Note 7, 8, and 10)
3.10.3 Loading of Topotecan and Measurement of Encapsulation Efficiency (Note 11 and 12)
3.11 Measurement of Particle Size, Zeta Potential, and Polydispersity Index (PDI, Note 6)
3.12 Transmission Electron Microscopy (TEM)
3.13 In Vitro Release of Vincristine or Topotecan (Note 20)
3.14 Cell Culture
3.15 Cytotoxicity to MDA-MB231 or MCF-7 Cells
3.16 Animal
3.17 In Vivo Inhibition of the Tumor Growth
3.18 Pharmacokinetics (Note 21, 22, and 23)
3.19 Tissue Distribution (Note 24)
3.20 Statistical Analysis
4 Notes
5 Conclusion
References
26 Application of Labeled Liposomes in Imaging and Biodistribution Observation
1 Introduction
2 Materials
2.1 Preparation of Liposomes Loaded with PET, MRI, and Optical Imaging Contrast
2.2 Loading of PET, MRI, and Optical Imaging Contrast and Encapsulation Efficiency
2.3 Measurement of Particle Size, Zeta Potential, and Polydispersity Index (PDI)
2.4 Transmission Electron Microscopy (TEM)
2.5 In Vitro Release
2.6 Cell Culture
2.7 Cytotoxicity to MDA-MB231, MDA-MB231-BR, or U87-Luc Cells
2.8 MRI Relaxivity
2.9 Establishment of the MDA-MB231 Tumor Model
2.10 Establishment of the Orthotopic Glioma Model
2.11 Establishment of the Breast Cancer Brain Metastasis Model
2.12 Bioluminescence Imaging
2.13 Optical Imaging
2.14 MRI
2.15 Pet/Ct
2.16 Imaging of Biodistribution
3 Methods
3.1 Preparation of Liposomes Loaded with PET, MRI, and Optical Imaging Contrast (Note1)
3.2 Loading of PET, MRI, and Optical Imaging Contrast and Encapsulation Efficiency (Note 2 and 3)
3.3 Measurement of Particle Size, Zeta Potential, and Polydispersity Index (PDI)
3.4 Transmission Electron Microscopy (TEM)
3.5 In Vitro Release
3.6 Cell Culture
3.7 Cytotoxicity to MDA-MB231, MDA-MB231-BR, or U87-Luc Cells
3.8 MRI Relaxivity (Note 4 and 5)
3.9 Animal
3.10 Establishment of the MDA-MB231 Tumor Model
3.11 Establishment of the Orthotopic Glioma Model
3.12 Establishment of the Breast Cancer Brain Metastasis Model (Note 6)
3.13 Bioluminescence Imaging (Note 7 and 8)
3.14 Optical Imaging (Fig. 1)
3.15 MRI (Note 9 and 10)
3.16 PET/CT Scan
3.17 Imaging of Biodistribution
3.18 Statistical Analysis
4 Notes
5 Conclusion
References
27 Application of Drug Liposomes in Gene Transfection
1 Introduction
2 Materials
2.1 Synthesis of MAL-PEG-DOPE
2.2 Preparation of the Liposomes
2.3 Characterization of the Liposomes
2.4 In Vitro Cell Assays
2.5 In Vivo Assays
3 Methods
3.1 Synthesis of MAL-PEG-DOPE (Fig. 2)
3.2 Preparation of the Liposomes
3.2.1 Preparation of DC-Chol/DOPE Liposomes
3.2.2 Preparation of siRNA/Liposome Complexes
3.2.3 Conjugation of AS1411 to the Surface of siRNA/Liposome Complexes
3.3 Characterization of the Liposomes
3.3.1 Confirm AS1411 Conjugation to Liposomes
3.3.2 Morphology of Liposomes
3.3.3 Particle Size and Zeta Potential
3.3.4 Evaluate the siRNA Loading Ability of Liposomes
3.4 In Vitro Cell Assays
3.4.1 Targeting Effects on Melanoma Cells
3.4.2 Cellular Uptakes in Melanoma Cells
3.4.3 Verify the Silencing Efficiency by Real-Time PCR
3.4.4 Assess the Downregulation of Protein by Western Blot Analysis
3.4.5 Cytotoxicity Assays
3.4.6 Antiproliferation Assays
3.5 In Vivo Assays
3.5.1 Establishing Melanoma Cancer-Bearing Nude Mice
3.5.2 Tissue Distribution Study
3.5.3 Tumor Uptake Study
3.5.4 Gene Silencing In Vivo
3.5.5 H&E Staining of Tumor Tissues
3.6 Statistical Analysis
4 Notes
References
28 Application of Drug Liposomes in the Hormone Therapy
1 Introduction
2 Materials and Apparatuses
2.1 Preparation of Dex-Loaded Liposomes (Dex-Lips)
2.2 Characterization of Dex-Loaded Liposomes (Dex-Lips)
2.3 In Vitro Release of Encapsulated Dex
2.4 Pharmacokinetic Studies of Dex-Lips in Healthy Rats
2.5 Establishment of the Adjuvant-Induced Arthritis (AIA) Rat Model
2.6 Biodistribution of DiD Loaded Liposomes (DiD-Lips) in Arthritic Rats
2.7 Therapeutic Efficacy of Dex-Lips In Vivo
2.8 Safety Evaluation in Arthritic Rats
2.9 Statistical Analysis
3 Methods
3.1 Preparation of Liposomes
3.1.1 Preparation of Dex-Loaded Liposomes (Dex-Lips)
3.1.2 Preparation of Fluorescence Labeling Liposomes
3.2 Characterization of Dex-Loaded Liposomes (Dex-Lips)
3.2.1 Size and Zeta Potential
3.2.2 Morphology Characterization with TEM
3.3 Encapsulation and Drug Loading Efficiencies
3.3.1 Methodology of the HPLC Determination
3.3.2 Determination of Encapsulation and Drug Loading Efficiencies
3.4 Stability of Dex-Lips
3.5 In Vitro Release of Encapsulated Dex
3.6 Pharmacokinetic Studies of Dex-Lips in Healthy Rats
3.7 Establishment of the Adjuvant-Induced Arthritis (AIA) Rat Model
3.8 Biodistribution of DiD Loaded Liposomes (DiD-Lips) in Arthritic Rats
3.9 Therapeutic Efficacy of Dex-Lips In Vivo
3.9.1 Paw Thickness Measurement in Dex-Treated Arthritic Rats
3.9.2 Cytokine Levels in Serum After Therapy
3.9.3 Histopathology of Ankle Joints from Arthritic Rats After Therapy
3.10 Safety Evaluation in Arthritic Rats
4 Notes
References
Index
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Biomaterial Engineering Series Editor: Youqing Shen
Wan-Liang Lu Xian-Rong Qi Editors
Liposome-Based Drug Delivery Systems
Biomaterial Engineering Series Editor Youqing Shen College of Chemical and Biological Engineering Zhejiang University Hangzhou, China
The use of human body compatible materials for medical diagnosis, treatment, and surgery is always critical in enhancing life-saving therapies and quality of life of patients. With the massive research efforts in this field in both universities and industries, there is a continuous growth in our knowledge/technique in design and use of such biomaterials to effectively deliver specific therapeutics, including DNA, protein, and contrast reagents, to the targeted cells and organs for treatment or diagnosis; to repair or replace tissues; or even to construct artificial organs like cartilages, bones, and skins to regain the body functions. The inherent interdisciplinary nature of biomaterials makes researchers often use methods to prepare a wide range of materials and various bioassays to evaluate their diverse bio-related properties. However, it is generally very difficult to find and tell reliable sources and detailed know-how for the needed methods and protocols, which are buried in the vast literature and only described in general lack of details. One still has to find the best by trial and error. The aim of this series is to provide a complete reference for researchers, engineers, and graduate students who are engaged in R&D of biomaterials. It provides newcomers the fundamental concepts and overall status of the areas they work on and provides researchers comprehensive, in-depth, and authoritative information including preparation/fabrication protocols of most biomaterials and bioassay protocols as well as a wide variety of advanced experimental methods used in practice, numerous examples, and practical applications. More information about this series at http://www.springer.com/series/13484
Wan-Liang Lu • Xian-Rong Qi Editors
Liposome-Based Drug Delivery Systems With 158 Figures and 17 Tables
Editors Wan-Liang Lu Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System Beijing, China
Xian-Rong Qi Department of Pharmaceutics School of Pharmaceutical Science Peking University Beijing, China Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System Beijing, China
ISSN 2523-8809 ISSN 2523-8817 (electronic) ISBN 978-3-662-49318-2 ISBN 978-3-662-49320-5 (eBook) ISBN 978-3-662-49319-9 (print and electronic bundle) https://doi.org/10.1007/978-3-662-49320-5 © Springer-Verlag GmbH Germany, part of Springer Nature 2021 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer-Verlag GmbH, DE part of Springer Nature. The registered company address is: Heidelberger Platz 3, 14197 Berlin, Germany
Series Preface
The use of human body compatible materials for medical diagnosis, treatment, and surgery is always critical in enhancing life-saving therapies and quality of life of patients. With the massive research efforts in this field in both universities and industries, there is a continuous growth in our knowledge/technique in design and use of such biomaterials to effectively deliver specific therapeutics, including DNA, protein, and contrast reagents, to the targeted cells and organs for treatment or diagnosis; to repair or replace tissues; or even to construct artificial organs like cartilages, bones, and skins to regain the body functions. The inherent interdisciplinary nature of biomaterials makes researchers often use methods to prepare a wide range of materials and various bioassays to evaluate their diverse bio-related properties. However, it is generally very difficult to find and tell reliable sources and detailed know-how for the needed methods and protocols, which are buried in the vast literature and only described in general lack of details. One still has to find the best by trial and error. The aim of this series is to provide a complete reference for researchers, engineers, and graduate students who are engaged in R&D of biomaterials. It provides newcomers the fundamental concepts and overall status of the areas they work on and provides researchers comprehensive, in-depth, and authoritative information including preparation/fabrication protocols of most biomaterials and bioassay protocols as well as a wide variety of advanced experimental methods used in practice, numerous examples, and practical applications. This reference work, tentatively composed of 10 volumes, covers the most popular topics of biomaterials, from drug delivery to gene and protein delivery, from biomedical biodetection to diagnosis, from cardiovascular diseases to tissue engineering. The content is created by leading scientists in each field and will evolve constantly, thus it presents both high-quality and up-to-date scientific and technical information. June, 2021
Youqing Shen
v
Volume Preface
Liposomes are the sealed vesicles that are generally formed by phospholipids, cholesterol, and other materials. Liposomes are not only a convenient platform for studying membrane kinetics and drug or biomolecule transport, but also a potential drug delivery carrier system. Liposomes have a unique role in overcoming biological barriers and carrying biological drugs. As a drug carrier, liposomes have a broad prospect in basic research and clinical application, such as delivering anticancer agents, anti-parasite drugs, hormones, and genes. This volume mainly describes the protocols for fabrication of liposomal drug delivery system, and consists of two parts. The first part emphasizes the basic protocols and concepts for fabrication of drug-loaded liposomes. For new investigators, laying the groundwork is the first step. This part focuses on the fabrication details in liposomes formulations, which are the so-called small tricks while usually not disclosed in most published research articles. However, these processing details are crucial for the preparation of liposomes. And the second part focuses on the strategies in modified and/or functionalized liposomes to adapt to pathophysiological environment, as well as their application in treating diseases. As new analytical and synthetic technologies become available, and improved understanding of pathophysiology, various functionalized drug liposomes are developed to fit the requirements of different therapeutic purposes, exhibiting a broad range of applications. Accordingly, the objectives of this part are aimed at deeply unearthing the formulation of drug-loaded liposomes, describing the detailed operation of liposomal formulation, and further demonstrating their potential applications. Therefore, this volume shows the features of experimental report or protocol for proving a guide to scientists, research students, and young investigators in pharmaceutical enterprises. Editorial members of this volume involve renowned experts and young scholars in the research field of liposomes, and some graduate students who have research experience in liposomes field also participate in the editing work. They are mainly from Peking University, Fudan University, Sichuan University, China Pharmaceutical University, Shenyang Pharmaceutical University, Second Military Medical University, Hebei Medical University, Beijing Institute of Petrochemical Technology, Shihezi University, Liaoning University of Traditional Chinese Medicine, Shanxi University of Traditional Chinese Medicine, Jilin Medical University, University of vii
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Volume Preface
North Carolina at Chapel Hill, University of Nebraska Medical Center, and Chapman University, as well as their affiliated hospitals. This volume is included in the reference series – Biomaterial Engineering, proposed by Prof. Youqing Shen. The volume is expected to be used by graduate students, postdoctoral researchers, and researchers using lipid-based systems in the fields of drug delivery, physical chemistry, and cellular and molecular biology. We are grateful to all the authors for their contributions. We are also very grateful to the series editor, Dr. Juby George, and the outstanding staff at Springer Nature for their support, help, and encouragement. School of Pharmaceutical Sciences Peking University June, 2021
Wan-Liang Lu Xian-Rong Qi
Contents
Part I
General Protocols for Fabrication of Drug Liposomes . . . . . .
1
1
Liposomes in Drug Delivery: Status and Advances . . . . . . . . . . . . Ying-Jie Hu, Rui-Jun Ju, Fan Zeng, Xian-Rong Qi, and Wan-Liang Lu
3
2
Preparation of Drug Liposomes by Thin-Film Hydration and Homogenization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bai Xiang and De-Ying Cao
25
3
Preparation of Drug Liposomes by Reverse-Phase Evaporation . . . Nian-Qiu Shi and Xian-Rong Qi
4
Preparation and Characterization of Drug Liposomes by pH-Gradient Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nian-Qiu Shi, Xian-Rong Qi, and Bai Xiang
47
Preparation and Characterization of Drug Liposomes by Ammonium Sulfate Gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rui-Jun Ju and Xue-Tao Li
59
5
6
Preparation of Drug Liposomes by EDTA Gradient Methods . . . . Yanzhi Song and Yihui Deng
7
Lipid-Coated Cisplatin Nanoparticles for Insoluble Drug Loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yang Liu and Leaf Huang
37
79
97
8
Purification Method of Drug-Loaded Liposome . . . . . . . . . . . . . . . Meng Lin and Xian-Rong Qi
111
9
Quality Evaluation of Drug-Loaded Liposomes . . . . . . . . . . . . . . . Meng Lin, Rudong Wang, and Xian-Rong Qi
123
ix
x
Contents
Part II Functionalized Liposome-Based Drug Delivery Systems and Potential Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
141
10
Coupling Methods of Antibodies and Ligands for Liposomes . . . . Ming Chen, Qiu-Ran Ma, and Wan-Liang Lu
11
Preparation and Evaluation of Folate Receptor Mediated Targeting Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wei Gao
167
Preparation of Cell Penetrating Peptides-Mediated Targeting Drug Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yang Wang and Qin He
179
Preparation of Multifunctional Paclitaxel Liposomes for Treatment of Brain Glioma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xiu-Ying Li
193
Preparation and Evaluation of Integrin Receptor-Mediated Targeting Drug Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fei Wang, Gang Wei, and Weiyue Lu
213
Preparation of Functional Vincristine Liposomes for Treatment of Invasive Breast Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fan Zeng
239
12
13
14
15
143
16
Preparation and Characterization of DNA Liposomes Vaccine . . . Ya-Fei Du, Ming Chen, Jia-Rui Xu, Qian Luo, and Wan-Liang Lu
17
Preparation and Evaluation of Biomineral-Binding Antibiotic Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xin-Ming Liu, Ke Ren, Geoffrey Wu, and Dong Wang
277
Dual-Modified siRNA-Loaded Liposomes for Prostate Cancer Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bai Xiang and De-Ying Cao
293
Fabrication and Evaluation of Dual Peptides-Modified Liposomes Coencapsulating siRNA and Docetaxel . . . . . . . . . . . . . . . . . . . . . . Zhenzhen Yang and Bai Xiang
309
Preparation and Evaluation of Rivastigmine Liposomes for Intranasal Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Zhenzhen Yang
325
Dequalinium-Mediated Mitochondria-Targeting Drug Liposomes for the Treatment of Drug-Resistant Lung Cancer . . . . . . . . . . . . Xue Ying
345
18
19
20
21
259
Contents
22
23
24
25
26
xi
Preparation of Anthracyclines Liposomes for Tumor-Targeting Drug Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xue Ying
365
Preparation and Characterization of pH Sensitive Drug Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Caoyun Ju and Can Zhang
385
HER2-Specific PEGylated Immunoliposomes Prepared by Lyophilization/Rehydration Method . . . . . . . . . . . . . . . . . . . . . . . . Jie Gao and Yanqiang Zhong
409
Preparation and Characterization of Drug Liposomes by Nigericin Ionophore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liang Zhang
423
Application of Labeled Liposomes in Imaging and Biodistribution Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liang Zhang
443
27
Application of Drug Liposomes in Gene Transfection . . . . . . . . . . Yao Xiao, Xin Wang, Min Fu, Jing-jing Liu, and Xue-tao Li
459
28
Application of Drug Liposomes in the Hormone Therapy . . . . . . . Yao Xiao, Xin Wang, Min Fu, Jing-jing Liu, and Xue-tao Li
475
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
489
About the Series Editor
Youqing Shen College of Chemical and Biological Engineering Zhejiang University Hangzhou, China Prof. Youqing Shen is a national Changjiang scholar chair professor in the College of Chemical and Biological Engineering and Deputy Dean of the Faculty of Engineering at Zhejiang University, China, and a fellow of the American Institute of Medical and Biological Engineering (AIMBE). He received his B.Sc. and Dr. Sc. degrees from Zhejiang University and Ph.D. Eng. degree from McMaster University in 2002. After working shortly at Akzo Nobel Canada, he joined the Department of Chemical Engineering at the University of Wyoming, Laramie, Wyoming, USA, as an assistant professor in 2002 and then was early promoted to tenured associate professor in 2007. In 2008, he moved back to Zhejiang University as a Qiushi chair professor and director of the Center for Bionanoengineering. His research, funded by the US and Chinese funding agencies, focuses on designing and synthesizing stimulusresponsive polymers as functional excipients for drug and gene nanomedicines. He has (co-)authored over 300 peer-reviewed articles and edited two books published by RSC and Wiley. Elsevier Scopus has listed him as a “Most Cited Chinese Scholar” in material science since 2014. He has received a number of awards, including the Distinguished Young Investigator Award from China National Natural Science Foundation and the Leading Young Innovator award from the Chinese Ministry of Science and Technology. He serves as editor-in-chief of Biomaterials Engineering, a Springer xiii
xiv
About the Series Editor
Nature book series, an associate editor for the American Chemical Society journal Industrial Engineering Chemistry Research, and an executive editor for Elsevier journal Advanced Drug Delivery Review. He is also vice chair of the nanomedicine committee in the Chinese Pharmaceutical Association and China Medicinal Biotechnology Association.
About the Volume Editors
Professor Dr. Wan-Liang Lu, a principal investigator in State Key Laboratory of Natural and Biomimetic Drugs, Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Systems, and School of Pharmaceutical Sciences, Peking University, Beijing, China
Professor Dr. Xian-Rong Qi, a principal investigator in Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Systems, Department of Pharmaceutics, and School of Pharmaceutical Sciences, Peking University, Beijing, China
xv
Contributors
De-Ying Cao Key Laboratory of Hebei Province for Innovative Drug Research and Evaluation, School of Pharmaceutical Sciences, Hebei Medical University, Shijiazhuang, China Ming Chen State Key Laboratory of Natural and Biomimetic Drugs, Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System, School of Pharmaceutical Sciences, Peking University, Beijing, China Yihui Deng College of Pharmacy, Shenyang Pharmaceutical University, Shenyang, China Ya-Fei Du State Key Laboratory of Natural and Biomimetic Drugs, Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System, School of Pharmaceutical Sciences, Peking University, Beijing, China Min Fu School of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian, China Jie Gao Department of Pharmaceutical Science, College of Pharmacy, The Second Military Medical University, Shanghai, China Wei Gao Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, West Lafayette, IN, USA Qin He Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Chengdu, Sichuan, China Ying-Jie Hu State Key Laboratory of Natural and Biomimetic Drugs, Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System, School of Pharmaceutical Sciences, Peking University, Beijing, China Leaf Huang Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Caoyun Ju Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Center of Drug Discovery, Academic Institute of Pharmaceutical Science, China Pharmaceutical University, Nanjing, China
xvii
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Contributors
Rui-Jun Ju Department of Pharmaceutical Engineering, Beijing Institute of Petrochemical Technology, Beijing, China Xiu-Ying Li Department of Traditional Chinese Materia Medica, Shanxi University of Chinese Medicine, Jinzhong, China Xue-tao Li School of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian, China Meng Lin Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System, School of Pharmaceutical Sciences, Peking University, Beijing, China Yang Liu Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Irvine, CA, USA Jing-jing Liu School of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian, China Xin-Ming Liu Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE, USA Wan-Liang Lu State Key Laboratory of Natural and Biomimetic Drugs, Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System, School of Pharmaceutical Sciences, Peking University, Beijing, China Weiyue Lu Department of Pharmaceutics, School of Pharmacy, Fudan University; Key Laboratory of Smart Drug Delivery, Ministry of Education (Fudan University), Shanghai, China Qian Luo State Key Laboratory of Natural and Biomimetic Drugs, Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System, School of Pharmaceutical Sciences, Peking University, Beijing, China Qiu-Ran Ma State Key Laboratory of Natural and Biomimetic Drugs, Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System, School of Pharmaceutical Sciences, Peking University, Beijing, China Xian-Rong Qi Department of Pharmaceutics, School of Pharmaceutical Science, Peking University, Beijing, China Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System, Beijing, China Ke Ren Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE, USA Nian-Qiu Shi Department of Pharmaceutics, School of Pharmacy, Jilin Medical University, Jilin, Jilin Province, China Yanzhi Song College of Pharmacy, Shenyang Pharmaceutical University, Shenyang, China
Contributors
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Dong Wang Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE, USA Fei Wang Department of Pharmaceutics, School of Pharmacy, Fudan University; Key Laboratory of Smart Drug Delivery, Ministry of Education (Fudan University), Shanghai, China Rudong Wang Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System, School of Pharmaceutical Sciences, Peking University, Beijing, China Xin Wang School of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian, China Yang Wang Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Chengdu, Sichuan, China Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Gang Wei Department of Pharmaceutics, School of Pharmacy, Fudan University; Key Laboratory of Smart Drug Delivery, Ministry of Education (Fudan University), Shanghai, China Geoffrey Wu Office of Lifecycle Drug Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA Bai Xiang Key Laboratory of Hebei Province for Innovative Drug Research and Evaluation, School of Pharmaceutical Sciences, Hebei Medical University, Shijiazhuang, China Yao Xiao School of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian, China Jia-Rui Xu State Key Laboratory of Natural and Biomimetic Drugs, Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System, School of Pharmaceutical Sciences, Peking University, Beijing, China Zhenzhen Yang Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery System, Department of pharmaceutics, School of Pharmaceutical Sciences, Peking University, Beijing, China Xue Ying School of Pharmaceutical Sciences/Key Laboratory of Sichuan Province for Specific Structure of Small Molecule Drugs, Chengdu Medical College, Chengdu, China Fan Zeng Beijing Neurosurgical Institute, Capital Medical University, Beijing, China Can Zhang Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Center of Drug Discovery, Academic Institute of Pharmaceutical Science, China Pharmaceutical University, Nanjing, China
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Contributors
Liang Zhang Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences, Beijing, China Yanqiang Zhong Department of Pharmaceutical Science, College of Pharmacy, The Second Military Medical University, Shanghai, China
Part I General Protocols for Fabrication of Drug Liposomes
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Liposomes in Drug Delivery: Status and Advances Ying-Jie Hu, Rui-Jun Ju, Fan Zeng, Xian-Rong Qi, and Wan-Liang Lu
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Features of Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Classification of Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Passive Targeting Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Active Targeting Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Physicochemical Targeting Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Multifunctional Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Preparation Materials and Methods of Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Preparation Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Preparation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Application of Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Y.-J. Hu · W.-L. Lu (*) State Key Laboratory of Natural and Biomimetic Drugs, Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System, School of Pharmaceutical Sciences, Peking University, Beijing, China e-mail: [email protected] R.-J. Ju Department of Pharmaceutical Engineering, Beijing Institute of Petrochemical Technology, Beijing, China F. Zeng Beijing Neurosurgical Institute, Capital Medical University, Beijing, China X.-R. Qi (*) Department of Pharmaceutics, School of Pharmaceutical Science, Peking University, Beijing, China Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System, Beijing, China e-mail: [email protected] © Springer-Verlag GmbH Germany, part of Springer Nature 2021 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49320-5_1
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Abstract
Liposomal drug delivery system has made evident breakthrough and innovation in the fields of drug treatment. The objectives of this chapter are to briefly review the features, classification, major preparation materials and methods, and application of drug liposomes. Liposomes have shown excellent biocompatibility; are able to protect drug from direct exposure in blood system; are capable of carrying hydrophilic, hydrophobic, and amphipathic agents; and have targeting natures in human body. According to different targeting strategies, liposomes are classified into passive, active, and physicochemical targeting liposomes. The major materials of liposomes are phospholipids and cholesterol. The manufacturing technology of drug liposomes is becoming mature now, consisting of film dispersion, reverse-phase evaporation, chemical gradient loading, and the other encapsulation methods. Tens of drug liposomes have been approved for clinical use meanwhile a number of drug liposomes are undergoing clinical trial evaluations. During clinical trials and uses, the liposomes have been evidenced to have an optimal drug delivery efficiency and better efficacy, despite the anticancer drug liposomes may lead to new side effects like hand-foot syndrome. The drug liposomes can be enriched into the tumor site, hence demonstrating a better efficacy and a reduced adverse reaction such as cardiotoxicity. Besides, the liposomal formulations are capable of potentiating efficacy of anticancer drugs by circumventing multidrug resistance of cancers and cancer stem cells and by transferring drug across the blood-brain barrier (BBB). These new functions have been evidenced in laboratory observations but need further clinical evaluations. The review demonstrates that the liposomes are promising drug delivery systems in the fields of anticancer, antiinfection, pain management, etc. Keywords
Liposomes · Drug delivery · Advance · Preparation · Review
1
Introduction
Liposomes are a kind of phospholipids-based spherical vesicles, which consist of one or more lipid bilayers surrounding a central aqueous core. When amphipathic phospholipids are dispersed in water, the hydrophobic parts on their molecules tend to gather together, while the hydrophilic parts expose towards water. As such, the phospholipids are able to form round-shape vesicles with bilayer structure (Fig. 1). Liposomes were first proposed as a biofilm model by Bangham in 1965 (Bangham et al. 1965a). Their experiment makes people aware of the biodegradability and biocompatibility of liposomes and lays a foundation for the liposomes to be used as drug carriers. In 1971, Gregoriadis et al. firstly encapsulated amyloglucosidase and
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Phospholipids Cholesterol
Local map of bilayer
Liposome
Fig. 1 Schematic representation of cholesterol and phospholipid in bilayer membrane
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I-albumin in liposomes. After intravenous injection in rats, liposomes were mainly distributed in liver and spleen (Gregoriadis and Ryman 1971). It indicated that the liposomes could be effectively loaded with bioactive substances and had a high content in specific tissues. These pioneering research studies inspired the development of liposomal drug delivery system. With favorable characteristics such as biocompatibility, lipid bilayer structure, and suitable particle sizes, liposomes are able to encapsulate different kinds of pharmaceutical agents, protect drug from degradation, improve unfavorable pharmacokinetics of drug, and reduce side effects, exhibiting great advantages as the drug carriers. Therefore, liposomal drug delivery system has become one of the most well-studied and widely used delivery system for pharmaceutical applications, especially in the fields of antitumor, anti-infection, and pain treatment. Nowadays, a number of drug liposomes have been applied to clinical treatments of multiple diseases, such as Doxil for treatment of cancer (Barenholz 2012), AmBisome for treatment of fungal infection (Chopra et al. 1991), and DepoDur for relief of pain (Gambling et al. 2005). As the laboratory research progresses, liposomal drug delivery system is continually improved, from the regular liposomes to the long circulation liposomes or from the passive targeting liposomes to the active targeting liposomes. More and more liposomes modified with special materials have been fabricated in the laboratory studies to reach unique purposes, such as transferring drug across the physiological barrier, enhancing the uptake of drug-resistant cancer cells, killing the “dormant” cancer stem cells, and interfering the critical life signaling pathways or targets. Some of these advanced liposomal drug delivery systems have been approved to be evaluated in clinical trials. These advanced liposomal drug delivery systems are expected to provide promising new strategies in clinical treatments of cancer and other diseases.
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Features of Liposomes
As the drug carriers, liposomes exhibit a number of unique favorable features: 1. Liposomes have excellent biocompatibility and biodegradability, without toxicity and immunogenicity. There are because liposomes are mainly composed of phospholipids and cholesterol. These two materials are also natural components of mammalian cell membranes. 2. Liposomes are able to protect drug from direct exposure in blood system, hence lowering drug degradation and blood toxicity. 3. Liposomes are capable of carrying hydrophilic, hydrophobic, and amphipathic agents, including enzymes, hormones, vitamins, antibiotics and cytokines, etc. The hydrophilic agents can be incorporated into the aqueous compartment of liposomes, while the hydrophobic and amphipathic can be incorporated into the lipid bilayers. 4. Liposomes have passive targeting nature in human body. The varied particle sizes of liposomes change the distribution of loaded drug in tissues by a physical retention due to the difference in compact degrees of each organ tissue. As some lesions such as cancer tissues are less compact than normal tissues, drug loaded liposomes are able to maintain a higher concentration in cancer tissue and a lower concentration in normal tissue, thereby improving therapeutic index and lowering side effect of drug. Moreover, the pegylated liposomes provide a prolonged circulation in blood system by avoiding the rapid clearance of reticuloendothelial system (RES) (Woodle and Lasic 1992), hence accumulating more drug into cancer tissue by the enhanced permeability and retention (EPR) effects (Zucker and Barenholz 2010; Qiao et al. 2011). 5. Liposomes can be assigned active targeting or physicochemical targeting nature in human body. These are because liposomes are apt to be modified with functional ligands, monoclonal antibodies, or microenvironment responsive materials to achieve particular purposes of targeting cancer cells, transferring drug across the physiological barrier, controlling drug release, and so on.
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Classification of Liposomes
With the progress of the studies, liposomes have shown diverse classifications and various action mechanisms. According to different targeting mechanisms, liposomes can be classified as passive targeting liposomes, active targeting liposomes, physicochemical targeting liposomes, and multifunctional liposomes.
3.1
Passive Targeting Liposomes
Passive targeting liposomes are able to escape from nonspecific trapping by other tissues but passively accumulate in target tissues with the circulation of blood. The targeting mechanism is based on the physicochemical properties of liposomes, such
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Fig. 2 EPR effect of nanostructured liposomes in the fenestrated tumor tissue
as a suitable particle size. As cancer tissues are characterized by high interstitial pressure, enhanced vascular permeability and retention (EPR effect, Fig. 2), and the lack of functional lymphatic drainage, the passive targeting liposomes are more easily to accumulate in cancer tissue, thus providing a promising strategy for anticancer treatment.
3.1.1 Conventional Liposomes As the name suggested, conventional liposomes are the simplest liposomes mainly composed of phospholipid and cholesterol, without any modification. After injection, the conventional drug liposomes are mainly concentrated in the liver, spleen, lung, lymph node, bone marrow, and other reticuloendothelial rich locations, and also aggregated in the locations of inflammation, infection, and solid tumors, exhibiting passive targeting effect. Based on laboratory evaluations, conventional liposomes have shown multipotentials in the prevention and treatment of cancers. Imanaka et al. reported that beta-sitosterol loaded conventional liposomes were able to enhance the immune surveillance activity of mice and prevent the metastasis of tumor by oral administration (Imanaka et al. 2008). Igarashi et al. developed a conventional photosensitizer liposome that could enhance the therapeutic efficacy of photodynamic therapy for gastrointestinal tumors (Igarashi et al. 2003). 3.1.2 Long Circulating Liposomes Since the conventional liposomes are easily trapped by the reticuloendothelial system (RES), it is necessary to avoid the rapid clearance by RES and to prolong the circulation time of liposomes so that liposomes can target tumor sites or any other target tissue. Therefore, long circulating liposomes, also known as stealth liposomes
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or sterically stabilized liposomes (Awasthi et al. 2003), have been developed for reaching such a purpose. Long circulating liposomes can be prepared by modifying with ganglioside, phosphatidylinositol, or polyethylene glycol (PEG) lipid derivatives to form a steric hydrophilic layer, which is able to prevent the recognition of liposomes by opsonin, reduces the rapid clearance by RES, and prolongs the circulation time of liposomes in blood system (Crosasso et al. 2000; Yokoe et al. 2008). Because of the RES-escaping effect and EPR effect, long circulating liposomes could passively accumulate in tumor tissues, exhibiting the potentials for tumor imaging and therapy. Cogswell et al. reported that the long-circulating econazole liposomes had a superior efficacy in treatment of breast cancer by parenteral administration (Cogswell et al. 2006). Furthermore, Fanciullino et al. developed a kind of pegylated liposomes of 20 -deoxyinosine (d-Ino), which displayed a sevenfold long-circulating effect, and exhibited a strong anticancer potentiation effect on colon cancer cells by a combination use with 5-fluorouracil (Fanciullino et al. 2005). In recent years, the long-circulating liposomes have been further assigned new functions, such as temperature sensitive long-circulating liposomes, as discussed below.
3.2
Active Targeting Liposomes
With the rise of precision medicine, drug delivery systems are expected to selectively deliver drugs into specific tissue, cells, or even organelles to improve targeting specificity and to diminish systemic adverse effects. Passive targeting liposomes cannot reach a precise selectivity, while active targeting liposomes can provide a strategy to address this problem. Active targeting liposomes refer to the liposomes modified with the receptor ligands or antibodies as homing “devices” to target specific cells or organelles.
3.2.1 Ligand-Mediated Liposomes There are many receptors that are overexpressed on the specific cells but less expressed or nonexpressed on the other cells. For example, folate receptor is overexpressed on many epithelial cancers, and it has been exploited as the action target on cancer cells. Based on that, liposomes modified with specific folate are able to selectively bind with cancer cells which overexpress folate receptors, hence reaching an enhanced cellular internalization of liposomes by a folate receptormediated mechanism. Recently, researchers have been made efforts on the development of ligandmediated liposomes. Zeng et al. developed a kind of functional vincristine plus dasatinib liposomes modified with a targeting molecule DSPE-PEG2000-c(RGDyK) for eradicating triple-negative breast cancer (TNBC). C(RGDyK) is a cyclic peptide that has a specific affinity with integrin receptor. It functions as a targeting molecule to bind liposomes with integrin receptor overexpressed TNBC cells (Zeng et al. 2015). Moreover, multiple ligands-mediated liposomes are developed to avoid the
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a
b
Paracellular aqueous pathway
Transcellular Transport proteins lipophilic pathway Vinca alkaloids, Lipid-soluble Glucose, amino acids, Cyclosporin A, agents nucleosides AZT
Water-soluble agents
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Receptor-mediated transcytosis
Adsorptive transcytosis
Insulin, transferrin
Albumin, other plasma proteins
Blood
Tight junction
Endothelium Brain
Astrocyte
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Fig. 3 Pathways across the blood–brain barrier (Abbott et al. 2006)
heterogeneity of cancer cells. Sriraman et al. investigated pegylated doxorubicin liposomes modified with folate (F), transferrin (Tf), or both (F + Tf). The two-ligand targeted liposomes (F + Tf) showed a sevenfold increase in cell association compared to either of the single-ligand targeted ones in human cervical carcinoma (HeLa) cells (Sriraman et al. 2016).
3.2.2 Antibody-Mediated Liposomes In addition to receptor ligands, monoclonal antibodies (mAbs) and their derivatives are often used as targeting molecules in the active targeting liposomes. Due to interaction of antigens with monoclonal antibodies, the liposomes modified with mAbs or their derivatives could actively target cells with specific antigens. The antibody-mediated liposomes are also defined as immunoliposomes, which can be designed to improve pharmacological properties of conventional anticancer drugs. A number of methods have been reported for modifying antibodies onto the surface of the drug liposomes. Huwyler et al. developed a kind of daunorubicin immunoliposomes by introducing antitransferrin receptor OX26 antibody onto the liposomes, demonstrating a successful result for the enhanced accumulation at brain glioma site (Huwyler et al. 1996). Besides, Loureiro et al. designed and prepared a kind of immounliposomes by modifying two antibodies, namely, the OX26 antibody and the antiamyloid beta peptide antibody (19B8MAb), for treatment of Alzheimer’s disease. Results showed that the established immounliposomes could effectively cross the blood brain barrier (BBB) (Fig. 3) (Abbott et al. 2006) and concentrate at the Alzheimer’s area (Loureiro et al. 2015).
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Physicochemical Targeting Liposomes
Physicochemical targeting liposomes are liposomes with particular physicochemical characterizations that could actively respond to the environment by different strategies. Such strategies include utilization of pH, temperature, enzyme, or electric responsive property, etc. The particular physicochemical characteristics are usually achieved by modification on the liposome membrane or by using particular phospholipids.
3.3.1 pH-Sensitive Liposomes pH-sensitive liposomes are able to maintain stability at physiological pH (pH = 7.4) but undergo destabilization under acidic conditions, thus leading to the selective release and accumulation of the loaded drug. Since tumor microenvironment has been confirmed to have mildly acidic (pH 6.0–7.0) condition due to the glycolytic metabolism of glucose to lactate in tumor tissues, pH-sensitive liposomes have been especially useful for anticancer therapy (Cardone et al. 2005). Wang et al. developed a pH sensitive octylamine-graft-poly aspartic acid modified liposomes (OPLPs) that demonstrated a slow release of drug in the physiological pH 7.4 environment, while providing a fast release in subacid environment (pH 6.0 of resembled tumor tissues), therefore exhibiting significantly enhanced anticancer efficacy (Wang et al. 2014). To prolong the circulation time of pH sensitive liposomes in blood system, stealth pH-sensitive liposomes modified with PEG were developed. Júnior et al. evaluated the tissue distribution of stealth pH sensitive liposomes containing cisplatin in solid Ehrlich tumor-bearing mice, and their results demonstrated that the liposomes had a longer circulation in blood and a higher accumulation of drug in tumor as compared with free cisplatin (Júnior et al. 2007). 3.3.2 Temperature-Sensitive Liposomes Temperature-sensitive liposomes are composed of lipids that could undergo a gel-toliquid phase transition at a critical temperature (transition temperature, Tm). When temperature is lower than Tm, liposomes could maintain stability and prevent the encapsulated drug from release. When the temperature is higher than Tm, double molecular chain of phospholipids would gain a higher degree of disorder and activity, hence resulting in the release of drug from the liposome vesicles. Tumor tissues are usually exhibiting hyperthermia due to the rapid metabolism, and accordingly, the fever often occurred in the tumor sites similar to the inflammatory response. Therefore, the temperature-sensitive liposomes could be used in anticancer therapy and anti-inflammatory therapy. Yatvin et al. described a type of temperature-sensitive liposomes consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-snglycero-3- phosphocholine (DSPC), and the liposomes were able to release drug rapidly when the temperature was increased a few of degrees above physiological temperature (Yatvin et al. 1978). Kakinuma et al. developed a kind of temperaturesensitive liposomes containing cis-platinum for treatment of brain glioma-bearing
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rats, and the results showed a significantly increased concentration of cis-platinum in the brain glioma sites (Kakinuma et al. 1996).
3.3.3 Enzyme-Sensitive Liposomes Enzyme-sensitive liposomes are composed of lipids that could be degraded by specific enzyme, leading to rapid drug release under the enzyme-rich environment. The over-expressed enzyme systems in particular tissue microenvironment have been utilized to trigger the release of drug from enzyme-sensitive liposomes. Matrix metalloproteinases (MMP) are overexpressed in tumor microenvironment and therefore could be used for the design of enzyme-sensitive anticancer drug liposomes. Mura et al. established enzyme-sensitive liposomes by coupling a monoclonal antibody 2C5 with the polyethylene glycol chain via an MMP2-cleavable linker (Mura et al. 2013). Similarly, the over-secretion of phospholipase A2 (sPLA2) has been found in tumor site and can be used to initiate drug release of enzymesensitive liposomes. Hansen et al. evidenced that the activity of human sPLA2 was highly sensitive to phospholipid acyl-chain length and negative surface charge density of the liposomes (Hansen et al. 2015), thereby triggering drug release from the enzyme-sensitive liposomes. 3.3.4 Physical Adsorption-Mediated Liposomes Physical adsorption-mediated liposomes are modified with cationic material on the surface to produce a positive potential to adsorb onto the electronegative membrane of cancer cells, therefore achieving a kind of physical adsorption targeting effect. Furthermore, after uptake by cancer cells, the cationic liposomes can further accumulate into mitochondria of living cells in response to mitochondrial membrane potential (Fig. 4) (Mu et al. 2017). Wang et al. developed mitochondrial targeting resveratrol liposomes by modifying a conjugate of dequalinium (DQA) with polyethylene glycol distearoylphosphatidylethanolamine (PEG2000-DSPE). The results exhibited a significant antitumor efficacy in either cancer cells or drug-resistant cancer cells (Wang et al. 2011). In addition, Ma et al. developed mitochondrial targeting berberine liposomes by modifying DQA-PEG2000-DSPE (Ma et al. 2013). The mitochondrial targeting berberine liposomes could transport across cancer stem cell membrane and selectively accumulate into the mitochondria of cancer cells. When co-treated with paclitaxel liposomes, mitochondrial targeting berberine liposomes significantly potentiated the anticancer efficacy in human breast cancer stem cells xenografts in nude mice.
3.4
Multifunctional Liposomes
In the latest years, multifunctional liposomes have been developed to achieve multiple purposes by which one liposome formulation is able to reach a
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Fig. 4 An illustration for targeting mitochondria by liposomal drug delivery system (Mu et al. 2017)
comprehensive objective by combing passive, active, and physicochemical targeting effects. For example, Li et al. developed a kind of multifunctional targeting paclitaxel plus artemether liposomes for treatment of brain glioma (Li et al. 2014). In this construct, paclitaxel was used as the anticancer drug and artemether used as the regulator of apoptosis and inhibitor of vasculogenic mimicry channels. Two functional materials, mannose-vitamin E derivative conjugate (MAN-TPGS1000) and dequalinium-lipid derivative conjugate (DQA-PEG2000-DSPE), were used to enhance the capabilities of liposomes in transferring drug across blood-brain barrier (BBB), eliminating brain glioma stem cells and destroying vasculogenic mimicry channels (Fig. 5) (Li et al. 2014). The transport mechanism of the liposomes across the BBB was associated with receptor-mediated endocytosis by MAN-TPGS1000 conjugate via glucose transporters and adsorption-mediated endocytosis by DQA-PEG2000-DSPE conjugate via electric charge-based interaction. Furthermore, Kono et al. designed a kind of multifunctional liposomes which combined the properties of active targeting and temperature-sensitive liposomes and were used for treatment and diagnosis of human epidermal growth factor 2 (Her-2) positive cancer by imaging, such as ovarian cancer and breast cancer (Kono et al. 2015). The liposomes were functionalized with thermo-sensitive poly [2-(2-ethoxy)ethoxyethyl vinyl ether] chains for triggering drug release from liposomes (approximately 38 C), with conjugation of antibody trastuzumab for targeting Her-2 positive cancer, and with entrapment of indocyanine green for
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Fig. 5 Characterization of targeting molecular materials and liposomes (Li et al. 2014). (Notes: MALDI-TOF-MS spectra of TPGS1000 (A1) and MAN-TPGS1000 conjugate (A2); a schematic representation of the functional targeting paclitaxel plus artemether liposomes (B); AFM images of paclitaxel liposomes (C1) and functional targeting paclitaxel plus artemether liposomes (C2))
diagnosis by near-infrared fluorescence imaging. The liposomes could retain drug under physiological temperature, while release drug immediately at a slightly higher temperature in tumor, and exhibited significant ability in targeting Her-2 positive cancer cells.
4
Preparation Materials and Methods of Liposomes
4.1
Preparation Materials
Most lipids or lipid mixtures can be used for the preparation of liposomes, among them, phospholipids and cholesterol are the most common materials.
4.1.1 Phospholipids Phospholipids are a class of lipids that are the major components of cell membranes. They can form lipid bilayers because of their amphiphilic characteristic. The structure of the phospholipid molecule generally consists of two hydrophobic fatty acids “tails” and a hydrophilic “head” consisting of a phosphate group. The two
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components are joined together by a glycerol molecule. The phosphate groups can be modified with simple organic molecules such as choline. According to different electric properties, phospholipids are divided into neutral phospholipids and negatively charged phospholipids. Neutral phospholipids are the most commonly used phospholipids in the preparation of liposomes, including phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylethanolamine (PE), and so on. PC is the most common neutral phospholipid that incorporates choline as a head group. It is a major component of biological membrane and can be easily obtained from a variety of readily available sources, such as egg yolk or soybean. Natural PC are mixture composed of several PC, and each kind of PC contains fatty acid chains in varying length and varying saturation. The saturation of fatty acid chains is closely related to the stability of liposomes. While saturated fatty acid chains could be arranged densely to form stable liposomes, unsaturated fatty acid chains are arranged incompactly, leading to unstable liposomes and drug leakage. Compared with other lipids, PC has advantages of low price, electric neutrality, and chemical inertness. Another neutral phospholipid SM is a type of sphingolipid found in animal cell membrane, especially in the membranous myelin sheath that surrounds some nerve cell axons. It usually consists of phosphocholine and ceramide or a phosphoethanolamine head group. Due to the hydrogen bond between hydroxyl and acylamino, SM has a higher ordered colloidal phase than PC. PE is also a common neutral phospholipid, which consists of a combination of glycerol esterified with two fatty acids and phosphoric acid. Its phosphate group is combined with ethanolamine. As a polar head group, PE creates a more viscous lipid membrane as compared to PC. Furthermore, negatively charged phospholipids are also used in the liposome preparation, such as phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and dicetylphosphate (DCP). In negatively charged phospholipids, steric hindrance, hydrogen bond, and static charge regulate the interaction between the hydrophilic “head” in a joint manner.
4.1.2 Cholesterol Cholesterol (Ch) is a type of lipid molecule that exists in animal cell membrane as an essential structural component to maintain both membrane structural integrity and fluidity. It composes about 30% of animal cell membrane. The hydroxyl group on cholesterol interacts with the polar heads of the membrane phospholipids and sphingolipids, while the bulky steroid and the hydrocarbon chain are embedded into the bilayer of membrane. Through the interaction with the phospholipid fattyacid chains, cholesterol increases membrane packing and thereby altering membrane fluidity and maintaining membrane integrity. 4.1.3 Other Materials Positively charged lipids are commonly used in the DNA delivery liposomes. Currently, positively charged lipids for liposome preparation are all synthetic, including sterylamine (SA) and cholesteryl acetate.
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Soybean-derived sterols, such as β-sitosterol, campesterol, stigmasterol, and brassicasterol, have the similar structure with cholesterol but a better membrane stabilizing activity.
4.2
Preparation Methods
4.2.1 Film Dispersion Method Film disperse method is also known as the Bangham method or the thin film hydration method, and it is one of the most widely used technique for the formation of liposomes (Bangham et al. 1965a, b). The general process of this method could be described as the following. Firstly, phospholipids with lipophilic drugs are dissolved in appropriate amount of chloroform or other solvents, and then the solvent is evaporated to form a lipid film. A buffer solution containing water soluble drug is added to the lipid film with shaking, yielding drug-loaded liposomes with a particle size range of 1–5 μm. The liposome suspensions need to be further treated by ultrasound or through the membrane extrusion to make the particle size of liposomes uniform. To lower down and homogenize the particle size of liposomes, several methods can be selected, including ultrasonic method, film extrusion, and French film extrusion method, etc. 4.2.2 Reverse-Phase Evaporation Method The reverse-phase evaporation process was first described by Szoka and Papahadjopoulos, and it is based on the formation of water drops that are surrounded by lipid and dispersed in an organic solvent, referred to as inverted micelles (Szoka and Papahadjopoulos 1978). The general process is as follows. Phospholipid membrane material is dissolved in an organic solvent (such as chloroform and ether), and then aqueous drug solution is added to form W/O emulsions under ultrasonic treatment. The organic solvent is removed by vacuum evaporation to yield liposomes. The liposomes prepared by reverse-phase evaporation method are usually large unilamellar liposomes. The problem of residual organic solvents can be solved by using supercritical CO2, instead of organic solvents, known as supercritical reverse evaporation method (Sakai et al. 2008; Garg et al. 2008). 4.2.3 Chemical Gradient Methods Chemical gradient methods are active encapsulation methods by which drugs could be actively loaded into liposomes. They are also known as the remote loading methods, consisting of pH gradient method, ammonium sulfate transmembrane gradient method, and calcium acetate gradient method, etc. pH Gradient Method pH gradient method is briefly described as the following procedures. Blank liposomes are firstly prepared by film dispersion method, then the pH value of the aqueous phase of liposome vesicles is adjusted to form a pH gradient difference between internal and external vesicles, and the weak acid or alkaline agents may be encapsulated in the internal phase of liposomes in the form of ions by using the pH
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Fig. 6 Illustration of liposomal fabrication in pH gradient method
DH+
DH+
DH+
DH+
H+
H+ D D D
D
Vm
Vm Δ pH
gradient (Fig. 6). This method makes it possible for preparing liposomes with a high drug entrapment efficiency. However, as the method is dependent on drug structure, it cannot be applied to the drugs with arbitrary structures. Ammonium Sulfate Gradient Method Ammonium sulfate gradient method is designed according to the principle of chemical equilibrium. The general process of the method could be described as the following procedures (Fig. 7). Firstly, blank liposomes are prepared by using film dispersion method with ammonium sulfate solution as the hydration solution. Secondly, the blank liposomes are dialyzed in the dialysis tubing with phosphate buffered saline to remove the ammonium sulfate outside, thus forming an ammonium sulfate gradient between two sides of liposome vesicles, namely, the inside has a high concentration of ammonium sulfate, while the outside has lower one. Finally, active encapsulation is achieved by incubation with weak bases solution in water bath at 60 C with continually shaking for 20 min (Men et al. 2011). In this method, the stability of the ammonium ion gradient is related to the low permeability of its counter ion, the sulfate, which also stabilizes anthracycline accumulation for prolonged storage periods (>6 months) due to the aggregation and gelation of anthracycline sulfate salt (Haran et al. 1993). Calcium Acetate Gradient Method The fabrication procedures in calcium acetate gradient method are described as the following. Blank liposomes containing calcium acetate solution are prepared by thin film dispersion method, and then a calcium acetate concentration gradient is formed by removing the external calcium acetate of liposomes. As the internal concentration is higher than the external one, acetic acid moves across the membrane of liposome. Accordingly, a large number of protons transfer from the liposome interior to the outside, thereby forming a pH gradient. By incubating the liposomes with weak acidic drug solution, the drug can bind with the liposome internal calcium ions to form less soluble calcium salt, which prevents the drugs from passing through the
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Liposomes in Drug Delivery: Status and Advances
2NH3
(NH4)2SO4
2NH3+2H+
17
2DOX-NH3 · Cl
2NH4++SO422DOX-NH3++2Cl-
(DOX-NH2)2SO4 (gelation)
2DOX-NH2
2DOX-NH2+2HCl
Fig. 7 Illustration of liposomal fabrication in ammonium sulfate gradient method
phospholipid bilayer, thus improving the entrapment efficiency and reducing drug leakage (Qin et al. 2008; Kheirolomoom et al. 2010; Li et al. 2009).
4.2.4 Additional Methods There are many other methods in the preparation of liposomes. The ethanol and ether injection method can be used for dissolving the lipids into an organic phase, followed by the injection of the lipid solution into aqueous media, hence forming liposomes (Batzri and Korn 1973; Deamer 1978). Besides, the heating method has been developed to produce blank liposomes by hydration of phospholipids in an aqueous solution containing 3% glycerol through raising the temperature to 60 C or 120 C (Mozafari 2005). During preparation, the drug was incorporated into blank liposomes using the heating method by addition of drug to the solution at different temperature stages, including the beginning, above the transition temperature of the lipids and the ambient temperature. In addition, the freeze-drying of mono-phase solution method for encapsulation of heat-sensitive drugs such as DNA, and the produced drug liposomes can be stored for a long time in a sealed container (Li and Deng 2004).
5
Application of Liposomes
In 1995, the first marketed liposomal drug, doxorubicin liposomes (Doxil ®), was approved by Food and Drug Administration (FDA) for treatment of various types of cancer (Barenholz 2012). It is a kind of decorated liposomes with hydrophilic
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polymer, which is conjugated by polyethylene glycol with distearoylphosphatidyl ethanolamine (PEG2000-DSPE). The use of PEG2000-DSPE conjugate is used to prevent the adsorption of plasma proteins onto liposomes or to prevent opsonization, thereby avoiding the rapid clearance of liposomes by reticuloendothelial system. The pegylated liposomes are able to extend the circulating time in blood circulation and to accumulate more into the specific tissues such as solid tumor tissue. Because of the decreased distribution in heart tissue, doxorubicin liposomes could evidently lower the cardiotoxicity after use. Nevertheless, an unexpectedly hand-foot syndrome (HFS) has been experienced during clinical application of doxorubicin liposomes. Actually, the pegylated doxorubicin liposomes have become one of the most common causes of HFS. The risk of developing HFS appears to be doxorubicin dose-dependent. Drug formulations that prolong serum drug levels or that concentrate drug at affected sites have higher rates. This may be one reason why doxorubicin liposomes are associated with a higher HFS incidence than the standard, nonencapsulated formulation (Miller et al. 2014). In spite of this, pegylated doxorubicin liposomes are still considered as an efficient drug in tumor therapy. The success of Doxil ® has inspired the research and development of liposomal drug delivery systems (Table 1) (Barenholz 2012; Pratt et al. 1998; Benesch and Urban 2008; Zhang et al. 2009; Taiwanese Gynecologic Oncology Group et al. 2006; Rodriguez et al. 2009; Swenson et al. 2001; Poon and Borys 2009; Harrington et al. 2001; Seetharamu et al. 2010; Sarris et al. 2000; Dragovich et al. 2006; Seiden et al. 2004; Sankhala et al. 2009; Wetzler et al. 2013; Rudin et al. 2004; Zuckerman et al. 2014; Matsumura et al. 2004; Senzer et al. 2013). With the gradual study of the liposomes in-depth, a number of drug liposomes have been approved for clinical uses, such as daunorubicin liposomes (DaunoXome ®) (Pratt et al. 1998), cytarabine liposome (Depocyt ®) (Benesch and Urban 2008), vincristine sulfate liposomes (Marqibo ®) (Rodriguez et al. 2009), and nonpegylated doxorubicin liposomes (Myocet ®) (Swenson et al. 2001). Meanwhile, several regular liposomes in clinical trials are expected to be approved by drug administration authority soon. A phase 2 clinical trial showed that the single-agent nanomolecular liposomal annamycin appeared to be well tolerated and exhibited a significant clinical activity as a single agent in treatment of the refractory adult acute lymphoblastic leukemia (Wetzler et al. 2013). Another phase 2 study indicated that L-NDDP (Aroplatin), a liposomal formulation of a structural analogue of oxaliplatin, was well tolerated in treatment of the refractory patients with advanced colorectal cancer, and demonstrated a positive antitumor activity. Further studies of L-NDDP, preferably in combination with other agents such as fluoropyrimidines, are warranted (Dragovich et al. 2006). In addition to these, several active targeting liposomes are undergoing clinical evaluation. MBP-426, a transferrin-mediated liposomes containing oxaliplatin, is now in phase II trial as second line treatment for gastric, gastroesophageal, and esophageal adenocarcinomas (Sankhala et al. 2009). The other transferrin-mediated liposomes that contain P53 plasmid DNA are now in phase Ib trial to treat solid tumor (Senzer et al. 2013). Besides, immunoliposomes also have made a lot of progress. MCC-465, a type of pegylated doxorubicin liposomes functionalized with
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Table 1 Liposomal anticancer drugs used in clinical uses or in clinical trials Drug liposomes Doxorubicin long circulating liposome (Doxil ®) Daunorubicin liposome (DaunoXome ®) Cytarabine liposome (Depocyt ®) Paclitaxel liposome (Lipusu) Doxorubicin long circulating liposome (LipoDox) Vincristine sulfate liposomes (Marqibo ®) Doxorubicin liposomes (Myocet ®) Thermosensitive liposomes (ThermoDox) Cisplatin liposomes (SPI-77)
Vincristine liposomes (Onco TCS) Oxaliplatin analogue liposomes (Aroplatin/ L-NDDP) Lurtotecan liposomes (OSI-211) Oxaliplatin liposomes (MBP-426)
Main materials PEG-DSPE/ phospholipids
Anticancer spectrum Various cancers
Phospholipids
Research unit Ortho Biotech; Zeneus Pharm
Current state 1995 sale in the USA Barenholz (2012)
Various cancers
Gilead
Phospholipids
Meningeal lymphoma
Enzon
Phospholipids
Ovarian cancer
Luye Pharma Group
1996 sale in the USA Pratt et al. (1998) 1999 sale in the USA and Canada Benesch and Urban (2008) 2004 sale in China Zhang et al. (2009)
PEG-DSPE/ phospholipids
Various cancers
Fudan Zhangjiang biomedical
Phospholipids
Hodgkin’s lymphoma and leukemia Metastatic breast cancer
Talon
Phospholipids
Elan
Phospholipids
Hepatocellular carcinoma
Celsion
PEG-DSPE/ phospholipids
Head and neck cancer, nonsmall-cell lung cancer
ALZA
Phospholipids
NonHodgkin’s lymphoma Colorectal cancer
Inex Pharmaceuticals Aronex Pharmaceuticals
Phospholipids
Various cancers
OSI Pharmaceuticals
Transferrin/ phospholipids
Various cancers
Mebiopharm Co., Ltd
Phospholipids
2009 sale in China Taiwanese Gynecologic Oncology Group et al. (2006) 2012 sale in the USA Rodriguez et al. (2009) 2000 sale in EU Swenson et al. (2001) USA III clinical trials Poon and Borys (2009) USA II clinical trials Harrington et al. (2001), Seetharamu et al. (2010) USA II clinical trials Sarris et al. (2000) USA II clinical trials Dragovich et al. (2006)
USA II clinical trials Seiden et al. (2004) USA II clinical trials Sankhala et al. (2009) (continued)
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Table 1 (continued) Drug liposomes Annamycin liposomes (L-annamycin) C-Raf AON cationic liposomes (LErafAONETU) Sirna liposomes (CALAA-01) Doxorubicin liposomes (MCC-465) P53 plasmid DNA liposomes (SGT-53)
Main materials Phospholipids
Phospholipids
Anticancer spectrum Acute lymphoblastic leukemia Solid tumors
Research unit Callisto Pharmaceuticals INSYS Therapeutics Inc.
Transferrin/ phospholipids
Solid tumors
Calando Pharmaceuticals
Mcab-GAHphospholipids
Gastric carcinoma
Mitsubishi
Transferrin/ phospholipids
Solid tumors
SynerGene Therapeutics, Inc.
Current state USA I/II clinical trials Wetzler et al. (2013) USA I clinical trials Rudin et al. (2004)
USA I clinical trials Zuckerman et al. (2014) Japan I clinical trials Matsumura et al. (2004) USA I clinical trials Senzer et al. (2013)
the F(ab0 )2 of GAH antibody that shows a significant anticancer activity against GAH-positive colorectal and gastric cancer cells, is progressed to phase I clinical trial (Matsumura et al. 2004). However, clinical trials also revealed some unexpected adverse events and results. According to early results of an ongoing phase II trial, liposomal vincristine (Onco-TCS) was active and well tolerated in this heavily pretreated population with relapsed non-Hodgkin’s lymphomas but was neurotoxic in a fraction of patients heavily exposed to prior neurotoxic agents (Sarris et al. 2000). The clinical study of pegylated liposomal cisplatin (SPI-077) showed that SPI-077 was essentially inactive against squamous cancers of head and neck and only modestly active in patients with non-small-cell lung cancer (Harrington et al. 2001; Seetharamu et al. 2010). ThermoDox, a thermally sensitive liposomal doxorubicin, was considered as an efficient agent against liver cancer. However, it was announced that ThermoDox failed in the phase 3 study, and it was unable to demonstrate a significant improvement in progression-free survival. Nonetheless, ThermoDox is not given up and a phase 3 study (OPTIMA) is conducted now to determine whether ThermoDox is effective in the treatment of nonresectable hepatocellular carcinoma when used in conjunction with standardized radiofrequency ablation (sRFA).
6
Concluding Remarks
Liposomes have shown suitable biocompatibility, are able to protect drug from direct exposure in blood system, are capable of carrying hydrophilic, hydrophobic, and amphipathic agents, and have targeting natures in human body. According to
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different targeting strategies, liposomes are classified into passive, active, and physicochemical targeting liposomes. The major materials of liposomes are phospholipids and cholesterol. The manufacturing methods mainly consist of film dispersion, reverse-phase evaporation, chemical gradient loading, and the other encapsulation methods. Tens of drug liposomes have been approved for clinical use meanwhile a number of drug liposomes are undergoing clinical trial evaluations. During clinical trials and uses, the liposomes have been evidenced having an optimal drug delivery efficiency and better efficacy, despite the anticancer drug liposomes may lead to new side effects like hand-foot syndrome. The drug liposomes can be enriched into the tumor site, hence demonstrating a better efficacy and a reduced adverse reaction such as cardiotoxicity. Besides, the liposomal formulations are capable of enhancing the efficacy of anticancer drugs by circumventing multidrug resistance of cancers and cancer stem cells and by transferring drug across the bloodbrain barrier (BBB). These new functions have been evidenced in laboratory observations but need further clinical evaluations. In view of current status, there are still a few limitations in industry production and clinical application of advanced liposomal drug delivery systems in the following aspects: approval difficulty of excipients by drug administration authority for pharmaceutical use, complicated manufacturing procedure or formulation, instability of liposomes in long-term storage, and robust targeting efficacy in human body. It is believed that the liposomal drug delivery systems would have more applications in treatments of cancer and other diseases with the progress of science and the development of pharmaceutical technology, a new type of liposomes, dualfunctional drug liposomes have been proposed and studied in laboratory investigation, and the strongest strength of dual-functional drug liposomes exists in their extended function, which could reach the specific treatment purpose including eliminating drug-resistant cancer, killing cancer stem cells, destroying mitochondria, inducing apoptosis, regulating autophagy, destructing supply channels, utilizing microenvironment, and silencing genes of resistant cancers (Mu et al. 2017). The continuous development of liposomal drug delivery systems could provide promising therapeutic strategy and wide clinical applications.
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Preparation of Drug Liposomes by Thin-Film Hydration and Homogenization Bai Xiang and De-Ying Cao
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Thin-Film Hydration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Homogenization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Preparation of DTX-Encapsulating Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Preparation of siRNA-Encapsulating Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Preparation of DTX-Encapsulating Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Preparation of siRNA-Incorporating Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Among the methods for liposome preparation, thin-film hydration is one of the most commonly used methods, which will produce heterogeneous multilamellar vesicles (MLVs). Depending on this process, two types of model molecules, including lipophilic drugs and hydrophilic cargoes, have been mainly reported to be incorporated into liposome. The former can be dissolved together with the lipids prior to the formation of thin film, and the latter, such as oligonucleotidebased hydrophilic ingredients, can be dissolved in the hydration mediums, and then passively incorporated into liposomes via hydration procedure. Following the operation of thin-film hydration, two homogenization methods, sonication and extrusion, have been most usually applied to generate liposomes with optimal B. Xiang (*) · D.-Y. Cao Key Laboratory of Hebei Province for Innovative Drug Research and Evaluation, School of Pharmaceutical Sciences, Hebei Medical University, Shijiazhuang, China e-mail: [email protected]; [email protected] © Springer-Verlag GmbH Germany, part of Springer Nature 2021 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49320-5_2
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size and polydispersity, large unilamellar vesicles (LUVs) or small unilamellar vesicles (SUVs). Here, we initially introduce the thin-film hydration and homogenization, and describe the preparation methods for liposomal products entrapping two types of various cargoes above. Keywords
Liposomes · Thin-film hydration · Homogenization · Sonication · Extrusion
1
Introduction
It has been nearly half of century since it was observed that intense dispersal of purified phospholipids in water led to the formation of microscopic vesicles with closed membrane (Bangham 1968). These artificial membranes were referred to as liposomes in which one or more aqueous compartments are completely enclosed by amphipathic lipid molecules (Sezer 2012). In the lipid bilayer, molecules line up with their polar head groups being exposed towards the water space. Parts of hydrophobic hydrocarbon adhere together and form close, concentric, bimolecular lipid leaflets which separate aqueous compartments (Ranade and Hollinger 2005). In terms of the special structure of liposomes, incorporating both hydrophobic and hydrophilic layers alternatively, hydrophobic materials can be contained within the bilayer and water-soluble molecules within the aqueous compartments. As a delivery system for drugs, liposomes can encapsulate highly nonpolar drugs within the nonpolar bilayer, whereas entrapping more polar molecules within the aqueous core (Wang 2005).
1.1
Thin-Film Hydration
Thin-film hydration is one of the most commonly used methods for the liposome preparation. The standard process to prepare liposomes in the laboratory scale, starting from the selection of the lipids, making a film of the mixed ingredients (such as lipids and drugs) by organic solvent evaporation, drying the film under reduced pressure, dispersing the film, and the subsequent procedures (homogenization, sterilization, etc.) (Douroumis and Fahr 2013). Strategy for loading drug into liposomes relies on the properties of the drug. Lipophilic drugs can be dissolved together with the lipids during liposome preparation. Hydrophilic drugs can be passively incorporated into liposomes during liposome formation. On the basis of thin-film hydration, liposomes have been usually applied on encapsulation of two category active substances: lipophilic drug molecules (Chang et al. 2015; Manjappa et al. 2013; Zhai et al. 2010; Xu and Meng 2016; Luo et al. 2013; Jangde and Singh 2014; Shavi et al. 2016; Wang et al. 2015; Habib et al. 2014; Begum et al. 2012; Mattheolabakis et al. 2012; Chen et al. 2009; Vanaja
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et al. 2013; Umrethia et al. 2007; Hatziantoniou et al. 2006; Ramana et al. 2010; Kumar et al. 2011) and hydrophilic ingredients (i.e., small interfering RNA, siRNA) (Zhang et al. 2006; Mikhaylova et al. 2009; Buyens et al. 2009; Chen et al. 2011, 2013; Xiang et al. 2013). Representative examples of model molecules loaded into liposomes are presented in Table 1.
1.2
Homogenization
Liposomes are microparticulates containing phospholipid bilayers and the enclosed aqueous space. Based on their number of layers per particle and their average size, liposomes are classified into three various types: SUVs (15–100 nm in diameter), LUVs (100–1000 nm in diameter), and MLVs (1–5 μm in diameter) (Bangham 1968). According to thin-film hydration, lipid ingredients are first dissolved in a suitable solvent and dried into a thin film on a rotary evaporator and are then hydrated in an aqueous buffer above the phase-transition temperature (Thassu et al. 2007). This process will produce heterogeneous MLVs. Compared to this type of liposomes, LUVs and SUVs provide more surface area and have the potential to increase solubility, enhance bioavailability, extend the in vivo circulation time and enable accurate targeting of the encapsulated material to a greater extent. Accordingly, it is desirable for the reduction of liposome size. After the process of thin-film hydration, two methods, sonication and extrusion, have been most commonly applied to produce liposomes with optimal size and polydispersity (Handel 2005). Sonication, generally using a probe or bath-type sonicator, can reduce liposome particle size by inducing cavitation. When it comes to extrusion, the operation is performed with a high-pressure filter unit, containing a track-etched polycarbonate membrane above the phase-transition temperature. The polycarbonate membrane has well-defined pores of precise diameters and can withstand pressure of 3000 psi with suitable support. Liposomes extruded through the polycarbonate membranes have narrow particle size distribution (Hope et al. 1985).
1.2.1 Sonication It is Vander Waals and hydrophilic/hydrophobic interactions that drive the hydrated phospholipid molecules arranged in the form of bilayer structures (Weissig 2010a). However, formation of liposomes is not an absolutely spontaneous process and adequate amount of energy must be introduced into the system to overcome an energy barrier. After hydration of the dried lipid or drug-lipid mixture, the resulting MLVs can be treated with sonication. Input of energy gives rise to the arrangement of the lipid molecules, in the form of bilayer vesicles, to accomplish a thermodynamic equilibrium in the aqueous phase. Probe sonication can be used to downsize liposomes by inducing a mechanical rupture of membranes; however, this kind of sonication can result in lipid breakdown or contaminate the sample with pieces of titanium from the sonicator itself. An alternative method is a bath-type sonicator. Instead of inserting a probe into the
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Table 1 List of active substances that have been encapsulated in liposomes via thin-film hydration Encapsulated substance Lipophilic drugs Docetaxel (DTX)
Docetaxel (DTX) Docetaxel (DTX) Paclitaxel (PTX) Paclitaxel (PTX) Quercetin
Anastrozole (ANS) Polydatin (PLD) Cucurbitacin E(Cuc E)
Flurbiprofen (FP)
Phospho-ibuprofen (P-I)
Fenofibrate (FB) Resveratrol (RES) 6-Mercaptopurine (6-MP)
Sclareol Nevirapine Plumbagin (PLB)
Hydrophilic ingredients -nucleotides (targeted protein) siRNA (human double minute gene 2)
Research purpose
References
Development of a RGD-modified pH-sensitive liposome system to enhance the effectiveness of DTX treatment To construct DTX-contained liposomal injection resisting epimerization in vivo Evaluation of a transferrin receptor-targeted liposomal formulation for DTX Construct PTX-loaded stealth liposomes for a prolonged circulation To prepare NGR-modified sterically stabilized liposomes containing PTX Optimization of liposomal formulation for loading quercetin using response surface methodology Development of liposomes containing ANS for effective treatment of breast cancer Development of a novel PLD-loaded liposome
Chang et al. (2015)
To examine the effect of Cuc E on the membrane morphology and properties of lipid vesicles To prepare a new parenteral formulation for FP for prolonging the biologic half-life of the drug Formulation optimization of P-I incorporated liposomes to enhance antitumor activity in vitro and in vivo The development of liposomal FB to improve its oral bioavailability The effect of vitamin C on the antioxidant actions of RES following liposomal delivery To prepare a new parenteral formulation for 6-MP for prolonging the biologic half-life of the drug Construct liposome-incorporated sclareol to overcome its water insolubility Development of a liposome system containing nevirapine To encapsulate PLB as liposomal formulations to enhance its biological half-life and antitumor efficacy
A novel approach for siRNA cellular delivery using R8-modified liposomes
Manjappa et al. (2013) Zhai et al. (2010) Xu and Meng (2016) Luo et al. (2013) Jangde and Singh (2014) Shavi et al. (2016) Wang et al. (2015) Habib et al. (2014) Begum et al. (2012) Mattheolabakis et al. (2012) Chen et al. (2009) Vanaja et al. (2013) Umrethia et al. (2007) Hatziantoniou et al. (2006) Ramana et al. (2010) Kumar et al. (2011)
Zhang et al. (2006) (continued)
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Table 1 (continued) Encapsulated substance siRNA (cyclooxygenase-2, COX-2) siRNA (pGL3 firefly luciferase) siRNA
siRNA (vascular endothelial growth factor) siRNA (polo-like kinase 1)
Research purpose To investigate loading COX-2-specific siRNA into cationic liposomes entrapping MR contrast agents To elucidate the encapsulation of siRNA in pegylated cationic liposomes Preparation of RGD-lipid conjugate-modified liposomes to enhance siRNA delivery in human retinal pigment epithelial cells To prepare RGD motif peptide modified liposomes for enhancing RNAi and cytotoxicity To construct a PSA-responsive and PSMAmediated liposomes for targeted therapy of prostate cancer
References Mikhaylova et al. (2009) Buyens et al. (2009) Chen et al. (2011) Chen et al. (2013) Xiang et al. (2013)
sample, a water bath is applied to transfer the energy to a container. Glass or plastic tubes can be chosen and the sample can also be protected by an inert gas such as argon or nitrogen (Weissig 2010b). Following sonication, the milky suspension should change from cloudy to clear with the decrease of particle size.
1.2.2 Extrusion Another useful technique to generate liposomes of a desired size involves the extrusion through membranes containing pores of a defined size. These orifices in the membranes can range from more than 1 mm down to 50 nm. Several passes through the polycarbonate membrane can also be introduced to control the size distribution of the liposomes tightly (Sims and Larose 1962). Papahadjopolous and coworkers observed that sequential extrusion of MLVs through successively smaller defined pore polycarbonate filters gave rise to LUV systems (Olson et al. 1979). In laboratory, two types of devices from Canada allow both small- (0.2 ml) and large-scale extrusions (10 ml). A simple extruder for small-scale preparation is a handheld model. Under pressure exerted by hand on a syringe, this model allows the operator to pass the liquid system through a membrane back and forth. Extrusion should also be performed above the phase transition temperature of the lipids, which can be accomplished via a heating block. Following about 10–15 passes through the membrane, the solution should achieve a narrow-size distribution close to the size of membrane pore (Magotoshi et al. 1983). Another type of extruder can be equipped with a tank of inert gas, which can push the lipid suspension through a chamber containing polycarbonate membranes. Owing to the fact that constant pressures can be kept on liposomes, this type of extruder is desirable. Also, there are no moving parts, and temperature-controlled water can be cycled through the unit. In the following section of this chapter, the general methodology for encapsulation of hydrophobic drugs and hydrophilic ingredients (i.e., siRNA) will be provided in detail.
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2
Materials
2.1
Preparation of DTX-Encapsulating Liposomes
1. Soy phosphatidylcholine (SPC, >98% purity). SPC is used in solid state (powder), and stored at 20 C. 2. Cholesterol (Chol, pure). Chol is used in solid state (powder), and stored at 20 C. 3. Docetaxel (DTX, >98% purity). 4. Round bottom flasks (50–100 mL). 5. The water used in all solutions is deionized and then distilled [d.d. H2O]. 6. Chloroform. 7. Methanol. 8. Liposome hydration media can include the above-mentioned d.d. H2O or buffer solution. 9. Rotary evaporator (Eyela, Tokyo, Japan). 10. Bath sonicator (Shumei, Jiangsu, China) or probe sonicator (Scientz , Zhejiang, China).
2.2
Preparation of siRNA-Encapsulating Liposomes
1. SPC, see Sect. 2.1 (Bangham 1968). 2. 3β(N-(N0 , N0 -dimethylaminoethane) carbamoyl) cholesterol (DC-Chol). 3. N-(Carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoyl-sn-glycero-3phosphoethanolamine, sodium salt (DSPE-PEG 2000), stored at 80 C. 4. 6-Carboxyfluorescein-labeled negative control siRNA (FAM-siRNA) and siRNA targeting polo-like kinase 1 mRNA (PLK1-siRNA) were synthesized and stored at 20 C (see Note 7). 5. Hand-extruder (Avestin, Canada). 6. Nucleopore track-etch membranes of defined pore sizes: 400 and 200 nm (Whatman, Clifton, New Jersey, USA).
3
Methods
3.1
Preparation of DTX-Encapsulating Liposomes
3.1.1 Thin Film Hydration-MLVs Formation 1. Weigh the required lipid phase composed of phospholipids (i.e., SPC)/cholesterol (2,1, mol/mol), and DTX (0.375 mg) at a drug-to-lipid ratio of 1:20 (w/w), and transfer them to a round-bottom flask. 2. Codissolve the lipids and DTX in either chloroform or in chloroform-methanol mixture (usually 2:1 or 3:1, v/v). Alternatively, place the appropriate volumes of preformed lipid solutions in the flask and mix.
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3. Attach the flask to a rotary evaporator and evaporate the organic solvent under vacuum, until formation of a thin lipid film on the inner walls of the flask. 4. Blow the resulting thin film with an inert gas, such as nitrogen or argon, for at least 10 min and keep the flask overnight under vacuum for total removal of residual solvents. 5. Add 1 mL of a suitable aqueous phase, such as d.d. H2O or buffer, to the flask containing the dry thin film (see Note 3). 6. If phospholipids with high Tc are used as hydrogenated soy phosphatidylcholine (HSPC), dipalmitoylphosphatidylcholine (DPPC), etc., the hydration medium has to be preheated above the lipid Tc, and the hydration procedure should be performed at that temperature, in a heated water bath. 7. Disperse the dried lipids into the hydration fluid by hand shaking the flask or repeated vortex agitation. MLV type liposomes formed at this stage.
3.1.2 Homogenization (Sonication)-SUVs Formation 1. Place the flask containing MLV suspension produced above, either in a bath-type sonicator or a probe sonicator for vesicle size reduction. 2. For bath sonication, suspend the MLVs flask in the bath sonicator with enough preheated water, which is on a liquid level with that of inside the flask. Employ sonication for a time period of 5–15 min. 3. An alternative method is probe sonication performed to downsize the MLVs. Place the probe of the sonicator in the MLVs flask below the liquid level and subject the sample to high intensity sonication with 10 s ON, 10 s OFF intervals, for a total period of 15 min at least, or until the vesicle dispersion becomes semitransparent with light-blue opalescence. At this stage, the resulting liposomes are predominantly in the form of SUVs (see Note 4). 4. Following sonication, incubate the SUVs product at a temperature over the Tc of the lipid used, under an inert atmosphere such as nitrogen or argon for 1 h, in order to amend any structural defects of the vesicles. 5. Remove the unloaded drug, titanium fragments (from the probe), residual MLVs, or liposomal aggregates in the SUVs system produced, by centrifugation at 10,000 rpm for 15 min.
3.2
Preparation of siRNA-Incorporating Liposomes
Under certain circumstances, the objective of producing various liposome systems entrapping drugs of different properties can be achieved by simply altering lipid composition. The introduction of positively charged lipids, such as DC-Chol, into lipid film formulation, is applicable to liposomalization of siRNA, which consists of 21 nt short double-stranded and strongly negatively charged RNA (Zuckerman and Davis 2015). The siRNA molecules can be passively encapsulated during the hydration step and the general procedures are described in the following section.
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3.2.1 Thin Film Hydration- MLVs Formation 1. Weigh the appropriate quantities of lipid ingredients containing normal phospholipids and cationic lipids, such as SPC/cholesterol/DC-Chol/DSPE-PEG2000 (48,8:40:3, mol/mol), and then place them in a round-bottom flask. 2. Prepare a solution by dissolving the lipids in an organic phase mixture of chloroform and methanol (3:1, v/v). Alternatively, place the appropriate volumes of a preformed lipid stock solutions (see Note 7) in the flask and blend. 3. Drive the organic solvent away under reduced pressure by rotary evaporator, forming a thin lipid film at the bottom of the flask, and keep the flask under vacuum for an additional 1–2 h, in order to remove the traces of solvent totally. 4. Introduce a suitable mount of diethyl pyrocarbonate (DEPC)-treated aqueous system, such as d.d. H2O and 5% dextrose (w/v) with PLK1-siRNA or FAM-siRNA (1.25 μM), onto the resulting dry thin film. 5. Hydrate the dried lipid film by hand shaking the flask or repeated vortex agitation, if necessary, in a heated water bath at a temperature above the specific Tc of the most stable lipid used. Thus, MLV type liposome complexes formed in a charge ratio of 5. 3.2.2 Homogenization (Extrusion)-SUVs Formation 1. Downsize and homogenize the siRNA-loaded MLVs by extrusion of 10 cycles through a hand-extruder at 40 C. The extrusion should be performed sequentially through polycarbonate nuclepore track-etch membranes with pore sizes of 0.4 μm (5 times) and 0.2 μm (5 times), resulting in a disperse system with a uniform size distribution of approximate 200 nm. 2. Incubate the final product in a water bath at temperatures above Tc under nitrogen for 1 h, to allow for the sample maturation. 3. Gas the liposomes-contained tube with nitrogen carefully, flushing air out, and close the tube tightly followed by the storage at 4 C.
4
Notes
1. Instead of the use of one phospholipid in liposome formulation, the application of phospholipid mixture, such as HSPC/DPPC/dipalmitoyl phosphatidyl glycerol/ Chol, is preferred to, which would achieve an enhanced loading of the hydrophobic drug by preventing imperfect packing of lipid with varied dimensional characteristics(Chen et al. 2006; Kan et al. 2011). 2. The addition of an organic acid has been proved to helpful to enhance the stability of DTX even after heating at higher temperature. At present, the most preferred acid is citric acid anhydrous, which can be added during film formation and exhibits better controls over the formation of impurity such as 7-epidocetaxel, a well-known degradation product of DTX(Manjappa et al. 2013). 3. For the hydration of dried lipid film, other appropriate dispersion mediums are saline or nonelectrolytes such as a sugar solution. In preparation of liposomes for the in vivo application, physiological osmolality (290 m Osmol/kg) is
2
4.
5.
6.
7.
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recommended and can be obtained using 0.6% saline, 5% dextrose, or 10% sucrose solution (Weissig 2010a). The preferred wares for sonication are the more resistant glass tubes with a thick wall. The sonication probe should be immersed as deep as possible in the test tube, without touching the inner wall or bottom. The energy given should be sufficient to promote homogenization of the lipid suspension, without producing excessive cavitation. If necessary, an ice bath should be employed to keep the lipids and/or drugs from being disrupted by the high heat. In liposome samples, the loaded DTX locates in the membrane of liposomes due to its poor aqueous solubility; however, the unloaded parts emerge in the crystal forms in outer water phase. The crystal can be removed from liposomes suspension by centrifugation (Zhang et al. 2014). Make sure that all aqueous solutions and wares (including glass and plastic container) are DNase- and RNase-free grade. Also, the inner cavity of extruder should be cleaned entirely, followed by rinse with DEPC-treated double-distilled water. In this case, the protection of fluorescently labeled siRNA (FAM-siRNA) from light should be kept throughout the whole process; otherwise, its fluorescence intensity would decrease with time.
Conclusion
Herein, the general preparation methods for liposome encapsulating hydrophobic ingredients (DTX) and hydrophilic cargoes (siRNA) were given in detail. In summary, the progress of thin-film hydration accomplishes drug loading and gives rise to MLVs. The subsequent sonication and extrusion turn the dispersed system into liposomal suspension with a uniform size distribution, obtaining DTX-loaded and siRNA-loaded SUVs products, respectively.
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Preparation of Drug Liposomes by Reverse-Phase Evaporation Nian-Qiu Shi and Xian-Rong Qi
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Preparation of Liposomes by Reverse-Phase Evaporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 The Determination of Encapsulation Efficiency (%) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Observation of Particle Size and Electron Micrograph . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Measurement of Surface Charge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Diffusional Exchange Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
38 39 39 40 40 41 41 42 42 44 45 45
Abstract
Liposomes display increased therapies for a series of biomedical application by stabilizing loaded payloads, overcoming shortcomings to cellular and tissue uptake, and enhancing biodistribution of payloads to target sites in vivo. The Bangham method or thin lipid film hydration technology was the first described
N.-Q. Shi (*) Department of Pharmaceutics, School of Pharmacy, Jilin Medical University, Jilin, Jilin Province, China e-mail: [email protected] X.-R. Qi Department of Pharmaceutics, School of Pharmaceutical Science, Peking University, Beijing, China Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System, Beijing, China e-mail: [email protected] © Springer-Verlag GmbH Germany, part of Springer Nature 2021 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49320-5_3
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method for constructing liposomes. The drawbacks of this method involve sonicator contact with the liposomes, and risk of high-temperature exposure may lead to phospholipid/drug damage and deceased encapsulation. A generally adopted preparative alternative is the reverse-phase evaporation technique that tends to form inverted micelles or water-in-oil emulsions. The water phase carries the drug, and organic phase is made up of lipids for forming the liposome bilayer. The lipid mixture is dissolved in solvents, and the lipid solvents are evaporated. Formed lipid film after evaporation is redissolved in an organic phase. Under reduced pressure condition, the organic solvent can be slowly evaporated, initially resulting in the conversion of the dispersion into a viscous gel and finally generating an aqueous suspension containing the liposomes. Similar to other preparation methods, the size of liposome generated by reverse-phase evaporation technique needs to be reduced by multiple extrusions through a polycarbonate membrane. The number of extrusion cycles and the size of polycarbonate membrane pore influence the degree of size reduction and the final particle size and distribution. This chapter summarized the preparation procedure and formation principle of various payload-loaded liposomes by reverse-phase evaporation technique. Keywords
Liposomes · Reverse- phase evaporation · Encapsulation efficiency · Sizes · Electron micrograph
1
Introduction
Liposomes were described first in the 1960s by Bangham et al. (1965) and have been used to be the most common and well-investigated nanocarriers for targeted drug delivery (Xiang et al. 2013; Zhao et al. 2011; Yang et al. 2014). They display increased therapies for a series of biomedical application by stabilizing loaded payloads, overcoming shortcomings to cellular and tissue uptake, and enhancing biodistribution of payloads to target sites in vivo (Bozzuto and Molinari 2015; Koning and Storm 2003; Metselaar and Storm 2005). Over five decades of researches in the field of liposomes have suggested their promise in the medical and cosmetic as well as the food industry (Mu and Sprando 2010; Mozafari et al. 2008; Madni et al. 2014). Liposomes consist of phospholipids, which self-assemble to form sphere-shaped vesicles of lipid bilayer and an aqueous core within the bilayers (Qi et al. 1995a, b, 1997). Liposomes seem to be an almost ideal carrier, because their structure is similar to that of cellular membrane and they can incorporate many substances. Hydrophilic as well as lipophilic compounds could be encapsulated into an aqueous core and a lipid bilayer of liposomes through hydrogen bonding, van der Waals forces, and other electrostatic interactions (Israelachvili et al. 1980; Lasic 1998). Many advantages of liposomes have been displayed including solubility enhancement of the encapsulated drugs, prevention of chemical and biological degradation,
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reduction of the nonspecific side effects and toxicity, versatile modification with various functional moiety, and improved biocompatibility in vivo (Madni et al. 2014; Qi et al. 1995a, b). These merits of liposomes have resulted in many successful basic and clinical applications. Currently approved commercial liposomal drug products include Myocet ®, Lipodox ®, DaunoXome ®, AmBisome®, DepoDur ®, Inflexal V ®, etc. for the therapy of various cancers, fungal infections, pain, and influenza. More and more liposome-based formulations are being tested in phase I, II, or III stages (Pattni et al. 2015; Sercombe et al. 2015). The Bangham method or thin lipid film hydration technology was the first described method for constructing liposomes. The drawbacks of this method involve sonicator contact with the liposomes, and risk of high-temperature exposure may lead to phospholipid/drug damage and deceased encapsulation (Mozafari 2005). A generally adopted preparative alternative is the reverse-phase evaporation technique that tends to form inverted micelles or water-in-oil emulsions. The water phase carries the drug, and organic phase is made up of the lipids for forming the liposome bilayer. Briefly, the lipid mixture is dissolved in solvents, and the lipid solvents are evaporated. Formed lipid film after evaporation is redissolved in an organic phase comprised of diethyl ether or isopropyl ether. A two-phase system may generate after the addition of the aqueous phase through forming a homogeneous dispersion by sonication. Under reduced pressure condition, the organic solvent can be slowly evaporated, initially resulting in the conversion of the dispersion into a viscous gel and finally generating an aqueous suspension containing the liposomes. Compared with the thin film hydration method, this method causes a higher internal aqueous loading. The residual solvent can be removed by centrifugation, dialysis, or passage via a Sepharose gel column. The disadvantage is trace remaining organic solvent that can probably interrupt the chemical or biological stability of lipid or loaded drugs/genes. Similar to other preparation methods, the size of liposome generated by reverse-phase evaporation technique needs to be reduced by multiple extrusions through a polycarbonate membrane. The number of extrusion cycles and the size of polycarbonate membrane pore influence the degree of size reduction and the final particle size and distribution (Szoka and Papahadjopoulos 1978; Elorza et al. 1993; Cortesi et al. 1999; Qi et al. 1995c).
2
Materials
2.1
Chemicals
1. 2. 3. 4. 5. 6. 7. 8.
Cholesterol Palmitic acid Phosphatidylcholine (PtdCho) Phosphatidylglycerol (PtdGro) Phosphatidylserine (PtdSer) Phosphatidic acid Dipalmitoyl phosphatidylcholine (Pal2PtdCho) Sphingomyelin (bovine brain)
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9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
N.-Q. Shi and X.-R. Qi
[3H] poly(A) Ferritin Albumin Rabbit Ig G Porcine insulin Alkaline phosphatase Ribonuclease (RNase) 125 I-labeled sucrose 22 Na-labeled sucrose 14 C-labeled sucrose [3H]Cytosine arabinoside (araC) 25S[32P]RNA 50-ml round-bottom flask Nitrogen Ether Chloroform Methanol Phosphate-buffered saline Isopropyl ether 0.1% Triton X-100 Tirfluorotrichloroethane
3
Methods
3.1
Preparation of Liposomes by Reverse-Phase Evaporation
1. Add lipid mixtures (several phospholipids, cholesterol, and long-chain alcohols) to a 50-ml round-bottom flask with a long extension neck. 2. Remove the solvent under reduced pressure by a rotary evaporator. 3. Purges nitrogen into the system for protecting the lipid mixtures from degradation. 4. Redissolve lipids in the organic phase such as diethyl ether, isopropyl ether, halothane, and trifluorotrichloroethane. 5. Increase the solubility of lipids also using ether, chloroform, or methanol. 6. Add the aqueous phase and keep the system continuously under nitrogen. 7. Sonicate the resulting two-phase system briefly (2–5 min) in a bath-type sonicator until the mixture becomes either a clear one-phase dispersion or a homogeneous opalescent dispersion. 8. Place the mixture on the rotary evaporator, and remove the organic solvent under reduced pressure at 20–25 C, rotating at approximately 200 rpm. 9. A viscous gel forms and an aqueous suspension appears. 10. Add excess water or buffer, and evaporate the suspension for an additional 15 min at 20 C to remove traces of solvent.
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11. Dialyze the preparation, and pass through a Sepharose 4B column, or centrifuged.
3.2
The Determination of Encapsulation Efficiency (%)
1. Dialyze the vesicles overnight against 300 vol of phosphate-buffered saline at 4 C to determine the amount of encapsulated small smalls such as sodium, sucrose, or [3H] araC (as shown in Table 1, Figs. 1 and 2). 2. Apply column chromatography on Sepharose 4B (1.5 42 cm) to separate encapsulated iodinated proteins from unencapsulated proteins. 3. Use centrifugation (1000 g for 30 min) to separate encapsulated [3H] poly (A) from unencapsulated materials. 4. Separate encapsulated 25S[32P]RNA from unencapsulated materials by first treating with RNase (5 μg) and alkaline phosphatase (10 μg) and separating the encapsulated materials from the hydrolyzed RNA on a Sepharose column (Table 2). 5. Degrade the unencapsulated RNA totally by above procedure. Establish the latency of alkaline phosphatase by measuring enzyme activity in the presence and absence of 0.1% Triton X-100.
3.3
Observation of Particle Size and Electron Micrograph
1. Observe electron micrographs picture of liposomes through negative stain method (as shown in Fig. 1). 2. After diluting liposomes to an appropriate volume with water, determine the particle size of liposomes using a dynamic light scattering instrument (Table 3). Table 1 Effect of lipid composition on encapsulation of araC and sucrose in liposomes prepared through reverse-phase evaporation method Lipid composition 1. PtdGro/PtdCho/Chol (1:4:5) 2. PtdGro/PtdCho (1:4) 3. PtdGro/PtdCho (1:4) 4. SA/PtdCho (1:4) 5. PtdGro 6. PtdGro 7. Pal2PtdCho 8. PtdGro/PtdCho/Chol (1:4:5) MLV 9. PtdGro/PtdCho/Chol (1:4:5) SUV
Captured volume, μl/mg 13.7 9.2 8.1 15.6 10.5 8.7 11.7 4.1 0.5
% encapsulation araC Sucrose 55.0 3.9 64.6 24.2 0.5 30.1 42.8 0.6 ND 59.7 2.7 63.0 27.7 2.4 18.7 46.7 2.7 ND 28.9 2.7 35.5 16.5 ND 1.8 ND
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Fig. 1 (a) Freeze-fracture electron micrograph of PtdGro/PtdCho/cholesterol (1/4/5) liposomes containing ferritin prepared by reverse-phase evaporation technique. Caliper indicated 100 nm. (b–d) Negative-stain electron micrographs of liposomes prepared by the standard procedure: (b) PtdGro/PtdCho/cholesterol (1/4/5), a typical field of an unfiltered preparation; (c) PtdGro/PtdCho/ cholesterol (1/4/5), a typical field of preparation filtered through a 0.2-μm Unipore filter; (d) PtdGro/PtdCho (1/4), a typical field of an unfiltered preparation. Bar indicates 200 nm
3.4
Measurement of Surface Charge
1. Measure the electrophoretic mobility of phospholipid particles in 130 mM KCl containing Tris-HC1 (15 mM, pH 7.4). 2. Convert the electrophoretic mobility to ξ-potential using the formula: ξ = 12.9 V/ Eapp, where V is the electrophoretic mobility in μ.sec 1 and Eapp is the applied voltage in V-cm 1 and ξ in mV.
3.5
Diffusional Exchange Measurements
1. Separate bulk-phase tracer ions from those contained in the lipid particles either by dialysis or by Sephadex filtration. 2. For direct dialysis, pipette 0.5-ml aliquots of a dispersion, each containing 2–10 μmol of phosphorus into dialysis bags (0. 9 6 cm, previously rinsed with the
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Fig. 2 Formation of liposomes prepared by reverse-phase evaporation technique. Dissolved lipids in appropriate solvents, lipids indicated by lollipop structures; addition of aqueous phase containing compound to be encapsulated, indicated by filled square
Table 2 Encapsulation of various molecules in PtdGro/PtdCho/ cholesterol (1:4:5) liposomes prepared through reverse-phase evaporation method
Encapsulation materials Sodium Carboxyfluorescein Poly(A) Poly(A) 25S RNA Insulin Ferritin Alkaline phosphatase Albumin IgG
Buffer PBS 1/10 PBS PBS 1/10 PBS 1/10 PBS PBS 1/10 PBS PBS PBS 1/10 PBS
% encapsulation 42 57 24 43 40 34 54 34 38 28–40
exchange medium) and dialyze them against 5–30-min changes of 500 ml of the exchange fluid which was the same as the dispersing medium but without tracer. 3. Accomplish more rapid removal of the bulk tracer (5 min) by the passage of the dispersion (1–2 ml) through a column of Sephadex (3 g, G-50, coarse grade), packed in the “cold” exchange medium.
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Table 3 Summary of size, encapsulation efficiency, and internal volume of different liposome preparations
Liposomes Liposomes prepared by reverse evaporation method (PtdGro/PtdCho/Chol, 1:4:5) Liposomes prepared by reverse evaporation method (PtdGro/PtdCho, 1:4) Multilamellar liposomes (PtdGro/PtdCho/Chol, 1:4:5) Sonicated unilamellar liposomes (PtdGro/ PtdCho/Chol, 1:4:5) Large unilamellar liposomes (PtdSer)
Encapsulation efficiency 35–65
Captured volume, μl/mg 13.7
Diameter, nm (range) 200–1000
30–45
8.1
100–300
5–15
4.1
400–3500
0.5
20–50
9.1
200–1000
0.5–1 5–15
4. Wash the column with the exchange medium and collect the main lipid peak (usually 5 ml, between 13 and 17 ml of eluted volume). 5. Pipette aliquots (0.5 ml) into rinsed dialysis bags. 6. Transfer dialysis bags into duplicate stoppered tubes each containing 10 ml of the exchange medium after removal of bulk-phase tracer. Rotate the tubes gently at a speed of a rev./min. 7. Determine the tracer ions in each 10-ml dialysate, and those remaining inside the bags, were d with the appropriate counting equipment. 8. Take the amount of tracer appearing in the dialysate during the first hour after the removal of unincorporated ions to represent the diffusion rate constant, for a concentration difference of 145 mM at zero time. 9. Self-diffusion rate is expressed both as mequiv of ions/mole of lipid per h and as a percentage of the ions present inside the particles at zero time. The total amount of ions present at zero time, referred to as capture, is expressed as equiv/mole of lipid. 10. Estimate the amount of lipid present in each dialysis tube by phosphorus analysis. 11. Calculate activation energies from the Arrhenius equation by plotting the natural log of self-diffusion rates vs. the reciprocal of absolute temperature.
4
Notes
1. All above lipids need to be purified on silicic acid column to ensure the quality of lipids. The purity may be verified by thin-layer chromatography. 2. To avoid the degradation or damage of lipids, all mentioned lipids should be stored in chloroform in sealed ampules under nitrogen at 50 C until use. 3. Redistill diethyl ether led from sodium bisulfite immediately before use to eliminate any peroxides for some preparations.
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4. Keep the stabilization of the lipids using the nitrogen protection in the preparation of Rev. 5. Bath-type sonicator, instead of probe-type sonicator, is suitable to prepare reverse evaporation liposomes because metal contamination probably appears through sonicating by metal probe. 6. The sonication process will result in the increase of temperature. Thus, control the temperature at 0~5 C. 7. Change the determination methods of encapsulation efficiency (%) for various encapsulated substances. It is necessary to ensure the removal or degradation of unencapsulated substances. 8. Reverse-phase evaporation method results in the formation of “inverted micelles” that are collapsed into a viscous gel-like state when the organic phase is removed by evaporation. The diagram of the formation of liposomes by reverse-phase evaporation is shown in Fig. 2. 9. The introduction of cholesterol in the liposome preparations will cause the higher encapsulation efficiency generally. 10. Using reverse-phase evaporation method, a substantial fraction of the aqueous phase is up to 65% in liposomes. This preparative method of liposomes has unique advantage for encapsulating valuable water-soluble materials such as drugs, proteins, nucleic acids, and other biochemical reagents.
5
Conclusion
Among the methods for preparing liposomes, reverse-phase evaporation is a typical and representative approach. The lipid mixtures are dispersed and dissolved in solvents, and the lipid solvents are evaporated. Yield lipid film after evaporation is redissolved in an organic phase. By reduced pressure method, the organic solvent can be slowly removed, initially leading to the conversion of the dispersion into a viscous gel and finally forming an aqueous suspension containing the liposomes. A substantial fraction of the aqueous phase (up to 65% at low salt concentrations) is entrapped within the vesicles, encapsulating even large macromolecular assemblies with high efficiency. Thus, this relatively simple technique has unique advantages for encapsulating valuable water-soluble materials such as drugs, proteins, nucleic acids, and other biochemical reagents. The preparation and properties of the liposomes are described.
References Bangham AD, Standish MM, Watkins JC (1965) Diffusion of univalent ions across the lamellae of swollen phospholipids. J Mol Biol 13(1):238–252 Bozzuto G, Molinari A (2015) Liposomes as nanomedical devices. Int J Nanomed 10:975–999 Cortesi R, Esposito E, Gambarin S, Telloli P, Menegatti E, Nastruzzi C (1999) Preparation of liposomes by reverse-phase evaporation using alternative organic solvents. J Microencapsul 16 (2):251–256
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Elorza B, Elorza MA, Frutos G, Chantres JR (1993) Characterization of 5-fluorouracil loaded liposomes prepared by reverse-phase evaporation or freezing-thawing extrusion methods: study of drug release. Biochim Biophys Acta 1153(2):135–142 Israelachvili JN, Marcelja S, Horn RG (1980) Physical principles of membrane organization. Q Rev Biophys 13(2):121–200 Koning GA, Storm G (2003) Targeted drug delivery systems for the intracellular delivery of macromolecular drugs. Drug Discov Today 8(11):482–483 Lasic DD (1998) Novel applications of liposomes. Trends Biotechnol 16(7):307–321 Madni A, Sarfraz M, Rehman M, Ahmad M, Akhtar N, Ahmad S, Tahir N, Ijaz S, Al-Kassas R, Löbenberg R (2014) Liposomal drug delivery: a versatile platform for challenging clinical applications. J Pharm Pharm Sci 17(3):401–426 Metselaar JM, Storm G (2005) Liposomes in the treatment of inflammatory disorders. Expert Opin Drug Deliv 2(3):465–476 Mozafari MR (2005) Liposomes: an overview of manufacturing techniques. Cell Mol Biol Lett 10(4):711–719 Mozafari MR, Johnson C, Hatziantoniou S, Demetzos C (2008) Nanoliposomes and their applications in food nanotechnology. J Liposome Res 18(4):309–327 Mu L, Sprando RL (2010) Application of nanotechnology in cosmetics. Pharm Res 27(8):1746–1749 Pattni BS, Chupin VV, Torchilin VP (2015) New developments in liposomal drug delivery. Chem Rev 115(19):10938–10966 Qi XR, Maitani Y, Nagai T (1995a) Effect of soybean-derived sterols on the in vitro stability and the blood circulation of liposomes in mice. Int J Pharm 114(1):33–41 Qi XR, Maitani Y, Nagai T (1995b) Rates of systemic degradation and reticuloendothelial system uptake of calcein in the dipalmitoylphosphatidylcholine liposomes with soybean-derived sterols in mice. Pharm Res 12(1):49–52 Qi XR, Maitani Y, Shimoda N, Sakaguchi K, Nagai T (1995c) Evaluation of liposomal erythropoietin prepared with reverse-phase evaporation vesicle method by subcutaneous administration in rats. Chem Pharm Bull 43:295–299 Qi XR, Maitani Y, Nagai T, Wei SL (1997) Comparative pharmacokinetics and antitumor efficacy of doxorubicin encapsulated in soybean-derived sterols and poly(ethylene glycol) liposomes in mice. Int J Pharm 146(1):31–39 Sercombe L, Veerati T, Moheimani F, Wu SY, Sood AK, Hua S (2015) Advances and challenges of liposome assisted drug delivery. Front Pharmacol 6:286 Szoka F Jr, Papahadjopoulos D (1978) Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation. Proc Natl Acad Sci USA 75(9):4194–4198 Xiang B, Dong DW, Shi NQ, Gao W, Yang ZZ, Cui Y, Cao DY, Qi XR (2013) PSA-responsive and PSMA-mediated multifunctional liposomes for targeted therapy of prostate cancer. Biomaterials 34(28):6976–6991 Yang ZZ, Li JQ, Wang ZZ, Dong DW, Qi XR (2014) Tumor-targeting dual peptides-modified cationic liposomes for delivery of siRNA and docetaxel to gliomas. Biomaterials 35(19):5226–5239 Zhao W, Zhuang S, Qi XR (2011) Comparative study of the in vitro and in vivo characteristics of cationic and neutral liposomes. Int J Nanomed 6:3087–3098
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Preparation and Characterization of Drug Liposomes by pH-Gradient Method Nian-Qiu Shi, Xian-Rong Qi, and Bai Xiang
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Preparation of Liposomes Loaded by Fluorescent Probes or Adriamycin . . . . . . . . . . . . 3.2 Fluorescence and Release Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
48 49 50 50 51 51 55 56
Abstract
During last five decades, liposomes are well developed as delivery carriers for various molecules and can form unilamellar or multilamellar lipid vesicles. To design liposomal formulations for tumor therapy, administration by intravenous injection is a main option. Considering that sometimes anticancer drug needs higher dose (e.g., ~50 mg/m2 for doxorubicin), the stable drug-loading method must be explored for
N.-Q. Shi (*) Department of Pharmaceutics, School of Pharmacy, Jilin Medical University, Jilin, Jilin Province, China e-mail: [email protected] X.-R. Qi Department of Pharmaceutics, School of Pharmaceutical Science, Peking University, Beijing, China Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System, Beijing, China e-mail: [email protected] B. Xiang Key Laboratory of Hebei Province for Innovative Drug Research and Evaluation, School of Pharmaceutical Sciences, Hebei Medical University, Shijiazhuang, China e-mail: [email protected] © Springer-Verlag GmbH Germany, part of Springer Nature 2021 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49320-5_18
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the practical use of drug liposomes during storage and in circulation. Conventional preparation approaches are impossible to reach higher drug encapsulation because of the nature of the poor drug solubility. The issue of low drug-loading rate would make therapeutic potential of drugs in low levels and cannot reach clinic use. The development of remote loading can also overcome the instability when loading with high internal concentrations of drugs. Transmembrane pH gradients in liposomal carrier can be helpful to promote the encapsulation of various commonly used drugs and fluorescent probes. In fact, pH-gradient method is the first use of remote loading approaches. The transmembrane ion gradients can be constructed by adopting salts composes either weak bases or weak acids. The degree of ionization of these drugs is pH dependent. Their ionized forms have a very low permeability coefficient, while their unionized species have high permeability and can diffuse relatively fast across liposome lipid bilayer to reach the intraliposomes. pH-gradient method provides a basic insight to develop other remote loading methods. Keywords
Liposomes · Preparation methods · pH gradient · Remote loading technique · Anticancer drug
1
Introduction
During last five decades, liposomes are well developed as delivery carriers for various molecules and can form unilamellar or multilamellar lipid vesicles (Bangham et al. 1965; Satish and Surolia 2002; Pantos et al. 2005; Sideratou et al. 2009; Xing et al. 2016). There is increased evidence that liposomal drug delivery systems can obviously decrease the side effect of loading drug molecules without weakening drug activity. Some drugs such as amphotericin B and antitumor compounds (e.g., cytosine, arabinoside, and adriamycin) have been entrapped into liposomes (Shimizu et al. 2010; Schwendener and Schott 2005; Sehgal and Rogers 1995; Zhao et al. 2011). Owing to the enhanced permeability and retention (EPR) effect (Russell et al. 2017; Greish 2007), liposomes can accumulate into disease tissues and promote the therapeutic effect of some problematical drugs after they are administrated intravenously. A leaky microvasculature as well as an insufficient lymphatic drainage can result in the accumulation of drug-loaded liposomes into tumor sites (Brown and Giaccia 1998; Brown 2002). A classic anticancer compound, doxorubicin-related liposomal formulations have been investigated for many years (Cagel et al. 2017a, b; Burade et al. 2017). Liposomal encapsulation for doxorubicin has decreased tumor exposure. The decoration of liposomes by poly (ethylene glycol) (PEG) can cause long-circulation effect of encapsulated drugs and avoid its premature uptake by the reticuloendothelial system (RES) through prolonging its serum half-life (Qi et al. 2005; Shi et al. 2005; Mineart et al. 2017). Thus, the first FDA-approved nano-drug was marketed as Doxil ® in 1995 (Gabizon et al. 2002; Hadjidemetriou et al. 2016). To design liposomal formulations for tumor therapy, administration by intravenous injection is a main option (Ichihara et al. 2012). Considering that sometimes
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anticancer drugs need higher dose (e.g., ~50 mg/m2 for doxorubicin), the stable drug-loading method must be explored for the practical use of drug liposomes during storage and in circulation. Conventional preparation approaches involve thin-membrane dispersion technique, organic solvent injection method, and reverse-phase evaporation et al. (Mozafari 2005; Perrie et al. 2017; Giddam et al. 2012). These loading technologies are impossible to reach higher drug encapsulation because of the nature of the poor drug solubility (Elizondo et al. 2011; Sercombe et al. 2015). The issue of low drug-loading rates would make therapeutic potential of drugs in low levels and cannot reach clinic use. The administration of such liposomes will require very large amounts of lipids. Moreover, removal of unencapsulated drugs is necessary due to inefficient loading (Yang et al. 2013; Barenholz 2012). A comprehensive survey indicated that the remote loading method is the main choice to obtain a viable or high drug-loading formulation among available loading approaches at early time (1980~1990) (Redelmeier et al. 1989). The development of remote loading can also overcome the instability when loading with high internal concentrations of drugs (Sur et al. 2014). Some earlier works have found that transmembrane pH gradients in liposomal carrier can be helpful to promote the encapsulation of various commonly used drugs, certain ions, and peptides. In fact, pH-gradient method is the first use of remote loading approaches (Deamer et al. 1972; Mayer et al. 1986). Deamer and coauthors (Mayer et al. 1986) initially report remote loading of amphipathic weak bases (such as catecholamines) by a pH-gradient. pH-gradient method was extensively adopted by Cullis group for developing liposomes loaded by many amphipathic weak bases including doxorubicin (Redelmeier et al. 1989). Bally et al. (Dos Santos et al. 2004) used ethanol to regulate drug-loading rates for pH-gradient loading of anthracyclines into cholesterol-free liposomes. As a representative method of liposomal preparation, pH-gradient method on remote loading of doxorubicin into liposomes resulted in the development of Myocet® by The Liposome Company (TLC) in Princeton (Shapiro et al. 1999). The transmembrane ion gradients can be constructed by adopting salts composes either weak bases or weak acids. The degree of ionization of these drugs is pH dependent. Their ionized forms have a very low permeability coefficient, while their unionized species have high permeability and can diffuse relatively fast across liposome lipid bilayer to reach the intraliposomes aqueous phase (Barenholz 2012; Cern et al. 2012). Fig. 1 indicates the model and principle of weakly basic compounds into liposomes. pH-gradient method provides a basic insight to develop other remote loading methods (Haran et al. 1993).
2 1. 2. 3. 4. 5. 6. 7.
Materials Egg lecithin Hens eggs Alumina Silica acid Dicetyl phosphate Bacteriochlorophyll Atebrin
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Fig. 1 Model and principle for the uptake of weakly basic compounds into liposomes in response to Δ pH (pH gradient). For compounds with appropriate pKa values, a neutral exterior pH results in a mixture of both the protonated AH+ (membrane impermeable) and unprotonated A (membrane permeable) forms of compounds
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
9-aminoacridine Tricine, 2-(N-morpholino) ethanesulfonic acid (MES) N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES) A buffer containing 0.1 M sodium phosphate and 0.1 M sodium pyrophosphate H2SO4 Cholesterol Adriamycin Egg yolk phosphatidylcholine Dipalmitoylphosphatidylcholine (DPPC) Tritiated DPPC Sephadex G-50 KOH Glutamic acid NaCl Triton X-100 3 [H] DPPC Nitrogen K3Fe(CN)6 K2SO4 EDTA Sucrose
3
Methods
3.1
Preparation of Liposomes Loaded by Fluorescent Probes or Adriamycin
1. Produce liposomes by evaporating chloroform solutions of varying amounts of egg lecithin plus 5% dicetyl phosphate under nitrogen.
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2. Sonicate the lipid for 1 min with 2 ml of the medium to be trapped inside the liposome volume. 3. The solutions to be trapped contained either phosphate-pyrophosphate buffer at pH 5.0 to 9.0, 0.1 M K3Fe(CN)6, or varying concentrations of K2SO4. All solutions include 1 mM EDTA. 4. Place the sonicated suspension (1.5 ml) on a 15 cm 1 cm column of Sephadex G-50. 5. Collect the turbid liposome suspension from the column in 3–4 ml of the eluate and adjust to a known volume (3–5 ml) with sucrose solution. 6. In some experiments, 2 mole % bacteriochlorophyll is included in the lipid prior to evaporation and sonication. All experiments are carried out within 6 h of sonication. 7. For adriamycin-loaded liposomes, vesicles are prepared according to the extrusion procedure employing a freeze-thaw protocol and 100 nm pore size polycarbonate filters. These LUVs have an average diameter of 103 nm and a trapped volume of approx. 1.5 μl/μmol phospholipid. These buffer compositions yield transmembrane pH gradients which are stable during the uptake process.
3.2
Fluorescence and Release Measurements
1. Measure fluorescence of the amines in a fluorimeter with the photomultiplier at 90 to the actinic light path. 2. Provide the actinic beam by a quartz-iodine bulb (Philips 12,258/99 12 V 55 W) supplied by a stabilized power supply (Coutant Electronics Ltd., Reading, England). 3. Select blue light either by a monochromator (Hilger and Watt Ltd., London, England Type D 292) or by a Wratten 36 gelatin filter (Kodak Ltd., London) together with a Corning 9782 glass filter. 4. Screen the photomultiplier (EMI Electronics Ltd., Hayes, Middlesex, England, Type 9601 B) by a Wratten 61 filter, together with a Corning 9782 glass filter. 5. The output is fed to a recorder (Toa Polyrecorder EPR-2 TB, T.E.M. Sales Ltd., Crawley, Sussex, England) by way of a simple amplifier and backing circuit. 6. For release measurement, adriamycin release is monitored under flow dialysis conditions as follows. Vesicles (5 mM lipid) containing adriamycin are placed in a flow dialysis apparatus equilibrated at 37 C. Flow rates are adjusted to achieve total exchange of the sample compartment volume (50 ml) in 10 min. Aliquots (0.15 ml) are removed at various times, and untrapped material is removed employing 1 ml gel filtration columns. The sample is then assayed for adriamycin and lipid.
3.3
Data Analysis
1. Intrinsic fluorescence is affected by pH value. Atebrin fluorescence is pH dependent and increased nearly twofold as pH increased from 6 to 9 (as shown in Fig. 2). This is related to the first pK of this diamine (pK = 7.9, 10.5), and the fluorescence change is half maximal at pH 7.9. There is little variation in 9aminoacridine fluorescence (pK = 10.0) in inorganic buffers over the same pH range.
Fig. 2 Dependence of amine fluorescence on pH. The fluorescence (arbitrary units) of atebrin and 9aminoacridine is measured in organic buffers. The organic buffers (———) are 0.1 M MES (pH 5 and 6), 0.1 M TES (pH 7), and 0.1 M Tricine (pH 8 and 9). The inorganic buffer (—) is the phosphatepyrophosphate buffer described in Methods. ■ and □, 4 μM 9-aminoacridine in organic and inorganic buffers, respectively; ● and ○, 0.4 μM atebrin in organic and inorganic buffers, respectively
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FLUORESCENCE
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6
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8
9
pH
2. Fluorescence quenching and enhancement are dependent on pH gradients. Fig. 3 shows a typical experiment in which liposomes are added to media containing atebrin or 9-aminoacridine. When the liposomes contain buffers at pH lower than that of the medium, atebrin fluorescence is enhanced rather than quenched (Fig. 3a). At higher lipid concentrations, the enhancement may be 250%. Triton (0.1 mM) or NH4Cl (5 mM) completely reverses the enhancement. However, if 2 mole % bacteriochlorophyll is included in the lipid phase, quenching of fluorescence occurs under the same conditions, and again the quenching is reversible with Triton or NH4Cl (Fig. 3b). Quenching of 9-aminoacridine fluorescence occurs both with and without bacteriochlorophyll, although there is somewhat increased quenching when bacteriochlorophyll is present (Fig. 3c and 3d). 3. The effect of a range of pH gradients on atebrin and 9-aminoacridine fluorescence quenching by liposomes is shown in Fig. 4. In both cases, the inner phase is maintained at pH 5, and the external pH is varied from 5 to 9. It is apparent that quenching of the fluorescence of the amines in liposome suspensions is strongly dependent on pH gradients across the liposome membrane. In the absence of bacteriochlorophyll, the enhancement of atebrin fluorescence is maximal at pH 6.2 (ΔpH = 1.2) and then decreases. If bacteriochlorophyll is present, tile quenching curve of atebrin fluorescence is similar to that of 9-aminoacridine fluorescence, but as noted previously, atebrin is more responsive to pH gradients.
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a b T
T
50%
L
L
c
L
T
d
L T
2 min Fig. 3 Amine fluorescence changes in liposome suspensions. Liposomes (L) containing buffer at pH 5.0 are added to solutions of atebrin and 9-aminoacridine, followed by Triton X-100 (T) to a final concentration of 0.1 mM. Final lipid concentration is 0.08 mg/ml. (a) Fluorescence enhancement occurs when liposomes are added to 4 μM atebrin in inorganic buffer (pH 7.0). No bacteriochlorophyll is present. (b) Fluorescence quenching occurs under conditions for (a) but with 2 mole % bacteriochlorophyll in the liposome membranes. (c) Fluorescence quenching occurs when liposomes are added to 4 μM 9-aminoacridine in inorganic buffer (pH 8.0). No bacteriochlorophyll is present. (d) Increased fluorescence quenching occurs under conditions for (c) but with 2 mole % bacteriochlorophyll in the liposome membranes
4. The first set of experiments is aimed at showing that LUVs composed of egg yolk phosphatidylcholine (egg PC), and egg PC/cholesterol (1:1) could efficiently accumulate adriamycin in response to a pH gradient. As shown in Fig. 5 for a Δ pH of 2.9 (interior pH 4.6), adriamycin is rapidly accumulated into both systems, in the absence of the proton ionophore CCCP, at both 20 C and 37 C. The rate of uptake at 37 C is somewhat faster, particularly for the cholesterol-containing systems. The uptake levels thus achieve (70–100 nmol adriamycin/~mol lipid) correspond to trapping efficiencies of 70–98%, as calculated from the percentage of available drug which is encapsulated into the vesicles. 5. As shown in Fig. 6a, T50 values of approx. 24 h are observed for adriamycin sequestered into egg PC/cholesterol (1:1) LUVs, which are reduced to approx. 4 h when the H+ ion gradient is eliminated (Fig. 6b). Similarly, retention times of 4 h for adriamycin actively entrapped into EPC vesicles are reduced to approx. 30 min when the ΔpH is eliminated.
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Fig. 4 Effect of varying pH gradients on fluorescence changes. O—O, enhancement of atebrin fluorescence in the presence of liposomes lacking pigment; ●—●, quenching of atebrin fluorescence by liposome containing 2 mole % bacteriochlorophyll; ■—■, quenching of 9-aminoacridine fluorescence by liposomes containing 2 mole % bacteriochlorophyll. Liposomes contain buffer at pH 5.0, lipid concentration, 0.08 mg/ml; and amine concentration, 4 μM
Quenching
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1 Δ pH Enhancement
FLUORESCENCE (%CHANGE)
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Fig. 5 Uptake of adriamycin (ADM) into LUVs exhibiting a transmembrane pH gradient at 20 C (A) and 37 C (B). Experimental conditions are 2 mM lipid and 0.2 mM adriamycin. Lipid compositions are egg PC (●) and egg PC/cholesterol at a molar ratio of 1: 1 (■)
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PERCENT ADM ENTRAPPED
PERCENT ADM ENTRAPPED
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Fig. 6 Release of ΔpH-dependent accumulated adriamycin (ADM) from egg PC (●,○) and egg PC/cholesterol at a molar ratio of 1: 1 (■, □) LUVs at 37 C. Free adriamycin is separated from vesicle-associated drug by gel filtration chromatography employing columns equilibrated in buffers adjusted to pH 7.5 (a) or pH 4.6 (b) which contains 150 mM K+ (closed symbols) or 180 mM Na+ (open symbols)
4
Notes
1. During sonication for preparing liposomes, the temperature of the solution rises to approximately 40 C, and this presumably aided formation of liposome vesicles. 2. In some experiments, sonication time is varied from 0 to 30 min, and the amount of trapped anion (phosphate or ferricyanide) is measured. 3. It is found that the amount of trapped material is maximal after 30–60 s sonication. 4. 1 min seems a reasonable time interval in view of the point that prolonging sonication causes some degradation of phospholipid. 5. Sephadex G-50 is equilibrated with sucrose solution containing 10 mM buffer at the original pH. Sucrose is added to osmotically balance the vesicles during gel filtration. For instance, 0.1 M K3Fe(CN) would be balanced with 0. 4 M sucrose. 6. Examination of the preparation by phase-contrast microscopy shows a mixed population of liposome. The major species are 0.1–1 μm vesicles apparently with single membranes but with occasional larger multilayered liposomes. 7. It is important to note the marked quenching effect of organic buffers on 9aminoacridine fluorescence (Fig. 1). Atebrin fluorescence is not significantly affected by organic buffers. 8. Quenching of 9-aminoacridine fluorescence is used to measure the development of pH gradients in liposomes. In one system, an oxidation-reduction reaction mediated by a dye which accepts H+ upon reduction is established across
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liposome membranes. It is found that gradients of at least 4 pH units could develop under these conditions. In a second system, nigericin mediated the exchange of K+ for H+ across the liposome membranes, and it is found that the pH gradient developed depended upon the original K+ gradient and could be at least 2.2 pH units. 9. It may be noted that in some cases, the presence of a K+ ion gradient (K+ buffer inside, Na+ buffer outside), in addition to the ΔpH, results in extended adriamycin retention times. This is particularly noticeable for the egg PC systems, where the T50 values are increased from 3 to 16 h when the exterior K+ buffer is exchanged for a Na+ buffer prior to dialysis. 10. Adriamycin can be efficiently and rapidly sequestered into LUV systems in response to transmembrane pH gradients (interior acidic), in the absence of any extraneous ionophores. The trapping efficiencies, drug retention times, and concentrations of entrapped drug thus achieved are superior to those achievable by passive trapping procedures. As a large proportion of commonly used drugs are hydrophobic amines, it is likely that liposomal drug loading in response to pH gradients is of general utility.
References Bangham AD, Standish MM, Watkins JC (1965) Diffusion of univalent ions across the lamellae of swollen phospholipids. J Mol Biol 13(1):238–252 Barenholz Y (2012) Doxil ® – the first FDA-approved nano-drug: lessons learned. J Control Release 160(2):117–134 Brown JM (2002) Tumor microenvironment and the response to anticancer therapy. Cancer Biol Ther 1(5):453–458 Brown JM, Giaccia AJ (1998) The unique physiology of solid tumors: opportunities (and problems) for cancer therapy. Cancer Res 58(7):1408–1416 Burade V, Bhowmick S, Maiti K, Zalawadia R, Ruan H, Thennati R (2017) Lipodox ® (generic doxorubicin hydrochloride liposome injection): in vivo efficacy and bioequivalence versus Caelyx ® (doxorubicin hydrochloride liposome injection) in human mammary carcinoma (MX1) xenograft and syngeneic fibrosarcoma (WEHI 164) mouse models. BMC Cancer 17(1):405 Cagel M, Grotz E, Bernabeu E, Moretton MA, Chiappetta DA (2017a) Doxorubicin: nanotechnological overviews from bench to bedside. Drug Discov Today 22(2):270–281 Cagel M, Grotz E, Bernabeu E, Moretton MA, Chiappetta DA (2017b) Doxorubicin: nanotechnological overviews from bench to bedside. Drug Discov Today 22(2):270–281 Cern A, Golbraikh A, Sedykh A, Tropsha A, Barenholz Y, Goldblum A (2012) Quantitative structure-property relationship modeling of remote liposome loading of drugs. J Control Release 160(2):147–157 Deamer DW, Prince RC, Crofts AR (1972) The response of fluorescent amines to pH gradients across liposome membranes. Biochim Biophys Acta 274(2):323–335 Dos Santos N, Cox KA, McKenzie CA, van Baarda F, Gallagher RC, Karlsson G, Edwards K, Mayer LD, Allen C, Bally MB (2004) pH gradient loading of anthracyclines into cholesterolfree liposomes: enhancing drug loading rates through use of ethanol. Biochim Biophys Acta 1661(1):47–60 Elizondo E, Moreno E, Cabrera I, Córdoba A, Sala S, Veciana J, Ventosa N (2011) Liposomes and other vesicular systems: structural characteristics, methods of preparation, and use in nanomedicine. Prog Mol Biol Transl Sci 104:1–52
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Gabizon A, Tzemach D, Mak L, Bronstein M, Horowitz AT (2002) Dose dependency of pharmacokinetics and therapeutic efficacy of pegylated liposomal doxorubicin (DOXIL) in murine models. J Drug Target 10(7):539–548 Giddam AK, Zaman M, Skwarczynski M, Toth I (2012) Liposome-based delivery system for vaccine candidates: constructing an effective formulation. Nanomedicine (Lond) 7(12):1877–1893 Greish K (2007) Enhanced permeability and retention of macromolecular drugs in solid tumors: a royal gate for targeted anticancer nanomedicines. J Drug Target 15(7–8):457–464 Hadjidemetriou M, Al-Ahmady Z, Kostarelos K (2016) Time-evolution of in vivo protein corona onto blood-circulating PEGylated liposomal doxorubicin (DOXIL) nanoparticles. Nanoscale 8(13):6948–6957 Haran G, Cohen R, Bar LK, Barenholz Y (1993) Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases. Biochim Biophys Acta 1151(2):201–215 Ichihara H, Hino M, Umebayashi M, Matsumoto Y, Ueoka R (2012) Intravenous injection of hybrid liposomes suppresses the liver metastases in xenograft mouse models of colorectal cancer in vivo. Eur J Med Chem 57:143–148 Mayer LD, Bally MB, Cullis PR (1986) Uptake of adriamycin into large unilamellar vesicles in response to a pH gradient. Biochim Biophys Acta 857(1):123–126 Mineart KP, Kelley EG, Nagao M, Prabhu VM (2017) Processing-structure relationships of poly (ethylene glycol)-modified liposomes. Soft Matter 13(31):5228–5232 Mozafari MR (2005) Liposomes: an overview of manufacturing techniques. Cell Mol Biol Lett 10(4):711–719 Pantos A, Tsiourvas D, Paleos CM, Nounesis G (2005) Enhanced drug transport from unilamellar to multilamellar liposomes induced by molecular recognition of their lipid membranes. Langmuir 21(15):6696–6702 Perrie Y, Kastner E, Khadke S, Roces CB, Stone P (2017) Manufacturing methods for liposome adjuvants. Methods Mol Biol 1494:127–144 Qi XR, Yan WW, Shi J (2005) Hepatocytes targeting of cationic liposomes modified with soybean sterylglucoside and polyethylene glycol. World J Gastroenterol 11(32):4947–4952 Redelmeier TE, Mayer LD, Wong KF, Bally MB, Cullis PR (1989) Proton flux in large unilamellar vesicles in response to membrane potentials and pH gradients. Biophys J 56(2):385–393 Russell LM, Dawidczyk CM, Searson PC (2017) Quantitative evaluation of the enhanced permeability and retention (EPR) effect. Methods Mol Biol 1530:247–254 Satish PR, Surolia A (2002) Preparation and characterization of glycolipid-bearing multilamellar and unilamellar liposomes. Methods Mol Biol 199:193–202 Schwendener R, Schott H (2005) Lipophilic arabinofuranosyl cytosine derivatives in liposomes. Methods Enzymol 391:58–70 Sehgal S, Rogers JA (1995) Polymer-coated liposomes: improved liposome stability and release of cytosine arabinoside (Ara-C). J Microencapsul 12(1):37–47 Sercombe L, Veerati T, Moheimani F, Wu SY, Sood AK, Hua S (2015) Advances and challenges of liposome assisted drug delivery. Front Pharmacol 6:286 Shapiro CL, Ervin T, Welles L, Azarnia N, Keating J, Hayes DF (1999) Phase II trial of high-dose liposome-encapsulated doxorubicin with granulocyte colony-stimulating factor in metastatic breast cancer. TLC D-99 study group. J Clin Oncol 17(5):1435–1441 Shi J, Yan WW, Qi XR, Maitani Y, Nagai T (2005) Characteristics and biodistribution of soybean sterylglucoside and polyethylene glycol-modified cationic liposomes and their complexes with antisense oligodeoxynucleotide. Drug Deliv 12(6):349–356 Shimizu K, Osada M, Takemoto K, Yamamoto Y, Asai T, Oku N (2010) Temperature-dependent transfer of amphotericin B from liposomal membrane of AmBisome to fungal cell membrane. J Control Release 141(2):208–215 Sideratou Z, Sterioti N, Tsiourvas D, Paleos CM (2009) Structural features of interacting complementary liposomes promoting formation of multicompartment structures. Chem Phys Chem 10(17):3083–3089
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Sur S, Fries AC, Kinzler KW, Zhou S, Vogelstein B (2014) Remote loading of preencapsulated drugs into stealth liposomes. Proc Natl Acad Sci U S A 111(6):2283–2288 Xing H, Hwang K, Lu Y (2016) Recent developments of liposomes as nanocarriers for theranostic applications. Theranostics 6(9):1336–1352 Yang Q, Ma Y, Zhao Y, She Z, Wang L, Li J, Wang C, Deng Y (2013) Accelerated drug release and clearance of PEGylated epirubicin liposomes following repeated injections: a new challenge for sequential low-dose chemotherapy. Int J Nanomedicine 8:1257–1268 Zhao W, Zhuang S, Qi XR (2011) Comparative study of the in vitro and in vivo characteristics of cationic and neutral liposomes. Int J Nanomedicine 6:3087–3098
5
Preparation and Characterization of Drug Liposomes by Ammonium Sulfate Gradient Rui-Jun Ju and Xue-Tao Li
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Materials and Apparatuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Synthesis of Targeting Molecule (DSPE-PEG2000-PTDHIV-1 Conjugate) . . . . . . . . . . . . . 2.2 Preparation of the Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Determination of Epirubicin and Celecoxib . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Characterization of the Targeting Epirubicin Plus Celecoxib Liposomes . . . . . . . . . . . . . 2.5 Cell Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Evaluations on Breast Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7 Evaluations on Breast Cancer Spheroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8 Evaluations on Breast Cancer Bearing Nude Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9 Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Procedures and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Synthesis of DSPE-PEG2000-PTDHIV-1 Conjugate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Preparation of the Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Determination of Epirubicin and Celecoxib by HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Characterization with DLS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Morphology Characterization with AFM and TEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 In Vitro Release of Epirubicin and Celecoxib . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Cell Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 Evaluations on Breast Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9 Evaluations on Breast Cancer Cell Spheroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.10 Evaluations on Breast Cancer–Bearing Nude Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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R.-J. Ju (*) Department of Pharmaceutical Engineering, Beijing Institute of Petrochemical Technology, Beijing, China e-mail: [email protected] X.-T. Li School of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian, China e-mail: [email protected] © Springer-Verlag GmbH Germany, part of Springer Nature 2021 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49320-5_4
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Abstract
Liposomes have been extensively studied and widely used for drug delivery in the past several decades. Various types of liposomal formulations have been used in anticancer therapies due to their distinctive advantages: versatility, biocompatibility, and flexibility. Liposomal formulations have been considered promising and encouraging dosage forms for novel anticancer chemotherapies. A high and stable encapsulation efficiency is the basis and premise for the preparation of the liposomal formulations. An active loading methods mediated by sulfate gradient is included in this protocol with an anthracycline, epirubicin, as the model drug. Principle of the ammonium sulfate gradient method is related to low permeability of ammonium counterion, the sulfate, which also stabilizes the drugs of weak alkaline accumulation in the aqueous phase due to the aggregation and gelation of anthracycline sulfate salt. The procedure of liposome preparation by the ammonium sulfate gradient is described step by step, while further anticancer effects are also concisely stated in the protocol. Keywords
Liposomes · Sulfate gradient · Encapsulation efficiency · Epirubicin · Breast cancer
1
Introduction
Liposomes have been extensively studied and widely used for drug delivery in the past several decades, especially in anticancer treatments (Tila et al. 2015; Drummond et al. 1999). Liposomal formulations exhibit significant advantages in many aspects, including good biocompatibility, easy biodegradability, and lower toxicity and so on (Simone et al. 2008; Coune 1988). In addition, drug-loaded liposomes could improve distribution characteristics, which lead to a reduced systemic toxicity and a better prognosis as compared to normal chemotherapies (Durymanov et al. 2015). Mechanism involved in this phenomenon of liposome formulation mainly attributes to the enhanced permeation and retention effect (EPR), which behaves as blood vessels nearby tumor tissue are relatively loose along with larger leakage pores (Maruyama 2011). Nanostructured liposomes could be leaked through these pores into the tumor tissues. In addition, hydration shell on the surface of liposomes renders the liposomes a long circulation time in the blood. Several studies have certified that high encapsulation efficiency (EE) is closely related to the anticancer efficacy (Lee et al. 2012). Hence, achieving a higher EE is the basis and premise for preparing liposomal formulations. Liposome ingredients and loaded drugs themselves could influence the EE due to their intrinsic properties, such as lipid-drug ratio and hydrophilic-lipophilic property. In addition to these liposome formulation factors, a loading process could greatly influence the EE to a large extent. The loading process is relatively simple for lipophilic drug, as the lipophilic drug could be directly mixed with the lipid to prepare thin films. However, as for hydrophilic drug, the loading methods could be complicated and varied
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(Eloy et al. 2014). Thus active drug-loading methods have been introduced, such methods typically employ pH, manganese, sulfate, citrate, or phosphate-gradient established across the liposome bilayer (Ong et al. 2011). In the numerous active loading methods, sulfate gradient-mediated drug loading is the most commonly used. The ammonium sulfate gradient approach differs from other loading approaches, since it neither requires preparation of the liposomes in acidic pH, nor alkalinizes extra liposome aqueous phase. Stability of the ammonium ion gradient is related to low permeability of its counterion, the sulfate, which also stabilizes drugs of weak alkaline accumulation for prolonged storage periods (>6 months) due to the aggregation and gelation of anthracycline sulfate salt (Haran et al. 1993). Briefly, general process of ammonium sulfate gradient drug-loading method could be described as the following procedures. Firstly, blank liposomes are prepared by using thin-film dispersion with ammonium sulfate solution as hydration solution. Secondly, the blank liposomes are dialyzed in the dialysis bag with phosphatebuffered saline to form ammonium sulfate gradient intra and outside the liposomes. Finally, active encapsulation is achieved by incubation with drugs of weak alkaline solution in water bath at 40 C with continually shaking for 20 min. To offer more detailed instructions, ammonium sulfate gradient-mediated encapsulation is provided in this chapter (see Fig. 1). The model drug is a kind of an amphipathic weak alkaline, epirubicin, and the model liposomes is a kind of targeting liposomes incorporating epirubicin and modulator. Epirubicin is an anthracycline anticancer agent with a broad cancer spectrum (Mulligan et al. 2014). It acts through intercalation with DNA strands and inhibition to the syntheses of DNA and RNA. In addition, it also triggers DNA cleavage by topoisomerase II and generates free radicals to kill cancer cells (Cowell and Austin 2012). Epirubicin
Fig. 1 Schematic diagram of ammonium sulfate gradient-mediated encapsulation
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hydrochloride is used in the liposomal formulation due to its enhanced water solubility. The procedure of liposome preparation is described step by step. And the further anticancer effect on breast cancer is also concisely stated in the protocol (Ju et al. 2014).
2
Materials and Apparatuses
2.1
Synthesis of Targeting Molecule (DSPE-PEG2000-PTDHIV-1 Conjugate)
1. PTDHIV-1 peptide (Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg- Cys-Gly-NH2) 2. N-hydroxysuccinimidyl-polyethylene glycol distearoylphosphatidyl ethanolamine (DSPE-PEG2000-NHS), 3. Anhydrous dimethylformamide (DMF) 4. N-methyl morphine 5. Regenerated cellulose dialysis tubing (molecular weight cut-off point, 3500) 6. Magnetic stirrer 7. Matrix-assisted laser desorption/ionization time of flight mass spectrometry
2.2 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
2.3
Preparation of the Liposomes Egg phosphatidylcholine (EPC) Cholesterol Polyethylene glycol distearoylphosphosphatidylethanolamine (DSPE-PEG2000) Epirubicin hydrochloride Celecoxib Chloroform Hepes Phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4 and 2 mM KH2PO4, PBS pH 7.4). Polycarbonate membranes (400 and 200 nm) 250 mM ammonium sulfate solution 100 ml pear-shaped flask Rotary vacuum evaporator Circulating water multipurpose vacuum pump Bath-type sonicator Ultrasonic cell disruptor 1,10 -dioctadecyl-3,3,30 ,30 -tetramethyl indotricarbocyanine Iodide Coumarin
Determination of Epirubicin and Celecoxib
1. Hundred thousandth analytical balance 2. High performance liquid chromatography system with UV detector
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Eclipse XDB-C18 column (5 μm, 4.6 250 mm). Sephadex G-50 Centrifuge Glass column (15 mm in diameter) Acetonitrile was of chromatographic grade Distilled water Sodium dihydrogen phosphate (NaH2PO4) was of analytical grade Triethylamine was of analytical grade Phosphoric acid was of analytical grade Methanol was of analytical grade
Characterization of the Targeting Epirubicin Plus Celecoxib Liposomes
Zetasizer 3000HSA for dynamic light scattering (DLS) analysis Atomic force microscope Transmission electron microscopy Polished silicon wafer Fetal bovine serum (FBS) Shaker Polished silicon wafer Phosphate buffered saline
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Cell Cultures Invasive human breast cancer MDA-MB-435S cells Human breast cancer MCF-7 cells Dulbecco’s modification of Eagle’s medium (DMEM) Roswell park memorial institute-1640 (RPMI-1640) Fetal bovine serum (FBS) Trypsin EDTA Penicillin-streptomycin solution 100 Cell culture flask CO2 incubator Vertical flow clean bench Optical microscope
Evaluations on Breast Cancer Cells
Flow cytometer Confocal laser scanning fluorescent microscopy 96-Well culture plates Trichloroacetic acid
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Sulforhodamine B Acetic acid in analytically pure Tris base Hoechst 33258 Microplate reader
2.7
Evaluations on Breast Cancer Spheroids
1. Agarose 2. Confocal laser scanning fluorescent microscopy 3. Scanning electron microscope
2.8 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
2.9
Evaluations on Breast Cancer Bearing Nude Mice Female BALB/c nude mice (16–18 g) Kodak multimodal imaging system Weighing scale Electronic digital vernier caliper Isoflurane Mouse monoclonal anti-CD34 1% sodium periodate PAS Staining Kit Hematoxylin Triton-X100 Freezing microtome EVOS microscope
Statistics
1. Data are presented as the mean standard deviation (SD). 2. One-way analysis of variance (ANOVA) was used to determine significance among groups, after which post hoc tests with the Bonferroni correction were used for comparison between individual groups. 3. A value of P < 0.05 was considered to be significant.
3
Procedures and Methods
3.1
Synthesis of DSPE-PEG2000-PTDHIV-1 Conjugate
1. Dissolve DSPE-PEG2000-NHS (2 mmol) and PTDHIV-1 peptide (2 mmol) in anhydrous DMF (see note 1 and 2). 2. Adjust the pH value of the reaction solution to 9.0 by using N-methyl morphine.
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3. Stir at room temperature for 36 h. 4. Transfer the mixture into dialysis tubing and dialyzed against deionized water for another 36 h to remove the DMF solvent. 5. Lyophilize the mixture and store at 20 C. 6. Use MALDI-TOF mass spectrometer to confirm conjugation of DSPE-PEG2000PTDHIV-1 (Fig. 2).
3.2
Preparation of the Liposomes
3.2.1 Preparation of Targeting Celecoxib Liposomes 1. Dissolve EPC, cholesterol, DSPE-PEG2000, DSPE-PEG2000-PTDHIV-1, and celecoxib in chloroform at a ratio of 70:30:2:3:2.8 (molecular ratio) into a pearshaped flask. 2. Remove chloroform by a rotary vacuum evaporator to form the lipid film (see note 3). 3. Add 250 mM ammonium sulfate solution followed by sonication in the water bath for 5 min. 4. Treat with an ultrasonic cell disruptor for 10 min (200 W) subsequently. 5. Extrude through polycarbonate membranes thrice using 400 nm pore sizes, and then thrice using those of 200 nm pore sizes. 3.2.2 Preparation of Targeting Epirubicin Plus Celecoxib Liposomes 1. Dialyze the newly prepared targeting celecoxib liposomes in the HBS for 24 h and refresh the dialysate twice. 3
JRJ
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Fig. 2 MALDI-TOF-MS spectrum of DSPE-PEG2000-PTDHIV-1 conjugate. Where, 1. PTDHIV-1 peptide; 2. DSPE-PEG2000-NHS; 3. DSPE-PEG2000-PTDHIV-1. Results indicate that DSPEPEG2000-PTDHIV-1 conjugate is successfully synthesized
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Fig. 3 Schematic drawing of targeting epirubicin plus celecoxib liposomes
2. Incubate the targeting celecoxib liposomes with epirubicin solution in water bath at 40 C with continually shaking for 20 min (lipids: drug 20:1, weight ratio). 3. Extrude the liposomes through polycarbonate membranes thrice with the pore size of 400 nm, and then thrice with 200 nm to target epirubicin plus celecoxib liposomes (Fig. 3). 4. Store at 4 C in refrigerator.
3.2.3 Preparation of Epirubicin Liposomes 1. Dissolve EPC, cholesterol, DSPE-PEG2000 in chloroform at a ratio of 70:30:5 (molecular ratio) into a pear-shaped flask. 2. Remove chloroform by a rotary vacuum evaporator to form the lipid film. 3. Hydrate with 250 mM ammonium sulfate solution followed by sonication in the water bath for 5 min. 4. Treat the suspensions were with an ultrasonic cell disruptor for 10 min (200 W). 5. Extrude the obtained suspensions through polycarbonate membranes thrice using 400 nm pore sizes, and then thrice using those of 200 nm pore sizes. 6. Dialyze the newly prepared blank liposomes in the HBS for 24 h and refresh the dialysate twice. 7. Incubate the blank liposomes with epirubicin solution in water bath at 40 C via continually shaking for 20 min (lipids: drug 20:1, weight ratio). 8. Then extrude the epirubicin-loaded liposomes through polycarbonate membranes thrice with the pore size of 400 nm, and then thrice with those of 200 nm. 3.2.4 Preparation of Targeting Epirubicin Liposomes 1. Dissolve EPC, cholesterol, DSPE-PEG2000, and DSPE-PEG2000-PTDHIV-1 in chloroform at a ratio of 70:30:2:3 (molecular ratio) in a pear-shaped flask. 2. Remove chloroform by a rotary vacuum evaporator to form the lipid film.
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3. Hydrate the newly formed lipid film with 250 mM ammonium sulfate solution followed by sonication in the water bath for 5 min. 4. Treat the suspensions using an ultrasonic cell disruptor for 10 min (200 W). 5. Extrude the suspensions through polycarbonate membranes thrice using 400 nm pore sizes, and then thrice using 200 nm pore sizes. 6. Dialyze the newly prepared blank liposomes in the HBS for 24 h and refresh the dialysate twice. 7. Incubate the blank liposomes with epirubicin solution in water bath at 40 C with continually shaking for 20 min (lipids: drug 20:1, weight ratio). 8. Extrude the targeting epirubicin liposomes through polycarbonate membranes thrice with the pore size of 400 nm, and then thrice with those of 200 nm.
3.2.5 Preparation of Fluorescence Labeling Liposomes 1. Conduct all the preparation procedures in dark light. 2. Dissolve the EPC, cholesterol, DSPE-PEG2000, DSPE-PEG2000-PTDHIV-1, and appropriate coumarin (DiR) in chloroform at a ratio of 70:30:2:3 (molecular ratio) into a pear-shaped flask. Where (lipids: coumarin = 500:1, weight ratio) for coumarin liposomes (without DSPE-PEG2000-PTDHIV-1) and targeting coumarin liposomes, and (lipids: DiR = 200:1, weight ratio) DiR liposomes (without DSPE-PEG2000-PTDHIV-1) and targeting DiR liposomes. 3. Remove the chloroform by a rotary vacuum evaporator to form the lipid film. 4. Hydrate the newly formed lipid film with 250 mM ammonium sulfate solution followed by sonication in the water bath for 5 min. 5. Treat the suspensions using an ultrasonic cell disruptor for 10 min (200 W). 6. Extrude the obtained suspensions through polycarbonate membranes thrice using 400 nm pore sizes, and then thrice using those of 200 nm. 7. Store in darkness.
3.3
Determination of Epirubicin and Celecoxib by HPLC
3.3.1 Methodology of the HPLC Determination 1. Weigh precisely epirubicin and celecoxib, and dissolve them in methanol rendering the epirubicin to celecoxib is 5:3 (weight ratio) (see note 4). 2. Determine both epirubicin and celecoxib simultaneously measured by HPLC at a UV wavelength of 254 nm with a gradient elution (Table 1). 3. Aqueous phase is consisted of 0.02 M NaH2PO4 water solution with triethylamine and phosphoric acid adjusting the pH value to 4. The retention time for epirubicin and celecoxib are about 4.1 min and 10.6 min, respectively. 4. Linearities of epirubicin and celecoxib (1) Weigh epirubicin and celecoxib precisely, and dissolve in methanol, rendering the epirubicin in serial concentrations (0.5, 1.0, 2.5, 5.0, 10.0, 25.0, and 50.0 μg/ml), the celecoxib in serial concentrations (0.3, 0.6, 1.5, 3.0, 6.0, 15.0, and 30.0 μg/ml). (2) Measure these samples under the above HPLC condition.
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Table 1 Gradient elution of epirubicin and celecoxib measured by HPLC Time (min) 0 8 8.01 15 15.01 20
Aqueous phase (%) 75 70 20 20 75 75
Acetonitrile (%) 25 30 80 80 25 25
Flow (ml/min) 1.0 1.0 1.0 1.0 1.0 1.0
(3) Prepare standard solutions of epirubicin and celecoxib at different concentration levels. The standard calibration curve is generated to evaluate the linearity. The slope, intercept and linear correlation coefficient (r2) are calculated to carry out linear regression analysis. 5. Precisions of epirubicin and celecoxib (1) Dilute epirubicin and celecoxib with methanol at concentrations of 2.0, 10.0, and 50.0 μg/ml epirubicin, with celecoxib concentrations of 1.2, 6.0, and 30.0 μg/ml. (2) Measure the methanol solutions by HPLC to evaluate the coefficients of variation thrice on the same day and inter days, respectively. (3) Evaluate the intra- and inter-day coefficients of variation by calculating RSD for a statistically significant number between replicate measurements. The obtained inter-day and intra-day precisions are RSD < 2, indicating that the proposed HPLC method is well precise and reproducible for determining epirubicin and celecoxib. 6. Detection limits of epirubicin and celecoxib (1) Dilute the epirubicin and celecoxib in methanol properly followed by the same HPLC method. (2) Render the HPLC signals to reach noise ratio at 3:1, and the corresponding concentrations are considered as the detection limits for epirubicin and celecoxib, respectively.
3.3.2 EE Determination of Epirubicin and Celecoxib in the Liposomes 1. Add the sephadex G-50 in distilled water for at least 12 h before loading the column. 2. Saturate the sephadex G-50 column with a proper amount of blank liposomes (see note 5). 3. Pass over about 0.5 ml of drug-loaded liposomes through the column with HBS solution to remove the unloaded epirubicin and celecoxib. 4. Collect the liposome suspension, then add nine times volume of acetonitrile to disrupt the liposomes. 5. Shake for 1 min by a vortex instrument. 6. Centrifuge the mixture at a speed of 10,000 rpm for another 5 min, then collect the supernatant fluid which contains the drugs. 7. Measure the epirubicin and celecoxib with the established HPLC method.
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8. Calculate the EEs of epirubicin and celecoxib with the formula: EE% = (Wencap/ Wtotal) 100%, where Wencap is the measured amount of epirubicin or celecoxib in the liposome suspensions after passing over the column. Wtotal is the measured amount of epirubicin or celecoxib in the equal volume of original liposome suspensions.
3.4
Characterization with DLS
1. Dilute the liposomes distilled water to a proper concentration (100 μg/ml lipid) (see note 6). 2. Measure the particle sizes, zeta potential values, and polydispersity indexes (PDI) of all liposomes by using a dynamic light scatter.
3.5
Morphology Characterization with AFM and TEM
1. Dilute the liposome suspensions with distilled water, and filter through a polycarbonate membrane with the pore size of 200 nm. 2. Spread the liposome suspension (10 μl) on a silicon slice, dry at room temperature, and observe using AFM (Fig. 4a). 3. Coat copper grids with carbon films to absorb the liposome particles for 1 min in the liposome suspension, then take out and blot with filter paper. 4. Float these grids in 1% uranyl acetate solution for 1 min, remove, blot with filter paper, and dry at room temperature overnight. 5. Observe the specimens by TEM (Fig. 4b).
Fig. 4 AFM images (a) and TEM images (b) of the targeting liposomes. Results indicate that the targeting liposomes display a smooth surface with approximately 100 nm in diameter with a narrow size distribution
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3.6
In Vitro Release of Epirubicin and Celecoxib
1. Release medium is solution of pH 7.4 PBS containing 10% fetal calf serum. 2. Mix 2 ml liposomes with 2 ml release medium and seal in dialysis bags (molecular weight cut-off, 8,000–12,000). 3. Immerse dialysis bags in 20.0 ml release medium, followed by consecutively shaking at a speed of 100 rpm at 37 C in a shaker. 4. Take a volume of 0.2 ml release mediums at intervals of 0, 0.25, 0.5, 1, 2, 4, 8, 12, 18, 24, 36, 48, and 72 h, respectively, and replace immediately with the same volume of fresh release medium after each sampling. 5. Add 1.0 ml acetonitrile to the 0.2 ml sample and mix for 3 min with a vortex, followed by centrifugation at 10,000 rpm for 5 min. 6. Inject 20 μl supernatant into the HPLC system and record the signal peaks of epirubicin and celecoxib. 7. Calculate the accumulated release rate (Fig. 5) with the formula: RR = (Wi/Wtotal) 100%, where RR is the drug release rate (%), Wi is the measured amount of epirubicin or celecoxib at the time-point of i in release medium, and Wtotal is the total amount of epirubicin or celecoxib in the equal volume of liposome suspensions prior to dialysis.
3.7
Cell Cultures
1. Culture invasive human breast cancer MDA-MB-435S cells in DMEM medium supplemented with 10% FBS at 37 C under an atmosphere of 5% CO2.
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2. Culture human breast cancer MCF-7 cells in RPMI-1640 medium 10% FBS at 37 C under an atmosphere of 5% CO2.
3.8
Evaluations on Breast Cancer Cells
3.8.1 Cytotoxic Effects on Breast Cancer Cells 1. Seed MDA-MB-435S cells and MCF-7 cells at a density of 7200 cells/well in 96-well culture plates and culture for 24 h. 2. Treat the cells celecoxib liposomes, epirubicin liposomes, epirubicin plus celecoxib liposomes, and targeting epirubicin plus celecoxib liposomes. Both concentrations of epirubicin and celecoxib are in the range of 0–5.0 μM (epirubicin: celecoxib = 1:1, molecule ratio). 3. Incubate for another 48 h, and observe the inhibitory effects with sulforhodamine B (SRB) colorimetric assay. 4. Remove the culture medium, and then fix the cells with trichloroacetic acid followed by washing and staining with SRB. 5. Measure the absorbances of the stained SRB at 540 nm using a microplate reader. 6. Calculate survival rates using the following formula: Survival % = (A540 nm for treated cells/A540 nm for control cells) 100%, where A540 nm represents the absorbance value. 3.8.2 Cellular Uptakes in Breast Cancer Cells 1. Seed the MDA-MB-435S cells and MCF-7 cells into six-well plates at a density of 3 105 cells/well, and culture for 24 h. 2. Prior to the experiment, wash the cells twice with PBS to remove the remnant growth medium. 3. Incubate in serum-free medium containing free epirubicin, epirubicin liposomes, or targeting epirubicin liposome at a concentration of 15 μM epirubicin. The drug-free culture medium is used as the blank control. 4. After incubation for 4 h, wash the cells thrice with cold PBS (4 C). 5. Resuspend the cells in 300 μl PBS. 6. Measure the epirubicin fluorescence intensities by a flow cytometer (see note 7). 3.8.3 Targeting Effects on Breast Cancer Cells 1. Seed MDA-MB-435S cells into chambered coverslips at a density of 1.5 105 cell/well. 2. Incubate for 24 h, then treat the cells with free epirubicin, epirubicin liposomes, and targeting epirubicin liposomes at a concentration of 40 μM epirubicin for another 2 h. 3. Wash the cells with cold PBS for three times, followed by fix with 4% paraformaldehyde for 10 min.
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Fig. 6 Accumulation in the nuclei of breast cancer cells after incubation with the targeting epirubicin liposomes. (a) Epirubicin fluorescence. (b) Nuclei fluorescence. (c) Combined fluorescence. (d) Multioverlay images. Results indicate that the targeting epirubicin liposomes exhibit a higher fluorescence intensity in the cancer cells, displaying an evident affinity with cell membranes and cell nuclei
4. Stain cell nuclei with Hoechst 33,258 (2 μg/ml) for another 10 min. 5. Photograph the cells and analyze using a confocal laser scanning fluorescent microscopy (objective 25, Fig. 6).
3.9
Evaluations on Breast Cancer Cell Spheroids
3.9.1 Establishing Multicellular Cancer Cell Spheroids 1. Add agarose into serum FBS free DMEM culture medium. 2. Heat to 80 C for 30 min to form 2% (weight/volume) solution. 3. Coat each well of 48-well culture plates with the agarose solution (0.1 ml each). 4. Cool to room temperature, then seed the MDA-MB-435S cells with 100 μl growth medium at 1 103 cells/well.
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5. Shake culture the plates gently for 3 min to balance the cancer cells and incubate for 48 h to form cancer spheroids.
3.9.2 Penetrating Effects in Breast Cancer Cell Spheroids 1. Treat the MDA-MB-435S spheroids with free coumarin, coumarin liposomes, or targeting coumarin liposomes at a concentration of 10.0 μM coumarin. 2. Incubate for 12 h. 3. Wash the spheroids with PBS to remove the residual coumarin. 4. Scan the spheroids at the different layers from top to inside using a confocal laser scanning fluorescent microscope (Fig. 7a). 3.9.3 Destructing Effects on the Breast Cancer Spheroids 1. Collect MDA-MB-435S spheroids into six-well culture plates. 2. Treat with epirubicin liposomes, epirubicin plus celecoxib liposomes, and targeting epirubicin plus celecoxib liposomes at a concentration of 10 μM epirubicin, respectively. Drug-free culture medium is used as the blank control. 3. Incubate for 48 h, then fix the spheroids with 2.5% glutaraldehyde for 60 min. 4. Rinse three times in PBS, dehydrate, and embed. 5. Photograph the spheroids under a SEM (Fig. 7b).
Fig. 7 MDA-MB-435S spheroids. (a) Confocal images at different layers from top to middle of the spheroids; (b) SEM photographs of the spheroids. Results indicate that the targeting liposomes have the strongest penetrating ability and destructing effect on the breast cancer spheroids
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3.10
Evaluations on Breast Cancer–Bearing Nude Mice
3.10.1 Establishing Breast Cancer–Bearing Nude Mice Models 1. Female BALB/c nude mice (16–18 g) are included for the study (see note 8). 2. Re-suspend approximately 8 106 MDA-MB-435S cells in 200 μl serum-free DMEM culture medium. 3. Inject the cell suspensions subcutaneously into right flanks of nude mice. 4. Measure the solid tumor masses using a vernier caliper every day with a caliper, and monitor weight changes of nude mice every day with a weighing scale. 5. Calculate tumor volumes with the formula: V = length width2/2 (mm3). 3.10.2 1. 2.
3. 4.
Anticancer Evaluations of the Targeting Liposomes on Nude Mice Divide the tumor-bearing mice randomly into five groups (six per group) once the tumors reach about 100 mm3 in volume. Administer free epirubicin, epirubicin liposomes, epirubicin plus celecoxib liposomes, and targeting epirubicin plus celecoxib liposomes via tail vein at a dosage of 5 mg/kg epirubicin, respectively, at day 11, 14, 16, and 18 postinoculation. Administer physiological saline as a blank control. Calculate tumor volume ratios with the formula: R = Vith day/V11th day (see Fig. 8).
3.10.3 In Vivo Imaging in Mice 1. A total of 12 female BALB/c nude mice are inoculated with the MDA-MB-435S cells as above. 2. Divide the tumor-bearing mice randomly into four groups (three per group) once the tumors reach approximately 600 mm3 in volume. 3. Administer the tumor bearing mice with saline, free DiR, DiR liposomes, and targeting DiR liposomes (1 μg DiR each) via tail vein. 4. Anesthetize the mice with isoflurane. 5. Photograph the fluorescent images and X-ray images using a Kodak multimodal imaging system at predetermined time-points (1, 3, 6, 9, 12, 24, and 48 h) (Fig. 9a). 5
Tumor volume ratio
Fig. 8 Anticancer efficacy after treatment with the targeting liposomes. Data are presented as mean SD (n = 6). p < 0.05. a, vs saline; b, vs free epirubicin; c, vs epirubicin liposomes; d, vs epirubicin plus celecoxib liposomes. Results indicate that targeting epirubicin plus celecoxib liposomes could significantly inhibit tumor sizes
Saline Free epirubicin Epirubicin liposomes Epirubicin plus celecoxib liposomes Targeting epirubicin plus celecoxib liposomes
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Fig. 9 In vivo and ex vivo imagings after the administration of targeting liposomes in tumorbearing nude mice. (a) In vivo real-time images of targeting DiR liposomes or controls at predetermined times. DiR is used as fluorescent probe; (b) Ex vivo images of tumor masses and major organs at 48 h. Results indicate that the DiR-labeled targeting liposomes could effectively accumulate at the tumor areas and maintain up to 48 h
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6. Execute the mice by cervical dislocation at 48 h. 7. Dissect tumor masses, hearts, livers, spleens, lungs, and kidneys. 8. Photograph the fluorescent images and X-ray images for all ex vivo tissues (Fig. 9b).
4
Notes
1. The weighing operation should be as quick as possible, because the frozen materials could easily stick, especially for the PTDHIV-1 peptide. 2. The DMF must be anhydrous so as to make sure the success of the reaction. 3. The chloroform must be removed completely to create a lipid film without residual solvent. 4. The ratio of epirubicin to celecoxib is 5:3 (weight ratio), as so to be consistent with those in the targeting epirubicin plus celecoxib liposomes. 5. Sephadex G-50 is saturated with blank liposomes to prevent that the drug loaded liposomes were adsorbed in the glucan pores. 6. The liposome suspension should be diluted enough before characterization using DLS, AFM, or TEM. 7. The excitation wavelength of epirubicin is set at 488 nm, and the emission wavelength is in range 560–590 nm. 8. All procedures are performed according to guidelines of the Institutional Authority for Laboratory Animal Care of Peking University.
References Coune A (1988) Liposomes as drug delivery system in treatment of infectious diseases. Potential applications and clinical experience. Infection 16(3):141–147 Cowell IG, Austin CA (2012) Mechanism of generation of therapy related leukemia in response to anti-topoisomerase II agents. Int J Environ Res Public Health 9:2075–2091 Drummond DC, Meyer O, Hong K, Kirpotin DB, Papahadjopoulos D (1999) Optimizing liposomes for delivery of chemotherapeutic agents to solid tumors. Pharmacol Rev 51:691–743 Durymanov MO, Rosenkranz AA, Sobolev AS (2015) Current approaches for improving Intratumoral accumulation and distribution of nanomedicines. Theranostics 5(9):1007–1020 Eloy JO, Claro de Souza M, Petrilli R, Barcellos JP, Lee RJ, Marchetti JM (2014) Liposomes as carriers of hydrophilic small molecule drugs: strategies to enhance encapsulation and delivery. Colloids Surf B Biointerfaces 123:345–363 Haran G, Cohen R, Bar LK, Barenholz Y (1993) Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases. Biochim Biophys Acta 1151(2):201–215 Ju RJ, Li XT, Shi JF, Li XY, Sun MG, Zeng F, Zhou J, Liu L, Zhang CX, Zhao WY, Lu WL (2014) Liposomes, modified with PTD(HIV-1) peptide, containing epirubicin and celecoxib, to target vasculogenic mimicry channels in invasive breast cancer. Biomaterials 35(26):7610–7621 Lee P, Zhang R, Li V, Liu X, Sun RW, Che CM, Wong KK (2012) Enhancement of anticancer efficacy using modified lipophilic nanoparticle drug encapsulation. Int J Nanomedicine 7: 731–737
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Maruyama K (2011) Intracellular targeting delivery of liposomal drugs to solid tumors based on EPR effects. Adv Drug Deliv Rev 63(3):161–169 Mulligan JM, Hill LA, Deharo S, Irwin G, Boyle D, Keating KE, Raji OY, McDyer FA, O’Brien E, Bylesjo M, Quinn JE, Lindor NM, Mullan PB, James CR, Walker SM, Kerr P, James J, Davison TS, Proutski V, Salto-Tellez M, Johnston PG, Couch FJ, Paul Harkin D, Kennedy RD (2014) Identification and validation of an anthracycline/cyclophosphamide-based chemotherapy response assay in breast cancer. J Natl Cancer Inst 106:1–10 Ong JC, Sun F, Chan E (2011) Development of stealth liposome coencapsulating doxorubicin and fluoxetine. J Liposome Res 21(4):261–271 Simone EA, Dziubla TD, Muzykantov VR (2008) Polymeric carriers: role of geometry in drug delivery. Expert Opin Drug Deliv 5(12):1283–1300 Tila D, Ghasemi S, Yazdani-Arazi SN, Ghanbarzadeh S (2015) Functional liposomes in the cancertargeted drug delivery. J Biomater Appl 30(1):3–16
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Preparation of Drug Liposomes by EDTA Gradient Methods Yanzhi Song and Yihui Deng
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Materials for Preparation of Ammonium EDTA Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Materials for Preparation of Empty Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Materials for Preparation of EDTA Gradient Empty Liposomes . . . . . . . . . . . . . . . . . . . . . . 2.4 Materials for Preparation of Drug-Loaded EDTA Gradient Liposomes . . . . . . . . . . . . . . 2.5 Materials for Determination of Lipid Concentration Using a Phosphate Assay . . . . . . 3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Doxorubicin-Loaded EDTA Gradient Liposomes (Song et al. 2014) . . . . . . . . . . . . . . . . . 3.2 Preparation of Topotecan-Loaded EDTA Gradient Liposomes (Yang et al. 2012) . . . 3.3 Epirubicin-Loaded EDTA Gradient Liposomes (Yang et al. 2014) . . . . . . . . . . . . . . . . . . . 3.4 Idarubicin-Loaded EDTA Gradient Liposomes (Gubernator et al. 2010) . . . . . . . . . . . . . 3.5 Determination of Lipid Concentration Using a Phosphate Assay (Torchilin and Weissig 2003) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
The applications of ethylene diamine tetraacetic acid (EDTA) have expanded from the treatment for heavy metal poisoning to chelation therapies for atherosclerosis, heart disease, and cancer. In recent years, EDTA has been employed as a drug delivery system, in which the EDTA gradient method was used to load weakly amphiphilic base drugs into liposomes. Because EDTA can form low-solubility compounds with weakly amphiphilic base drugs such as anthracyclines, the liposomal formulations prepared by the EDTA gradient Y. Song · Y. Deng (*) College of Pharmacy, Shenyang Pharmaceutical University, Shenyang, China e-mail: [email protected]; [email protected] © Springer-Verlag GmbH Germany, part of Springer Nature 2021 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49320-5_6
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method have high drug-loading efficiency, good long-term stability, and delayed drug release. Furthermore, because of the chelation of EDTA, it can also reduce drug-related toxicity and liposome-related immune organ damage. In this chapter, the weakly amphiphilic base drugs (doxorubicin, topotecan, epirubicin, and idarubicin) were used as model drugs to introduce the EDTA gradient drugloading method. The method presented in this part may be helpful in obtaining reliable and reproducible liposomes and experimental results. Keywords
EDTA · Ion gradient · Liposomes · Doxorubicin · Topotecan · Epirubicin · Idarubicin
1
Introduction
Ethylene diamine tetraacetic acid (EDTA) is a general chelating agent that has been approved by the standard FDA for the treatment of heavy metal poisoning since the early 1950s (Domingo 1998; Flora and Pachauri 2010; Fulgenzi et al. 2015; Gordon 2003; Riordan et al. 1990; Waters et al. 2001). Since the earliest clinical trials, EDTA-related chelation therapy has consistently shown a remarkable ability to cleanse the human body of metals and other deposits responsible for atherosclerosis, heart disease, and cancer (Buss et al. 2003; Clarke 1960; Dans et al. 2002; Kitchell et al. 1963; Ouyang et al. 2015; Seely et al. 2005). In fact, EDTA can reduce the morbidity and mortality associated with the diseases mentioned above by chelating toxic metal ions. A study by Walter Blumer and Elmer Cranton showed that cancer mortality decreased by 90% in an 18-year follow-up of 59 patients treated with calcium EDTA (Blumer and Cranton 1989). In addition, EDTA can ameliorate hypercalcemia, soften the arteries, and reduce the incidence of osteoporosis by chelating calcium ions (Body 1992; Mundy and Guise 1997). The main reason for this is that EDTA and its analogs can inhibit calcium/calmodulin-dependent protein kinase II (CCPK II) and protein kinase C (PKC), which play important roles in the occurrence and development of numerous diseases. Furthermore, it was reported that EDTA could inhibit radical reactions and oxidation processes by chelating toxic metals responsible for cell membrane injury (Cranton and Frackelton 1998). In recent years, EDTA has also been widely applied in the field of pharmaceutical preparations. EDTA can improve the stability of preparations by acting as a fungistat to inhibit bacterial contamination by chelating divalent metal ions, which are essential for the survival of bacteria (Thompson and Goodale 2000). In the case of the ionophore-generated pH gradient method for liposomal preparation, EDTA is an external chelator that binds the released divalent cations, drives the uptake to completion, and helps prevent liposome aggregation (Erdahl et al. 1994, 1995; Fenske et al. 1998; Pressman 1976). Jerzy Gubernator et al. found that EDTA could form precipitates of low solubility with idarubicin (IDA) (Gubernator et al. 2010). The precipitates of IDA-EDTA
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showed very slow drug dissolution characteristics and slow drug-leakage. Additionally, disodium and diammonium EDTA salts had good buffering properties and an optimal initial pH. Based on these observations, the researchers developed a novel method of active anthracycline encapsulation based on an EDTA ion gradient. The results of the experiment showed that the efficiency of IDA encapsulation was close to 98% at a drug to lipid molar ratio of 1:5, and an in vitro long-term storage experiment confirmed the high stability of the liposomes. In vivo studies also indicated the superiority of the new IDA formulation over the current remote loading methods of citrate and ammonium sulfate gradients and showed that the plasma level of IDA was much higher when EDTA liposomes were used. Our research group also adopted the EDTA gradient method to load several drugs into liposomes, such as doxorubicin, topotecan, and epirubicin. Taking doxorubicin as an example (Song et al. 2014), compared with drug-loaded liposomes prepared by ammonium sulfate gradient, the liposomes prepared by the EDTA ammonium gradient showed improved long-term stability and delayed drug release. The in vivo studies showed much lower cardiotoxicity and less liposome-related immune organ damage. Similar results were also obtained with topotecan (Yang et al. 2012) and epirubicin (Gubernator et al. 2014; Yang et al. 2013) liposomes. On the basis of the above results, the loading of weakly amphiphilic base drugs into liposomes by using a transmembrane EDTA gradient method not only maintains the advantages of liposomes, such as high drug-loading efficiency, but also provides several other benefits. First, the intraliposomal EDTA will increase the stability of liposomal formulations by inhibiting bacterial contamination and preventing the catalytic effect of metal ions on the lipid. Secondly, EDTA will inhibit the generation of ROS by chelating the transition metal ions, thereby decreasing damage to the cardiomyocyte membrane and reducing the risk of drug-related cardiomyopathy. Thirdly, the liposomes prepared by a transmembrane EDTA gradient method can significantly increase the drug retention capability of the liposomes and decrease the liposome-related damage to the immune system, compared with liposomes prepared by other gradient methods. Lastly, EDTA can bind to extra calcium, decreasing the incidence rate of hypercalcinemia and improving the quality of life in patients with advanced cancer. The drug-loading mechanism of the EDTA gradient method is similar to that of the ammonium sulfate gradient method. So far, the most common hydrated medium is ammonium EDTA salt solution. Briefly, the drug of interest is incubated in the presence of unilamellar vesicles containing internal ammonium EDTA salt and external saline. A small amount of neutral ammonia will diffuse out of the vesicle, creating an unbuffered acidic interior. The neutral form of the externally added drug can diffuse into the vesicle interior, where it will consume a proton, and become charged and therefore trapped. As the incoming drug uses up the supply of protons, more neutral ammonia will diffuse out, generating more protons to drive drug uptake. This continues until the entire drug has been loaded, or until the internal proton supply is depleted. Like in the ammonium sulfate gradient method, the encapsulated drug could also form precipitates, crystals, or gel with EDTA in the vesicle interior, which provides an additional driving force for drug accumulation.
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In summary, the EDTA gradient method offered a possibility to load all of the anthracyclines and other weakly amphiphilic base drugs within liposomes and had specific advantages both in vitro and in vivo. However, since the EDTA gradient method started late, only four drugs have been encapsulated in liposomes using this method thus far. In this section, all of these drugs are taken as examples to provide a detailed protocol of this gradient method. This can be a useful reference for the preparation of liposomes by this gradient method.
2
Materials
2.1
Materials for Preparation of Ammonium EDTA Solution
1. Ethylene diamine tetraacetic acid (EDTA) (Damao Chemical Reagent Factory, China) 2. Diammonium EDTA salt (Sigma-Aldrich, Germany) 3. NH3H2O 4. Distilled water 5. Electronic analytical balance (BS124s, Sartorius, Germany) 6. Heating magnetic stirrer (DF-101S, Yuhua Instrument Co. Ltd., China) 7. pH meter (PB-10, Sartorius, Germany)
2.2
Materials for Preparation of Empty Liposomes
1. Hydrogenated soy phosphatidylcholine (HSPC, MW 785, Lucas Meyer, Germany, or Phospholipid GmbH, Germany) 2. Cholesterol (CH, Nanjing Xinbai Pharmaceutical Co. Ltd., China, or Northern Lipids Inc., Canada) 3. 1,2-Distearoyl-sn-glycero-3-phosphoethanolami-ne-N-[methoxy(polyethylene glycol)-2000] (mPEG2000-DSPE, Genzyme Co. Ltd. USA, or Northern Lipids Inc., Canada) 4. Ammonium EDTA solution (200 mM, pH 5.5) 5. Diammonium EDTA solution (300 mM, pH 5.5) 6. Absolute ethanol 7. Distilled water 8. Chloroform 9. Polycarbonate membrane (0.8, 0.45, and 0.22 μm pore size) 10. Nuclepore polycarbonate membrane (0.1 μm pore size; 25 mm diameter) 11. Electronic analytical balance (BS124s, Sartorius, Germany) 12. Heating magnetic stirrer (DF-101S, Yuhua Instrument Co. Ltd., China) 13. Rotary evaporator (RE-52, Shanghai Yarong Biochemical Instrument Factory, China)
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14. Vibra-cell probe sonicator (JY92-2D, Ningbo Xinzhi Biological Technology Co. Ltd., China) 15. Thermobarrel extruder (Lipex Biomembranes, Canada) 16. Circulating water bath 17. Vacuum drying oven (D2F-6020MBE, Shanghai Boxun Industrial Ltd., China)
2.3
Materials for Preparation of EDTA Gradient Empty Liposomes
1. 2. 3. 4. 5. 6.
Empty liposomes Phosphate-buffered saline (PBS, pH 7.4) 5% Glucose 300 mM Sucrose Distilled water ZB-1 Sodium cation-exchange fiber (Guilin Zhenghan Technology Development Co. Ltd., China) ZB-2 Chlorine anion-exchange fiber (Guilin Zhenghan Technology Development Co. Ltd., China) 732 Sodium cation-exchange resin (Sinopharm Chemical Reagent Co. Ltd., China) 717 Chlorine anion-exchange resin (Sinopharm Chemical Reagent Co. Ltd., China) Sephadex G-50 (Sigma, USA) Dialysis tubing (molecular weight of 10 kDa) 2.5-mL Disposable syringe (10 70 mm) and glass wool 10-mL Plastic Eppendorf (EP) tube (15 80 mm) Desktop centrifuge (TDL80-2B, Shanghai Anting Scientific Instrument Factory, China) High-performance liquid chromatography (HPLC) system equipped with a P230 pump and a UV230 UV/Vis Detector (Dalian Elite Analytical Instruments Co. Ltd., China)
7. 8. 9. 10. 11. 12. 13. 14. 15.
2.4 1. 2. 3. 4. 5. 6. 7. 8.
Materials for Preparation of Drug-Loaded EDTA Gradient Liposomes Doxorubicin (DOX, Huafeng Lianbo Technology Co. Ltd., China) Topotecan (TPT, Chengdu Tianyuan Natural Product Co. Ltd., China) Epirubicin (EPI, Olympic Star Pharmaceutical Co. Ltd., China) Idarubicin (IDA, Pharmaceutical Research Institute, Poland) EDTA gradient empty liposomes Distilled water 90% (v/v) Isopropyl alcohol containing 0.75 M HCl 70% (v/v) Ethanol containing 0.1 M NaOH
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2.5-mL Disposable syringe (10 70 mm) and glass wool 10-mL Plastic Eppendorf (EP) tube (15 80 mm) Ice/water bath Electronic analytical balance (BS124s, Sartorius, Germany) Heating magnetic stirrer (DF-101S, Yuhua Instrument Co. Ltd., China) Desktop centrifuge (TDL80-2B, Shanghai Anting Scientific Instrument Factory, China) 15. Submicron particle analyzer (Nicomp 380 HPL, Particle Sizing System, USA, or Zetasizer Nano-ZS, Malvern Instrument Ltd., UK) 16. UV-spectrophotometer (Uvikon 756MC, Shanghai Third Analysis Instrument Factory, China, or Shimad Zu, Japan) 9. 10. 11. 12. 13. 14.
2.5
Materials for Determination of Lipid Concentration Using a Phosphate Assay
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Ammonium molybdate Sodium bisulfite (NaHSO3) Sodium sulfite (Na2SO3) 1-Amino-2-naphthol-4-sulfonic acid (ANS) 70% Perchloric acid 1 mM Disodium hydrogen phosphate (Na2HPO4) Distilled water Buchner funnel and filter paper (No.1) Heat-resistant glass tube (Pyrex test tube, 16 150 mm, or 18 150 mm) Glass marble (20–30 mm diameter) Aluminum test tube block Thermometer (0–350 C) Boiling water bath (or frying pan) Nitrogen gas Electronic analytical balance (BS124s, Sartorius, Germany) Heating magnetic stirrer (DF-101S, Yuhua Instrument Co. Ltd., China) UV/Vis spectrophotometer (Uvikon 756MC, Shanghai Third Analysis Instrument Factory, China)
3
Methods
The general preparation process of liposomes by the EDTA gradient method is as follows: firstly, hydrate lipid in EDTA salt solution to form multilamellar vesicles. Transform multilamellar vesicles to unilamellar vesicles and downsize to desired size (this step can be omitted if no defined size is needed). Then, form an EDTA gradient by removal of the extraliposomal medium. Load weakly amphiphilic base drugs into liposomes. Lastly, remove non-entrapped drug (this step can be omitted in the case of the drug loading approaching 100%).
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3.1
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Doxorubicin-Loaded EDTA Gradient Liposomes (Song et al. 2014)
3.1.1 Preparation of Ammonium EDTA Solution (See Note 1) 1. Weigh 2.930 g of EDTA into a 100-mL beaker and disperse with 45 mL of distilled water. 2. Place the EDTA suspension into a water bath of 80 C, then add ammonia water dropwise into the suspension while stirring, until the formation of a clear solution. 3. Cool the EDTA solution to room temperature, then add ammonia water to adjust pH to 5.5 (see Note 2). 4. Transfer the EDTA solution (pH 5.5) to a volumetric flask and dilute with distilled water to a volume of 50 mL to obtain 200 mM ammonium EDTA buffer (see Note 2).
3.1.2 1.
2. 3.
4.
5.
Preparation of Empty Liposomes (Modified Ethanol Injection Method) (See Note 3) Add 200 mg of a lipid mixture containing HSPC, CH, and mPEG2000-DSPE (3:1:1, w/w/w) to a 10 mL penicillin bottle and dissolve in 0.5 mL of absolute ethanol at 65 C (see Notes 4 and 5). Remove the solvent at 65 C until the lipid solution becomes viscous or begins forming a lipid film (see Note 6). Prewarm 5 mL of the ammonium EDTA solution (Item 3.1.1, Step 4) to 65 C and hydrate the lipid film (Step 2) at the same temperature to form bulk liposomal suspension (see Note 7). Sonicate the bulk liposomal suspension (Step 3) with a Vibra-cell probe sonicator in a 2-min cycle (200 W; 3 s on, 3 s off) and additional 6-min cycle (400 W; 3 s on, 3 s off). The initially multilamellar vesicles are transformed into unilamellar vesicles (see Notes 8 and 9). Extrude the resulting liposomes (Step 4) through polycarbonate membranes with gradually decreasing pore sizes (0.8, then 0.45, then 0.22 μm) to remove large particles (see Notes 8 and 9).
3.1.3 1.
2. 3. 4.
Preparation of EDTA Gradient Empty Liposomes (Dialyzed Method) (See Note 10) Place 2 mL of empty liposomal suspension (Item 3.1.2, Step 5) in dialysis tubing with a molecular weight cutoff of 10 kDa, and dialyze against 500 mL of 5% glucose solution (see Note 11). Supersede the glucose solution at intervals of 30 min and cease the dialysis after 2 h (see Note 12). Determine lipid concentration of the dialyzed liposomes with a phosphate assay (see Item 3.5). Dilute the dialyzed liposomes with 5% glucose solution to a final lipid concentration of 20 mg/mL.
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3.1.4 Doxorubicin-Loaded EDTA Gradient Liposomes 1. Weigh 10 mg of doxorubicin hydrochloride (DOX) into a 5-mL volumetric flask, and dissolve in 3 mL of distilled water. 2. Dilute the solution to a volume of 5 mL with distilled water to obtain a 2 mg/mL DOX solution. 3. Add 1 mL of the DOX solution to 1 mL of the EDTA gradient liposomes (Item 3.1.3, Step 4) to achieve a drug to lipid ratio of 1:10 (w/w) (see Note 13). 4. Mix and incubate them at 60 C for 30 min, then cool rapidly in an ice/water bath (0–2 C) for 5 min (see Notes 14–16). 5. Determine the mean diameter of the liposomes using a NICOMP 380 HPL submicron particle analyzer operating at 632.8 nm (see Note 17). 6. Prepare a Sephadex G-50 mini-column (10 70 mm) (see Item 3.3.3, Steps 1–4). 7. Separate and remove liposomes from free DOX by size exclusion chromatography on a Sephadex G-50 mini-column (Step 6). Place 0.2 mL of DOX liposomal suspension (Step 4) on the top of the G-50 mini-column, then elute with distilled water (0.4 mL, four times). Alternatively, separate liposomes from free DOX by centrifuging G-50 mini-column in distilled water (0.1 mL, four times), and pool the fractions (see Notes 18–19). 8. Determine the DOX concentration with a UV-spectrophotometer at 480 nm after lysis of the liposomes with 90% (v/v) isopropyl alcohol containing 0.75 M HCl (see Note 20). The encapsulation efficiency is calculated as the percentage of DOX remaining in the liposomes following elution (Aliposomes), for the initial amount of drug used in liposomes (Atotal). Encapsulation efficiency ð%Þ ¼ Aliposomes =Atotal 9. The particle size and encapsulation efficiency of DOX-loaded liposomes is 79.4 1.87 nm and 95.5% 0.6%, respectively. 10. DOX forms a gel-like complex with the internal buffer of EDTA at pH 4; this complex cannot be precipitated by centrifugation. This complex is an important reason for the high drug encapsulation and slow drug release (see Fig. 1).
3.2
Preparation of Topotecan-Loaded EDTA Gradient Liposomes (Yang et al. 2012)
3.2.1 Preparation of Ammonium EDTA Solution The optimum concentration and pH of ammonium EDTA solution for topotecan liposomes is 200 mM and 5.5, respectively. The specific preparation process is shown in the item of 3.1.1. 3.2.2
Preparation of Empty Liposomes (Modified Ethanol Injection Method) The composition and preparation of empty liposomes is the same as that of DOX liposomes. The specific preparation process is shown in the item of 3.1.2.
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Fig. 1 Photographs of mixtures of DOX with (NH4)2SO4 and with NH4EDTA at 25 C and pH 4. (1) the mixture of (NH4)2SO4 and DOX, (2) the mixture of NH4EDTA and DOX, (A) the mixtures before centrifugation, (B) the mixtures after centrifugation, (C) the mixtures diluted with isochoric distilled water
3.2.3 1. 2. 3. 4.
5.
6. 7.
Preparation of EDTA Gradient Empty Liposomes (Anion and Cation Mixed Ion-Exchange Resin Mini-Column Method) Mix pretreated anion and cation ion-exchange resin with appropriate amount of distilled water at a ratio of 2:1 (v/v). Pack a tiny plug of glass wool into the end of a 2.5-mL disposable syringe, and then place into a 15 80 mm plastic EP tube. Swirl mixed ion-exchange resin slurry and fill syring with 2 mL resin using a plastic pipette. Place syring (in EP tube) into a desktop centrifuge, and pack the resin by centrifuging at 2000 rpm for 4 min. To prevent the resin mini-column from drying, cover the column with Parafilm and use within a day. Add 1 mL of the liposomes to the top of the resin mini-column (Step 4), and elute with distilled water (0.5 mL, three times) to replace the extraliposomal solution. Alternatively, form the gradient by centrifuging the resin mini-column prepared in distilled water (0.1 mL, four times), and pool the fractions (see Note 21). Determine lipid concentration of the gradient liposomes with a phosphate assay (see Item 3.5). Adjust the final phospholipid concentration of the gradient liposomes to 20 mg/ mL with distilled water.
3.2.4 Preparation of Topotecan-Loaded EDTA Gradient Liposomes 1. Prepare 2 mg/mL topotecan hydrochloride (TPT) solution. The preparation process is the same as that of DOX solution (see Item 3.1.4, Steps 1–2). 2. Add 1 mL of the TPT solution to 1 mL of the EDTA gradient liposomes (Item 3.2.3, Step 4) to achieve a drug to lipid ratio of 1:10 (w/w). 3. Mix and incubate them at 50 C for 10 min, then cool rapidly in an ice/water bath (0–2 C) for 5 min.
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4. Determine the mean diameter of the liposomes using a NICOMP 380 HPL submicron particle analyzer operating at 632.8 nm. 5. Separate and remove liposomes from free TPT by size exclusion chromatography on a Sephadex G-50 mini-column (10 70 mm) (see Item 3.1.4, Steps 6–7). 6. Determine the TPT concentration by a UV-spectrophotometer at 430 nm after lysis of the liposomes with 70% (v/v) ethanol containing 0.1 M NaOH. Calculate the encapsulation efficiency of TPT liposomes with the formula shown in the item of 3.1.4, Step 8. 7. The particle size and encapsulation efficiency of TPT-loaded liposomes is 84.8 3.8 nm and 92.0% 0.3%, respectively (see Note 22).
3.3
Epirubicin-Loaded EDTA Gradient Liposomes (Yang et al. 2014)
3.3.1 Preparation of Ammonium EDTA Solution The optimum concentration and pH of ammonium EDTA solution for epirubicin liposomes are 200 mM and 5.5, respectively, and the specific preparation process is shown in the item of 3.1.1.
3.3.2
Preparation of Empty Liposomes (Modified Ethanol Injection Method) The composition and preparation of empty liposomes are the same as that of DOX liposomes. The specific preparation process is shown in the item of 3.1.2.
3.3.3 1.
2. 3. 4. 5. 6. 7.
Preparation of EDTA Gradient Empty Liposomes (Sephadex G-50 Mini-Column Method) Pretreat Sephadex G-50 with PBS buffer (20 mM sodium phosphate, 150 mM NaCl). Add a small volume (2–3 mL) of dry G-50 to 200–300 mL PBS, and swirl. Add as necessary until settled G-50 occupies about half the aqueous volume. Let it sit overnight. Pack a tiny plug of glass wool into the end of a 2.5-mL disposable syringe, and then place it into a 15 80 mm plastic EP tube. Swirl the G-50 slurry and fill syringe with 2 mL gel using a plastic pipette. Place syringes (in EP tubes) into a desktop centrifuge, and pack the gel by centrifuging at 2000 rpm for 4 min. Pass the liposomes through the G-50 mini-column with 300 mM sucrose to replace the extraliposomal solution (see Item 3.2.3, Step 5). Determine lipid concentration of the gradient liposomes with a phosphate assay (see Item 3.5). Adjust the final phospholipid concentration of the gradient liposomes to 20 mg/ mL with 300 mM sucrose.
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3.3.4 Preparation of Epirubicin-Loaded EDTA Gradient Liposomes 1. Prepare 4 mg/mL epirubicin hydrochloride (EPI) solution. The preparation process is the same as that of DOX solution (see Item 3.1.4, Steps 1–2). 2. Add 1 mL of the EPI solution to 1 mL of the EDTA gradient liposomes (Item 3.3.3, Step 4) to achieve a drug to lipid ratio of 1:10 (w/w). 3. Mix and incubate them at 60 C for 20 min, then cool rapidly in an ice/water bath (0–2 C) for 5 min. 4. Determine the mean diameter of the liposomes using a NICOMP 380 HPL submicron particle analyzer operating at 632.8 nm. 5. Separate and remove liposomes from free EPI by ion exchange chromatography on a ZB-1 cation-exchange fiber mini-column (10 70 mm) (see Item 3.1.4, Step 7). The preparation of the fiber mini-column is similar to that of the ion-exchange resin (see Item 3.2.3, Steps 1–4). 6. Determine the EPI concentration with a UV-spectrophotometer at 480 nm after lysis of the liposomes with 90% (v/v) ethanol containing 0.75 M HCl. Calculate the encapsulation efficiency of EPI liposomes with the formula shown in the item of 3.1.4, Step 8. 7. The particle size and encapsulation efficiency of EPI-loaded liposomes are about 110 nm and 98.6% 0.2%, respectively.
3.4
Idarubicin-Loaded EDTA Gradient Liposomes (Gubernator et al. 2010)
3.4.1 Preparation of Diammonium EDTA Solution 1. Weigh 9.84 g of diammonium EDTA into a beaker and dissolve in 90 mL of distilled water. 2. Adjust pH to 4.5 or 5.5 with hydrochloric acid or ammonia water (see Note 23). 3. Transfer the solution to a volumetric flask, and dilute with distilled water to a volume of 100 mL to obtain 300 mM diammonium EDTA buffer. 3.4.2 Preparation of Empty Liposomes (Thin Lipid Film Method) 1. Weigh 30 mg of a lipid mixture containing HSPC, CH, and mPEG2000-DSPE (6.5:3:0.5, n/n/n) into a 100 mL round-bottom flask and dissolve in 3 mL of chloroform (10 mg/mL). 2. Remove the chloroform at 40 C with a rotary evaporator until a thin film of dried lipids forms. 3. Place rotary evaporator under high vacuum for a minimum of 1 h. 4. Prewarm 1.5 mL of the diammonium EDTA solution (300 mM, pH 4.5 or 5.5) to 64 C. Hydrate the lipid film at the same temperature, followed by seven freezethaw cycles to form multilamellar vesicles. 5. Extrude the resulting bulk liposomal suspensions through nucleopore polycarbonate filters with pore sizes of 100 nm (10 passes) on a thermobarrel extruder. The extruder is equilibrated to 64 C prior to liposome extrusion.
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3.4.3 1.
2. 3. 4.
Preparation of EDTA Gradient Empty Liposomes (Sephadex G-50 Mini-Column Method) Prepare a Sephadex G-50 mini-column (10 200 mm) equilibrated with PBS buffer (20 mM sodium phosphate, 150 mM NaCl) of pH 7.5 or 8.5. The preparation of the mini-column can reference item 3.3.3, Steps 1–4. Pass the liposomes through the G-50 mini-column with PBS buffer to replace the extraliposomal solution (see Item 3.2.3, Step 5). Determine lipid concentration of the gradient liposomes with an ammonium ferrothiocyanate assay (Stewart 1980) (see Note 24). Adjust the final phospholipid concentration of the gradient liposomes to 7.84 mg/ mL with PBS (10 mM).
3.4.4 Preparation of Idarubicin-Loaded EDTA Gradient Liposomes 1. Prepare 6 mg/mL idarubicin hydrochloride (IDA) solution. The preparation process is the same as that of DOX solution (see Item 3.1.4, Steps 1–2). 2. Add 0.18 mL of the IDA solution (6 mg/mL) to 1 mL of the EDTA gradient liposomes (Item 3.4.3, Step 4) to achieve a drug to lipid ratio of 1:5 (n/n). 3. Mix and incubate them at 60 C for 60 min. 4. Determine the mean diameter of the liposomes using a Zetasizer Nano-ZS submicron particle analyzer. 5. Separate and remove liposomes from free IDA by size exclusion chromatography on a Sephadex G-50 mini-column (5.5 70 mm) equilibrated with 150 mM sodium chloride solution (see Item 3.1.4, Step 7). 6. Determine the IDA concentration with a UV-spectrophotometer at 480 nm after solubilization of liposomes with Triton-X 100. Calculate the encapsulation efficiency of IDA liposomes with the formula shown in the item of 3.1.4, Step 8. 7. The particle size and encapsulation efficiency of IDA-loaded liposomes are about 114 nm and above 98%, respectively. 8. IDA forms a precipitate with the internal buffer of EDTA, which is the reason for the high drug encapsulation and slow drug release (Gubernator et al. 2010).
3.5
Determination of Lipid Concentration Using a Phosphate Assay (Torchilin and Weissig 2003)
3.5.1 Preparation of Solutions 1. Ammonium molybdate solution: Dissolve 4.4 g of ammonium molybdate in 1.5 L of distilled water. Slowly add 40 mL of sulfuric acid (conc.), and add distilled water for a final volume of 2 L. 2. Fiske-Subbarow solution: Dissolve 150 g of NaHSO3 and 5 g of Na2SO3 in 1 L of distilled water, then add 2.5 g of ANS and stir at 40 C in the dark for 1–2 h until dissolved (keep covered because of fumes). Store overnight in the dark at room temperature, then filter off any insoluble material with a Buchner funnel and Whatman (No.1) filter paper. The solution is stored in an amber glass bottle.
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3. The 1 mM Na2HPO4 standard: Dissolve 142 mg of Na2HPO4 with an appropriate amount of distilled water, and dilute to a total volume of 1 L.
3.5.2 Determination of Lipid Concentration 1. Prepare the standard curve. Pipette 0, 50, 100, 150, and 200 nmol of phosphate (from 1 mM Na2HPO4 standard) into a series of Pyrex tubes (in duplicate). 2. Prepare the samples. Pipet aliquots of samples (dilute if necessary) containing 100–200 nmol phosphate into Pyrex tubes (in duplicate). Samples can be aqueous or organic (for volumes in the μL range) (see Note 25). 3. Carefully add 0.7 mL perchloric acid into assay tubes, cover tubes with a marble, and vortex them. 4. Place tubes in heating block in fume hood for 1–2 h, ensuring the temperature remains between 180 C and 210 C. 5. Remove tubes from heating block and let them cool to room temperature. 6. Add 0.75 mL Fiske-Subbarow solution, followed by 7 mL ammonium molybdate solution, and vortex the tubes. 7. Place tubes (with marbles on top) in a boiling water bath in fume hood for 15 min, then place tubes in cold water bath to cool to room temperature. 8. Read the absorbance of all standards and samples at 815 nm. If possible, use the spectrophotometer to generate a standard curve so that samples can be quantified as nmol PO4/tube. 9. To determine the phosphate concentration of the original samples (mM), divide nmol PO4/tube by the volume of sample (in μL) added to the assay tube, accounting for any dilutions. 10. After reading absorbance values, contents of tubes are diluted down the sink with a large quantity of cold water. The Pyrex tubes are immediately rinsed several times with tap water followed by distilled water and allowed to dry. Phosphate assay tubes should never be cleaned with a detergent.
4
Notes
1. Ammonium EDTA solution is the most common hydration medium of the EDTA gradient method, and it can be prepared with EDTA and ammonia water. Besides, ammonium EDTA solution, other EDTA salt solutions such as disodium EDTA and other ammonium EDTA salt solutions prepared by ethylenediamine, triethylamine, ethanolamine, and trishydroxymethylaminomethane can also be used as the hydration medium for the EDTA gradient method. However, it should be noted that the type of EDTA salt solution has an important influence on the particle size, encapsulation efficiency, and stability of the liposomes, so it is better to choose an EDTA salt solution suitable for the drug. 2. The concentration and pH of the intraliposomal ammonium EDTA solution have great influence on the encapsulation efficiency of drug-loaded liposomes. In our previous studies, for doxorubicin, topotecan, and epirubicin, the optimum
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concentration and pH of ammonium EDTA were 200 mM and 5.5, respectively. Thus, we took these two conditions as examples to show the preparation process of ammonium EDTA solution. Other concentrations and pH of ammonium EDTA solution can be obtained by adjusting the amount of EDTA and ammonia water. Besides the modified ethanol injection method, other methods such as the thin lipid film method, lyophilized method, detergent dialysis, and reverse-phase evaporation technique can also be used to prepare empty liposomes. The liposomes prepared by the thin lipid film method are introduced in the item of 3.4.2. Phospholipid species have a great impact on the encapsulation efficiency of the liposomes. Compared with the phospholipid bilayer composed of unsaturated phospholipids (EPC or SPC), a bilayer composed of saturated phospholipids (HSPC) has high rigidity, and the drug encapsulated in the saturated phospholipid bilayer does not easily leak out, neither in storage in vitro nor in the circulation in vivo. Thus, in order to obtain a satisfactory encapsulation efficiency, it is better to use saturated phospholipids such as HSPC. Cholesterol is an important component of the membrane of liposomes, and the proportion of cholesterol and phospholipids has a great impact on the encapsulation efficiency of liposomes. In our previous studies with doxorubicin, topotecan, and epirubicin, when the quality ratio of phospholipids and cholesterol was 3:1, the encapsulation efficiencies of the three drug-loaded liposomes were higher than 90%. You can also refrain from removing the solvent of ethanol, but the ethanol concentration in liposomal suspensions should be less than 10% (v/v). In our previous studies, when the ethanol concentration was less than 10%, the encapsulation efficiency of liposomes had no significant change. When the ethanol concentration continued increasing, the encapsulation efficiency began to decline, and when the ethanol concentration reached 30% (v/v), the encapsulation efficiency decreased to below 25%. Hydration temperature should be higher than the transition temperature of the lipid. Because the transition temperature of HSPC is 50 C, the hydration temperature chosen is above that, by 65 C. Large unilamellar vesicles (LUVs) are helpful in drug loading, but the internal obtained bulk liposomal suspensions are multilamellar vesicles (MLVs). Thus, before establishing the gradient, the MLVs should first be transformed to LUVs by sonication or extrusion. For the liposomes prepared by the EDTA gradient method, the particle size of the drug-loaded liposomes mainly depends on the size of the empty liposomes. The size of empty liposomes can be regulated by the time and power of sonication and the pore size of polycarbonate membranes. The conditions mentioned in the item of 3.1.2, Steps 4–5 can help obtain the liposomes about 100 nm in diameter. Besides the dialyzed method, other methods such as the ion-exchange resin method, ion-exchange fiber method, and Sephadex G-50 method can be used to
6
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prepare empty gradient liposomes. The liposomes prepared by these methods are introduced in the items of 3.2.3, 3.3.3, and 3.4.3. Among these methods, the ion-exchange fiber method has the highest efficiency. Besides 5% glucose, the dialysis solution can be another isotonic solution such as 0.9% sodium chloride and PBS. However, it should be noted that the pH of extraliposomal solution has a dramatic effect on drug loading. When the △pH between extraliposomal solution and intraliposomal solution exceeds 3, the encapsulation efficiency can achieve a maximum value. However, in some cases, when the pH of extraliposomal solution is too high (above 8.5), the encapsulation efficiency may dramatically decrease, because of a decreased amount of the membrane-permeable electroneutral form of the drug. The EDTA absorbance of the dialysis solution is determined once every 30 min, and the dialysis can be ceased if the absorbance stays constant. Generally speaking, 2 h of dialysis is enough. The EDTA absorbance of the dialysis solution can be measured using an HPLC system equipped with a P230 pump, a UV230 UV/Vis Detector, and a C18 column (4.6 200 mm, 5 μm particle size). The mobile phase is 0.2% monopotassium phosphate (adjusted to pH 3.0 with phosphoric acid, and containing 0.2% tetrabutylammonium bromide)acetonitrile (90/10, v/v). Then, absorbance is detected at 258 nm, with a flow rate of 1 mL/min and a column temperature of 25 C. The drug-lipid ratio has great impact on the drug loading and encapsulation efficiency. With an increasing drug-lipid ratio, the drug loading increased and the encapsulation efficiency decreased. In order to obtain a high drug loading as well as optimum encapsulation efficiency under this condition, the best choice of DOX-lipid ratio is 1:10 (w/w). Incubation time also has great influence on the encapsulation efficiency of the liposomes. If the incubation time is too short, the drug loading process is incomplete, but if the incubation time is too long, the loaded drug may leak out and lead to a reduced encapsulation efficiency. In the process of drug loading, the samples can be taken at different times (0, 5, 15, 30 min) to detect whether the drug loading is complete. In the process of incubation, it is better to shake the bottle gently by hand at intervals or magnetically stir it at a low speed. The incubation temperature is another influential factor in encapsulation efficiency of the liposomes. It is better for the incubation temperature to be above the transition temperature of the lipid, because under this condition, the lipid molecular layer has good mobility and permeability, which is ideal for drug loading. However, the incubation temperature should not be too high, or the low solubility compound formed by DOX and EDTA will not form easily. The liposomal suspension should be diluted to an appropriate volume with distilled water before size determination. In addition, other particle analyzers also can be applied to determine the size of liposomes, such as Zetasizer NanoZS mentioned in the item of 3.4.4. Besides Sephadex G-50, cation-exchange resin and cation-exchange fiber methods can also be used to separate liposomes from free DOX. The samples
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should be gently added to the center of the G-50 mini-column, without blowing away the gel. Centrifugation can improve the efficiency of elution, but it should be noted that the liposomes would gather at the bottom of the EP tube after centrifugation, so the centrifuged liposomes should be blown up by pipette before transferred. Besides 90% (v/v) isopropyl alcohol containing 0.75 M HCl, other solvents such as 70% (v/v) ethanol containing 0.1 M NaOH, as well as Triton-X 100, can also be used to dissolve the liposomes (see Item 3.2.4 and 3.4.4). If the liposomes need to be isotonic, the eluted solution of distilled water can be changed to 5% glucose, 0.9% sodium chloride, or PBS with pH 7.4. Compared with the DOX liposomes, the encapsulation efficiency of TPT liposomes is slightly lower, which may be because the binding capability of TPT with EDTA is weaker than that of DOX with EDTA. For IDA liposomes, the intraliposomal solutions of both pH 4.5 and 5.5 are conducive to satisfactory encapsulation efficiency. The determination of lipid concentration can also be done using the method introduced in the item of 3.5. For larger volumes of organic samples, remove solvent under a stream of nitrogen gas. Samples containing sucrose should be dialyzed prior to analysis, and sample volume should not exceed 200 μL.
Conclusion
Use of the EDTA gradient method to load weakly base drugs into liposomes produced rapid and efficient drug encapsulation, and the liposomes showed slow drug release and good long-term stability. Furthermore, because of the chelation of EDTA, it can also reduce drug-related toxicity and liposome-related immune organ damage. The method presented in this part may be a helpful reference for the preparation of liposomes by this gradient method.
References Blumer W, Cranton E (1989) Ninety percent reduction in cancer mortality after chelation therapy with EDTA. J Adv Med 2:183–187 Body JJ (1992) Bone metastases and tumor-induced hypercalcemia. Curr Opin Oncol 4(4):624–631 Buss JL, Torti FM, Torti SV (2003) The role of iron chelation in cancer therapy. Curr Med Chem 10(12):1021–1034 Clarke NE (1960) Atherosclerosis, occlusive vascular disease and EDTA. Am J Cardiol 6(2):233–236 Cranton EM, Frackelton JP (1998) Free oxygen radical pathology and EDTA chelation therapy: mechanisms of action. J Adv Med 11(4):277–310 Dans AL, Tan FN, Villarruz-Sulit EC (2002) Chelation therapy for atherosclerotic cardiovascular disease. Cochrane Database Syst Rev 4(4):CD002785 Domingo JL (1998) Developmental toxicity of metal chelating agents. Reprod Toxicol 12(5):499–510
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Erdahl WL, Chapman CJ, Taylor RW, Pfeiffer DR (1994) Ca2+ transport properties of ionophores A23187, ionomycin, and 4-BrA23187 in a well defined model system. Biophy J 66(5):1678 Erdahl WL, Chapman CJ, Taylor RW, Pfeiffer DR (1995) Effects of pH conditions on Ca2+ transport catalyzed by ionophores A23187, 4-BrA23187, and ionomycin suggest problems with common applications of these compounds in biological systems. Biophy J 69(6):2350 Fenske DB, Wong KF, Maurer E, Maurer N, Leenhouts JM, Boman N, Amankwa L, Cullis PR (1998) Ionophore-mediated uptake of ciprofloxacin and vincristine into large unilamellar vesicles exhibiting transmembrane ion gradients. BBA-Biomembranes 1414(1):188–204 Flora SJ, Pachauri V (2010) Chelation in metal intoxication. Int J Environ Res Public Health 7(7):2745–2788 Fulgenzi A, De Giuseppe R, Bamonti F, Vietti D, Ferrero ME (2015) Efficacy of chelation therapy to remove aluminium intoxication. J Inorg Biochem 152:214–218 Gordon GF (2003) EDTA and chelation therapy: history and mechanisms of action. J Am Coll Cardiol 35:521 Gubernator J, Chwastek G, Korycińska M, Stasiuk M, Grynkiewicz G, Lewrick F, Süss R, Kozubek A (2010) The encapsulation of idarubicin within liposomes using the novel EDTA ion gradient method ensures improved drug retention in vitro and in vivo. J Control Release 146(1):68–75 Gubernator J, Lipka D, Korycińska M, Kempińska K, Milczarek M, Wietrzyk J, Hrynyk R, Barnert S, Süss R, Kozubek A (2014) Efficient human breast cancer xenograft regression after a single treatment with a novel liposomal formulation of Epirubicin prepared using the EDTA ion gradient method. PLoS One 9(3):e91487 Kitchell JR, Palmon F, Aytan N, Meltzer LE (1963) The treatment of coronary artery disease with disodium EDTA: a reappraisal. Am J Cardiol 11(4):501–506 Mundy GR, Guise TA (1997) Hypercalcemia of malignancy. Am J Med 103(2):134–145 Ouyang P, Gottlieb SH, Culotta VL, Navas-Acien A (2015) EDTA chelation therapy to reduce cardiovascular events in persons with diabetes. Curr Cardiol Rep 17(11):1–9 Pressman BC (1976) Biological applications of ionophores. Annu Rev Biochem 45(1):501–530 Riordan HD, Cheraskin E, Dirks M (1990) Mineral excretion associated with EDTA chelation therapy. J Adv Med 3(2):111–123 Seely DM, Wu P, Mills EJ (2005) EDTA chelation therapy for cardiovascular disease: a systematic review. BMC Cardiovasc Disord 5(1):32 Song Y, Huang Z, Song Y, Tian Q, Liu X, She Z, Jiao J, Lu E, Deng Y (2014) The application of EDTA in drug delivery systems: doxorubicin liposomes loaded via NH4EDTA gradient. Int J Nanomedicine 9:3611 Stewart JCM (1980) Colorimetric determination of phospholipids with ammonium ferrothiocyanate. Anal Biochem 104(1):10–14 Thompson KA, Goodale DB (2000) The recent development of propofol (DIPRIVAN ®). Intens Care Med 26(3):S400–S404 Torchilin V, Weissig V (2003) Liposomes: a practical approach. Oxford University Press, New York, pp 170–171 Waters RS, Bryden NA, Patterson KY, Veillon C, Anderson RA (2001) EDTA chelation effects on urinary losses of cadmium, calcium, chromium, cobalt, copper, lead, magnesium, and zinc. Biol Trace Elem Res 83(3):207–221 Yang Y, Ma Y, Wang SA (2012) Novel method to load topotecan into liposomes driven by a transmembrane NH 4 EDTA gradient. Eur J Pharm Biopharm 80(2):332–339 Yang Q, Ma Y, Zhao Y, She Z, Wang L, Li J, Wang C, Deng Y (2013) Accelerated drug release and clearance of PEGylated epirubicin liposomes following repeated injections: a new challenge for sequential low-dose chemotherapy. Int J Nanomedicine 8(1):1257 Yang Q, Zhang T, Wang C, Jiao J, Li J, Deng Y (2014) Coencapsulation of epirubicin and metformin in PEGylated liposomes inhibits the recurrence of murine sarcoma S180 existing CD133+ cancer stem-like cells. Eur J Pharm Biopharm 88(3):737–745
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Lipid-Coated Cisplatin Nanoparticles for Insoluble Drug Loading Yang Liu and Leaf Huang
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Synthesis of cis-[Pt(NH3)2(H2O)2](NO3)2 Precursor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Preparation of LPC NPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Characterization of Particle Size and Zeta Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Transmission Electron Microscopy (TEM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Characterization of Drug-Loading Capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7 In Vitro Release of Drug from NPs in Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8 In Vitro Release of Drug from NPs in Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Synthesis of Cis-[Pt(NH3)2(H2O)2](NO3)2 Precursor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Preparation of LPC NPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Characterization of Particle Size and Zeta Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Transmission Electron Microscopy (TEM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Characterization of Drug-Loading Capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 In Vitro Release of Drug from NPs in Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 In Vitro Release of Drug from NPs in Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
98 100 100 100 100 101 101 101 101 101 102 102 102 103 103 104 104 104 104 107 108
Y. Liu (*) Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Irvine, CA, USA e-mail: [email protected] L. Huang Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA e-mail: [email protected] © Springer-Verlag GmbH Germany, part of Springer Nature 2021 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49320-5_7
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Abstract
The large number of water insoluble drugs on the market and in the development pipeline provides challenges as well as opportunities for scientists to develop advanced drug delivery systems to meet the clinical needs. Liposomal formulations as drug carriers for insoluble drugs have not been extensively exploited. This chapter described a conceptually novel method using an insoluble drug cisplatin (CDDP) as a building material for the preparation of nanoparticles (NPs). The formulation was done using a reverse microemulsion method. The formation of the NPs was first initiated by mixing KCl and a highly soluble precursor of CDDP. The insoluble CDDP was precipitated and formed the nanosized core. Dioleoylphosphatydic acid (DOPA), an anionic lipid, was employed as the inner leaflet lipid to stabilize and coat the nano-size CDDP precipitates. A suitable cationic lipid was then added as the outer leaflet lipid to form an asymmetric lipid bilayer structure, and PEGylation of NP was done by including a PEG-phospholipid conjugate in the outer leaflet lipid mixture. The resulting NPs were named lipid-Pt-Cl (LPC) NPs. LPC NPs were characterized with a controllable size (in the range of 12–75 nm) and high drug loading capacity (approximately 80 wt.%). LPC NPs exhibited sustained release profile in vitro drug release studies in medium and in cells. This method might be applicable in formulating other insoluble drugs. Keywords
Lipid · Nanoparticles · Drug delivery · Cisplatin · Precipitate · Microemulsions
1
Introduction
An estimated 40% of the approved drugs and nearly 90% of molecules under development are poorly water-soluble (Kalepu and Nekkanti 2015). There is an increasing number of new chemical entities (NCEs) selected from combinatorial chemistry and high-throughput screening are quite lipophilic, since the biological screenings are often based on the interaction between the drug candidates and membrane-associated proteins. This presents a challenge to the parenteral administration of poorly water-soluble compounds. Traditional formulation approaches include the use of cosolvents and surfactants for solubilization, pH adjustment, adding cyclodextrins, or forming micelles. These approaches pose the risk of excipients-related toxicity and potential precipitation of the poorly water-soluble compounds upon dilution in the blood circulation. Encapsulation of insoluble drugs in nanoparticles (NPs) permits the dispersion in aqueous solution and improves the pharmacokinetics. Several strategies have been successfully developed to prepare NPs containing insoluble drugs. Biodegradable polymers, such as polylactides (PLA), polycaprolactone (PCL), and poly (D,L-lactide-co-glycolide) (PLGA), encapsulate hydrophobic drugs through hydrophobic interactions (Bala et al. 2004; Smith
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and Hunneyball 1986; Chen et al. 2011). Liposomes, which have been established as one of the most successful drug carriers, played a significant role in drug delivery systems for hydrophilic drugs such as doxorubicin (Sharma and Sharma 1997). Water-soluble drugs are usually incorporated into liposomes with acidic interior by active (remote) loading method. Previous study suggested that the quantity of loaded drugs was proportional to (Kalepu and Nekkanti 2015) the volume of interior aqueous phase of liposomes (Bala et al. 2004), the extraliposomal concentration of neutral drug, and (Smith and Hunneyball 1986) the transmembrane proton gradient with a favorable interior concentration of H+ (Zhang et al. 2015). Poorly watersoluble drug cannot be readily encapsulated via this method due to its low solubility. Its application in the delivery of insoluble drugs, therefore, has received limited attention. Alternatively, the hydrophobic drugs can be successfully incorporated into the phospholipid bilayer. The encapsulation efficiency of hydrophobic drugs is known to be influenced by physicochemical properties of the drug (i.e., lipophilicity), composition of lipid bilayer, and the method of preparation (Sharma and Sharma 1997). This chapter describes a conceptually novel method which utilized an insoluble drug cisplatin (CDDP) to form the NP cores and further coated the nano-size cores with a lipid bilayer. The uniqueness of this method lies in the formation of CDDP precipitates stabilized by a lipid bilayer, resulting in homogenous size distribution and high drug loading efficiency. CDDP is a widely used chemotherapy drug in the clinic to treat a variety of cancers including lung, ovarian, breast and bladder cancers (Wang and Lippard 2005). Unfortunately, a maximum tolerated dose of CDDP therapy is significantly limited by its severe side effects such as nephrotoxicity (Pabla and Dong 2008). A promising approach to improve the therapeutic index of CDDP is to load the drug into nanoparticles (NPs). Several advantages could be achieved through this approach, for example, encapsulation of CDDP could prevent the undesirable plasma protein binding, which is believed to be responsible for deactivation of the drug (Andrews et al. 1984). The NPs could preferentially deliver the drug to the tumor via the enhanced permeability and retention (EPR) effect and potentially via ligand-mediated active targeting (Guo et al. 2013). Recent advances on nanoparticulate formulations of CDDP include chelating CDDP with polymeric materials and encapsulating CDDP into PLGA NPs or liposomes (Plummer et al. 2011; Baba et al. 2012; Sengupta et al. 2012; Zamboni et al. 2003; Avgoustakis et al. 2002). However, the encapsulation and loading efficiency were usually not optimal and burst release often occurred (Avgoustakis et al. 2002). For example, LipoplatinTM, a liposomal formulation of CDDP, employed electrostatic interaction to load positively charged platinum into negatively charged 1,2-dipalmitoyl-snglycero-3phosphoglycerol sodium salt (DPPG)-lipid micelles (Boulikas 2009). Drug loading of LipoplatinTM was reported to be 8.9 wt.% (Boulikas 2009). The barriers for the development of nanoparticulate formulations of CDDP can largely be attributed to the poor solubility of CDDP in both water and organic solvents (Aryal et al. 2010). Prodrugs which increased the hydrophobicity of CDDP have been reported to improve the encapsulation of CDDP into NPs. These methods require chemical modification of CDDP (Dhar et al. 2011; Kolishetti et al. 2010).
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A novel method was developed to prepare NPs using the unmodified CDDP, which takes advantage of its poor solubility. The formulation was prepared in two steps. A highly soluble precursor of CDDP was first synthesized. By mixing two reverse microemulsions containing KCl and the precursor, nano-size CDDP precipitates were formed in the microemulsions. Then lipid components were added in sequence to coat the CDDP precipitates. The final NPs, lipid-Pt-Cl (LPC) NPs, contain a lipid bilayer coating and can be well dispersed in aqueous solution. LPC NPs exhibited potent antitumor activity both in vitro and in vivo.
2
Materials
2.1
Materials
1. CDDP (Sigma, St. Louis, MO). 2. Dioleoylphosphatydic acid (DOPA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol)2000] (DSPE-PEG2000), Cholesterol (Avanti Polar Lipids, Alabaster, AL). 3. Chloroform (Sigma, St. Louis, MO)
2.2 1. 2. 3. 4. 5. 6.
AgNO3 (Sigma, St. Louis, MO) 100-mL round-bottomed spherical flasks Aluminum foil A Beckman ultracentrifuge 0.2 μm syringe filters Inductively coupled plasma mass spectrometry (ICP-MS) Varian 820-MS (Palo Alto, CA)
2.3 1. 2. 3. 4. 5. 6. 7. 8.
Synthesis of cis-[Pt(NH3)2(H2O)2](NO3)2 Precursor
Preparation of LPC NPs
Cyclohexane, Igepal CO-520, triton-X100, hexanol (Sigma, St. Louis, MO) 100 mL round-bottomed spherical flasks 800 mM potassium chloride (KCl) A Beckman ultracentrifuge Ethanol, 200 proof (100%), USP 7 mL borosilicate glass scintillation vials A bath type sonicator A vacuum desiccator
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Characterization of Particle Size and Zeta Potential
1. A Malvern ZetaSizer Nano series (Westborough, MA).
2.5
Transmission Electron Microscopy (TEM)
1. 200 mesh carbon-coated copper grids (Ted Pella, Inc., Redding, CA) 2. 2% uranyl acetate 3. A JEOL 100CX II TEM (JEOL, Japan)
2.6
Characterization of Drug-Loading Capacity
1. 70% HNO3 2. A ICP-MS Varian 820-MS (Palo Alto, CA).
2.7 1. 2. 3. 4. 5.
In Vitro Release of Drug from NPs in Medium
Fetal bovine serum (FBS) (Gibco™) 50 mL Corning tubes A bench-top shaker A Beckman ultracentrifuge An ICP-MS Varian 820-MS (Palo Alto, CA).
2.8 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
In Vitro Release of Drug from NPs in Cells Fetal bovine serum (FBS) (Gibco™) Dulbecco’s Modified Eagle Medium (DMEM) (Gibco™) Penicillin G sodium (Gibco™) Streptomycin (Gibco™) Opti-MEM reduced serum medium (Gibco™) A bench-top shaker A375M cells (the American Type Culture Collection (ATCC), Manassas, VA) 24-well plates (Corning Inc., Corning, NY) A Beckman ultracentrifuge An ICP-MS Varian 820-MS (Palo Alto, CA).
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Methods
3.1
Synthesis of Cis-[Pt(NH3)2(H2O)2](NO3)2 Precursor
1. Add CDDP (60 mg, 0.20 mmol) to 1.0 mL water to make a suspension. 2. Add AgNO3 (66.2 mg, 0.39 mmol) to the CDDP suspension. 3. Heat the mixture at 60 C for 3 h and then stir overnight in a flask protected from light with aluminum foil. 4. Afterward, centrifuge the mixture was at 16,000 rpm for 15 min to remove the AgCl precipitate. 5. Then filter the solution using a 0.2 μm syringe filter. 6. Measure the concentration of cis-[Pt(NH3)2(H2O)2](NO3)2 using ICP-MS Varian 820-MS and adjust to 200 mM.
3.2
Preparation of LPC NPs
1. Mix cyclohexane/Igepal CO-520 (71:29, V:V) and cyclohexane/triton-X100/ hexanol (75:15:10, V:V:V) (3:1) to make the oil phase. 2. Disperse 100 μL of 200 mM cis-[Pt(NH3)2(H2O)2](NO3)2 in 8.0 mL oil phase to form a well dispersed, water-in-oil reverse microemulsion. 3. Then add 100 μL of DOPA (20 mM) in chloroform to the CDDP precursor phase and stirred the mixture. 4. Prepare another emulsion containing KCl by adding 100 μL of 800 mM KCl in water into a separate 8.0 mL oil phase. 5. Twenty minutes later, mix the two emulsions and let the reaction to proceed for another 30 min. 6. After that, add 16.0 mL of ethanol to the microemulsion to break the emulsion. 7. Centrifuge the mixture at 12,000 g for at least 15 min to precipitate the CDDP cores. 8. Wash the precipitated pellets with ethanol 2–3 times to remove cyclohexane and surfactants. 9. After being extensively washed, redisperse the pellets in 3.0 mL of chloroform and store in a glass vial for further modification. 10. To create the outer leaflet lipid coating, mix 1.0 mL of CDDP cores and 100 μL of 20 mM. 11. DOTAP/Cholesterol (molar ratio 1:1) and 50 μL of 10 mM DSPE-PEG2000 in a glass vial. 11. Dry the mixture with N2 gas to remove the chloroform solvent. 12. Vacuum desiccate the dried film for at least 10 min. 13. Add a small volume of ethanol and 1.0 mL water to the vial, then hydrate the film and suspended the particles under vortexing. 14. Sonicate the sample in a bath type sonicator (Fig. 1).
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Fig. 1 Illustration for the preparation of LPC NPs. (1) Prepare two emulsions containing precursor and KCl separately; (2) add DOPA to the CDDP precursor and stir the mixture; (3) mix the two emulsions and let the reaction to proceed for another 30 min; (4) add ethanol to the micro-emulsion to break the emulsion; (5) centrifuge the mixture to precipitate the CDDP cores; (6) wash the precipitated pellets with ethanol 2–3 times to remove cyclohexane and surfactants; (7) mix CDDP cores, DOTAP, cholesterol, and DSPE-PEG2000 in a glass vial and remove the chloroform solvent; (8) hydrate the film and suspend the particles under vortexing
3.3
Characterization of Particle Size and Zeta Potential
1. Dilute a dispersion of LPC NPs into an appropriate concentration with water. 2. Use the dynamic light scattering (DLS) method to determine the particle size and zeta potential of LPC NPs using a Malvern ZetaSizer Nano series.
3.4
Transmission Electron Microscopy (TEM)
1. Place a drop of 5 μL dispersion of LPC NPs onto the grid and allowed to deposit for 5 min. 2. Blot the excess liquid by touching with a piece of filter paper. 3. Stain the sample with 2% uranyl acetate (5 μL), followed by overnight air drying at room temperature. 4. Acquire TEM images using a JEOL 100CX II TEM.
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Characterization of Drug-Loading Capacity
1. Digest the samples in 70% HNO3 2. Dilute the samples with water to a final acid content of 2%. 3. Determine the platinum concentration using the 195Pt isotope and measure by ICP-MS using Varian 820-MS. 4. Calculate the drug loading yield as the weight ratio of the cisplatin payload to the total NPs including both lipid excipients and cisplatin.
3.6
In Vitro Release of Drug from NPs in Medium
1. Add LPC NPs containing 200 μg Pt into 50% FBS 2. Incubate the mixture at 37 C on a shaker at 300 rpm. 3. At different time points, withdraw 1 ml of the corresponding samples and replenish the medium 4. Centrifuge the samples at 16,000 g for 20 min and obtain the supernatant liquid 5. Measure the Pt-based drug released into the supernatant liquid by using ICP-MS
3.7
In Vitro Release of Drug from NPs in Cells
1. Seed A375M cells in 24-well plates at a density of 3 104 cells per well and incubate for 20 h in 10% FBS of DMEM containing 100 U/mL penicillin, and 100 mg/mL streptomycin. 2. Remove the medium and replace by Opti-MEM containing 100 μM of LPC NPs. All transfections were performed in triplicate. 3. After incubation for 4 h at 37 C in a 5% CO2, humidified atmosphere, aspirate the medium, and wash the cells. 4. To study of the release of Pt drugs from cells, add fresh medium at different time points and harvest the medium containing the released drugs. 5. Separate the intact NPs and free drug released into the medium by centrifugation at 16,000 g for 20 min. 6. Measure the quantity of Pt in the supernatant and pellets by using ICP-MS.
4
Notes
1. Unless stated otherwise, water and all solutions should be used in Milli Q water. 2. Unless stated otherwise, all lipids were dissolved in chloroform to prepare the stock solutions at specific concentrations. 3. The classic synthesis route of CDDP is shown in Scheme 1. 4. To maximize the yield of CDDP NPs, an excess of KCl was used to inhibit hydrolysis equilibrium.
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Scheme 1 Classic synthesis route of CDDP (Guo et al. 2014)
5. After CDDP was precipitated, CDDP cores were then coated with a hydrophobic layer of DOPA. The nano-size precipitates in the microemulsions were stabilized by DOPA, and DOPA-coated CDDP precipitates collected by centrifugation can be readily dispersed in organic solvents such as chloroform, toluene or hexane. 6. The composition of DOPA-coated CDDP cores was determined using X-ray photoelectron spectroscopy (XPS) and NMR. The ratio of N:Pt:Cl was 2:1:1.8. A small amount of phosphorous element was also present, which is expected due to the presence of DOPA. No potassium element was detected. Additional confirmation of the composition of DOPA-coated CDDP cores was obtained using 1H NMR spectra in DMF-d7. The major peaks of DOPA-coated NPs were consistent with those of CDDP. Therefore, we can conclude that DOPA-coated CDDP cores have a core-shell structure with CDDP as the core and DOPA as the coating lipid layer (Guo et al. 2014). 7. DOPA is known to strongly interact with the platinum cation at the interface (Burger et al. 2002). Such strong interaction provides increased stability for the supported bilayer, allowing a high amount of incorporated DSPE-PEG2000. 8. To disperse the hydrophobic DOPA-coated CDDP cores in an aqueous solution, additional lipids composed of DOTAP, cholesterol, and DSPE-PEG2000 were used. When the lipid film was hydrated, these lipids self-assembled in water into the outer leaflet of the bilayer through a hydrophobic interaction with DOPAcoated CDDP cores as a template (Chen et al. 2012; Li et al. 2010). 9. DOPA-coated CDDP cores dispersed in organic solvents allow easy surface modification for a variety of purposes, in a manner similar to quantum dots and iron NPs. 10. The final LPC NPs could be further purified via centrifugation to remove free liposomes formed as the result of excess outer leaflet lipids. 11. As shown in Fig. 2, DLS results indicated that the hydrodynamic diameter of LPC NPs was approximately 30 nm. The size of the particles determined using TEM was approximately 20 nm, which was slightly smaller than the results obtained from DLS. DLS results showed that size distribution of LPC NPs was narrow, with a PDI of 0.15 0.02.
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a
b 25 By number
20 15 10 5 0 1
10
100
1000
Size (nm) Fig. 2 LPC NPs were characterized by a small size and narrow dispersity. (a) Characterization of LPC NPs using TEM. LPC NPs were negatively stained with uranyl acetate. Scale bar represents 50 nm. (b) Characterization of LPC NPs using dynamic light scattering (DLS). (Data is taken from (Guo et al. 2014) with permission)
12. The size of DOPA-coated CDDP cores could be altered between 12 and 75 nm in diameter by adjusting the composition of the surfactant systems. The oil phase used to create the microemulsions was a mixture of Igepal-520 system (Igepal520: cyclohexane = 30:70 (v/v)) and Triton X-100 system (Triton X-100: Hexanol: cyclohexane = 15:10:75 (v/v/v). When using an oil phase with a volume ratio of Igepal-520 system to Triton X-100 system at 6:2, 2:6, and 0:8, the size of the resulting DOPA-coated CDDP cores was 15, 50, and 75 nm, respectively (Guo et al. 2014). 13. Composition of outer leaflet lipids was carefully chosen to contain steric lipid (DSPEmPEG) for prolonged circulation of NPs in the bloodstream. Zeta potential of the LPC NPs was 15 0.3 mV, which was an indication of PEG modification on the NPs, since the zeta potential of non-PEGylated DOTAP liposomes were around 70 mV (Guo et al. 2014; Liu et al. 2014). 14. ICP-MS was used to measure the content of Pt. The drug loading capacity of LPC NPs was approximately 82 5 wt% (Guo et al. 2014). 15. The optimal size of LPC NPs facilitated the EPR effect and resulted in the accumulation of LPC NPs in tumor cells. In vivo study compared the biodistribution of LPC NPs with free CDDP in tumor-bearing mice. Twentyfour hours post-iv injection, 10.5% of the injected dose (ID) per gram of LPC NPs accumulated in the xenograft tumors, which was significantly higher than the 1.2% of the injected dose per gram of free CDDP. LPC NPs exhibited enhanced anticancer therapeutic effect at a low dose and less dosing frequency than free CDDP (Guo et al. 2013). 16. The kinetics of the Pt-based drugs release from LPC NPs was determined in 50% FBS medium. As shown in Fig. 3a, LPC NPs exhibited a sustained release of Pt over time with a half-life of 3.0 h. 17. Preliminary in vivo study suggested that the massive apoptosis occurred in the tumor cells might be induced by the small fraction of cells that took up the LPC NPs. This is a phenomenon known as the neighboring effect, which is
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b 90
100
Percentage (%)
Cumulative release
a 120
80 60 40 20 0 0
4
8
12
Time (h)
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80 70 60 50 40 30 20 10 0
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60.59 45.51
24.32
2
4
16
Time (h)
Fig. 3 The drug release profile of LPC NPs in medium and in cells. (a) In vitro release kinetics of encapsulated Pt in 50% FBS medium at 37 C and the cellular release of Pt from LPC NPs treated cells. (b) Percentages of Pt in the released medium that were pelletable (green) and unpelletable (red) are shown. Pelletable represents intact NPs and unpelletable represents free drugs. (Data are taken from (Guo et al. 2013) with permission)
characterized as the uptake of NPs by a small population of tumor cells that become in situ drug depots and release active drugs to induce apoptosis in the surrounding cells. The cellular release of the drug in this process is diffusion dependent. To test the hypothesis of neighboring effect in vitro, cellular release of Pt-based drugs from LPC NP was investigated. Cells were first incubated with LPC NPs for 2, 4, or 16 h and then washed and incubated with fresh medium. At different time points, the medium was collected, and the released NPs and free drugs in the medium were separated by centrifugation. The results suggested that LPC NPs were taken up by the cells and subsequently released Pt-based drugs in a sustained manner. Free drug composed the major fraction of Pt-based drugs present in the medium (Fig. 3b). Furthermore, the activity of the released drugs was tested. The medium harvested from the cellular release study at different time points was transferred and incubated with fresh, untreated cells. After 48 h, the viability of the tumor cells was assayed. The results showed that the medium containing more released drugs was more toxic, indicating that the free CDDP released from the cells retained its bioactivity (data not shown here) (Guo et al. 2013). The detailed mechanism of the drug release from the depot cells to neighboring cells still needs further investigation.
5
Conclusion
The poor water solubility of drugs often presents a major obstacle in the formulation development of traditional approaches. In this study, an NP formulation of CDDP was developed to make CDDP’s poor solubility to our advantage, promoting the synthesis of a pure CDDP core with a high drug loading capacity. Engineered via the microemulsion method, the LPC NPs was coated with DOPA and an additional outer
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leaflet to enhance the stability and in vivo performance. Successfully fabricated LPC NPs were characterized with optimal drug loading capacity and aqueous dispensability. By adjusting the preparation parameters, the size of LPC NPs can also be altered between 12 and 75 nm for optimal tumor accumulation. The CDDP is the major component of LPC NPs (82 5 wt%). To our best knowledge, this is the highest loading capacity for cisplatin NPs that has been reported in the literature. This is a good example of using an unmodified drug as a building material for the assembly of NPs. Synthesis of LPC NPs represents a platform technology that could be transferred to formulate other water-insoluble drugs. For example, we have also shown that etoposide can be precipitated within and forms insoluble cores. After lipid coating, the NPs showed growth inhibition in a human lung cancer model (Srinivas et al. 2015).
References Andrews PA, Wung WE, Howell SBA (1984) High-performance liquid chromatographic assay with improved selectivity for cisplatin and active platinum (II) complexes in plasma ultrafiltrate. Anal Biochem 143(1):46–56 Aryal S, C-MJ H, Zhang L (2010) Polymer cisplatin conjugate nanoparticles for acid-responsive drug delivery. ACS Nano 4(1):251–258 Avgoustakis K, Beletsi A, Panagi Z, Klepetsanis P, Karydas AG, Ithakissios DS (2002) PLGA–mPEG nanoparticles of cisplatin: in vitro nanoparticle degradation, in vitro drug release and in vivo drug residence in blood properties. J Control Release 79(1–3):123–135 Baba M, Matsumoto Y, Kashio A, Cabral H, Nishiyama N, Kataoka K, Yamasoba T (2012) Micellization of cisplatin (NC-6004) reduces its ototoxicity in guinea pigs. J Control Release 157(1):112–117 Bala I, Hariharan S, Kumar MNVR (2004) PLGA Nanoparticles in drug delivery: the state of the art. Crit Rev Ther Drug Carrier Syst 21(5):387–422 Boulikas T (2009) Clinical overview on Lipoplatin™: a successful liposomal formulation of cisplatin. Expert Opin Investig Drugs 18(8):1197–1218 Burger KNJ, Staffhorst RWHM, de Vijlder HC, Velinova MJ, Bomans PH, Frederik PM, de Kruijff B (2002) Nanocapsules: lipid-coated aggregates of cisplatin with high cytotoxicity. Nat Med 8(1):81–84 Chen H, Khemtong C, Yang X, Chang X, Gao J (2011) Nanonization strategies for poorly watersoluble drugs. Drug Discov Today 16(7–8):354–360 Chen T, Öçsoy I, Yuan Q, Wang R, You M, Zhao Z, Song E, Zhang X, Tan W (2012) One-step facile surface engineering of hydrophobic nanocrystals with designer molecular recognition. J Am Chem Soc 134(32):13164–13167 Dhar S, Kolishetti N, Lippard SJ, Farokhzad OC (2011) Targeted delivery of a cisplatin prodrug for safer and more effective prostate cancer therapy in vivo. Proc Natl Acad Sci 108(5):1850–1855 Guo S, Wang Y, Miao L, Xu Z, Lin CM, Zhang Y, Huang L (2013) Lipid-coated cisplatin nanoparticles induce neighboring effect and exhibit enhanced anticancer efficacy. ACS Nano 7(11):9896–9904 Guo S, Miao L, Wang Y, Huang L (2014) Unmodified drug used as a material to construct nanoparticles: delivery of cisplatin for enhanced anti-cancer therapy. J Control Release 174:137–142 Kalepu S, Nekkanti V (2015) Insoluble drug delivery strategies: review of recent advances and business prospects. Acta Pharm Sin B 5(5):442–453
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Kolishetti N, Dhar S, Valencia PM, Lin LQ, Karnik R, Lippard SJ, Langer R, Farokhzad OC (2010) Engineering of self-assembled nanoparticle platform for precisely controlled combination drug therapy. Proc Natl Acad Sci 107(42):17939–17944 Li J, Chen Y-C, Tseng Y-C, Mozumdar S, Huang L (2010) Biodegradable calcium phosphate nanoparticle with lipid coating for systemic siRNA delivery. J Control Release 142(3):416–421 Liu Y, Hu Y, Huang L (2014) Influence of polyethylene glycol density and surface lipid on pharmacokinetics and biodistribution of lipid-calcium-phosphate nanoparticles. Biomaterials 35(9):3027–3034 Pabla N, Dong Z (2008) Cisplatin nephrotoxicity: mechanisms and renoprotective strategies. Kidney Int 73(9):994–1007 Plummer R, Wilson RH, Calvert H, Boddy AV, Griffin M, Sludden J, Tilby MJ, Eatock M, Pearson DG, Ottley CJ et al. (2011) A phase I clinical study of cisplatin-incorporated polymeric micelles (NC-6004) in patients with solid tumours. Br J Cancer 104(4):593–598 Sengupta P, Basu S, Soni S, Pandey A, Roy B, Oh MS, Chin KT, Paraskar AS, Sarangi S, Connor Y et al. 2012 Cholesterol-tethered platinum II-based supramolecular nanoparticle increases antitumor efficacy and reduces nephrotoxicity. Proc Natl Acad Sci 109(28):11294–11299 Sharma A, Sharma US (1997) Liposomes in drug delivery: progress and limitations. Int J Pharm 154(2):123–140 Smith A, Hunneyball IM (1986) Evaluation of poly(lactic acid) as a biodegradable drug delivery system for parenteral administration. Int J Pharm 30(2):215–220 Srinivas R, Satterlee A, Wang Y, Zhang Y, Wang Y, Huang L (2015) Theranostic etoposide phosphate/indium nanoparticles for cancer therapy and imaging. Nanoscale 7(44):18542–18551 Wang D, Lippard SJ (2005) Cellular processing of platinum anticancer drugs. Nat Rev Drug Discov 4(4):307–320 Zamboni WC, Gervais AC, Egorin MJ, Schellens JHM, Zuhowski EG, Pluim D, Joseph E, Hamburger DR, Working PK, Colbern G et al. (2003) Systemic and tumor disposition of platinum after administration of cisplatin or STEALTH liposomal-cisplatin formulations (SPI-077 and SPI-077 B103) in a preclinical tumor model of melanoma. Cancer Chemother Pharmacol 53(4):329–336 Zhang W, Wang G, Falconer JR, Baguley BC, Shaw JP, Liu J, Xu H, See E, Sun J, Aa J et al.(2015) Strategies to maximize liposomal drug loading for a poorly water-soluble anticancer drug. Pharm Res 32(4):1451–1461
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Purification Method of Drug-Loaded Liposome Meng Lin and Xian-Rong Qi
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Dialysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Column Chromatographic Separation Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Centrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Microcentrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Ion-Exchange Resin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Ultrafiltration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Protamine Aggregation Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Liposomes are lipid-based bilayer vesicles, which can encapsulate drugs to improve their pharmacokinetic performance and thus are widely used as drug delivery system. Drugs loaded in liposomes have distinct pharmacokinetic characteristics from non-encapsulated drugs. Thus, the purification of liposomes,
M. Lin Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System, School of Pharmaceutical Sciences, Peking University, Beijing, China e-mail: [email protected] X.-R. Qi (*) Department of Pharmaceutics, School of Pharmaceutical Science, Peking University, Beijing, China Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System, Beijing, China e-mail: [email protected] © Springer-Verlag GmbH Germany, part of Springer Nature 2021 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49320-5_24
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i.e., separating or removing nonencapsulated drugs as well as detergent used in the preparation process, is important for quality control of liposomal products. From a pharmaceutical point of view, well-defined liposome preparations should use wellcharacterized lipids and would avoid the use of organic solvents and detergents (which are difficult to remove). In this chapter, we reviewed some methods for liposome purification, including dialysis, column chromatographic separation method, centrifugation, protamine aggregation method, ion-exchange resin, and ultrafiltration method. For each method, its basic principle and characteristics are introduced, and some examples are also given. To achieve effective purification, suitable methods should be chosen based on the characteristics of every individual liposome and optimization of separation condition is also demanded. And more efforts needed to be put into the liposome purification field to improve these methods as well as develop other new techniques with a wide range of application. Keywords
Liposomes · Purification · Dialysis · Column chromatography · Centrifugation · Protamine aggregation method · Ion-exchange resin · Ultrafiltration
1
Introduction
Liposomes are widely used drug delivery systems in pharmaceutical fields, which are of lipid bilayer structures capable of entrapping both hydrophobic drugs in the bilayer and hydrophilic drugs in the interior core simultaneously. By incorporation of plentiful lipids in membranes, liposomes with various properties can be obtained. For instance, by modification of ligands on the membrane surface, liposomes can achieve active targeting through recognition between ligands and receptors. Through these means, pharmacokinetic performance of the encapsulated drugs can be significantly improved: enlargement of the drug therapeutic index, enhancement of drug’s pharmaceutical properties, and stimulus-triggered drug release. Currently, the methods used for liposome preparation include the use of solvent injection, reverse-phase evaporation, and emulsification methods (Dario et al. 2016). To remove excessive components in the liposome preparation process, it is necessary to take some measures to obtain homogenous liposome products. Besides, elimination of non-entrapped drugs is also important to ensure the liposome product quality; therefore, further purification is demanded during liposome manufacturing. Generally speaking, for hydrophilic drugs, only a small number of drugs are incorporated into the aqueous interior core, while most are left in the liposomal suspensions. Thus, it is essential to remove these free drugs by some specific means. As the molecular weight of most of the drugs is much lower than that of liposomes, based on their size difference, dialysis, centrifugal, and column chromatographic separation methods can be utilized for liposome purification. Although these methods are useful for liposome purification, they may be tedious or time-consuming and may even diminish product yield or dilute liposomes, which limit their use in large-scale purification. Other methods for elimination of nonencapsulated drugs include ion-exchange chromatography, microcentrifugation, and ultrafiltration.
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Till now, purification remains a challenge in the development of liposomal products (Dimov et al. 2017). In this chapter, we reviewed some of the methods used for liposome purification, focusing on their principles, advantages, and disadvantages as well as their application field.
2
Dialysis
Dialysis is a common technique adopted to remove small molecules from liposomal suspensions with the use of a semi-permeable membrane, whose pore allows small molecules to pass through freely while the diffusion of large molecules through the sample chamber is restricted (Fig. 1). Thus, it is suitable for separation of compounds with low molecular weight that can pass through the pore of membrane. The key to dialysis is the choice of suitable dialysis bag as well as dialysis media. Generally speaking, an ideal dialysis bag is supposed to have an appropriate pore size, which allows free diffusion of drug molecules while preventing liposomes from passing through. As for the choice of dialysis media, it is required that the drugs be highly soluble in the chosen media and the structure of liposomes is well preserved as well. Thus, apart from hydrophilic drugs, hydrophobic drugs can also be isolated via dialysis if an appropriate media is used. The main advantages of this method include: (1) it is simple to operate and no expensive equipment is required; (2) large quantities of samples can be processed and nearly all of the free drugs can be removed using this method; and (3) the obtained result is accurate and well reproducible (Jin et al. 2015). However, to achieve a better purification efficacy, a relatively long time is demanded to remove the nontrapped drugs as completely as possible and the dialysis media needs to be replaced every once in a while to maintain the concentration gradient for drug diffusion. In general, at room temperature, it takes more than 24 h to ensure that 95% of the free drugs can be removed using this method. It is also noted that the osmotic pressure of the media should be consistent with that of the liposome-contained suspensions, otherwise the structure of liposomes may be disrupted, leading to the leakage of drugs from liposomes. Moreover, in this separation process, the adhesion of liposomes to some containers (e.g., dialysis bag and
Fig. 1 Schematic representation of dialysis
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test tube) may cause product loss especially for liposomes with a high concentration. Yang et al. used dialysis method to purify liposomes prior to HPLC analysis (Yang et al. 2018). The drug-loaded formulations were filtered using an Amicon dialysis tube and the filtrate was used to measure the unloaded drugs. Apart from eliminating non-encapsulated drugs from liposomes, dialysis can also be used to eliminate remaining components of the liposomes. For example, to enhance the intracellular delivery, Yoon et al. modified the RIPL peptide on the surface of liposomes. First, RIPL peptide was added to the liposomal solution and incubated for 12 h at room temperature. Then the unreacted RIPL was separated from modified liposomes by dialysis against distilled water for 24 h at 4 C with the use of a cellulose ester membrane (Yoon et al. 2017). And here is an example showing the procedure of dialysis in detail (Jin et al. 2015): first, 2 mL of liposomal suspensions is put into a dialysis bag and then it is fixed with a dialysis tubing closure and placed in 200 mL of dialysis media at room temperature under magnetic stirring for 10 h. During the dialysis process, every 1 h, 1 mL of dialysate is taken out and analyzed for drug concentration measurement, and another 1 mL of fresh media is added. This process is continued till the drug concentration shows no significant change, which indicates the dialysis has reached a state of equilibrium.
3
Column Chromatographic Separation Method
Column chromatographic separation method (Fig. 2) is useful for isolation of free drugs from drug-loaded liposomes in the drug delivery field. The most commonly used chromatographic columns are Sephadex column and Sepharose
Fig. 2 Schematic representation of column chromatographic separation method
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column, and thus, it is also called gel filtration method. The basic separation principle of this method is that the difference in molecular weight between liposomes and nontrapped drugs will lead to different retention time. Usually, Separation occurs in a column which are packed with porous beads, such as agarose gel and Sephadex. In the elution process, particles with a relatively low molecular weight (free drugs) are capable of entering the pores easily and therefore spend more time in these pores, whereas larger particles (liposomes) do not enter the pores and can be eluted more quickly. Taking advantage of the difference in retention time, non-encapsulated drugs can be separated from the drug-encapsulated liposomes in this way. The choice of gel particle with a suitable size is the key to efficient separation. If the diameter of gel particle is too small or the column contains many fine particles, sometimes, large liposomes (>0.4 μm) may be retained in the column. Usually, Sephadex G-50 can be used when isolating small molecules, while for macromolecules, Sepharose 4B is more commonly used. Besides, it is worth noting that on the surface of dextran exist many reactive groups, which are prone to interacting with liposomal membrane. Although this kind of interaction does not interfere with the elution process, it may cause losses of a small quantity of liposomes. Especially when the liposome concentration is low, its impact will be significant. Thus, it is recommended to pretreat the column with blank liposomes which are of the same components as the samples to presaturate these reactive sites so that the losses of liposomes caused by adsorption can be minimized. Here is the detailed procedure of column chromatographic separation method: first, 0.5 mL of liposomal suspension is added to the Sephadex G-50 column, and then phosphate buffer solution (PBS, pH 7.4) is used as eluent for liposome purification at a flow rate of 0.5 mL/min. Every 2mL of eluate is collected in one tube. The liposomes are collected by the monitoring the turbidity of the eluate or by determining drug concentrations and lipid concentrations in the eluate. For example, to determine the content of rivastigmine in liposomes, Yang et al. used column chromatographic method for liposome purification prior to encapsulation efficiency analysis (Yang et al. 2013). First, the liposomes were separated from the free rivastigmine by gel filtration through a SephadexG-50 column and eluted with PBS (pH 6.5). Then the separated liposome suspensions were destroyed by methanol and HPLC method was used to determine the rivastigmine concentration. The main advantages of this method are that the separation condition is quite stable and the chromatographic column can be reused, which is cost effective. But with the use of large quantities of eluents in the purification process, liposome dilution and drug leakage from liposomes is a major concern, which may have a significant impact on the product quality. Another drawback of this method is that it is not applicable for lipophilic drugs, which are unlikely to be eluted with hydrophilic eluents (Jin et al. 2015). Using this method, Assanhou et al. purified the liposomes prior to HPLC analysis and the amount of drug before and after filtration through the Sephadex G-50 column is regarded as the total amount of drug and the amount of the encapsulated drug respectively. And the result indicated that the encapsulation efficiency of paclitaxel was more than 90% in all the liposome formulations (Assanhou et al. 2015).
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Centrifugation
Centrifugation is a technique which involves the application of centrifugal force to separate particles from a solution according to their size, shape, density, viscosity of the medium, and rotor speed. In drug delivery system field, taking advantage of the density difference between liposomes and free drugs, it can be utilized for isolation of free drugs. Under the centrifugal force, liposomes are left in the bottom of centrifuge tube whereas the non-trapped dugs remain in the supernatant. However, sometimes the density difference between the liposomes and dispersing media is not significant; therefore, the rotating speed should be up to 10,000 r/min to achieve effective separation, which may cause particle aggregation and drug leakage caused by membrane fusion. In some cases where solutions of high molecular weight compound are used at concentration iso-osmolar with physiological saline or the drug concentrations are very high, the density of the suspending medium may be higher than that of the liposome; therefore, it will be difficult to precipitate the liposomes. To overcome this problem, a medium of a much lower density can be used to dilute the liposomes, and thus, the liposomes can be precipitated in such a low-density medium. Another limitation of centrifugation method is the requirement for expensive equipment. The typical types of centrifugations for liposome purification include differential centrifugation, density gradient centrifugation, and centrifugation through molecular sieves (Torchilin and Weissing 2002). Differential high speed centrifugation is quite useful for separating large liposomes from liposome mixtures. Usually, large liposomes can be precipitated under the condition of 100,000–160,000 g for 15–30 min while small liposomes remain in the supernatant. As the separation efficacy is highly dependent on the centrifugation condition, such as the component of the liposomes, the temperature, and the buffer solution, it should be optimized for each individual case. Apart from producing homogeneous liposomes, differential high speed centrifugation can also be utilized to remove non-encapsulated drugs. As for glycerol density gradient centrifugation, it can be used to yield homogenous liposomes in large quantities and high concentrations. Ultracentrifugation method is a common procedure in microbiology and cytology for separation of organelles from cells. And it can be adopted to separate molecules in batches or continuous flow systems. It works by taking advantage of the different sedimentation rates caused by the difference of samples’ density and centrifugal force they are subjected to. Similar to organelle separation, it can also be used to purify liposomes. But one limitation of this method is that high-speed centrifuge is often accompanied by an increase in temperature. Also, it is noted that the high speed may cause damage to the liposomal membrane and lead to drug leakage; therefore, an optimal rotational speed is highly demanded and attention should be paid to protecting the liposomes in this process. Khatri et al. separated free siRNA using ultracentrifugation and then the content of unloaded siRNA was measured by UV spectrophotometry at 260 nm for entrapment efficiency determination. And the result showed that more than 80% siRNA entrapment was achieved (Khatri et al. 2014).
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Microcentrifugation
Microcentrifugation method is fast, simple, and quite suitable for separation of low molecular weight compounds (98%) as well as effective removal of non-encapsulated drugs or proteins and organic solvents (ethanol >95% reduction) within 4 min, which met high throughput requirements of early stage development processes (Dimov et al. 2017).
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Conclusion
The purification of liposome is important in their manufacturing process as well as quality control, which requires effective means to remove the free drugs that are not entrapped into the liposomes. Different kinds of purification methods have been adopted to eliminate the unwanted materials in the liposomal suspensions to yield purified liposomes, including dialysis, column chromatographic separation, centrifugation, protamine aggregation method, cation-exchange resin, and ultrafiltration. All of these methods have their own advantages and disadvantages, and according to the characteristics of liposomes to be purified, suitable methods should be chosen for effective purification. For example, for negatively charged liposomes, cation-exchange resin method can be utilized to separate the free drugs which are positively charged. As for low molecular weight drugs which are hydrophilic, methods such as dialysis, centrifugation, and ultrafiltration can be used to remove the free drugs. And the optimization of separation conditions is also important to achieve effective purification; hence, this should be taken into consideration in the procedure. In addition to the efficacy of separation, other factors such as cost, time, quantities of the samples should be taken into account as well. Moreover, although various methods have been developed for liposome purification, more efforts need to be put into this field to improve these procedures. Take centrifugation, for example, mechanical stress during the process may lead to drug leakage from liposomes, which is harmful for the liposome products and causes inaccurate measurement of encapsulation efficiency. Thus, how to separate the liposomes from other unwanted materials effectively without disrupting their structures is a demanding issue to be addressed. Moreover, more new methods are demanded to promote the liposome purification efficacy for quality control.
References Assanhou AG, Li W, Zhang L, Xue LJ, Kong LY, Sun HB, Mo R, Zhang C (2015) Reversal of multidrug resistance by co-delivery of paclitaxel and lonidamine using a TPGS and hyaluronic acid dual-functionalized liposome for cancer treatment. Biomaterials 73:284–295 Calle D, Negri V, Ballesteros P, Cerdan S (2015) Magnetoliposomes loaded with poly-unsaturated fatty acids as novel theranostic anti-inflammatory formulations. Theranostics 5(5):489–503
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Chen JT, Pan H, Yang YN, Xiong SH, Duan HL, Yang XG, Pan WS (2018) Self-assembled liposome from multi-layered fibrous mucoadhesive membrane for buccal delivery of drugs having high first-pass metabolism. Int J Pharm 547(1–2):303–314 Dario C, Elisabetta B, Joshua O, Eleanor S, Claudio N (2016) Liposome production by microfluidics: potential and limiting factors. Sci Rep 6:25876 Dimov N, Kastner E, Hussain M, Perrie Y, Szita N (2017) Formation and purification of tailored liposomes for drug delivery using a module-based micro continuous-flow system. Sci Rep 7 (1):12045–12057 Dipali SR, Kulkarni SB, Betageri GV (1996) Comparative study of separation of non-encapsulated drug from unilamellar liposomes by various methods. J Pharm Pharmacol 48(11):1112 Jin YG, Du LN, Chen Y, Ling PX (2015) Application of nanotechnology in drug delivery, 1st edn, Chemical Industry Press, Beijing, pp 11–12, pp 175–177 Khatri N, Baradia D, Vhora I, Rathi M, Misra A (2014) cRGD grafted liposomes containing inorganic nano-precipitate complexed siRNA for intracellular delivery in cancer cells. J Control Release 182(10):45–57 Li HR, Li SF (2007) The measurement of the entrapment efficiency of drugs in liposome. Chin J Pharm Anal 27(11):1844–1848 Torchilin VP, Weissing V (2002) Liposomes, 2nd edn, Oxford University Press, New York, pp 45–46, pp 149–164 Yang ZZ, Zhang YQ, Wang ZZ, Wu K, Lou JN, Qi XR (2013) Enhanced brain distribution and pharmacodynamics of rivastigmine by liposomes following intranasal administration. Int J Pharm 452(1–2):344–354 Yang WT, Hu Q, Xu YM, Liu HL, Zhong L (2018) Antibody fragment-conjugated gemcitabine and paclitaxel-based liposome for effective therapeutic efficacy in pancreatic cancer. Mater Sci Eng C-Mater 89:328–335 Yoon HY, Kwak SS, Jang MH, Kang MH, Sung SW, Kim CH, Kim SR, Yeom DW, Kang MJ, Choi YW (2017) Docetaxel-loaded RIPL peptide (IPLVVPLRRRRRRRRC)-conjugated liposomes: drug release, cytotoxicity, and antitumor efficacy. Int J Pharm 523(1):229–237
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Quality Evaluation of Drug-Loaded Liposomes Meng Lin, Rudong Wang, and Xian-Rong Qi
Contents 1 2 3 4 5 6
7 8 9 10 11 12 13
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Particle Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Size Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Zeta Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Entrapment Efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Gel Column Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Dialysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Ultracentrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 Ultrafiltration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5 Protamine Aggregation Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Vitro Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Osmotic Pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sterilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidation Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lysophospholipid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Drug Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Leakage Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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M. Lin · R. Wang (*) Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System, School of Pharmaceutical Sciences, Peking University, Beijing, China X.-R. Qi (*) Department of Pharmaceutics, School of Pharmaceutical Science, Peking University, Beijing, China Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System, Beijing, China e-mail: [email protected] © Springer-Verlag GmbH Germany, part of Springer Nature 2021 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49320-5_25
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14 Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Pharmacokinetics and Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Assessment of Efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Safety and Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
The quality of liposomes is highly dependent on their starting materials. The characteristics of liposomal formulations, including particle size, entrapment efficiency, polydispersity index, morphology, zeta potential, stability, release of drugs from liposomes, etc., is crucial for their therapeutic efficacy. Techniques and methods to measure the characteristics of liposomal formulations have their own pros and cons, and thus on basis of the properties of liposomal formulation, a suitable method for measurement should be adopted. In addition to their in vitro characterization, in vivo studies are essential to evaluate their biological behaviors, such as pharmacology, pharmacokinetics, therapeutic efficacy, safety, and toxicity study, which provides detailed information about the quality of liposomes.
Keywords
Liposomes · Characterization · Quality control · Techniques
1
Introduction
Liposomes have demonstrated great potential in the field of drug delivery to improve drug dissolution, stability, and bioavailability (Abolfazl et al. 2013; Bahari and Hamishehkar 2016). Due to their unique structure, they are capable of entrapping both hydrophobic drugs in the bilayer and hydrophilic drugs in the core simultaneously, or absorbing agents on the liposomal membranes. For drugs with a relatively low solubility (especially chemotherapeutic drugs), their pharmacokinetic profiles will be improved after entrapment, including lower toxicity and less side effect. Besides, PEG or ligands can be modified on the surface of liposomes to achieve long circulation time or active targeting (Fig. 1). In spite of the rapid development of liposomes, their stability remains the key challenge within the field (Kapoor et al. 2017). As the physical, chemical, and thermodynamic properties have a significant effect on liposomes’ safety and effectiveness, characterization and quality control should be taken into consideration. Of course, a prerequisite for the preparation of well-defined liposomes is the use of well-characterized lipids. In this chapter, we will introduce some key parameters involved in quality control of liposomes and some related techniques for evaluation.
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Fig. 1 Representation of a typical lipid bilayer indicating entrapment of hydrophilic drug in inner core and hydrophobic drug in bilayer region. Liposome surface shown to be decorated with targeting ligands
2
Particle Size
Liposomes can be formulated using various techniques (sonication, extrusion, homogenization, and/or freeze-thawing) to differ in terms of composition, size, charge, and lamellarity. All preparation processes affect the particle size of the liposome. Size plays a critical role in liposomal properties, including the stability, encapsulation efficiency, drug release, cellular uptake, and bio-distribution (Bahari and Hamishehkar 2016). For example, liposomes with a diameter less than 200 nm are capable of entering the tumor via the enhanced permeability and retention (EPR) effect, while those within the size of 50 nm in diameter can significantly reduce clearance mediated by mononuclear phagocyte system (MPS) in mice models (Gabizon et al. 1993; Proffitt et al. 1983). To achieve better bio-distribution and effective therapeutic effects, optimization of particle size must be paid attention to. There have been varieties of techniques to determine the size of liposomes, including microscopy (e.g., optical microscopy, negative stain electron microscopy, cryo-transmission electron microscopy, scanning electron microscopy, confocal microscopy, and scanning probe microscopy), diffraction and scattering techniques (laser light scattering and photon correlation spectroscopy), and hydrodynamic techniques (field flow fractionation, gel permeation chromatography, ultracentrifugation, and centrifugal sedimentation) (Danaei et al. 2018). Ideal methods for nanoparticles size determination should bear the following characteristics: simple, rapid, and reproducible. Currently, the most commonly used methods are microscopic techniques and light scattering. The former can provide the detailed information of the particles, including size, morphology, lamellarity, and surface
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characteristics, while the latter measures numbers of particles simultaneously and gives the result of size distribution based on the statistical analysis. However, the main disadvantage of microscopic techniques lies in the tedious process of sample preparation and the limited numbers of particles analyzed in the field of vision. Take transmission electron microscopy (TEM), for example, a thin sample (1000 nm), and multilamellar vesicles (MLVs, >500 nm) (Kuntsche et al. 2011). As for shape, the typical type of liposomes is spherical with some being oval or tubular. Liposomes with different morphologies differ in in vivo fate and thus lead to variations of bioavailability. So determination and optimization of liposomes’ morphology is a factor to be considered. Moreover,
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the measurement of morphology is beneficial to distinguish various types of liposomes, such as conventional liposomes and ligand-targeted liposomes. Scanning electron microscopes (SEM) and transmission electron microscopy (TEM) are the most frequently used imaging method to evaluate the structure of nanoparticles. SEM can provide general information of the particles, including size, shape, and structure of the different lipid bilayers. But prior to imaging, the samples must be dried or fixed, which is likely to disturb the structure of the liposomes. Compared with SEM, TEM allows visualization of small vesicles with a much higher magnification. Based on the method utilized for sample preparation, it can be categorized to three types: namely, negative staining microscope, freeze–fracture electron microscope (FFEM), and cryo-electron microscope (CEM). As for negative staining microscope, a proper electron-dense material is needed to provide high contrast so that particles can be viewed against a dark-stained background. Uranyl acetate or phosphotungstic acid are commonly used heavy metal salts, with the former suitable for cationic liposomes staining and the latter for anionic liposomes. For FFEM, the sample does not undergo drying process and thus the naive state of preparation is well reserved. Usually, the sample is vitrified via rapid freezing with liquid propane or liquid nitrogen before being fractured. Then a thin platinum or carbon layer is used to form a “negative” replica of the fracture (Kuntsche et al. 2011; Severs 2007). Prior to imaging, organic solvent is used to clean the replica. CEM is also a useful tool to give comprehensive information of liposomes, such as shape, structure, and lamellarity, and it is by far the best technique to analyze particles in its naive form. But its main limitations are low resolution and only 2D images available. Apart from electron microscopy techniques, atomic force microscopy (AFM) and light microscopy can be utilized for morphology analysis. And depending on morphological characteristics and research objectives, one or more proper techniques should be chosen.
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Zeta Potential
It is well documented that the zeta potential of liposomes influences their stability, uptake, and clearance in vivo. Usually, poorly charged or uncharged particles (zeta potential between 30 mV and +30 mV) are prone to coagulation as a result of their interactions, whereas particles with a zeta potential below 30 mVor above +30 mV are considered to be acceptable and indicate a relatively stable state (Lin et al. 2014). Certain physiological processes such as cellular uptake are highly dependent on the surface properties of liposomes. In comparison to ionic liposomes, those with a positive charge are more likely to enter the cells and show a much higher cellular uptake. On the other hand, during the circulation, cationic liposomes are more likely to be cleared by reticular endothelial system (RES), and thus, their circulation time is largely reduced. So optimization of the surface charges is demanded to provide the particles with good stability as well as good bioavailability. Light-scattering techniques such as dynamic light scattering (DLS) and electrophoresis light scattering (ELS) are currently used to determine zeta-potential and
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they can report the measurement result quickly (Manaia et al. 2017). For example, we used DLS for liposome characterization, and the results showed their size ranges from 110 to 150 nm and showed a uniform size distribution (polydispersity index 5), their losses during storage are relatively low. In addition, it is necessary to prepare liposomes with saturated lipids which can form
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Fig. 2 Schematic representation of saturated lipids (left) and unsaturated lipids (right)
stabler bilayer structures compared with unsaturated lipids (Fig. 2). For all drugs, entrapment stability is better in MLVs than in SUVs. Stability of liposomes is enhanced when the lipid portion of the phospholipids includes long acyl chains, when liposomes are maintained at low temperature, and when phospholipids are in the gel state. Drugs can be loaded to liposomes by adsorption, inclusion, and incorporation. Liposomes, drugs, and the surrounding solutions are in an equilibrium state. Influenced by factors such as the concentration gradient of the solution or the binding of plasma proteins, drugs in the liposome suspensions can be percolated. Therefore, the stability of the liposomes in the PBS solution or the plasma protein solution needs to be evaluated and the leakage rate can be used to reflect their stability. The leakage rate refers to the change of entrapment efficiency during storage and can be calculated as follows (Jin et al. 2015): The amount of drug percolated into the medium 100% The amount of drug entrapped before storage ¼ Entrapment efficiency ðbefore storageÞ
Leakage rate ¼
entrapment efficiency ðstorage for some timeÞ Similar to the entrapment efficiency, the leakage of drugs in liposomes also depends primarily on the characteristics of the drugs and the type of liposomal structure. And methods used for entrapment efficiency determination can be applied to evaluate leakage rate as well.
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Stability
Stability studies should address the microbiological, physical, and chemical stability of the liposome drug product, including the integrity of the liposomes in the drug product. Liposomal stability studies include drug leakage, liposome structural
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changes (including particle size distribution, liposome aggregation, fusion, and liposome precipitation), liposome and drugs’ chemical stability, pH, ionic strength, buffer composition, and solvent system. Some of these (particle size, drug leakage, etc.) have been discussed above. The quality and purity of the lipid starting materials has a determining effect on liposome quality; therefore, it is essential to characterize these lipid starting materials such as characterization of lipid bilayer phase transition behavior (e.g., temperature and enthalpy of transitions). In detail, the physical stability of liposome drug products can be affected by the liposome integrity, the size distribution of the lipid vesicles, unsaturation of the fatty acid groups, etc. Some liposomes are susceptible to fusion (i.e., irreversible coalition of smaller liposomes to form larger liposomes), aggregation (i.e., reversible conglomeration or pooling of two or more liposomes without fusion), and leakage of the contained drug substance during storage. Fusion, aggregation, or leakage can be affected by the lipid components in the liposome or by the contained drug substance. Stability testing should include tests to assess liposome size distribution and integrity. The chemical stability of the lipid components in the liposome as well as the chemical stability of the contained drug substance also needs to be evaluated. Lipids with unsaturated fatty acids are subject to oxidative degradation, while both saturated and unsaturated lipids are subject to hydrolysis to form lysolipids and free fatty acids. It may be appropriate to conduct stress testing of unloaded liposomes to assess possible degradation or other reaction processes unique to the liposomes. For each lipid used for liposome production, stability studies and stress testing (e.g., high (e.g., 50 C) and low (e.g., freezing) temperatures, light, pH, and oxygen) should be performed to establish appropriate storage conditions and retest period, determine the degradation profile, and develop an appropriate stability-indicating analytical procedure (Guidance for Industry). There have been many methods to study the stability of liposomes. For example, water-soluble fluorescent dyes can serve as markers for drugs encapsulated in liposomes, such as carboxyfluorescein (CF) and calceins. The common feature of these two kinds of fluoresceins is that they are not likely to excite fluorescence at high concentrations. However, when the liposomes leak or are damaged, leading to the release of drug into the surrounding medium or into the cytoplasm, the concentration of fluorescein will decrease and it will show fluorescence. Besides, fluorescence self-quenching (FSQ) is widely used to determine the manner in which liposomal contents enter cells; to determine the effect of serum, protein, lipoprotein, enzyme, or phase transition temperature on the stability of liposomes; and to determine the liposomal elimination rate in vivo.
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Pharmacokinetics and Pharmacology
Drugs encapsulated in liposomes can be protected from degradation, but their biological activity may be restricted as well, for only when the drugs are released from liposomes can they exert their pharmaceutical effects. Thus, it is of vital
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importance to individually quantify encapsulated and unencapsulated drugs in blood/plasma and unencapsulated drugs in tissue. In addition to traditional methods for total drug and metabolite in blood/plasma and tissues, reliable separation methods are demanded for encapsulated and unencapsulated drugs and special attention should be paid to methodology verification. As liposomes are likely to be disrupted in tissue processing, making it challenging to quantify drugs in tissue, one should take care to separate these drugs prior to steps during which liposome integrity may be damaged and employ methodologies to verify the suitability and interpretability of all bioanalytical results (Committee for Human Medicinal Products). Pharmacokinetic study is quite useful for developing dose-concentrationresponse relationships and establishing dosing regimens. In some cases, animalbased models can be used to predict the performance of liposomes in humans. For nonclinical pharmacokinetic studies, the choice of appropriate models and species is vital, and special emphasis should be on accumulation and retention in target organs, pharmacokinetics, and distribution. When it comes to liposome biodistribution, as there are large numbers of mononuclear macrophages in the liver and spleen, two vital organs for lipid metabolism, liposomes are prone to accumulating at these sites after intravenous administration. Under pathological conditions, some other sites such as tumor may also be target sites for liposomal products due to the enhanced permeability and retention (EPR) effect. Most of the animal models for scientific use can be used for liposome in vivo study such as dog, cat, rabbit, mouse, and rat, but mouse and rat are most common. Due to the differences among species, the clearance rate and mechanism may vary, and thus, the explanation for obtained results needs to be cautioned. As it is impossible to have a full view of liposomal drug product performance from blood/plasma data alone, similarities in the distribution and elimination should be demonstrated as well, which provides useful information about the comparability of disposition of liposomal drug products. If feasible, the kinetics (including tissue distribution and excretion) of the unencapsulated drug and the encapsulated drug should be investigated separately (Committee for Human Medicinal Products). As for clinical pharmacokinetic studies, the commonly used parameters for the study include area under the plasma concentration versus time curve (AUC), peak plasma concentration, time to peak plasma concentration, elimination half-life, volume of distribution, total clearance, renal clearance, and accumulation for both free and total drug. Generally, the 90% confidence intervals for Cmax should be within the range of 80–125% (Committee for Human Medicinal Products). Usually, pharmacokinetic characteristics of drugs can be significantly changed when administered in a liposomal formulation, i.e., volume of distribution and clearance may be reduced and half-life prolonged. It is recommended by FDA to compare the differences of liposomal drug products with their nonliposomal formulation in absorption, distribution, metabolism, and excretion (ADME) (Tochilin and Weissing 2002). Different doses of liposomes and nonliposomal products may be used based on the drugs to be investigated.
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Assessment of Efficacy
Generally, besides obligatory clinical pharmacokinetic studies, a clinical efficacy trial is not always demanded and dependent on the nonclinical models and clinical PK data to detect differences between active substances and their corresponding liposomal products as well as the complexity of the formulation. In cases where formulations are of different qualitative composition, additional therapeutic equivalence studies are very likely to be required. But usually it is not preferred to carry out such trials due to their insensitivity to detect differences in efficacy (Committee for Human Medicinal Products). Therefore, efforts should be made to demonstrate bioequivalence of the developed liposomal products to their reference products as well as similarity in nonclinical pharmacokinetic and pharmacodynamic and clinical pharmacokinetic studies.
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Safety and Toxicity
Acute infusion reactions are relatively common with liposomal formulations. To minimize the risk of acute infusion reactions, it is required in vitro and in vivo immune reactogenicity assays be used such as complement (and/or macrophage/ basophil activation assays). And to determine the extent of potential adverse event, testing for complement activation-related pseudoallergy (CARPA) in sensitive animal models should be adopted. If a liposomal product shows any sign of increased risk, then its development ought to be re-evaluated until reasons are classified. Besides, infusion reactions should be carefully evaluated in bioequivalence studies, and if a new liposomal product shows any differences, its development should be re-evaluated (Committee for Human Medicinal Products). Furthermore, to support equivalence in the context of known target organ toxicity, appropriate organ function tests may be required. For example, for heart toxicity, it is appropriate to assess cardiac function by measurement of left ventricular end-diastolic pressure in a rodent model.
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Conclusion
As the properties of liposomes (size, zeta potential, PDI, morphology, entrapment efficiency, etc.) have a dramatic impact on their performances in vitro and in vivo, such as stability, circulation time, biodistribution, and bioavailability, eventually leading to variation in their therapeutic effect, it is of great importance to have a comprehensive evaluation of the preparations and take corresponding measures for quality control. And choice of appropriate methods for characterization is crucial as well, which is supposed to be based on the study objectives and specified for a certain formulation. Furthermore, with the rapid development of techniques, more accurate and reproducible methods will emerge to facilitate the evaluation process.
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References Abolfazl A, Rogaie RS, Soodabeh D, Sang WJ, Nosratollah Z, Younes H, Mohammad S, Mohammad K, Kazem NK (2013) Liposome: classification, preparation, and applications. Nanoscale Res Lett 8(1):102–110 Arouri A, Mouritsen OG (2013) Membrane-perturbing effect of fatty acids and lysolipids. Prog Lipid Res 52(1):130–140 Bahari LAS, Hamishehkar H (2016) The impact of variables on particle size of solid lipid nanoparticles and nanostructured lipid carriers; a comparative literature review. Adv Pharm Bull 6(2):143–151 Bera B (2015) Nanoporous silicon prepared by vapour phase strain etch and sacrificial technique. In: Proceedings of the international conference on Microelectronic Circuit and System (Micro), Kolkata, pp 42–45 Burgess DJ, Hussain AS, Ingallinera TS, Chen ML (2002) Assuring quality and performance of sustained and controlled release parenterals: workshop report. AAPS PharmSci 4(2):13–23 Committee for Human Medicinal Products. Reflection paper on the data requirements for intravenous liposomal products developed with reference to an innovator liposomal. European Medicines Agency. https://www.ema.europa.eu/documents/scientific-guideline/reflection-paperdata-requirements-intravenous-liposomal-products-developed-reference-innovator_en-0.pdf Danaei M, Dehghankhold M, Ataei S, Davarani FH, Javanmard R, Dokhani A, Khorasani S, Mozafari MR (2018) Impact of particle size and polydispersity index on the clinical applications of lipidic nanocarrier systems. Pharmaceutics 10(2):57–73 Gabizon AA, Barenholz Y, Bialer M (1993) Prolongation of the circulation time of doxorubicin encapsulated in liposomes containing a polyethylene glycol-derivatized phospholipid: pharmacokinetic studies in rodents and dogs. Pharm Res 10(5):703–708 Guidance for Industry. Liposome drug products, chemistry, manufacturing, and controls; human pharmacokinetics and bioavailability; and labeling documentation. U.S. Food and Drug Administration. http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guid ances/ucm070570.pdf. Accessed Apr 2018 Jiang G, Su JF, Fu H, Xing JX, Dong YX, Wu CB (2012) Content determination of lysophosphatidylcholine in seal oil based lipid emulsion for parenteral nutrition by HPLC-ELSD. Chin J Mod Appl Pharm 29(5):440–444 Jin YG, Du LN, Chen Y, Ling PX (2015) Application of nanotechnology in drug delivery, 1st edn, pp 174–179 Kapoor M, Lee SL, Tyner KM (2017) Liposomal drug product development and quality: current us experience and perspective. AAPS J 19(3):632–641 Kuntsche J, Horst JC, Bunjes H (2011) Cryogenic transmission electron microscopy (cryo-TEM) for studying the morphology of colloidal drug delivery systems. Int J Pharm 417(1–2):120–137 Li H, Gong W, Zhang H, Meng XG (2014) Determination of lysolipid in vinorelbine liposome for injection by HPLC-charged aerosol detector. Chin J New Drug 23(13):1493–1496 Lin PC, Lin S, Wang PC, Sridhar R (2014) Techniques for physicochemical characterization of nanomaterials. Biotechnol Adv 32(4):711–726 Manaia EB, Abuçafy MP, Chiariandréo BG, Silva BL, Oshiro-Júnior JA, Chiavacci LA (2017) Physicochemical characterization of drug nanocarriers. Int J Nanomedicine 12:4991–5011 Mills JK, Needham D (2005) Lysolipid incorporation in dipalmi toylphosphatidylcholine bilayer membranes enhances the ion permeability and drug release rates at the membrane phase transition. BBA-Biomenbranes 1716(2):77–96 Proffitt RT, Williams LE, Presant CA, Tin GW, Uliana JA, Gamble RC, Baldeschwieler JD (1983) Tumor-imaging potential of liposomes loaded with In-111-NTA: biodistribution in mice. J Nucl Med 24(1):45–51 Putri DCA, Dwiastuti R, Marchaban M, Nugroho AK (2017) Optimization of mixing temperature and sonication duration in liposome preparation. J Pharm Sci Community 14(2):79–85 Severs NJ (2007) Freeze-fracture electron microscopy. Nat Protoc 2(3):547–576
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Solomon D, Gupta N, Mulla NS, Shukla S, Guerrero YA, Gupta V (2017) Role of in vitro release methods in liposomal formulation development: challenges and regulatory perspective. AAPS J 19(6):1669–1681 Tochilin VP, Weissing V (2002) Liposomes, 2nd edn, Oxford University Press, New York, pp 45–46, 149–164 Yang ZZ, Gao W, Liu YJ, Pang N, Qi XR (2017) Delivering siRNA and chemotherapeutic molecules across BBB and BTB for intracranial glioblastoma therapy. Mol Pharm 14(4):1012–1022
Part II Functionalized Liposome-Based Drug Delivery Systems and Potential Application
Coupling Methods of Antibodies and Ligands for Liposomes
10
Ming Chen, Qiu-Ran Ma, and Wan-Liang Lu
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Coupling Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Targeted Antibody-Drug Conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Targeted Drug Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 ADC Conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Preparation of Mitochondrial Targeting Topotecan-Loaded Liposomes (Yu et al. 2012) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 ADC Conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Preparation of Mitochondrial Targeting Topotecan-Loaded Liposomes (Yu et al. 2012) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
This chapter describes the preparations of coupling methods of antibodies and ligands for targeted drug conjugates and for targeted drug liposomes. To prepare antibody-mediated targeting drug (referring to as antibody-drug conjugate, ADC), or antibody-mediated targeting drug liposomes, there are many approaches of coupling methods. According to the types of coupling agents,
M. Chen · Q.-R. Ma · W.-L. Lu (*) State Key Laboratory of Natural and Biomimetic Drugs, Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System, School of Pharmaceutical Sciences, Peking University, Beijing, China e-mail: [email protected] © Springer-Verlag GmbH Germany, part of Springer Nature 2021 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49320-5_22
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the methods usually consist of homobifunctional and heterobifunctional crosslinking approaches. To prepare ligand-mediated targeting drug or ligandmediated targeting drug liposomes, various chemical modification and synthesis method can be used, depending on the clinical treatment purposes and material structures. The following will describe the typical protocol examples for preparing the targeted drug conjugates and targeted drug liposomes.
Keywords
ADCs · mAbs · Coupling method · Linker · Targeted drug liposomes
1
Introduction
1.1
Coupling Methods
Antibody-drug coupling process is of great importance in the process of preparing both the targeted antibody-drug conjugates and the targeted drug liposomes. There are many approaches of coupling methods according to the types of coupling agents described as the following. The homobifunctional cross-linking approach is used as a coupling method which establishes connection between the same agent with a dual function. For example, glutaraldehyde can be used to conjugate a drug with an antibody. In this conjugation, one of two aldehyde groups on glutaraldehyde is able to link the amino group of antibody, while another aldehyde is to conjugate the amino group of the drug, thus forming an antibody-drug conjugate (ADC) (Fig. 1). Apart from this, there is another method using carbodiimide to link the antibody with a drug. In this process, carbodiimide (R – N = C = N R0 ) can act as a dehydrating agent. It reacts with the carboxyl group on the drug and an antibody to form two intermediate products, respectively. And then two intermediate products form a conjugate between antibody and drug (Fig. 2). Ellman method can also be used for conjugation through 5,50 -dithiobis (2-nitrobenzoic acid) (DTNB) linking (Fig. 3). Ellman agent can react with the sulfydryl group on the antibody, and the subsequent product can react with drug
Fig. 1 Glutaraldehyde method for linking amino group containing drug and antibody
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R' R'
H . C O
. O
O +
N C N
N C N
H
OH
Dicyclohexylcarbodiimide (DCC) O OH
O + mAB
H C
C
OH
C mAb + H Antibody-drug conjugate R'
C
O H N C N H
R': carboxyl group containing drug mAB: monoclonal antibody
Fig. 2 Carbodiimide method for linking carboxyl group containing drug and antibody
HOOC
NO2
+ R' SH
COOH
COOH
S S
NO2 + mAb-SH
mAb S S
NO2
mAb S S R' mAb: monoclonal antibody R': sulfydryl group containing drug
Fig. 3 Ellman reagent for conjugation of ADC
by replacing one part of benzene structure with mAb, thus establishing the linking between antibody and drug. The heterobifunctional cross-linker can also be used a dual function agent for establishing connection between antibody and drug. For example, N-succinimidyl3-(2-pyridyldithiol) propionate (SPDP) reagent can react with amino groups on the antibody and on the drug, respectively, and form two kinds of ADC products, depending on the amino or sulfydryl group containing drug (Fig. 4). Besides, maleimide can be used as a cross-liker to produce ADC, as the double bond on maleimide is often active. The amino group on the antibody is able to react with the active ester group of maleimide, while the double bond on maleimide can react with the sulfydryl group on the drug, thereby establishing a connection of antibody and sulfydryl containing drug (Fig. 5). Therefore, both homobifunctional cross-linker and heterobifunctional crosslinker are able to establish direct connection between antibody and drug.
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O
SPDP
mAB NH2
R NH2
mAB NHCO(CH2)2S S
R NHCO(CH2)2S S
DT
T
+
+ mAb H2NCO(CH2)2SH
R SH
mAb NHCO(CH2)2S S R
mAb NHCO(CH2)2 S S (CH2)2CONH R ADC2
ADC1 DTT:dithiothreitol mAb: monoclonal antibody
R-SH: sulfydryl group containing drug R-NH2: amino group containing drug
Fig. 4 SPDP method for linking amino or sulfydryl group containing drug and antibody
O mAb
NH2 +
O
O mAb
N(CH2)2COON O
NHCO(H2C)2N O
O O S R
+
HS R
mAb NHCO(H2C)2N O
HS-R: sulfydryl group containing drug mAb: monoclonal antibody Fig. 5 Maleimide method for linking amino or sulfydryl group containing drug and antibody
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1.2
Coupling Methods of Antibodies and Ligands for Liposomes
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Targeted Antibody-Drug Conjugates
An approach to enhance drug concentration in the pathological site is to prepare antibody-drug conjugate (ADC), which allows drug to concentrate on the action site and minimizes systemic toxicity. ADC is often used for treatment of cancer aimed at targeting cancer cells. To develop a suitable ADC drug, the formulation factors should be fully considered, including action target, antigen, monoclonal antibody (mAb), linker, and cytotoxic payload, in addition to the disease condition of cancer. For example, a suitable ADC drug should not affect both the targeting capability of monoclonal antibody and the killing ability of a highly potent cytotoxic drug. On the contrary, ADC drug should enhance the treatment efficacy of anticancer drug through the antibody-mediated targeting effect. In the process of ADC drug delivery, the antibody is able to track a specific cancer antigen and bind onto the surface of cancer cells. After internalizing and processing by endosomes or lysosomes in the cells, drug can release from ADC as a lethal agent and takes effect to kill cancer cells (Fig. 6). Due to the specific recognition of antigen
Fig. 6 Schematic representation for action mechanism of antibody drug conjugate (ADC) (Peters and Brown 2015)
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on the target cancer cells, ADC drug is expected to provide higher efficacy and a wider therapeutic window than its parent anticancer drug. ADC drug can be divided into three main structural units: mAb (or ligand), linker, and drug. High-affinity mAb, stable linker, and potent cytotoxic agent enable the development of a safe and efficient ADC (Peters and Brown 2015). The selection of mAb used in ADC drug depends on the expressed antigen on cancer cells. An appropriate antigen-binding site is critical for the eventual success of an antibodydrug conjugate. It is required that the monoclonal antibody on the ADC is able to bind with a target antigen on the surface of cancer cells after administration via the bloodstream. Further, the ADC complex should be able to be rapidly internalized by cancer cells and allow the release of the cytotoxic agent within the cell (Jaracz and Chen 2005; Chari 2008). To develop a suitable ADC drug, a proper antigen should be well characterized and is demonstrated to be abundant expressed and accessible on cancer cells in contrast to the surrounding normal cells (Shefet-Carasso and Benhar 2015; Parakh and Parslow 2015). This condition enables a preferential binding of ADC with malignant cancer cells and reduces toxicity of ADC to normal cells. Moreover, the selection of antibody is also an important factor that should be considered because the therapy requires ADC drug that can be efficiently internalized into the target cells, followed by releasing small molecule from the conjugate. Usually, the cellular uptake of ADC is limited by the internalization of ADC-antigen complex (Teicher and Chari 2011). The antibody selected should have high affinity with the antigen and be able to be internalized after forming ADC-antigen complex. Many mAbs themselves, such as avastin, rituximab, and cetuximab, are used as standard therapeutic drugs for treatment of solid tumors and hematological cancers (Scott and Wolchok 2012). These mAbs can be used as targeting molecules for preparing ADCs by conjugating with anticancer agents. These are to overcome the problems of anticancer agents encountered in the clinical treatment. For instance, vinblastine, doxorubicin, and paclitaxel are main anticancer drugs used in the clinical therapy but are limitedly used because of their nonspecific toxicity, narrow therapeutic window, and multidrug resistance (Chabner and Roberts 2005; Szakacs and Paterson 2006). If these anticancer agents are formulated into the ADCs, there will decrease the nonspecific toxicity to normal tissues, expand the therapeutic window, and potentially circumvent the drug resistance. The first generation of ADC drugs were developed mainly focusing on the clinically approved cytotoxic agents. Doxorubicin, 5-fluorouracil, and methotrexate have been regarded as advantageous choice for preparing ADC drugs. However, ADCs of these agents did not achieve clinical benefit due to the moderate cytotoxic potential, lack of selectivity, and low intracellular drug concentration (Hartley 2011). Likely one of the reasons may be due to inappropriate preparation procedure of ADC, in addition to the reasons of antibody used and the specific antigen overexpressed on the cancer cells. To be a useful procedure, the following coupling method can be used for preparing anthracycline anticancer agents like doxorubicin (Fig. 7). The selection of anticancer drug is also a critical factor to be considered during designing ADC. The commonly selected cytotoxic agents involve two kinds of
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Coupling Methods of Antibodies and Ligands for Liposomes CH2OH O
R
CH2OH O
OH
+
OH
NaIO4
CH2OH O
–H2O
+ H2N mAb
N
OH mAb
CH2OH O OH NaBH2
+
O
R HO
O
OH
+
R +H2O
CH2OH O OH
OH
R O
149
N
mAb
R N HO mAb
mAb: monoclonal antibody R: saccharides, carbohydrate or O-glycol hydroxyl group
Fig. 7 Periodate coupling method for doxorubicin containing ADC. Notes: Alcoholic hydroxyl in the structure of doxorubicin can be oxidized by sodium periodate. After hydroxyl reacts with amine on the antibody, it produces a stable ADC conjugate by a reduction reaction in the presence of sodium borohydride
drugs: tubulin-targeting agents and DNA-damaging drugs. For example, tubulintargeting anticancer agents, maytansinoid and auristatin, have been selected for preparing ADCs at the stage of development, showing a promising preclinical result (Hartley 2011; Sutherland and Walter 2013). The linker plays a key role in producing ADC drug because its characteristics substantially impact the therapeutic index, efficacy, and pharmacokinetics of the final product (Hughes 2010; Hamblett and Senter 2004). A linker is used to establish covalent connection between the cytotoxic compound and the antibody. The linker influences the stability of drug, antibody, and final product of ADC. A stable linker in an ADC can maintain the mAb concentration in the blood circulation and ensures the stability of the cytotoxic agent without release before reaching the treatment site, making off-target effects minimized. However, the linker should become labile once the ADC is internalized by the cancer cells in order to guarantee a rapid release of the cytotoxic drug (Teicher and Chari 2011).
1.3
Targeted Drug Liposomes
The targeted drug liposome is a drug delivery system that selectively localizes a drug to a target tissue, target organ, target cell, or intracellular structure by topical administration or systemic administration. The targeted drug liposome consists of antibody-mediated targeting drug liposome and ligand-mediated targeting drug liposome.
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To prepare antibody-mediated drug-targeting liposomes, Maruyama et al. develop a simple approach of conjugating antibodies directly to the distal end of polyethylene glycol (PEG) which is already bound to the liposome membrane. In this research, distearoyl-N-(3-carboxypropionoyl poly(ethylene glycol) succinyl) phosphatidylethanolamine (DSPE-PEG-COOH) is used to prepare immunoliposomes carrying monoclonal antibody at the distal ends of the PEG chains (Maruyama et al. 1995). To prepare ligand-mediated drug targeting liposomes, a chemical ligand is often used for the targeting purpose by modifying drug liposomes. The ligand is a substance, usually a chemical compound, which forms a complex with a specific protein on cells to serve the targeting purpose. In the protein-ligand binding, the ligand is usually a molecule which produces a signal by binding to a site on a target protein. Based on this consideration, Yu et al. establish mitochondrial targeting topotecanloaded liposomes by modifying dequalinium (DQA) as a ligand. The mitochondrial probe, DQA, is used as the ligand by conjugating with a lipid derivative, polyethylene glycol-distearoyl phosphosphatidylethanolamine(PEG2000-DSPE). After conjugation, D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS1000) is also conjugated as an inhibitor of ABC (ATP-binding cassette transporters) protein. TPGS1000 and PEG-DSPE2000 anchored to the surface of mitochondrial targeting liposomes, which enables apoptosis of drug-resistant cancer cells at the mitochondrial level (Fig. 8).
2
Materials
2.1
ADC Conjugates
2.1.1 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Preparation of Conjugate P/PEG(5%)GH-DOX (Rameshwar and Jose 2012) N-Hydroxysuccinimide (NHS) N,N0 -Dicyclohexylcarbodiimide (DCC) N,N-Dimethylformamide (DMF) Poly(β-L-malic acid) (PMLA) Anhydrous acetone mPEG5000-NH2 Triethylamine (TEA) Thin layer chromatography (TLC) Glycine-boc-hydrazide (GBH) Sephadex column Doxorubicin (DOX) HCl Phosphate-buffered saline (PBS) Liquid nitrogen
2.1.2 Preparation of mAb-taxoid Conjugates (Ojima and Geng 2002) 1. Pyridine 2. Acetonitrile
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a. Dequalinium (DQA) chloride O CH3
N
O
H2C
N CH3
O HO
CH2
N
OH
H3C
b. Topotecan, TPT O H
O
O
O n
CH3 O CH3
CH3
CH3
CH3
O
H3C
CH3
CH3
c. D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS1000)
d. Mitochondrial targeting topotecan-loaded liposomes Fig. 8 Illustration of mitochondrial targeting topotecan-loaded liposomes (Yu et al. 2012). (a) Dequalinium (DQA) chloride. (b) Topotecan, TPT. (c) D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS1000). (d) Mitochondrial targeting topotecan-loaded liposomes
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Ethyl acetate (EtOAc) Ethanol (EtOH) NaHCO3 CuSO4 Water Brine Anhydrous MgSO4 Column chromatography Hexane Bruker AC-250 NMR spectrometer Dithiothreitol (DTT) N-Succinimidyl-4-(2-pyridyldithio) pentanoate (SPP) Potassium phosphate buffer NaCl Ethylenediaminetetraacetic acid (EDTA) Ethanol Dithiothreitol (DTT) Sephadex G25 column
2.1.3 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Preparation of cAC10-Valine-Citrulline-MMAE (Russell and Sanderson 2004) Monomethyl auristatin E (MMAE) N,N-Dimethylvaline Valine-citrulline linker 1-Hydroxybenzotriazole (HOBt) Anhydrous dimethylformamide (DMF) Pyridine High performance liquid chromatography (HPLC) Trifluoroacetic acid (TFA) Methyl cyanide (MeCN) Methylene chloride Hexane NaCl Dithiothreitol (DTT) Sephadex G-25 Phosphate buffered saline (PBS pH 7.4) Diethylenetriaminepentaacetic acid (DTPA) Thiol-antibody 5,50 -dithiobis(2-nitrobenzoic acid) (DTNB) Dimethyl sulfoxide (DMSO) Cysteine Maleimide
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Preparation of b-Galactosidase-Sensitive Antibody Drug Conjugates (Sergii and Chloe 2017) O-(2-aminoethyl)-O0 -(2-azidoethyl)nonaethylene glycol Cu(MeCN)4PF6 Ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) H 2O CH2Cl2 MgSO4 Column chromatography Methanol (MeOH) Lithium hydroxide monohydrate IRC-50 acidic resin Dimethyl sulfoxide (DMSO) N-hydroxysuccinimide ester Preparative-reverse phase HPLC Trastuzumab PBS (100 mM with 2 mM EDTA, pH 7.4) Tris(2-carboxyethyl)phosphine (TCEP) Size-exclusion chromatography Sodium succinate buffer (10 mM, pH 5.0)
2.1.4 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
2.2 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Preparation of Mitochondrial Targeting Topotecan-Loaded Liposomes (Yu et al. 2012) Egg phosphatidylcholine (EPC) Cholesterol (Chol) Polyethylene glycol distearoylphosphosphatidylethanolamine (PEG2000-DSPE) D-a-tocopheryl polyethylene glycol 1000 succinate (TPGS1000) Dequalinium Chloroform Hepes buffered saline (HBS, 25 nM Hepes/150 nM NaCl) Topotecan hydrochloride Phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) Sephedex G-50 BCA kit Polycarbon ester filter Dialysis tube Phenomenex ODS-C18 reversed-phase column Fetal bovine serum (FBS) Methanol Acetonitrile
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3
Methods
3.1
ADC Conjugates
3.1.1
Preparation of Conjugate P/PEG(5%)GH-DOX (Fig. 9, Notes 1, 2, 3)
Preparation of Conjugate P/PEG(5%)GH 1. Dissolve 1 mmol N-hydroxysuccinimide (NHS) and 1 mmol DCC in 2 mL DMF as solution 1. Dissolve 116 mg of PMLA (1 mmol with regard to malyl units) in 2 mL anhydrous acetone as solution 2. Add solution 1 to solution 2 under vigorous stirring at RT for 3 h to complete the activation of carboxyl groups. 2. Add 0.05 mmol of triethylamine (TEA) and 0.05 mmol of mPEG5000-NH2 (in 1 mL of DMF, 5 mol% with regard to malyl units) to the reaction solution. Indicate the reaction process by TLC. 3. Dissolve 0.05 mmol GBH in 1 mL DMF (5 mol% with regard to malyl units). Add this solution and 0.05 mmol TEA to the reaction solution at room temperature. Indicate the reaction process by TLC. 4. Add 5–6 mL 100 mM sodium phosphate buffer containing 150 mM NaCl (pH 5.5) to reaction solution and stir for 1 h at RT to remove Boc. After centrifugation at 1500 g for 10 min, pass the clear supernatant containing glycine hydrazide (GH) over a Sephadex column (PD-10, GE Healthcare, Piscataway, NJ, USA) preequilibrated with deionized (DI) water. 5. Combine and lyophilize the fractions to obtain a white solid. Preparation of Conjugate P/PEG(5%)GH-DOX(5%) 1. Dissolve 10 mg P/PEG(5%)/GH(5%) in 2 mL DMF as solution 3. Dissolve 2 mg DOX in 1 mL DMF as DOX excess solution. Add the excess solution to solution 3 along with activated molecular sieves (4 Å, 10–18 mesh). 2. Stir the reaction mixture in the dark for 16 h. Then add 6 mL phosphate-buffered saline (PBS, pH 7.4) to the reaction. 3. Pass the clear supernatant over a Sephadex column (PD-10, GE Healthcare, Piscataway, NJ, USA) pre-equilibrated with PBS pH 7.4. Collect the P/PEG (5%)/GH-DOX(5%) fractions and immediately freeze using liquid nitrogen. 4. After lyophilization, conjugate P/PEG(5%)/GH-DOX(5%) is obtained as a pinkish red solid.
3.1.2
Preparation of mAb-taxoid Conjugates (Fig. 10, Note 4)
Preparation of 30 -Dephenyl-30 -(2-methylprop-1-enyl)-10(3-methyldisulfanylopropa- noyl-docetaxel) 1. Dissolve 52 mg (0.044 mol) 30 -dephenyl-30 -(2-methylprop-1-enyl)-10(3-methyldisulfanyl- propanoyl)-7-triethylsily-20 -triisopropylsily-docetaxel in
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Fig. 9 The synthetic route of P/PEG(5%)GH-DOX. Note: (i) Mixture of NHS and DCC, acetone, DMF, RT 3 h, followed by addition of mPEG5000-NH2, RT 45 min, and by addition of glycine hydrazide, 2 h yield 67%; (ii) DOX, HCl, molecular sieves 4 Å, 10–18 mesh, DMF, RT 16 h, yield 72%. RT denotes room temperature
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Fig. 10 Preparations of mAb-taxoid conjugates. Note: (i) Dithiothreitol (DTT); (ii) N-succinimidyl-4-(2-pyridyldithio)-pentanoate (SPP, 10 equiv in ethanol), 50 mM potassium phosphate buffer, pH 6.5, NaCl (50 mM), EDTA (2 mM), 90 min; (iii) 50 mM potassium phosphate buffer, pH 6.5, NaCl (50 mM), EDTA (2 mM), 15 (1.7 equiv per dithiopyridyl group in EtOH), 24 h
1 mL pyridine as solution 1. Add 1 mL acetonitrile and 0.6 mL HF/pyridine (70:30, 0.60 mL) to solution 1 drop by drop at 0 C. Then wait until the mixture’s temperature rise to ambient temperature, stir for 17 h.
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2. Dilute the reaction mixture with EtOAc and wash it with saturated 10 mL NaHCO3, saturated 10 mL * 4 CuSO4 solution, 10 mL * 3 water and 10 mL brine. 3. Dry the organic layer with anhydrous MgSO4. Remove the solvent by reduced pressure vacuum and purify the residue by column chromatography (hexanes: EtOAc = 1:1) to afford 30 -dephenyl-30 -(2-methylprop-1-enyl)-10-(3-methyldisulfanylpropanoy) docetaxel, white solid. 4. Confirm the structure of the product with 1H NMR. Preparation of Antibody-Taxoid Conjugates 1. Reduce the 30 -dephenyl-30 -(2-methylprop-1-enyl)-10-(3-methyldisulfanylpropanoy) docetaxel with dithiothreitol to give a thiol-containing taxiod (HS-taxoid) and purify it by HPLC. Modify the antibody with N-succinimidyl4-(2-pyridyldithio) pentanoate (SPP) to introduce dithiopyridyl groups. Dissolve antibody in 50 mM potassium phosphate buffer (pH 6.5) containing 50 mM NaCl and 2 mM EDTA and add SPP (11 molar equiv in ethanol) to the potassium phosphate buffer. The final ethanol concentration was 1.4% (v/v). 2. After 90 min at ambient temperature, add 50 mM lysine to remove any noncovalently bound SPP. Allow the reaction for 2 h, then purify it by gel filtration through a Sephadex G25 column equilibrated in the above buffer. Collect the antibody-containing fractions, determine the degree of modification by treating a sample with dithiothreitol, and measure the change in absorbance at 343 nm. 3. Dilute the modified antibody with 50 mM potassium phosphate buffer (pH 6.5) containing 50 mM NaCl and 2 mM EDTA. Then add HS-taxoid in ethanol (10% v/v in final reaction mixture) to modify antibody solution. 4. Last the reaction at ambient temperature under argon for 24 h. Monitor the progress of the reaction spectrophotometrically at 343 nm for release of pyridine-2-thiol caused by disulfide exchange between the HS-taxoid and the dithiopyridyl groups on the antibody. The increase in absorbance at 343 nm indicates that the taxoid had linked to all of the SPP sites in the antibody. 5. Load the reaction mixture on a Sephacryl S300 gel filtration column equilibrated with phosphate-buffered saline (PBS, pH 6.5) containing 20% propylene glycol. 6. The major peak comprises the antibody-taxoid conjugate peak. The recovery of the conjugate is 65–70%. Determine the concentration of the conjugate by measuring the absorbance at 280 nm.
3.1.3
Preparation of cAC10-Valine-Citrulline-MMAE (Fig. 11, Notes 5, 6)
Preparation of Valine-Citrulline-MMAE 1. Dissolve the activated linker (60 mg, 84 μmol, 1.1 equiv), MMAE (56 mg, 76 μmol, 1.0 equiv), and HOBt (10 mg, 1.0 equiv) in anhydrous 2 mL DMF and 0.5 mL pyridine. 2. Stir the contents during monitoring with high performance liquid chromatography (HPLC). Inject the reaction mixture onto a reverse-phase preparative-HPLC column (SynergiMAX-RP; C12 column 21.2 mm * 25 cm, 10 μ, 80 Å, using a
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Fig. 11 cAC10 ADCs and cathepsin B reaction products. Note: Enzyme-mediated hydrolysis of the protease-sensitive valine citrulline dipeptide linker followed by fragmentation of the p-aminobenzylcarbamatering generates free MMAE and cAC10-(vcOH)n. The amount of released drug(n) per mAb following complete hydrolysis of each dipeptide linker is direct function of cAC10-(val-cit-MMAE)n
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gradient run of MeCN and 0.1% TFA at 25 mL/min from 10% to 100% over 40 min, followed by 100% MeCN for 20 min). 3. Analysis, pool, and concentrate the fractions immediately to a pale yellow solid. The addition of methylene chloride and hexanes (1:1) is followed by evaporation led to the production of vcMMAE as an off-white powder.
Preparation of cAC10-Valine-Citrulline-MMAE 1. Add 600 μL of 100 mM dithiothreitol (DTT) in water and add 600 μL of 500 mM sodium borate/500 mM NaCl, pH 8.0 to 4.8 mL cAC10 (10 mg/mL). After incubation at 37 C for 30 min, change the buffer to PBS containing 1 mM diethylenetriaminepentacetic acid (DTPA) and elute it through Sephadex G-25 resin. Determine the thiol-antibody value by determining the reduced mAb concentration at 280 nm, and determine the thiol concentration after reaction with DTNB (5,50 -dithiobis(2-nitrobenzoic acid)) at 412 nm. 2. Prepare the drug-linker solution to be used in the conjugation by diluting drug-linker from a frozen dimethyl sulfoxide (DMSO) stock solution at a known concentration (approximately 10 mM) in sufficient acetonitrile to make the conjugation reaction mixture of 20% organic and 80% aqueous, and freeze the solution once. 3. Calculate the volume of drug-linker stock solution containing 9.5 mol druglinker/1 mol antibody. Add the drug-linker solution rapidly while mixing it to the cold-reduced antibody solution, and leave the mixture on ice for 1 h. Add a 20 fold excess of cysteine over maleimide from a freshly prepared 100 mM solution in PBS to quench the conjugate reaction. Maintain the temperature at 4 C, concentrate the reaction mixture by centrifugal ultrafiltration and change the buffer by eluting through Sephadex G25 equilibrate in PBS. 4. Filter the conjugate through a 0.2 μm filter under sterile conditions. Store the conjugate at 80 C for analyzing and testing. Analyze ADCs’ concentration by UV absorbance, aggregate by size exclusion chromatography, measure drug, antibody, unreacted thiols, and residual free drug by reverse-phase HPLC. In these methods, all mAbs and ADCs contain exceed 98% monomeric protein.
3.1.4
Preparation of b-Galactosidase-Sensitive Antibody Drug Conjugates (Figs. 12 and 13, Notes 7, 8)
Preparation of Payloads 1. Dissolve compound 1 (28 mg, 0.0218 mmol) and O-(2-aminoethyl)-O0 (2-azidoethyl)nona-ethylene glycol (12.6 mg, 0.024 mmol, 1.1 equiv) in 1.8 mL CH2Cl2 as a solution compound 1. Add Cu(MeCN)4PF6 (12.2 mg, 0.0327 mmol) to the solution compound 1. Stir the mixture at ambient temperature for 12 h. Add a solution of disodium EDTA(0.13 g) in 2.1 mL H2O to the mixture. Stir the mixture for 2 h and extract it with 3 * 50 mL CH2Cl2. Combine the organic layers and dry it over MgSO4 and concentrate it in vacuum. Purify the crude product by column chromatography over silica gel (gradient elution 2–10%
O
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Fig. 12 Synthesis of galactoside payloads compound 5a and 5b and structure of valine-citrulline payloads compound 5c. (a) NH2(CH2CH2O)10CH2CH2N3, Cu (CH3CN)PF6, CH2Cl2, RT, 12 h, 48%; (b) LiOH, MeOH, 0 C, 20 min; (c) Compound 4a or 4b, DMSO, 16 h, 38% and 71% respectively
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MMAE =
HO
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Fig. 13 Structures of ADCs: T-MC-Gal-MMAE, T-APN-Gal-MMAE, and T-MC-VC-MMAE
MMAE
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MeOH in CH2Cl2) to give the instable amine compound 2 (18.9 mg, 0.0104 mmol, 48%). 2. Dissolve compound 2 in 0.8 mL MeOH. Cool the mixture at 0 C. Drop a solution of lithium hydroxide monohydrate (3.9 mg, 0.0915 mmol) in 0.8 mL water to the mixture. Stir the mixture for 15 min, neutralize it with IRC-50 acidic resin, filtrate and concentrate it in vacuum. Dissolve the crude product in 0.5 mL DMSO and add the N-hydroxysuccinimideester compound 4a (1.2 equiv, 3.9 mg) to it. Stir the mixture at ambient temperature for 12 h and remove the solvent under reduced pressure. 3. Purify the payload compound 5a by preparative-reverse phase HPLC. Confirm the structure of the product with 1H NMR. 4. Compound 5b was obtained using the protocol described for the compound 5a and replace reagent 4a by the reagent 4b. Compound 5b was obtained in 71% yield after purification by preparative-reverse phase HPLC. Preparation of ADCs Dissolve trastuzumab (5 mg/ml, 1 mL) in PBS (100 mM with 2 mM EDTA, pH 7.4) and add a solution of tris(2-carboxyethyl)phosphine(TCEP) (6.88 mL, 2 equiv, 10 mM in water) to it. Incubate the mixture at 37 C for 2 h and then cool it to 4 C. Add a solution of the payload (20.63 mL, 12 equiv, 20 mM in DMSO) to reduce trastuzumab. Incubate the mixture at 4 C for 2 h and then centrifuge it at 5000 g for 2 min to remove the insoluble excess of the payload. Purify the supernatant by size-exclusion chromatography on PD-10 columns equilibrated with sodium succinate buffer (10 mM, pH 5.0) to give solutions of pure ADC (a purity of >95% was confirmed by size exclusion chromatography).
3.2
Preparation of Mitochondrial Targeting Topotecan-Loaded Liposomes (Yu et al. 2012)
1. Dissolve lecithin (EPC), cholesterol (CHOL), PEG2000-DSPE, TPGS1000, and decaquinium chloride (60/29/2/2/7, μmol/μmol) in solution of chloroform and methanol mixture. 2. Evaporate and dry the solution in water bath at 40 C to remove organic reagent. 3. Hydrate the product with 250 mM (NH4)2SO4 solution. Treat the suspensions with ultrasonic in water bath for 20 min till liposomes become white and uniform. 4. Transfer the product to JY92-IID ultrasonic cell disruptor and treat it for 7 min at 35 C to get blue fluorescent translucent solution. Set the ultrasonic working time to 10 s and the intermittent time to 10 s (200 W). 5. Extrude the solution through 400 nm, 200 nm polyestercarbon membrane for three times. Encapsulate the product into dialysis membrane tubing. Dialysis it in Hepes buffer (HBS, 25 Mm Hepes/150 mM NaCl) for 36 h. Change the buffer every 12 h. 6. Mix the product with topotecan and vibrate them in water bath at 60 C for 20 min (1:15, w/w).
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Notes
1. In this conjugate, the attachment of DOX via an acid-cleavable hydrazone linkage has been an effective way to enhance the delivery of DOX (Ulbrich and Etrych 2003, 2004; Lee and Gillies 2006), because the hydrazone linkage can be cleaved under the mild acidic conditions in late endosomes/lysosomes to yield free DOX molecules. 2. Acid labile linkers used for drug attachment include hydrazones (Dubowchik and Walker 1999), hydrazines and thiosemicarbazones (Monkovic and Knipe 1991), trityl groups (Patel and Hardin 1995), cis-aconityl spacers (Shen and Ryser 1981), orthoesters, acetals, and ketals (Srinivasachar and Neville 1989). The most extensively exploited cleavable linker system contains the hydrazone functionality. Several methods are available for producing mAb-drug conjugates through hydrazone bond formation. The simplest method is to introduce the hydrazone functionality as an appendage of the drug to attach the entire complex to reactive functionalities on mAbs. Beside this method, aldehydes and ketones can be introduced onto the mAb through treatment with sodium periodate, thus oxidize diols on mAb sugars. Adding hydrazido drug derivatives can lead to the formation of hydrazones and satisfy the need to be relatively stable at neutral pH but labile under acidic conditions (Laguzza and Nichols 1989; Apelgren and Zimmerman 1990; Hinman and Hamann 1993). 3. One advantage of this methodology is that the mAbs are modified region specifically since the carbohydrates on mAbs are largely restricted to the Fc region. However, the problem is that the oxidation method may lead to a variety of reactive species and result in the poor definition of the final hydrazone. In addition, the oxidative conditions used for hydrazone formation can also lead to methionine oxidation, which can be detrimental to mAb binding activity (Hamann and Hinman 2002a). To circumvent this problem, bifunctional crosslinking reagents that contain aldehyde or ketone functionalities can be attached to mAb lysine residues and can then be used for subsequent modification. This approach gets much greater control over the relative hydrolysis rates of the hydrazone bond. Aromatic ketones are the most useful in this approach because the resultant hydrazones are stable for several days under neutral pH conditions while the stability is much less at pH 5 (Apelgren and Bailey 1993; Hamann and Hinman 2002b). 4. Using an appropriate linker between a taxoid and a mAb is crucial for high efficacy of the resulting immune conjugate. It is required that the linker be stable for an extended period of time in order to guarantee storage and circulation in vivo, while it is also expected to be readily cleavable inside cancer cells. Disulfide linker unit has its favorable characteristics (Chari 1998; Liu and Tadayoni 1996; Chari and Jackel 1995). 5. It is expected that the mAb component of the conjugate should bind to the specific antigens tumor surfaces and then the whole conjugate is internalized via endocytosis. The disulfide bond is then cleaved by an intracellular thiol such as glutathione to release taxoid in its active form. To synthesize a mAb-taxoid conjugate, both a taxoid and a mAb need to be modified to form a disulfide linkage by disulfide-thiol exchange reaction.
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6. Conjugation of monomethyl auristatin E (MMAE) to the anti-CD30 monoclonal antibody (mAb), cAC10, produced a selective and potent ADC against CD30+ an aplastic large cell lymphoma and Hodgkin’s disease models. This ADC, cAC10valine-citrulline-MMAE, uses a protease-sensitive dipeptide linker designed to release MMAE by lysosomal cathepsin B in target cells but maintain a stable linkage and attenuate drug potency in circulation. 7. Peptide (lysosomal protease-sensitive) linker is more stable than the other forms of cleavable linker. It enables improved control of drug release by attaching to the cytotoxic drug with mAbs (Sanderson and Hering 2005). The peptides are designed for high serum stability and rapid enzymatic hydrolysis once the mAb-conjugate is internalized into lysosomes of target cells. This approach is particularly attractive since the proteases leading to drug release are mainly intracellular, and they are not as active as them outside the cells, due to their pH optima and inhibition by serum protease inhibitors. 8. In the course of this study, the intracellular β-galactosidase-catalyzed drug release process was sufficiently efficient to produce a strong-by-stander effect. Glucuronide analogs, while selectively internalized inside folate receptor (FR)-positive cancer cells, did not conduct to the intracellular release of the drug. This observation suggested that intracellular activation of enzyme responsive drug delivery systems can be more efficient with galactoside linkers than with glucuronide linkers. 9. β-Glucuronide undergoes degradation and hydrolysis by β-glucuronidase, which is a lysosomal enzyme over expressed in many cancers and is utilized for selective payload release. Several galactoside pro-drugs were developed for the selective delivery of potent anti-cancer drugs in the course of antibody-directed enzyme pro-drug therapy (ADEPT).
5
Summary
This protocol introduces the preparations of coupling methods of antibodies and ligands for targeted drug conjugates and for targeted drug liposomes. The methods mainly consist of homobifunctional and heterobifunctional cross-linking approaches, involving various chemical modification and synthesis methods. To reach a targeting purpose, the process of antibody or ligand coupling is critical step. During selection of the coupling methods, the following factors should be considered: (i) the yield rate of the coupling reaction, (ii) the simplicity of coupling reaction, (iii) the difficulty in purifying conjugates, (iv) repeatability and reproducibility of coupling reaction, and (v) whether the bond can be biodegraded or not. An ideal coupling method should have high yield rate and homogeneity ratio. Simple operation and easy purification are preferred. So far there is no perfect coupling method that can completely meet all the requirements above. Therefore, it is important to select the appropriate coupling method based on the intended use and expected effect in actual operation process.
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References Apelgren LD, Bailey DL (1993) Chemo immunoconjugate development for ovarian carcinoma therapy: preclinical studies with vinca alkaloid- monoclonal antibody constructs. Bioconjug Chem 4(2):121–126 Apelgren LD, Zimmerman DL (1990) Antitumor activity of the monoclonal antibody-vinca alkaloid immuno conjugate LY203725 (KS1/4-4-desacetylvinblastine-3-carboxhydrazide) in a nude mouse model of human ovarian cancer. Cancer Res 50(12):3540–3544 Chabner A, Roberts TG (2005) Timeline: chemotherapy and the war on cancer. Nat Rev Cancer 5(1):65–72 Chari RVJ (1998) Targeted delivery of chemotherapeutics: tumor activated prodrug therapy. Adv Drug Deliv Rev 31(1–2):89–104 Chari RV (2008) Targeted cancer therapy: conferring specificity to cytotoxic drugs. Acc Chem Res 41(1):98–107 Chari RVJ, Jackel KA (1995) Enhancement of the selectivity and antitumor efficacy of a CC-1065 analog through immunoconjugate formation. Cancer Res 55(18):4079–4084 Dubowchik GM, Walker MA (1999) Receptor-mediated and enzyme-dependent targeting of cytotoxic anticancer drugs. Pharmacol Ther 83(2):67–123 Hamann PR, Hinman LM (2002a) An anti-CD33 antibody-calicheamicin conjugate for treatment of acute myeloid leukemia: choice of linker. Bioconjug Chem 13(1):40–46 Hamann PR, Hinman LM (2002b) Gemtuzumab ozogamicin, a potent and selective anti-CD33 antibody- calicheamicin conjugate for treatment of acute myeloid leukemia. Bioconjug Chem 13(1):47–58 Hamblett KJ, Senter PD (2004) Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res 10(20):7063–7070 Hartley JA (2011) The development of pyrrolobenzodiazepines as antitumour agents. Expert Opin Investig Drugs 20:733–744 Hinman LM, Hamann PR (1993) Preparation and characterization of monoclonal antibody conjugates of the calicheamicins: a novel and potent family of antitumor antibiotics. Cancer Res 53(14):3336–3342 Hughes B (2010) Antibody-drug conjugates for cancer: poised to deliver? Nat Rev Drug Discov 9(9):665–667 Jaracz S, Chen J (2005) Recent advances in tumor-targeting anticancer drug conjugates. Bioorg Med Chem 13(17):5043–5054 Laguzza BC, Nichols CL (1989) New antitumor monoclonal antibody-vinca conjugates LY203725 and related compounds: design, preparation, and representative in vivo activity. J Med Chem 32(3):548–555 Lee CC, Gillies ER (2006) A single dose of doxorubicin-functionalized bow-tie dendrimer cures mice bearing C-26 coloncarcinomas. Proc Natl Acad Sci 103(45):16649–16654 Liu CN, Tadayoni BM (1996) Eradication of large colon tumor xenografts by targeted delivery of maytansinoids. Proc Natl Acad Sci U S A 93(16):8618–8623 Maruyama K, Takizawa T, Yuda T, Kennel SJ, Huang L, Iwatsuru M (1995) Target ability of novel immunoliposomes modified with amphipathic poly(ethylene glycol)s conjugated at their distal terminals to monoclonal antibodies. Biochim Biophys Acta 1234(1):74 Monkovic I, Knipe JO (1991) New hydrazone derivatives of adriamycin and their immunoconjugates- a correlation between acid stability and cytotoxicity. Bioconjug Chem 2(3):133–141 Ojima I, Geng XD (2002) Tumor-specific noveltaxoid-monoclonal antibody conjugates. J Med Chem 45(2012):5620–5623 Parakh S, Parslow AC (2015) Antibody-mediated delivery of therapeutics for cancer therapy. Expert Opin Drug Deliv 13(3):401–419 Patel VF, Hardin JN (1995) Novel trityl linked drug immunoconjugates for cancer therapy. Bioorg Med Chem Lett 5(5):507–512
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Peters C, Brown S (2015) Antibody-drug conjugates as novel anti-cancer chemotherapeutics. Biosci Rep 35(4):e00225 Rameshwar P, Jose PA (2012) Cellular delivery of doxorubicin via pH-controlled hydrazone linkage using multifunctional nano ehicle based on poly(β-L-malic acid). Int J Mol Sci 13(2012):11681–11693 Russell J, Sanderson MA (2004) In vivo drug-linker stability of an anti-CD30 dipeptide-linked auristatin immune conjugate. Clin Caner Res 11(2005):843–852 Sanderson RJ, Hering MA (2005) In vivo drug-linker stability of an anti-CD30 dipeptide-linked auristatin immunoconjugate. Clin Cancer Res 11(2Pt1):843–852 Scott AM, Wolchok JD (2012) Antibody therapy of cancer. Nat Rev Cancer 12(2012):278–287 Sergii K, Chloe M (2017) Development and evaluation of β-galactosidase-sensitive antibody-drug conjugates. Eur J Med Chem 142(2017):376–382 Shefet-Carasso L, Benhar I (2015) Antibody-targeted drugs and drug resistance-challenges and solutions. Drug Resist Updat 18(2014):36–46 Shen WC, Ryser HJ (1981) Cis-aconityl spacer between daunomycin and macromolecular carriers: a model of pH-sensitive linkage releasing drug from a lysosomotropic conjugate. Biochem Bioph Res Commun 102(3):1048–1054 Srinivasachar K, Neville DM (1989) New protein cross-linking reagents that are cleaved by mild acid. Biochemistry 28(6):2501–2509 Sutherland MSK, Walter RB (2013) SGN-CD33A: a novel CD33-targeting antibody–drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML. Blood 122:1455–1463 Szakacs G, Paterson JK (2006) Targeting multidrug resistance in cancer. Nat Rev Drug Discov 5(3):219–234 Teicher BA, Chari RV (2011) Antibody conjugate therapeutics: challenges and potential. Clin Cancer Res 17(20):6389–6397 Ulbrich K, Etrych T (2003) HPMA copolymers with pH-controlled release of doxorubicin: in vitro cytotoxicity and in vivo antitumor activity. J Control Release 87(1–3):33–47 Ulbrich K, Etrych T (2004) Antibody-targeted polymer-doxorubicinconjugates with pH-controlled activation. J Drug Target 12(8):477–489 Yu Y, Wang Z-H, Zhang L, Yao H-J, Zhang Y, Li R-J, Ju R-J, Wang X-X, Zhou J, Li N, Lu W-L (2012) Mitochondrial targeting topotecan-loaded liposomes for treating drug-resistant breast cancer and inhibiting invasive metastases of melanoma. Biomaterials 33(6):1808–1820
Preparation and Evaluation of Folate Receptor Mediated Targeting Liposomes
11
Wei Gao
Contents 1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Rationale and Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
168 170 170 172 176 177
Abstract
Liposomes have been well used as an efficient delivery system for cancer therapy. Overexpressed folate receptor is a confirmed tumor-associated antigen that binds folate and folate-targeted liposomes with very high affinity. Folate receptor shuttles these bound molecules inside cells via an endocytic mechanism. It is described as an efficient folate-modified liposome preparation method in this chapter based on a post-insertion technique. Conjugate of 1,2-dipalmitoyl-snglycerophosphoethanolamine-polyethylene glycol-folate (DPPE-PEG-folate), derived from PEG with molecular masses of 2000 Da, is synthesized and characterized by 1H NMR and mass spectrometer. By micellization of folate conjugates and their controlled insertion into preformed liposomes, DOX-loaded folate-targeted liposome is prepared. The selectivity and specificity of folate target liposome is evaluated in KB, C6, and cortical cells. It was found that KB cells took up significant amounts of DOX relative to the C6 cells or relative to normal cortical tissue.
W. Gao (*) Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, West Lafayette, IN, USA e-mail: [email protected] © Springer-Verlag GmbH Germany, part of Springer Nature 2021 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49320-5_12
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Keywords
Folate · Ligand-modified targeting liposome · Cancer · Drug delivery · Post-insertion
1
Overview
Liposomes have shown considerable promise as nano-carrier for the delivery of small molecular chemotherapeutics, RNA interference agents, or imaging agent to tumors (Allen and Cullis 2013). It has been developed to accumulate selectively in the tumor tissues via a passive targeting mechanism termed the enhanced permeability and retention (EPR) effect (Fang et al. 2011; Maruyama 2011). Especially for the recent advances in the design of liposomes based on surface modification with poly(ethylene glycol) (PEG), which particle size is about 100 nm, long-circulating liposomes (Zalipsky et al. 1994; Zhang et al. 2007) have been shown to reduce recognition and phagocytosis. The first liposomal pharmaceutical product, Doxil ®, received US Food and Drug Administration (FDA) approval in 1995 for the treatment of chemotherapy refractory acquired immune deficiency syndrome (AIDS)related Kaposi’s sarcoma (Park 2002; Immordino et al. 2006). However, the liposomes still have displayed several intrinsic limitations due to the lack of specificity and selectivity to tumor tissues. The tumor cell membrane can also constitute a formidable barrier for those macromolecules that must enter their target cells to cause cell death. Lots of effects have been made to overcome the limitations. Some cancer cell-specific ligands as targeting moieties have been employed to improve delivery and retention of liposomes within the tumor tissue. Folate-based targeted liposome delivery presents an effective way of selectively delivering therapeutic or imaging agents to tumors (Lee and Low 1997). Folic acid is an essential vitamin for the synthesis of nucleotide bases, which is required in one carbon metabolic (Choi and Mason 2000). Uptake of folate into cells of the body is mediated by either the reduced folate carrier (Antony 1992; Matherly et al. 2007) or the proton-coupled folate transporter (Matherly et al. 2007). However, neither of these membrane-spanning transport proteins displays affinity for folate conjugates. Folate-linked drugs/cargos show no tendency to bind to most normal cells. Importantly, folic acid can also be internalized by a folate receptor (FR) that is expressed on surprisingly few cell types. FR consists of four well-characterized isoforms (FR-α, β, γ/γ0 , and -δ) that are 70–80 % identical in amino acid sequence but distinct in their expression patterns (Shen et al. 1994). Both FR-α and FR-β are membrane-associated proteins as a consequence of their attachment to a glycosylphosphatidylinositol (GPI) membrane anchor. FR-α is observed on the apical surfaces of several epithelial cells (Low et al. 2008) where it is inaccessible to parenterally administered folate and folate conjugates. FR- α is also overexpressed on 40 % of human cancers (Parker et al. 2005) where it is completely accessible to folate-linked drugs or folate-linked carrier. FR-β, in contrast, is expressed on activated macrophages, also on the surfaces of malignant cells of hematopoietic origin
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Cancer cell surface Direct activation of a cytotoxic function
Membrane fusion
Internalization H+ H+
Early endosomes
H+
H+
H+
H+
Recycling endosomes
H+
Acidified endosomes
Folate receptor Folic acid Linker Intracellularly active agents Extracellularly active agents
H+
H+ H+
Endosomal release
Fig. 1 Folate-mediated delivery of therapeutic agents to folate receptor-positive cancer cells (Lu and Low 2002)
(van der Heijden et al. 2009). FR-γ and FR-γ0 lack the GPI anchor and are constitutively secreted in barely detectable amounts as soluble forms of the human FR (Zhao et al. 2008). Folate and folate conjugates bind to FR-α and FR-β with high affinity (Kd 109 M) and enter FR-expressing cells by receptor-mediated endocytosis (Low et al. 2008). FR is located in caveolae and participates in the cellular accumulation of folates in many epithelial cells through the process of potocytosis (Kamen and Capdevila 1986; Kamen et al. 1988; Anderson et al. 1992a). In this process, receptor-bound ligand is sequestered in caveolae, internalized into post caveolar plasma vesicles, released from the receptor via an intravascular reduction in pH, and subsequently transported into cytoplasm for polyglutamation (Rothberg et al. 1990; Anderson et al. 1992b). The receptor is then recycled to the cell surface. As shown in Fig. 1, therapeutic agent that requires access to intracellular targets can be delivered in substantial quantities to cytosolic locations by the endocytic pathway, while drugs that can or must show function from an extracellular location will be enriched on cancer cell surfaces by the stationary population of the FR. FR is overexpressed in a wide variety of cancer types, such as lung, breast, kidney, and brain and hematopoietic cells of myelogenous origin, particularly in epithelial tumors, such as cancers of the ovary and endometrium. In addition, aggressive or undifferentiated tumors with advanced stage or grade appear to have an increased FR density (Toffoli et al. 1997). FR has been qualified as a tumor-
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specific target, since it generally becomes accessible to intravenous drugs only after malignant transformation. That is, because FR is selectively expressed on the apical membrane surface of certain epithelial cells (i.e., the membrane surface facing a body cavity), it is inaccessible to blood-borne reagents and therefore protected from FR-directed therapeutics delivered in the plasma. However, upon epithelial cell transformation, cell polarity is lost, and FR becomes accessible to targeted drugs in circulation. Because of this dual mechanism for tumor specificity, the receptor’s natural ligand, folic acid, has become a popular molecule for targeting attached drugs to cancer treatment (Lee and Low 1994; Yan and Qi 2008). The FR ligand, folate (or folic acid), is a vitamin that is used for the biosynthesis of nucleotides. High level of folate is utilized to meet the needs of proliferating cancer cells. The attractiveness of folate has been further enhanced by its high binding affinity, low immunogenicity, ease of modification, small size (MW 441.4), stability during storage, compatibility with a variety of organic and aqueous solvents, low cost, and ready availability. The attractiveness of liposomes as drug carriers was enhanced by the recent introduction of long-circulating liposomes coated with polyethylene glycol (PEG). By protecting the liposome surface from non-specific opsonization by certain plasma components, the PEG coating inhibits the recognition of the liposomes by phagocytes of the reticuloendothelial system (Papahadjopoulos et al. 1991). However, the steric hindrance introduced by the PEG coating on the liposome surface can inhibit ligand-mediated targeting of the liposome when the targeting ligand, e.g., an antibody, is directly conjugated to the lipid bilayer (Torchilin et al. 1992). While the liposomes can be efficiently targeted to receptor-bearing tumor cells when conjugated to folate via a long PEG spacer (Lee and Low 1995; Gabizon et al. 1999). In order to construct liposomal formulations bearing defined numbers of targeting ligands, the DPPE-PEG2000-folate vesicles will be incorporated into the lipid bilayer of preformed liposomes by the “post-insertion” technique (Uster et al. 1996; Iden and Allen 2001; Ishida et al. 1999) (see Fig. 2c). In this chapter, we will describe the synthesis, characterization of a 1,2-dipalmitoyl-sn-glycerophosphoethanolamine-PEG-Folate (DPPE-PEG2000-folate) conjugate (see Fig. 3), and folatemodified liposome preparation and evaluation (Saul et al. 2003).
2
Protocol
2.1
Materials
2.1.1 Synthesis of DPPE-PEG2000- Folate 1. Folic acid 2. Dicyclohexylcarbodiimide (DCC) 3. Triethylamine (TEA) 4. N-Hydroxysuccinimide (NHS) 5. 1,2-dipalmitoyl-sn-glycerophosphoethanolamine-polyethylene amine (DPPE- PEG2000-amine)
glycol2000-
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Fig. 2 Schemes for constructing targeted liposomes. Shown are three strategies for constructing targeted liposomes. (a) Traditional method of forming folate-targeted liposomes. A conjugate such as DPPE-PEG2000-folate (see Fig. 1) is synthesized and included in the thin film lipid formulation for hydration into liposomes. But the ligands are distributed in two sides of the bilayer. (b) The method of forming targeted liposomes in which targeting ligands are coupled to the termini of functionalized PEG chains on liposomes. The modification of the folate on the liposomes is not stable and efficient. (c) Post-insertion technique by which a conjugate is micellized and incubated with preformed liposomes to yield targeted liposomes. All targeting ligands are on the external leaflet of the liposomes and are available for targeting
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Methylsulfoxide (DMSO) Pyridine Sodium chloride Distilled water A dialysis bag (cutoff molecular weight 3500 Dalton) 25 mL round-bottomed flask Magnetic stirrers A vacuum pump A freeze dryer A rotary evaporator equipped with water bath 2 L glass reactor
2.1.2 Characterization of DPPE-PEG2000-Folate 1. DPPE-PEG2000-folate 2. DMSO-d6 deuterated solvent 3. Borosilicate glass tubes for nuclear magnetic resonance 4. Nuclear magnetic resonance (NMR) spectrometer 5. Quatro II triple quadrupole mass spectrometer 2.1.3 Preparation of Doxorubicin Liposomes 1. Distearoylphosphatidylcholine (DSPC) 2. Cholesterol (Chol) 3. Distearoyl phosphatidylethanolamine-monomethoxy (MW 2000) (mPEG2000-DSPE)
polyethylene
glycol
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4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Doxorubicin (DOX) DPPE-PEG2000- folate Hydration buffer: 200 mM ammonium sulfate buffer. Store at room temperature Distilled water DMSO Chloroform Sodium dodecyl sulfate (SDS) A 50 mL round-bottomed spherical quick-fit flask A rotary evaporator equipped with water bath Vacuum desiccators to remove solvent from the dried film 0.1 M sodium chloride Sephadex G-50 A dialysis bag (cutoff molecular weight 300 k MW) A vortex mixer A shaking water bath Mini-extruder UV-visible spectrometer
2.1.4 Characterization of Folate-Targeted Doxorubicin Liposomes 1. Folate-targeted doxorubicin liposomes 2. Distilled water 3. Malvern Zetasizer Nano ZS 4. Formvar-coated copper grid 5. Uranyl acetate solution (1 %, w/v) 6. Transmission electron microscope 2.1.5 1. 2. 3. 4. 5. 6. 7.
Cellular Uptake Evaluation of Folate-Targeted Doxorubicin Liposomes Cell growth medium: RPMI 1640 folate-free medium supplemented with 10 % fetal bovine serum (FBS) and 1 %(v/v) penicillin-streptomycin solution 100 C6 and KB and primary cortical neurons cell lines Cell culture flask (T-75) 0.25 % trypsin Collagen-coated 35 mm six-well plates CO2 incubator Fluorescence spectrometer
2.2
Rationale and Procedures
2.2.1 Synthesis of DPPE-PEG2000-Folate 1. Weigh out 17.7 mg of folate acid (0.038 mmol) and put it in a 25 mL roundbottomed flask. 2. Add 66.7 μL of DMSO to dissolve the folate acid in the flask.
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3. Weigh out 66.7 mg of DPPE-PEG-amine and 21.7 mg of DCC, dissolve in 333 μL of pyridine, and then add it into the reaction solution. 4. Keep the reaction solution stirred for 4 h at room temperature. 5. Remove the pyridine by rotary evaporation. 6. Add 16.7 mL of water to the solution. 7. Add the resulting reaction solutions into a dialysis bag (3500 MW), dialyze it in a 2 L glass reactor with 2 L of 50 mM sodium chloride, and change the outside sodium chloride solution every 6 h for 2 times; then dialyze it with 2 L of distilled water and change the water every 6 h for 3 times. 8. Freeze-dry the purified DPPE-PEG2000-folate using a freeze dryer, yielding 66.6 mg (92 % yield).
2.2.2 Characterization of DPPE-PEG2000-Folate 1. Add 2 mg of DPPE-PEG2000-folate into a borosilicate glass tube which is specially used for nuclear magnetic resonance. 2. Add 2 mL of DMSO-d6 deuterated solvent into the tube to dissolve the materials. 3. Characterize the materials by 1H-NMR. 4. Analyze the materials by mass spectroscopy on a Quatro II triple quadrupole mass spectrometer with ionization performed by electrospray ionization in the positive mode. Analysis of the product by H NMR showed peaks for DPPE [0.84 ppm (t), 1.2, 1.5 (d), 2.25(d), 2.9(t), 3.1(t), 5.04(m)], PEG [3.3 ppm], and folic acid [1.91, 2.03, 2.3 (t), 4.33 (m), 4.48(d), 6.5 (d), 6.93 (t), 7.64(d), 8.12 (d), 8.6 (s)]. Mass spectra for the starting materials found amass of 441 for folic acid and 2716 for DPPE-PEG-amine. The molecular weight of the product was 3144 mass units.
2.2.3
Preparation of Folate-Targeted Doxorubicin Liposomes
Liposome Formation 1. Weigh 16 mg DSPC, 3.92 mg Chol, 3.98 mg mPEG2000-DSPE (with mole ratio 2:1:0.14), dissolve in 10 mL ethanol in a 50 mL round-bottomed flask at 60 C. 2. Set the round-bottomed flask in a rotary evaporator. The organic solvent will evaporate under reduced pressure. 3. After a dried thin film formed on the wall of the flask, keep the flask further dried under vacuum for extra 4–6 h to eliminate the residual solvent. 4. Add 10 mL of ammonium sulfate buffer to the dry film, vortex for 5 min, and then shake for 1 h at 65 C to form the multilayer vesicles (MLV). 5. Extrude the liposomes on a 10 mL Lipex Thermoline extruder with five passes through a 0.2 μm nucleopore membrane (Whatman, Newton, MA) and ten passes through a 0.1 μm membrane. 6. Filter the liposomes over a Sephadex G-50 column, eluted with 0.1 M sodium chloride.
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H2 N
N
O
O
N
N
N H
O
HN
N
Folic Acid
OH
O
+
CH2
O
14
CH2 14
O
O O P O OH
NH O
NH2
45
O
OH
DPPE-PEG2000-amine DCC in DMSO/pyridine Room Temperature 4 hours
O CH2
O
14
CH2 14 O
O
O O P O OH
O NH O
N
45H
HO
O NH O
N H N
N
NH2
N N OH
Fig. 3 Schematic of synthesis of DPPE-PEG2000-folate. The compound was synthesized by dicyclohexylcarbodiimide (DCC) chemistry linking the amine group of DPPE-PEG-amine and the γ-carboxyl of folic acid
Micellization of DPPE-PEG2000-Folate and Insert the Micelle into Preformed Liposomes 1. Weigh out 2 mg of DPPE-PEG2000-folate and dissolve in 637 μL of DMSO. 2. Add 5.7 mL of distilled water in DPPE-PEG2000-folate DMSO solution, giving a solution with 10 % DMSO. 3. Add 100 μM of micelle suspension into a dialysis bag (30 k MW). Dialyze it with 2 L of distilled water and change the outside water every 4 h for three times to remove DMSO. 4. DPPE-PEG2000-folate micelles were lysed in 10 % SDS, and the UV absorbance was measured at 285 nm wavelength on a UV-visible spectrometer. The total amount of folate and the number of folate molecules per liposome were determined by comparison of the UV 285 reading to a standard curve of folic acid and the known lipid concentration. 5. Add 2 mL of 15 mm liposomes (10 mM DSPC, 5 mM cholesterol) solution in a 25 mL round-bottomed flask. Add micellized DPPE-PEG2000-folate at a lipid concentration of 100 μM to the liposomal suspension and heat at 60 C for 1 h. 6. Cool the liposomes on ice immediately. 7. Add the liposomes suspension into a dialysis bag (300 k MW). Dialyze it with 2 L of distilled water overnight to remove unincorporated DPPE-PEG2000-folate from the external volume of the liposome suspension. 8. The folate content of the liposomes was determined by lysing the liposomes with 10 % SDS and measuring the UV absorbance at 285 nm.
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Folate-Targeted Doxorubicin Liposomes 1. Mix folate liposomes and 2 mg/mL DOX at a ratio of 0.1 mg DOX per 1 mg of DSPC in the folate liposomes. 2. Heat the liposome/DOX suspension at 60 C for 1 h. 3. Cool the liposomes on ice immediately. 4. Dialyze the liposomes a dialysis bag (300 k MW) against 0.1 M sodium chloride to remove unencapsulated DOX.
2.2.4 Characterization of Folate-Targeted Doxorubicin Liposomes 1. Dilute 0.5 mL of folate-targeted doxorubicin liposomes with distilled water to 1 mL. 2. Measure the mean diameter, particle distribution, and Zeta potential of folatetargeted doxorubicin liposomes by dynamic light scattering using a Malvern Zetasizer Nano ZS (Zetasizer 3000HS, Malvern, Worcestershire, UK) at 25 C. The average diameter of extruded liposomes is about 130 nm. 3. Dilute 20 μL of folate-targeted doxorubicin liposomes with distilled water to 200 μL. 4. Deposit the droplets of dilution on the surface of a formvar-coated copper grid, followed by a negative staining method using uranyl acetate solution (1 %, w/v), before being air-dried overnight at room temperature. 5. Characterize the morphology of the folate-targeted doxorubicin liposomes by a transmission electron microscope (JEOL, JEM-200CX, Japan). 2.2.5 1.
2. 3. 4. 5.
6. 7. 8. 9. 10.
Cellular Uptake Evaluation of Folate-Targeted Doxorubicin Liposomes Culture C6 and KB cell lines in RPMI 1640 folate-free medium supplemented with 10 % FBS and 1 % pen-strep in T-75 flasks. Passage the cells with trypsin following by two washings with RPMI 1640 folate-free medium. Obtain the cortical neurons from day 9 (E9) chick embryos. Remove the two frontal lobes of the brain, mince, and dissociate with 0.25 % trypsin. Plate the cells into collagen-coated 35 mm six-well plates. Remove the C6 and KB cell lines from the culture medium. Wash the cells once with PBS, then add 1 mL RPMI 1640 folate-free medium to each well. Add free form DOX (free DOX) and folate-targeted doxorubicin liposomes with DOX amount of 100 μg to each well. A group of folate targeted DOX liposomes are coincubated with an excess of free folic acid at a 1 mM concentration. Incubate the cells for 2 h with the DOX mixture at 37 C in a 5 % CO2 humidifier air. Place the cells on ice after incubated for 2 h. Wash three times with 1 mL of ice-cold PBS containing calcium and magnesium to remove extracellular DOX. Lyse the cells with 2 mL of 5 % triton X-100. Measure the fluorescence intensity of the lysed cell solution on a fluorescence spectrometer at an excitation/emission of 475/580 nm.
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Fig. 4 Competitive binding assay in three cell types with folate-targeted liposome and 1 mM free folic acid
1 mM free folic acid significantly reduced the DOX uptake in KB and C6 cells targeted with folate liposomes but had no significant effect in reducing DOX uptake in cortical cells targeted with folate liposomes. KB cells show a 78 % reduction in the amount of cell-associated DOX when incubated with 1 mM free folic acid. C6 cells show a reduction of 58 % in cell-associated DOX uptake. E9 cortical cells show no reduction in cell-associated DOX (Fig. 4).
3
Notes
1. The conjugate DPPE-PEG2000-folate is synthesized under an anhydrous condition, away from light. 2. All the lipids should be stored under 20 C. Prior to weighing, lipids should be brought to room temperature. 3. Add DMSO to dissolve folic acid completely before adding DCC. 4. Thin-layer chromatography could be used to supervise the reaction. 1.48 N ammonium hydroxide is prepared as mobile phase. DPPE-PEG-amine starting material and folic acid are run as controls along with the product (DPPE-PEGFolate). Samples are diluted in 0.3 N ammonium hydroxide and spotted on the plate. The presence of phospholipids can be determined by spraying cupric sulfate on the plate. The absence or presence of free amine is resolved separately by ninhydrin spray. 5. Store the dried DPPE-PEG-folate in a 20 C freezer.
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6. Use a water bath sonication if the vortex mixer can’t shake the lipid film off the flask. Set the temperature in the water bath at 60 C. 7. Conduct the extrusion progress as the temperature at 60 C. 8. During the liposome preparation, cool the liposomes on ice immediately after heating at 60 C for 1 h. 9. The procedures involving DOX should be performed away from light. 10. The folate-targeted doxorubicin liposomes should be suitably diluted with distilled water to avoid multi-scattering phenomena before measuring the particle size with the Malvern Zetasizer Nano ZS. 11. Dilute the folate-targeted doxorubicin liposomes with 20 mM phosphate buffer (PB) at pH 7.4 as a ratio of 1:1 before measuring the Zeta potential with the Malvern Zetasizer Nano ZS. Because the measurement is varied accordingly to the ionic strength. 12. Cortical cells need to be cultured on collagen-coated wells to attach, spread, and more accurately reflect their in vivo morphology. 13. Cortical cells need to be incubated for 12–20 h longer than C6 and KB cells after planting in the cell culture plates in the cellular uptake experiments. 14. Typically, about 3 106 cells are seeded in each well of a six-well cell culture plate. 15. Cellular uptake study is designed to saturate folate receptors to prevent receptorspecific binding by folate liposomes. Optimize the concentration of folic acid and pre-incubation time, depending on the experiment.
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Preparation of Cell Penetrating PeptidesMediated Targeting Drug Liposomes
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Yang Wang and Qin He
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Synthesis of DSPE-PEG2000-R8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Preparation of Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Cell Culture, Cellular Uptake, and Cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Rationale and Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Synthesis of DSPE-PEG2000-R8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Preparation of Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Determination of Particle Size and Zeta Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Morphological Examination of Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Stability of Liposomes in Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 PTX Release Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Cellular Uptake Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 Cytotoxicity Study In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Y. Wang Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Chengdu, Sichuan, China Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden e-mail: [email protected] Q. He (*) Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Chengdu, Sichuan, China e-mail: [email protected]; [email protected] © Springer-Verlag GmbH Germany, part of Springer Nature 2021 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49320-5_13
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Abstract
Biological membrane is the inevitable barrier needed to cross when liposomes try delivering drugs into cells, tissues, and organs for their biological actions. One of the most promising methods to overcome this barrier is based on the use of cell penetrating peptides (CPPs). Development of CPPs mediated targeting drug liposomes has focused on creating approaches for delivering bioactive molecules, especially for proteins and gene drugs, to cells and for enhancing their stability in vitro and in vivo. The most common type of various CPPs is cationic CPPs, which are positively charged and can enter cells when added exogenously. Here, we describe a mature approach based on thin film hydration method for cationic CPPs modified targeting drug liposomes (drug/CPPs-Lip) preparation. The formation of drug/CPPs-Lip depends on a number of experimental variables. The grafting method and location as well as the modification density of CPPs on liposomes strongly affect the activity and transmembrane capability of CPPsdrug/Lip. In addition, the control of experimental parameters such as temperature, hydration time, and sonicating frequency influences the final characteristics of the CPPs-drug/Lip, such as size, surface zeta potential, drug encapsulation efficiency, drug loading amount, stability, and reproducibility. The method presented in this chapter could be helpful to prepare reliable and reproducible drug/CPPs-Lip and according experimental results. Keywords
Cell penetrating peptides · Liposomes · Paclitaxel · Cellular uptake · Polyarginine · Thin film hydration method
1
Introduction
Over past years, the study field of cell penetrating peptides (CPPs) has been developed rapidly. CPPs represent one of the most promising tools for the delivery of biologically active molecules into cells and therefore play an important role in future exploration of therapeutics. It has been well-recognized that CPPs are peptides consisting of 5–30 amino acids that can cross cellular plasma membranes (Milletti 2012). Among various kinds of CPPs, cationic CPPs are the most common ones (Richard et al. 2003; Patel et al. 2007) which are always divided into two classes: the first class consists of amphipathic alpha-helical peptides, such as transportan and model amphipathic peptide (MAP), where lysine (Lys) is the main contributor to the positive charge, while the second class includes arginine (Arg)-rich peptides, such as R8 and TAT (Hallbrink et al. 2001; Torchilin 2008; Gupta et al. 2004). They themselves can serve as a vector to efficiently improve intracellular delivery of small molecules and various biomolecules, including plasmid DNA, siRNA (short interfering RNA), proteins, and peptides into cells and tissues both in vitro and in vivo (Huang et al. 2013; Farkhani et al. 2014; Said
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Hassane et al. 2010; Pappalardo et al. 2009). On the other hand, what researchers pay special attention to is that CPPs can also orient nanosized drug carrier systems, including liposomes, nanoparticles, micelles, into cells after proper surface modification, propelling the progress of novel drug carrier systems (Kuai et al. 2010; Vives 2005). Among all drug carriers, the most popular and well-investigated are liposomes (for the delivery of both water-soluble and poorly soluble drugs) (Zhang et al. 2013, 2015). The researches in the field of CPPs which mediated targeting drug liposomes (drug/CPPs-Lip) have been being conducted extensively – almost 400 research papers on this small subject were published from 2010 to 2015 (statistic data on Web of Science). With the evolution of liposomes in drug delivery, the appearance of long-circulating liposomes should be the milestone to promote liposomal drugs to clinical applications. They could be obtained by coating the liposomes surface with hydrophilic polymers, such as PEG, which form a protective corona over the liposome surface and slow down liposome recognition by opsonins. Nevertheless, these polymers seriously hinder the uptake of liposomes by targeting cells (Gullotti and Yeo 2009). Facing this dilemma, CPPs are used to be covalently attached to the liposome surface. For example, R8 was covalently attached to the distal end of PEG chains anchored to the surface of liposomes in several researches (Iwasa et al. 2006; Zhang et al. 2006; Liu et al. 2014). The cellular uptake of these R8 modified liposomes is generally increased significantly, improving the concentration of drugs loaded by liposomes within cells. Although charge adsorption method for CPPs modification on liposomes was also often used, the stability of liposomes and the modification amount of CPPs could not be well controlled. Thus, this covalently linked modification method has been widely used for studies of CPP delivery efficiency liposomes since its reliability. In this protocol, R8 was chosen as the model CPP to modify PGEylation liposomes, Paclitaxel (PTX) was used as a model drug, and the detailed preparation process was presented to ensure the reproduction of this kind of liposomes. This protocol can also be applied to prepare other CPPs-modified liposomes via covalent linking.
2
Materials
2.1
Materials
1. R8 peptide with a terminal cysteine (Cys-R8) (see Note 1). 2. Distearoyl-sn-glycero-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG2000-Mal). 3. Distearoyl-sn-glycero-phosphatidylethanolamine-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000-OCH3). 4. Soybean phospholipids (SPC). 5. Cholesterol. 6. 2-dioleoyl-snglycero-3-phosphoethanolamine-N-(carboxyfluorescein) (CFPE). 7. Paclitaxel. 8. All other reagents were of analytical grade.
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2.2 1. 2. 3. 4. 5.
Synthesis of DSPE-PEG2000-R8
Chloroform, triethylamine, methanol, water (see Note 2) A 25-mL round-bottomed spherical Quickfit flask A magnetic stirrer, oil bath Bag filter (MWCO = 3500) (see Note 3) A lyophilizer
2.3 1. 2. 3. 4. 5. 6. 7.
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Preparation of Liposomes
25-mL eggplant-shaped bottles A rotary evaporator, water bath A probe sonicator Phosphate-buffered saline (PBS) Malvern Zetasizer Nano ZS90 HPLC system Transmission electron microscope (TEM)
2.4
Cell Culture, Cellular Uptake, and Cytotoxicity
1. For mice melanoma B16F10 cells, Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL streptomycin, and 100 U/mL penicillin 2. For mice breast cancer 4 T1 cells, RPMI-1640 medium supplemented with 10% FBS, 100 U/mL streptomycin, and 100 U/mL penicillin 3. A flow cytometer 4. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
3
Rationale and Procedures
3.1
Synthesis of DSPE-PEG2000-R8
DSPE-PEG2000-R8 was synthesized by conjugating the cysteine residue of Cys-R8 to maleimide group of DSPE-PEG2000-Mal via Michael addition reaction as shown in Fig. 1. Other CPPs such as TAT, GALA, and MAP could also be covalently linked with DSPE through this kind of reaction. 1. DSPE-PEG2000-Mal (10 mg) was dissolved in chloroform (4 mL), and triethylamine (2 μL) was added (see Note 4). 2. Cys-R8 (molar ratio of DSPE-PEG2000-Mal/Cys-R8 = 1/1.5) was dissolved in methanol (2 mL) (see Note 5).
Preparation of Cell Penetrating Peptides-Mediated Targeting Drug Liposomes
Fig. 1 Synthesis of DSPE-PEG2000-R8
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3. Methanol solution of Cys-R8 was added into the chloroform solution of DSPEPEG2000-Mal, and the mixture was gently stirring for 24 h in darkness at 25 C with the protection of nitrogen. 4. Thin layer chromatography is used to monitor the extent of the reaction. 5. After TLC showed the disappearance of DSPE-PEG2000-Mal, the organic solution was evaporated by rotary evaporation under vacuum. 6. The residue was redissolved by chloroform and the solution was dialyzed in water to purify production. 7. The purified solution was lyophilized to collect DSPE-PEG2000-Mal (see Note 6). 8. The existence of DSPE-PEG2000-Mal was confirmed by mass spectrometry (see Note 7) and nuclear magnetic resonance spectroscopy (see Note 8).
3.2
Preparation of Liposomes
Liposomes were prepared by thin film hydration method. Lipophilic drugs could be loaded within the lipid bilayer of liposomes during the film formation, while hydrophilic drugs could be encapsulated within the aqueous lumen of liposomes during the hydration of the film or after the formation of blank liposomes. Here, PTX, as a lipophilic drug, was chosen as the model drug for drug-containing liposomes preparation. 1. Cholesterol, SPC, DSPE-PEG2000, and DSPE-PEG2000-R8 (molar ratio = 33: 62: 4.2: 0.8) were dissolved in chloroform (see Note 9). 2. The organic solvent was removed by rotary evaporation to form a lipid film (see Note 10). 3. The film was further dried and stored in vacuum overnight. 4. The thin film was hydrated in PBS (pH 7.4) under 37 C for 20 min (see Note 11). 5. The hydrated film is further intermittently sonicated by a probe sonicator at 80 W for 80 s (see Note 12) to form the R8 modified liposome (R8-Lip) as shown in Fig. 2.
Fig. 2 Schematic illustration of traditional PEGylation liposomes (PEG-Lip), R8 modified liposomes (R8-Lip), and CFPE or PTX loaded R8-Lip (CFPE/R8-Lip or PTX/R8-Lip)
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6. The conventional PEGylated liposome (PEG-Lip) was prepared as described above with the DSPE-PEG2000-R8 in the lipid composition being replaced by DSPE-PEG2000. 7. For the preparation of CFPE-loaded liposomes, appropriate amount of CFPE was also added to the solution before the solvent evaporation (the final concentration of CFPE within the liposomes system was 20 μg/mL) (see Note 13). 8. PTX-loaded liposomes were prepared with PTX added to the lipid organic solution prior to the solvent evaporation. 9. The encapsulation efficiency of CFPE or PTX was measured by HPLC.
3.3
Determination of Particle Size and Zeta Potential
1. After diluting liposomes to an appropriate concentration with PBS (pH 7.4), the cumulative particle size of liposomes was measured by Malvern Zetasizer Nano ZS90 instrument (a dynamic light scattering instrument) as shown in Table 1. 2. After diluting liposomes to an appropriate concentration with PBS (pH 7.4), the zeta potential of liposomes was also measured by Malvern Zetasizer Nano ZS90 instrument as shown in Fig. 3. Table 1 Particle sizes of different liposomes (n = 3, mean SD) PEG-lip R8-lip CFPE/PEG-lip CFPE/R8-lip PTX/PEG-lip PTX/R8-lip
Size (nm) 101.62 0.5 101.91 1.3 109.36 5.6 110.36 7.5 107.26 7.2 113.56 5.2
Fig. 3 Zeta potentials of different liposomes under pH 7.4 (n = 3, mean SD). After the decoration of R8 on the surface of liposomes, the positive charge of R8 under pH 7.4 transformed the negative zeta potential of PEG-Lip, CFPE-Lip, and PTX/PEG-Lip to electropositivity
PDI 0.181 0.005 0.243 0.006 0.189 0.008 0.192 0.002 0.186 0.011 0.126 0.013
Entrapment efficiency (%) / / 96.36 2.06 97.12 2.11 94.62 1.98 93.26 2.71
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Fig. 4 Size image of PTX/ R8-Lip observed under TEM
3.4
Morphological Examination of Liposomes
1. Liposomes are negatively stained with sodium phosphotungstate solution. 2. Transmission electron microscope (TEM) is used for the morphological examination as shown in Fig. 4.
3.5
Stability of Liposomes in Serum
1. Liposomes were mixed with equal volume of FBS under 37 C with gentle shaking at 30 rpm. 2. At predetermined time points (1 h, 2 h, 4 h, 8 h, and 24 h), 200 μL of the sample was pipetted out and onto a 96-well plate to measure the transmittance at 750 nm by a microplate reader as shown in Fig. 5a. 3. Another 200 μl was diluted to 1 mL with PBS for the particle size measurements by Malvern Zetasizer Nano ZS90 instrument as shown in Fig. 5b.
3.6
PTX Release Study
The drug release of PTX-loaded liposomes was determined by the dialysis method. 1. The free PTX, PTX/PEG-Lip, or PTX/R8-Lip were dispersed in 1 mL of PBS and sealed tightly in dialysis tubes (MW = 8000). 2. The dialysis tubes were immersed in 40 mL of PBS containing 1% Tween-80 and incubated under 37 C with shaking of 75 rpm for 48 h. 3. A volume of 100 μL of release media was taken out and replaced with the same volume of fresh release media at predetermined time intervals. 4. The concentration of released PTX was determined by HPLC as shown in Fig. 6.
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Fig. 5 The fluctuation in particle sizes of liposomes in 50% FBS (a) and the variation in turbidity (represented by transmittance) of liposomes in 50% FBS (b) (n = 3, mean SD). The particle sizes and transmittance had hardly changed for liposomes over 24 h, indicating that there was no aggregation in the presence of serum. This should be owing to the PEGylation that stabilized the liposomes
Fig. 6 PTX release percentage of free PTX, PTX/PEG-Lip, and PTX/R8-Lip over 48 h (n = 3, mean SD). Free PTX exhibited a rapid release of drug in the media, while PTX/PEG-Lip and PTX/R8-Lip displayed a temperate release rate. After 48 h, the percentages of PTX released in PTX/ PEG-Lip and PTX/R8-Lip were close to 60%, and no statistical difference was obtained between PTX/PEG-Lip and PTX/R8-Lip
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Fig. 7 Cellular uptake of CFPE-labeled liposomes with different concentrations (0.2 μg/mL, 0.4 μg/mL, 0.6 μg/mL) on B16F10 cells and 4 T1 cells for 2 h detected by a flow cytometry (n = 3, mean SD). R8 increased the cellular uptake of R8-Lip on both cells compared to the PEGLip
3.7
Cellular Uptake Study
1. Cells are seeded onto 6-well plate at an appropriate density allowed for attachment to the plate under 37 C for 24 h. 2. Then CFPE-labeled liposomes are added at a final CFPE concentration of 2 μg/ mL, and liposome-free culture medium is applied as the control group. 3. After incubation under 37 C for 2 h, the cells are washed with cold PBS for three times, and are trypsinized and resuspended in 0.4 mL PBS. 4. The fluorescent intensity of cells is measured by a flow cytometer with the excitation wavelength at 495 nm and the emission wavelength at 515 nm. Ten thousand events were recorded for each sample. The resulted is shown in Fig. 7.
3.8
Cytotoxicity Study In Vitro
The cytotoxicity of PTX-loaded liposomes was measured through MTT assay. 1. B16F10 cells and 4 T1 cells were plated in 96-well plates at an appropriate density cultured for 48 h. 2. Free-PTX, PTX/PEG-Lip, and PTX/R8-Lip were diluted to predetermined concentrations with PBS, and added into each well for 24 h incubation. The final concentrations of PTX were in the range of 0.1–20 μg/ml. 3. 20 μL MTT (5 mg/ml in PBS) was added into each well and incubated for 4 h under 37 C.
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Fig. 8 Cytotoxicity study of blank liposomes and PTXloaded liposomes on B16F10 cells (a) and 4 T1 cells (b) (n = 3, mean SD). The cell viability was all above 85% after B16F10 cells and 4 T1 cells culturing with different concentrations of blank liposomes for 48 h, indicating that the materials constituting liposomes did not show obvious cytotoxicity to both cells. Compared with PTX/ PEG-Lip, PTX/R8-Lip showed a better efficacy to kill cells because of the cellular uptake promotion function of R8
4. The medium was removed and replaced with 150 ml dimethyl sulfoxide. Then the absorbance was measured by a microplate reader at 570 nm. The cells treated with medium were evaluated as controls. 5. Cell viability (Fig. 8) was calculated by the following formula: Cell viability (%) = Atreated/Acontrol 100, in which Atreated and Acontrol represented the absorbance of treated cells and control cells, respectively.
4
Notes
1. The hydrosulfide group ( SH) in the Cys of Cys-R8 was the active group for the next steps, and Cys-R8 was synthesized according to the standard solid phase peptide synthesis by professional biotech company. 2. Unless stated otherwise, water and all aqueous solution should be used in Milli-Q water. 3. The bag filter was not limited to that with MW = 3500. Bag filters with MW > MWR8 could be used.
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4. Triethylamine was added as a catalyst, and its amount could be adjusted according to situations but should not be more than 5 μL. 5. R8-Cys could not be well dissolved in chloroform, so it should be dissolve in other organic solvents such as methanol before its mixture with chloroform solution of DSPE-PEG2000-Mal. 6. The production DSPE-PEG2000-R8 was stored under 20 C. 7. MWDSPE-PEG2000-R8 calculated = 4312 Da. 8. The appearance of R8 peaks in the spectroscopy result of DSPE-PEG2000-R8 indicated the successful synthesis. 9. The modifying density of R8 on the surface of liposomes could be controlled via adjusting the molar ratio of (cholesterol + SPC + DSPE-PEG2000)/DSPEPEG2000-R8. 10. The temperature of water bath should be controlled between 25 C and 40 C, and the formed film should look uniform and transparent. The thickness of films could be adjusted via varying the total amount of lipodic materials. 11. This process was accomplished by a shaker, and the rotating speed was better to maintain within the range of 150 rpm–200 rpm. 12. Ultrasonic power and time could be appropriately changed according to the situations to ensure the opalescent status of final liposomal solutions. 13. CFPE is a kind of lipophilic fluorescent dye to label the lipid bilayer of liposomes, and the free CFPE not encapsulated into the lipid bilayer was separated by eluting the liposome aqueous system over a Sephadex G-50 column (1 cm 25 cm) with PBS.
5
Conclusion
Via using this protocol, CPP (here is R8) could be successfully modified on the surface of PGEylation liposomes. Further, drug-loaded liposomes could be armed by CPP to improve their cellular uptake ability. The method presented in this chapter could be helpful to prepare reliable and reproducible drug/CPPs-Lip and according experimental results. This protocol can also be applied to prepare other CPPsmodified liposomes via covalent linking.
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Huang Y, Jiang Y, Wang H, Wang J, Shin MC, Byun Y, He H, Liang Y, Yang VC (2013) Curb challenges of the “Trojan Horse” approach: smart strategies in achieving effective yet safe cell penetrating peptide-based drug delivery. Adv Drug Deliv Rev 65(10):1299–1315 Iwasa A, Akita H, Khalila I, Kogurea K, Futakic S, Harashimaa H (2006) Cellular uptake and subsequent intracellular trafficking of R8-liposomes introduced at low temperature. Biochim Biophys Acta Biomembr 1758(6):713–720 Kuai R, Yuan W, Qin Y, Chen H, Tang J, Yuan M, Zhang Z, He Q (2010) Efficient delivery of payload into tumor cells in a controlled manner by TAT and Thiolytic cleavable PEG comodified liposomes. Mol Pharm 7(5):1816–1826 Liu Y, Ran R, Chen J, Kuang Q, Tang J, Mei L, Zhang Q, Gao H, Zhang Z, He Q (2014) Paclitaxel loaded liposomes decorated with a multifunctional tandem peptide for glioma targeting. Biomaterials 35(17):4835–4847 Milletti F (2012) Cell-penetrating peptides: classes, origin, and current landscape. Drug Discov Today 17(15–16):850–860 Pappalardo J, Quattrocchi V, Langellotti C, Di Giacomo S, Gnazzo V, Olivera V, Calamante G, Zamorano P, Levchenko T, Torchilin VP (2009) Improved transfection of spleen-derived antigenpresenting cells in culture using TATp-liposomes. J Control Release 134(1):41–46 Patel LN, Zaro JL, Shen WC (2007) Cell penetrating peptides: intracellular pathways and pharmaceutical perspectives. Pharm Res 24(11):1977–1992 Richard JP, Melikov K, Vives E, Ramos C, Verbeure B, Gait MJ, Chernomordik LV, Lebleu B (2003) Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. J Biol Chem 278(1):585–590 Said Hassane F, Saleh AF, Abes R, Gait MJ, Lebleu B (2010) Cell penetrating peptides: overview and applications to the delivery of oligonucleotides. Cell Mol Life Sci 67(5):715–726 Torchilin VP (2008) Cell penetrating peptide-modified pharmaceutical nanocarriers for intracellular drug and gene delivery. Pept Sci 90(5):604–610 Vives E (2005) Present and future of cell-penetrating peptide mediated delivery systems: “is the Trojan horse too wild to go only to Troy?”. J Control Release 109(1–3):77–85 Zhang C, Tang N, Liu X, Liang W, Xu W, Torchilin VP (2006) siRNA-containing liposomes modified with polyarginine effectively silence the targeted gene. J Control Release 112(2): 229–239 Zhang Q, Tang J, Fu L, Ran R, Liu Y, Yuan M, He Q (2013) A pH-responsive α-helical cell penetrating peptide-mediated liposomal delivery system. Biomaterials 34(32):7980–7993 Zhang Q, Gao H, He Q (2015) Taming cell penetrating peptides: never too old to teach old dogs new tricks. Mol Pharm 12(9):3105–3118
Preparation of Multifunctional Paclitaxel Liposomes for Treatment of Brain Glioma
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Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Synthesis of Targeting Molecule Conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Preparation of Multifunctional Paclitaxel Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Measurement of Encapsulation Efficiency of Paclitaxel and Artemether . . . . . . . . . . 2.4 Measurement of Particle Size, Zeta Potential, and Polydispersity Index (PDI) . . . . 2.5 Morphology Characterization with Atomic Force Microscope (AFM) . . . . . . . . . . . . 2.6 In Vitro Release of Paclitaxel or Artemether . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8 Cytotoxic Effects on Brain Cancer Cells and Brain CSCs . . . . . . . . . . . . . . . . . . . . . . . . . 2.9 Transport Across the BBB and Targeting of Brain Cancer Cells . . . . . . . . . . . . . . . . . . . 2.10 In Vivo Imaging in Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.11 Anticancer Efficacy in Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Synthesis of Targeting Molecule Conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Method for Determination of Paclitaxel and Artemether . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Preparation of Multifunctional Paclitaxel Liposomes by Thin-Film Hydration . . . . 3.4 Measurement of Encapsulation Efficiency of Paclitaxel or Artemether . . . . . . . . . . . . 3.5 Measurement of Particle Size, Zeta Potential, and Polydispersity Index (PDI) . . . . 3.6 Morphology Characterization with AFM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 In Vitro Release of Paclitaxel or Artemether . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9 Cytotoxic Effects on Brain Cancer Cells or Brain CSCs . . . . . . . . . . . . . . . . . . . . . . . . . . .
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X.-Y. Li (*) Department of Traditional Chinese Materia Medica, Shanxi University of Chinese Medicine, Jinzhong, China e-mail: [email protected] © Springer-Verlag GmbH Germany, part of Springer Nature 2021 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49320-5_9
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3.10 Transport Across the BBB and Targeting of Brain Cancer Cells . . . . . . . . . . . . . . . . . . . 3.11 In Vivo Imaging in Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.12 Anticancer Efficacy in Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Invasive brain glioma is the most lethal type of cancer with highly infiltrating nature. It leads to an extremely poor prognosis and makes complete surgical removal of the tumor virtually impossible. Paclitaxel shows antitumor activities against invasive gliomas. However, the efficacy of paclitaxel against gliomas is limited by its poor solubility, non-penetration across the blood-brain barrier (BBB), and the resistance of brain glioma cells. A single medication cannot obtain optimal efficacy, while a combinational drug therapy may overcome this issue. This report focuses on liposomal formulations by incorporating paclitaxel and artemether and describes the preparation and characterizations of multifunctional paclitaxel liposomes, which are designed to treat brain glioma along with eliminating the cancer stem cells. In this liposomal drug delivery system, paclitaxel is used as the anticancer drug and artemether is used as the regulator; two functional materials (MAN-TPGS1000 and DQA-PEG2000-DSPE) are synthesized and used as functional materials by modifying onto the surface of the liposomes; egg phosphatidylcholine (EPC) and cholesterol are used as liposomal materials. The multifunctional paclitaxel liposomes, prepared by thin-film hydration, are about 80 nm, nearly electrically neutral and round with relatively smooth surfaces. The encapsulation efficiencies of paclitaxel or artemether are >80%. The multifunctional paclitaxel liposomes exhibit long circulation time, strong ability of transporting across BBB, and strong inhibitory effect in the brain glioma-bearing rats. Keywords
Multifunctional liposomes · MAN-TPGS1000 · DQA-PEG2000-DSPE · Paclitaxel · Artemether · Thin-film hydration · Brain glioma
1
Introduction
Invasive brain glioma is the most lethal type of cancer with highly infiltrating nature. It leads to an extremely poor prognosis and makes complete surgical removal of the tumor virtually impossible. The microtubule-targeting drug paclitaxel shows antitumor activities against various solid tumors, such as ovarian, lung, breast, head, and neck cancers, Kaposi’s sarcoma, and gliomas (Ai et al. 2016; Floyd et al. 2015; Zhan et al. 2010). However,
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the efficacy of commercial paclitaxel formulations against gliomas is far from satisfactory due to the following reasons. Firstly, glioma cells exhibit major resistance to paclitaxel by changing the cell microtubule protein, expressing the multidrug resistant gene and the excessive expression of ABC transporters family (especially P- glycoprotein) (Yao et al. 2011). Secondly, paclitaxel cannot penetrate the blood-brain barrier (BBB) (Postma et al. 2000). Thirdly, the poor solubility of paclitaxel and the nontargeted property of its formulations seriously affect its antitumor activity. In these regards, a single medication of paclitaxel cannot obtain optimal efficacy. Combination drug therapy has attempted to overcome this problem in the clinical treatment (Wang et al. 2006; Wiradharma et al. 2009). Artemether is a potent and rapid agent for the treatment of severe resistant malaria, including multiple drug-resistant falciparum malaria and cerebral malaria (Hien and White 1993). In addition, artemether also demonstrates cytotoxic activities against tumor cells (Nakase et al. 2008). Mechanism studies show that artemether exhibits the potential in downregulating the expression of matrix metalloproteinase (MMP)-9, hypoxia-inducible factor (HIF)-1α, VEGF, and other related proteins (Anfosso et al. 2006) and are able to inhibit the growth of embryonic stem cells (Wartenberg et al. 2003). The liposome is an artificially prepared vesicle comprising a lipid bilayer. Liposomes can be used as the vehicle for the administration of nutrients or pharmaceutical agents. In the present protocol, a multifunctional targeting liposomal drug system is designed for offering a comprehensive strategy to transport drugs across the BBB and to eradicate brain cancer cells along with resistant brain cancer stem cells (CSCs). In the functional targeting liposomal drug system, paclitaxel and artemether are incorporated into liposome vesicles as an anticancer agent and modulating agent, respectively. A synthesized mannose-vitamin E derivative (P-aminophenyl-a-D-mannopyranosideD-a-tocopheryl polyethylene glycol 1000 succinate, MAN-TPGS1000) conjugate is used as a targeting molecule for transporting drugs across the BBB. And a dequalinium-lipid derivative (dequalinium-polyethylene glycol 2000 distearoyl phosphatidyl ethanolamine, DQA-PEG2000-DSPE) conjugate is used as a functional molecule for targeting cancer cells, CSCs, and genotype-transformed cancer cells. Dequalinium (DQA) is a quaternary ammonium cation commonly available as a dichloride salt, as well as an amphiphile with delocalized cationic charge centers. DQA can accumulate selectively in mitochondria (Zhang et al. 2012) hence is used as a molecule targeting mitochondria. PAminophenyl-α-D-mannopyranoside (MAN) is a type of mannose analog. It can penetrate the brain efficiently via facilitative glucose transporters (GLUTs) because the BBB overexpresses GLUTs (Ying et al. 2010). Accordingly, MAN is used as a ligand targeting the BBB. The objectives of the present study are to develop a kind of functional targeting paclitaxel plus artemether liposomes and to improve the efficacy of paclitaxel for treatment of invasive brain gliomas by transporting across the BBB, followed by eliminating brain cancer cells and brain CSCs.
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2
Materials
2.1
Synthesis of Targeting Molecule Conjugates
2.1.1 MAN-TPGS1000 (See Note 1) 1. 4-Dimethylaminopyridine (DMAP) 2. Dicyclohexylcarbodiimide (DCC) 3. 4-Amino- phenyl-α-D-mannopyranoside (MAN) 4. D-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS1000) 5. N-Hydroxysuccinimide (NHS) 6. 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDCI) 7. Dimethyl sulfoxide (DMSO) 8. Glutaric acid 9. Pyridine 10. Regenerated cellulose dialysis tubing (molecular weight cutoff point, 1500) 11. Deionized water 12. N2 gas 13. Magnetic stirrer 14. Bath-type sonicator 15. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS instrument) 16. Nuclear magnetic resonance spectroscopy (400 MHz 1H NMR) 17. Freeze-drying machine 2.1.2 DQA-PEG2000-DSPE (See Note 2) 1. Dequalinium (DQA) 2. 1,2-Distearoyl-sn-glycero-3-phosphoethamolamine-N-[carboxy (polyethylene glycol) 2000] (COOH-PEG2000-DSPE) 3. 1-Hydroxy-1H-benzotriazole (HOBt) 4. DMAP 5. DCC 6. Distilled water 7. Acetonitrile 8. Regenerated cellulose dialysis tubing (molecular weight cutoff point, 2000) 9. Magnetic stirrer 10. Bath-type sonicator 11. Freeze-drying machine
2.2 1. 2. 3. 4.
Preparation of Multifunctional Paclitaxel Liposomes Paclitaxel Artemether Egg phosphatidylcholine (EPC) 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)2000] (PEG2000-DSPE)
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5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Cholesterol MAN-TPGS1000 DQA-PEG2000-DSPE Chloroform Methanol Hepes NaCl Pear-shaped flask Rotary evaporator Vacuum pump Bath-type sonicator Ultrasonic cell disruptor 400 nm and 200 nm polycarbonate membranes
2.3 1. 2. 3. 4. 5. 6.
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Measurement of Encapsulation Efficiency of Paclitaxel and Artemether
Diamonsil ODS-C18 column (200 4.6 mm, 5um) HPLC system with UV detector Sephadex G-50 column Centrifuge Acetonitrile Distilled water
2.4
Measurement of Particle Size, Zeta Potential, and Polydispersity Index (PDI)
1. Distilled water 2. Zetasizer 3000HSA for dynamic light scattering (DLS) analysis
2.5
Morphology Characterization with Atomic Force Microscope (AFM)
1. Polished silicon wafer 2. Atomic force microscope 3. Micropipette
2.6
In Vitro Release of Paclitaxel or Artemether
1. Dialysis tube 2. Fetal bovine serum 3. Shaker
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2.7 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Cell Culture Ham’s F10 medium Fetal bovine serum (FBS) Horse serum B27 Basic FGF Endothelial cell growth factor (EGF) Dulbecco’s minimum essential medium (DMEM) DMEM-F12 Phosphate-buffered saline (PBS) Trypsin EDTA Pen-Strep solution 100 L-Glutamine Heparin T75 cell culture flask Incubator
2.8 1. 2. 3. 4. 5. 6.
96-well culture plates Trichloroacetic acid Sulforhodamine B Acetic acid Tris base Microplate reader
2.9 1. 2. 3. 4.
Transport Across the BBB and Targeting of Brain Cancer Cells
Insert (0.4 μm pore size, 12 mm diameter, and 1.12 cm2 surface area) Gelation D-Hank’s solution Transendothelial electrical resistance (TEER) instrument
2.10 1. 2. 3. 4. 5. 6.
Cytotoxic Effects on Brain Cancer Cells and Brain CSCs
In Vivo Imaging in Mice
Kodak multimodal imaging system Stereotactic device 4% chloral hydrate 10 μl microinjector Alcohol cotton Bone wax
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7. 1.0 ml Syringes 8. ICR mice (initially weighing 19–21 g)
2.11 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Anticancer Efficacy in Rats
MRI 20 μl microinjector Stereotactic device Alcohol cotton Bone wax 1.0 ml syringes Male Sprague-Dawley rats (initially weighing 190–210 g) Physiological (0.9%) saline 4% paraformaldehyde solution CD133 antibodies Horseradish peroxidase Mouse monoclonal anti-CD34 1% sodium periodate Distilled water PAS Hematoxylin
3
Methods
3.1
Synthesis of Targeting Molecule Conjugates
3.1.1 Synthesis of MAN-TPGS1000 Conjugate 1. Dissolve the glutaric acid (0.2 mmol), DMAP (0.1 mmol), and DCC (0.24 mmol) completely in DMSO (2 ml). 2. Stir the mixture at room temperature under nitrogen gas protection for 2 h. 3. Add TPGS1000 (0.04 mmol). 4. Stir the reaction mixture using a magnetic stirrer at room temperature for 24 h. 5. Transfer the crude product to a regenerated cellulose dialysis tubing (molecular weight cutoff point, 1500), and then dialyze them against deionized water for 48 h to remove uncoupled glutaric acid, DMAP, DCC, N, N-dicyclohexylurea, and DMSO (see Note 3). 6. Transfer the solution in the dialysis tubing to a 10 ml vial. 7. Freeze-dry them to obtain the TPGS1000-COOH. 8. Dissolve TPGS1000-COOH (10 μmol), EDCI (40 μmol), and NHS (70 μmol) in pyridine-DMSO (1:1, 2 ml). 9. Stir the mixture under room temperature for 30 min. 10. Add MAN (10 μmol), and then stir the reaction mixture using a magnetic stirrer at room temperature for 12 h.
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11. Transfer the crude product to a regenerated cellulose dialysis tubing (molecular weight cutoff point, 1500), and then dialyze them against deionized water in the regenerated cellulose dialysis tubing for 48 h to remove uncoupled MAN, EDC, NHS, DMSO, and pyridine. 12. Transfer the solution in the dialysis tubing to a 10 ml vial. 13. Freeze-dry them to obtain the MAN-TPGS1000. 14. Identify and characterize the production mixture using matrix-assisted laser desorption/ionization time of flight mass spectrometry and nuclear magnetic resonance spectroscopy (Fig. 1).
3.1.2 Synthesis of DQA-PEG2000-DSPE Conjugate 1. Dissolve DQA (5.28 mg, 10 mmol) in acetonitrile and water (1:1, v/v, 2 ml) (see Note 4). 2. Dissolve 4-dimethylamiopryidine (DMAP) (6 mg, 49.2 mmol), N, N0 dicyclohexylcarbodiimide (DCC) (4.12 mg, 20 mmol), HOBt (2.7 mg, 20 mmol), and COOH-PEG2000-DSPE (6 mg, 2 mmol) in the above solution (see Note 5). 3. Stir the reaction mixture gently with a magnetic stirrer at ambient temperature for 12 h. 4. Transfer the crude product to a regenerated cellulose dialysis tubing (MWCO 2000), and then dialyze them against deionized water for 48 h to remove uncoupled DQA, DMAP, DCC, HOBt, and acetonitrile. 5. Lyophilize the residue to get dry powder. 6. Characterize the production mixture using nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption/ionization time of flight mass spectrometry (Fig. 2).
3.2
Method for Determination of Paclitaxel and Artemether
3.2.1 Linearity of Paclitaxel 1. Prepare paclitaxel with 99% purity of concentrations of 1.6, 3.1, 6.2, 12.5, 25, 50, and 100 μg/ml in acetonitrile and water (60:40, v/v) to generate an external calibration curve. 2. Analyze the samples by HPLC. 3.2.2 Linearity of Artemether 1. Prepare artemether of concentrations of 15.6, 31.2, 62.5, 125, 250, 500, and 1000 μg/ml in acetonitrile and water (45:55, v/v) to generate an external calibration curve. 2. Analyze the samples by HPLC. 3.2.3 HPLC Method for Measurement of Paclitaxel or Artemether 1. Perform the analysis of paclitaxel or artemether on ODS-C18 column (250 4.6 mm, 5.0 μm particle size).
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Fig. 1 Synthetic scheme for MAN-TPGS1000 conjugate
2. For paclitaxel, set the mobile phase with acetonitrile and water (60:40, v/v), the flow rate at 1.0 ml/min, the UV detection wavelength at 227 nm, the injection volume to 20 μl, and the column temperature at 30 C.
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Fig. 2 Synthetic scheme for DQA-PEG2000-DSPE conjugate
3. For artemether, set the mobile phase consisting of acetonitrile and water (45:55, v/ v), the flow rate at 1.0 ml/min, the UV detection wavelength at 210 nm, the injection volume to 20 μl, and the column temperature at 30 C.
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4. Validate the measurement methods by estimating the linearity, stability, and interday and intraday precision.
3.3
Preparation of Multifunctional Paclitaxel Liposomes by ThinFilm Hydration
1. Dissolve egg phosphatidylcholine(EPC), cholesterol, MAN-TPGS1000, and DQA-PEG2000-DSPE (88:3.5:3:3, molar ratio) in chloroform-methanol (3/1, v/ v) solution in a pear-shaped flask (see Note 6–9). 2. Add drugs (paclitaxel/artemether = 1:3, molar ratio) to the above flask with the drug (paclitaxel and artemether)-lipid ratio 1:25 (w/w) (see Note 10–11). 3. Evaporate the solvent (chloroform and methanol) under vacuum with a rotary evaporator (see Note 12). 4. Hydrate the formed lipid film with HBS buffer solution (25 mM Hepes/150 mM NaCl) by sonication in the water bath for 2 min. 5. Transfer the multilamellar lipid vesicles (MLV) dispersion into glass vial, and place it in a 4 C water bath. 6. Immerse the sonicator probe into the sample, and adjust it to at least 1 cm (0.4 in.) above the bottom of vial. 7. Set the sonication power at about 200 W. 8. Set the sonication time to about 10 min with 10 s on and 10 s off (see Note 13). 9. Extrude the above suspensions through polycarbonate membranes with the pore size of 400 nm for three times and then 200 nm for three times (Fig. 3; see Note 14).
3.4
Measurement of Encapsulation Efficiency of Paclitaxel or Artemether
1. Saturate a Sephadex G-50 column with blank multifunctional liposomes. 2. Load the liposomal dispersion on the column, and elute the column with HBS buffer solution (25 mM Hepes/150 mM NaCl) at a flow rate of 1 ml/min to remove unencapsulated paclitaxel or artemether. 3. Collect the drug-loaded liposomal suspension in glass vial. 4. Apply ten times volume of acetonitrile to disrupt the bilayer structure of drugloaded liposomes. 5. Shake for 1 min by a vortex instrument. 6. Centrifugate the mixture 5 min at a speed of 10,000 rpm. 7. Take the supernatant. 8. Measure the paclitaxel or artemether in liposome with high-performance liquid chromatography (HPLC) system with a UV detector. 9. Measure the encapsulation efficiency (EE) of paclitaxel or artemether with the formula: EE = (Wencap/Wtotal) 100%, where EE is the encapsulation efficiency of paclitaxel or artemether and Wencap is the measured amount of paclitaxel or
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Fig. 3 Schematic representation of multifunctional paclitaxel liposomes
artemether in the liposomal suspensions after passing over the column. Wtotal is the measured amount of paclitaxel or artemether in the equal volume of liposomal suspensions before passing over the column.
3.5
Measurement of Particle Size, Zeta Potential, and Polydispersity Index (PDI)
1. Dilute the liposomal suspension with distilled water to an appropriate volume. 2. Character the particle sizes, zeta potential values, and polydispersity indexes (PDI) of all liposomes using a dynamic scatter.
3.6
Morphology Characterization with AFM
1. Dilute the liposomes with distilled water. 2. Filter them through a 200 nm micropore filter membrane. 3. Spread a 10 μL volume of liposome suspension on a silicon slice, and dry it at room temperature. 4. Observe using AFM.
3.7
In Vitro Release of Paclitaxel or Artemether
1. Perform the in vitro release of paclitaxel or artemether in the liposomes by the dialysis against the release medium containing serum protein (pH 7.4 phosphatebuffered saline containing 10% fetal calf serum).
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2. Mix a volume of 1 ml liposomes with 1 ml the release medium, and seal the mixture in dialysis tubing (molecular weight cutoff point, 12,000–14,000). 3. Immerse the tubing in a glass vial with 20.0 ml of the release medium, shaking the vial at a speed of 100 times per minute at 37 C. 4. Take a volume of 0.2 ml release medium at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 h, respectively, and immediately replace them with the same volume of fresh release medium after each sampling. 5. Add a 1.0 ml volume of acetonitrile to the 0.2 ml release medium. 6. Mix the mixture for 3 min with a vortex, and then centrifugate at 10,000 rpm for 5 min. 7. Inject a 20 μl volume of supernatant into the HPLC system. 8. Determine the paclitaxel or artemether content in the release medium by HPLC as above. 9. Calculate the release rate with the formula: RR = (Wi/Wtotal) 100%, where RR is the drug release rate (%), Wi is the measured amount of paclitaxel or artemether at the time point of ith h in release medium, and Wtotal is the total amount of paclitaxel or artemether in the equal volume of liposome suspensions prior to dialysis.
3.8
Cell Culture
1. Maintain the glioma C6 cells in serum-containing medium comprising Ham’s F10 medium supplemented with 5% fetal bovine serum and 15% horse serum. 2. Grow the glioma C6 CSCs in serum-free DMEM-F12supplemented with 2% B27 20 ng/mL basic fibroblast growth factor and 20 ng/mL epidermal growth factor. After continuous culture of glioma C6 cells in a serum-free medium under 5% CO2 at 37 C for 3 weeks, brain CSC mammospheres are formed. 3. Passage the murine brain microvascular endothelial cells (BMVECs) in the endothelial cell culture medium (DMEM, 20% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, 2 mmol/L L-glutamine, 100 mg/mL endothelial cell growth factor, 40 U/mL heparin).
3.9
Cytotoxic Effects on Brain Cancer Cells or Brain CSCs
3.9.1 Cytotoxic Effects on Brain Cancer Cells 1. Seed C6 cells into 96-well culture plates at a density of 5.0 103 cells per well, and grow them in serum-containing culture medium in the incubator at 37 C for 24 h under an atmosphere of 5% CO2. 2. Replace the medium with fresh culture media containing varying concentrations of drugs (free paclitaxel, free artemether, free paclitaxel plus a fixed concentration of free artemether, free paclitaxel plus free artemether, paclitaxel liposomes, paclitaxel plus artemether liposomes, MAN-targeting paclitaxel plus artemether liposomes, DQA-targeting paclitaxel plus artemether liposomes, or the
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multifunctional paclitaxel liposomes). The final concentration of paclitaxel is 0.5 mM. Use the blank culture medium as a blank control. 3. After 48 h of incubation, fix the cells with 10% (w/v) trichloroacetic acid, stained by the sulforhodamine B (SRB) for 30 min, and then remove the excess by washing repeatedly with 1% (v/v) acetic acid. Finally, dry at room temperature. 4. Dissolve the protein-bound dye in 10 mM Tris base solution. Read the absorbance on a microplate reader at 540 nm. 5. Calculate the survival percentages using the following formula: Survival % = (A540nm for the treated cells/A540nm for the control cells) 100%, where A540 nm is the absorbance value. Each assay was repeated in triplicate. Finally, the dose-effect curves were plotted.
3.9.2 Cytotoxic Effects on Brain CSCs 1. Collect the brain CSCs mammospheres, dissociate the mammospheres by enzymatic means, and wash them in PBS using gentle agitation. 2. Seed single CSC suspensions at 1 104 cells/well and grow them in serumcontaining culture medium in an incubator at 37 C for 24 h under an atmosphere of 5% CO2. 3. The subsequent procedure is similar to that of the brain cancer cells (see Sect. 3.9.1, Steps 2–5).
3.10
Transport Across the BBB and Targeting of Brain Cancer Cells
To assess potential dual-targeting effects, a BMVEC/C6 co-culture BBB model is established. 1. Coat the upper side of the inserts (0.4 μm pore size, 12 mm diameter, and 1.12 cm2 surface area) by 2% gelation D-Hank’s solution, and then place the inserts in the incubator at 37 C for 0.5 h. 2. Seed BMVECs on the upper side of 2% gelatin-coated culture insert at 1.0 104 cells per insert. 3. Replace the culture medium every 2 days. 4. After 6 days, assess the tightness of the monolayer by measuring the transendothelial electrical resistance (TEER) using a TEER instrument, and select cell monolayers with TEER above 200 Ωcm2 for the following experiment. 5. Seed glioma C6 cells on the basolateral compartment of the insert at 2000 cells/compartment. 6. After incubation for 5 days, use this model for experiments. 7. Add drugs into the apical compartment. The final concentrations of paclitaxel and artemether are 5 mM and 15 mM, respectively. 8. After incubation for 48 h, determine the percentage of surviving glioma C6 cells in the basolateral compartment by the sulforhodamine-B staining assay as above.
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3.11
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In Vivo Imaging in Mice
Noninvasive optical imaging systems are used to observe the real-time distribution and tumor accumulation ability of multifunctional DiR liposomes in brain tumorbearing ICR mice. 1. Anesthetize ICR mice with 4% chloral hydrate and then fix the mice into a stereotactic device. 2. Make an incision and drill a burr hole in the skull 2 mm to the right and 0.5 mm anterior to the bregma. 3. Inject glioma C6 cells (2 105 cells/3 ml F10 culture medium) into the right striatum of the brain to a depth of 3 mm at a rate of 0.2 ml/min. 4. Cover the craniotomy with dental cement and close the incision with sutures. 5. After 14 days, divide the mice into six groups (six each group) and inject injection with physiological saline, free DiR, DiR liposomes, MAN-targeting DiR liposomes, DQA-targeting DiR liposomes, or multifunctional DiR liposomes via tail vein. 6. Scan the mice at 1, 3, 6, 12, 24, and 48 h using a noninvasive optical imaging system.
3.12
Anticancer Efficacy in Rats
1. Use male Sprague-Dawley rats (initially weighing 190–210 g) for investigating the anticancer efficacy in vivo. 2. Anesthetize SD rats with 4% chloral hydrate and then fix the rats into stereotactic device. 3. Make a sagittal incision through the skin to expose the cranium. 4. Drill a burr hole in the skull 1.0 mm anterior and 3.0 mm lateral from the bregma. 5. Stereotaxically implant approximately 1 106 glioma C6 cells/10 μl in serumfree F10 medium into the right forebrain of each rat at a rate of 2 μl/min and a depth of 5.0 mm from the brain surface. 6. Eight days after tumor inoculation, divide rats randomly into seven groups of seven (ten rats per group). 7. Inject physiological (0.9%) saline into animals as the blank control. 8. Treat rats in the other six groups with taxol, paclitaxel liposomes, paclitaxel plus artemether liposomes, MAN-targeting paclitaxel plus artemether liposomes, DQA-targeting paclitaxel plus artemether liposomes, or the multifunctional paclitaxel liposomes via the tail vein at 5 mg/kg paclitaxel and 10 mg/kg artemether. Administration is every 2 days with a total of four doses per rat. 9. At day 16, anesthetize four rats from each group and assess brain cancer by MRI for measurement of tumor diameter.
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10. Calculate cancer inhibition using the formula: Rv = (Vdrug/Vsaline) 100%, where Vdrug is the glioma volume after treatment with drug and Vsaline is the glioma volume after treatment with physiological saline. 11. Monitor survival using the remaining six rats in each group. 12. Calculate survival time from day 0 (tumor inoculation) to the day of death. Plot the Kaplan-Meier survival curves for each group. 13. After MRI assay, sacrifice the hearts of the anesthetized rats by perfusion with 100 ml PBS and then with 100 ml 4% paraformaldehyde solution. 14. Remove the brains to make cryosections at 20 μm per slice. 15. Use the immunoperoxidase method to perform immunohistochemical staining with antibodies for CD133, which are used at a dilution of 1:300. 16. Brain cancer VM channels: Mouse monoclonal anti-CD34 is used at a dilution of 1:300. After immunohistochemical staining, sections are exposure to 1% sodium periodate for 10 min. The sections are then rinsed with distilled water for 5 min and incubated with PAS for 2 min. Finally, all of the sections are counterstained with hematoxylin, dehydrated, and mounted. Positive and negative controls without primary antibody are included.
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Notes
1. TPGS1000 is a water-soluble form of a vitamin-E derivative. It has a long halflife in blood, can circumvent the multidrug resistance of cancer mediated by adenosine triphosphate-binding cassette transporters, and has anticancer as well as neuroprotective effects. In the present study, MAN-TPGS1000 conjugate is synthesized. It is incorporated onto drug-loaded liposomes for transporting drugs across the BBB and then targeting VM-capable cancer cells, cancer cells, and CSCs in response to the GLUTs that are overexpressed in brain microvessels and brain cancer cells. 2. Adsorptive-mediated endocytosis (AME) is also an important mechanism for transporting drugs across the cerebral endothelium. AME relies on nonspecific charge-based interactions between the ligands and membrane of endothelial cells in the BBB. These interactions can be initiated by polycationic molecules binding to negative charges on the membrane or by binding to extracellular lectins. DQA-PEG2000-DSPE conjugate is a type of cationic amphiphilic material of phospholipids with delocalized cationic charge centers that can selectively accumulate in mitochondria. This feature makes it easier to cross the BBB and target cancer cells, CSCs, and genotype-transformed VM-capable cancer cells by AME. Our previous investigations demonstrated that DQA-PEG2000DSPE conjugate-modified liposomes can target the mitochondria of cancer cells or CSCs and then trigger apoptosis. In the present study, DQA-PEG2000-DSPE conjugate was incorporated onto liposomes for the same purposes. Furthermore, DQA-PEG2000-DSPE conjugate is used as material which remains in the circulation for a long time because it can escape the rapid uptake of the reticuloendothelial system due to the action of PEG. In this way, it exhibits biologically
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stable properties in the circulation and decreases the immediate distribution and clearance. Replace the dialysis media at a certain time, and set the replacement time at 2, 4, 8, 12, and 24 h. The dissolution rate of DQA in 50% acetonitrile is slow, so it must be completely dissolved, or the yield will be reduced or even failed. In this case, a bath-type sonicator is often used to dissolve it completely. These substances are added and dissolved in accordance with the order. As the ratios of phospholipids to cholesterol increase, the encapsulation efficiency of paclitaxel or artemether increases. Cholesterol is a general constituent of liposomal formulations due to its ability to modulate membrane permeability and biological stability. The amount of cholesterol being incorporated can affect the membrane permeability of liposomes. DQA-PEG2000-DSPE as a cationic lipid material has great impact on the targeting effect of the liposomes. With an increasing DQA-PEG2000-DSPE concentration (1–5%), the targeting effect increases and its toxicity increases as well. In order to obtain a high target effect as well as low toxicity effect under this condition, the best concentration of DQA-PEG2000-DSPE is about 3% (molar ratio). MAN-TPGS1000, as a functional material, shows the P-gp inhibition effect and targeting effect to both brain glioma cells and BMVECs. In view of the total quantity of the long circulation materials (DQA-PEG2000-DSPE and MAN-TPGS1000), which should be less than 7%, the best choice of the concentration of MAN-TPGS1000 in the liposomes is about 3% (molar ratio). The choice of the ratio of paclitaxel to artemether is based on the following facts: firstly, the cytotoxic effect on brain cancer cells increases with the increase of the ratio of paclitaxel to artemether; secondly, when the concentration of artemether is 15 μmol, the survival of brain glioma U87 cells is above 90%; thirdly, in the multifunctional liposomes, artemether is used as a regulator, not a cytotoxic drug; fourthly, the final concentration of paclitaxel is 5 μmol; and finally, the ratio of paclitaxel to artemether is chosen 1:3 (molar ratio). The drug-lipid ratio has great impact on the encapsulation efficiency and drug loading rate. With an increasing drug-lipid ratio, the encapsulation efficiency decreases, while the drug loading rate increases. In order to obtain a high drug loading rate as well as optimum encapsulation efficiency under this condition, the best choice of drugs (paclitaxel and artemether)-lipid ratio is 1:25 (w/w). All the lipid materials must be dissolved in the solvent (chloroform and methanol), and ultrasonic operation can help this procedure. The speed of evaporating chloroform and that of the methanol should be proper; otherwise, DQA- PEG2000-DSPE will precipitate and it will not form a uniform lipid film. The solvent (chloroform and methanol) must be removed completely to create a lipid film. Followed by a quickly transfer to a vacuum pump, the sample is kept for several hours to ensure the removal of residual solvent. The sonication time depends on the sample volume and concentration, and the sonication keeps a suitable time until the dispersion turn from milky into opalescent.
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14. The changes of particle size may indicate liposome instability. If the particle size distribution is less than expected, the sample may be re-extruded to obtain the desired size range.
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Conclusion
The multifunctional paclitaxel liposomes are prepared by thin-film hydration method, followed by hydration with sonication and membrane extrusion. The final drug liposomes are approximately 80 nm in size. The fabricated multifunctional paclitaxel liposomes have evidenced the potential in the treatment of brain glioma in vitro and in brain glioma-bearing animals. The combinational use of artemether and paclitaxel are able to circumvent the drug resistance of brain glioma, thereby increasing the treatment efficacy. Two conjugates (DQA-PEG2000-DSPE and MAN-TPGS1000) modified on the liposomes demonstrate the capability of transporting drug across the BBB and exhibit targeting effects of the multifunctional liposomes to brain glioma and glioma stem cells. The multifunctional paclitaxel liposomes deserve further development for the potential clinical treatment of brain glioma.
References Ai B, Bie Z, Zhang S, Li A (2016) Paclitaxel targets VEGF-mediated angiogenesis in ovarian cancer treatment. Am J Cancer Res 6(8):1624–1635 Anfosso L, Efferth T, Albini A, Pfeffer U (2006) Microarray expression profiles of angiogenesisrelated genes predict tumor cell response to artemisinins. Pharmacogenomics J 6:269–278 Floyd JA, Galperin A, Ratner BD (2015) Drug encapsulated polymeric microspheres for intracranial tumor therapy: a review of the literature. Adv Drug Deliv Rev 91:23–37 Hien TT, White NJ (1993) Qinghaosu. Lancet 341:603–608 Nakase I, Lai H, Singh NP, Sasaki T (2008) Anticancer properties of artemisinin derivatives and their targeted delivery by transferrin conjugation. Int J Pharm 345:28–33 Postma TJ, Heimans JJ, Luykx SA, Groeningen CJ, Beenen LF, Hoekstra OS, Taphoorn MJ, Zonnenberg BA, Klein M, Vermorken JB (2000) A phase II study of paclitaxel in chemonaïve patients with recurrent high-grade glioma. Ann Oncol 11:409–413 Wang Y, Gao S, Ye WH, Yoon HS, Yang YY (2006) Co-delivery of drugs and DNA from cationic core-shell nanoparticles self-assembled from a biodegradable copolymer. Nat Mater 5(10):791–796 Wartenberg M, Wolf S, Budde P, Grünheck F, Acker H, Hescheler J, Wartenberg G, Sauer H (2003) The antimalaria agent artemisinin exerts antiangiogenic effects in mouse embryonic stem cellderived embryoid bodies. Lab Invest 83:1647–1655 Wiradharma N, Tong YW, Yang YY (2009) Self-assembled oligopeptide nanostructures for codelivery of drug and gene with synergistic therapeutic effect. Biomaterials 30(17):3100–3109 Yao HJ, Ju RJ, Wang XX, Zhang Y, Li RJ, Yu Y, Zhang L, Lu WL (2011) The antitumor efficacy of functional paclitaxel nanomicelles in treating resistant breast cancers by oral delivery. Biomaterials 32:3285–3302 Ying X, Wen H, Lu WL, Du J, Guo J, Tian W et al (2010) Dual-targeting daunorubicin liposomes improve the therapeutic efficacy of brain glioma in animals. J Control Release 141:183–192
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Zhan C, Gu B, Xie C, Li J, Liu Y, Lu W (2010) Cyclic RGD conjugated poly(ethylene glycol)-copoly(lactic acid) micelle enhances paclitaxel anti-glioblastoma effect. J Control Release 143:136–142 Zhang L, Yao HJ, Yu Y, Zhang Y, Li RJ, Ju RJ et al (2012) Mitochondrial targeting liposomes incorporating daunorubicin and quinacrine for treatment of relapsed breast cancer arising from cancer stem cells. Biomaterials 33:565–582
Preparation and Evaluation of Integrin Receptor-Mediated Targeting Drug Liposomes
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Fei Wang, Gang Wei, and Weiyue Lu
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Synthesis of RGD Peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Preparation of RGD Conjugated Polyethylene Glycol-Lipid (RGD-PEG-DSPE) . . . 2.3 Characterization of RGD-PEG-DSPE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Preparation of RGD-Modified Liposome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Characterization of RGD-Modified Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Tumor Targeting Ability of RGD-Modified Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7 Antitumor Activity of RGD-Modified Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Synthesis of RGD Peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Preparation of RGD-PEG-DSPE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Characterization of RGD-PEG-DSPE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Preparation of RGD-Modified Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Characterization of RGD-Modified Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Tumor Targeting Ability of RGD-Modified Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Antitumor Activity of RGD-Modified Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
To promote the therapeutic efficiency of drug liposomes, ligand-modified targeting liposomes have received extensive attentions in recent years. RGD peptide (Arginine-Glycine-Aspartic acid), which is a ligand of the integrin F. Wang · G. Wei (*) · W. Lu Department of Pharmaceutics, School of Pharmacy, Fudan University; Key Laboratory of Smart Drug Delivery, Ministry of Education (Fudan University), Shanghai, China e-mail: [email protected] © Springer-Verlag GmbH Germany, part of Springer Nature 2021 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49320-5_15
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receptors, has been widely used to enhance the interaction between nanocarriers and integrin-overexpressed tumor tissues. It has been generally validated that RGD modification can significantly boost the antitumor effect of drug delivery systems. In this chapter, the methods how to prepare and evaluate a RGDmediated targeting drug liposome are presented. In order to facilitate longer circulation time in vivo, polyethylene glycol (PEG)-modified lipids are usually employed to construct the active targeting liposomes. Firstly, the protocols to synthesize the RGD-conjugated liposomal membrane materials based on the PEGylated lipids and the approaches to confirm successful conjugation of RGD to the lipids are described. Then the preparation methods of RGD-modified liposomes are introduced, including the thin film hydration, reverse evaporation, and extrusion method. Here two kinds of antitumor agents are chosen as model drugs and are described in detail how to prepare their respective liposomes. Doxorubicin (DOX), an antitumor anthracycline antibiotic from Streptomyces bacteria, can be loaded into liposomes using the pH-gradient method. pDP peptide, an antitumor D-peptide working by inhibiting the p53-MDM2 interaction, can be loaded into liposomes using the reverse evaporation method. The presence of RGD on the liposomal surface and the physicochemical properties of the resultant liposomes are characterized. Finally, the tumor targeting ability and antitumor efficacy of the liposomes are assessed in vitro and in vivo to exhibit the benefits of RGD modification. Keywords
Integrin · RGD peptide · Ligand-modified targeting liposome · Doxorubicin · pDP peptide · Liposome characterization · Antitumor efficiency
1
Introduction
In the last few decades, liposomes have been widely used for passive tumor targeting, by virtue of the enhanced permeability and retention (EPR) effect (Fang et al. 2011; Maeda et al. 2000). In clinic, several liposomal antitumor drugs have afforded markedly improved treatment efficacy (Gabizon et al. 2003; Hiemenz and Walsh 1996; Rodriguez et al. 2009). To achieve better targeting efficiency of nanomedicines, numerous active targeting approaches have been developed by conjugating nanocarriers with molecules that can specially bind to receptors overexpressed on tumor cells and/or other related cells (Sudimack and Lee 2000; Choi et al. 2010; Zhan et al. 2011, 2015). Integrins are a family of transmembrane adhesion glycoproteins composed of an α and a β subunit. In the case of cancer, integrins play an important role in cell proliferation, differentiation, angiogenesis, migration, invasion, and metastasis, by interfacing with their ligands in extracellular matrix (ECM) proteins (Avraamides et al. 2008; Alghisi and Ruegg 2006; Desgrosellier and Cheresh 2010). Integrin ανβ3 is one of the most investigated subtype of integrins. While it is overexpressed on several tumor cells and endothelial cells involved in cancer
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angiogenesis, it is hard to detect ανβ3 in normal endothelium (Arosio and Casagrande 2016). The amino acid sequence RGD (Arg-Gly-Asp) is recognized as the widely expressed sequence of the ECM proteins that specifically bind to integrins (Ruoslahti 2003). A variety of high-affinity peptide ligands mimicking the RGD moiety have been developed, including various linear RGD (Kang and Jhon 2000), cyclic RGD (Belvisi et al. 2006), N-methylated RGD (Chatterjee et al. 2013), and other peptides (Arap et al. 1998) or peptidomimetics (Manzoni et al. 2009). Integrin-targeted delivery of chemotherapy (Murphy et al. 2008; Eldar-Boock et al. 2011) or gene therapy (Oba et al. 2008; Vachutinsky et al. 2011) have been frequently reported using RGD-modified liposomes (Murphy et al. 2008), micelles (Oba et al. 2008), poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) (Danhier et al. 2009), proteinbased NPs (Ji et al. 2012), or metallic NPs (Yang et al. 2011). Eric A. Murphy designed a cyclic RGDfK-modified (Arginine-Glycine-Aspartic acid-D-phenylalanine-Lysine) liposome loading the cytotoxic drug doxorubicin (DOX) for ανβ3targeted drug delivery to tumor vasculature (Murphy et al. 2008). They observed a 15-fold increase in antimetastatic activity relative to animals intravenously administrated with free drug. Compared with cyclic RGD peptides, linear RGDs are less effective to bind with integrins (Samanen et al. 1991) and more susceptible to enzymatic degradation (Bogdanowich-Knipp et al. 1999). Therefore, cyclic RGD peptides are more frequently used. The cyclic RGD peptide c(RGDyK) (ArginineGlycine-Aspartic acid-D-tyrosine-Lysine) is one of the most investigated RGD peptides. It has been extensively utilized to mediate the interaction between drug delivery systems and integrin-overexpressed tumor cells and tumor vasculature (Gao et al. 2016; Zhan et al. 2010, 2012; Li et al. 2012). As a peptide, it is easy to design a chemical synthesis protocol for conjugation of c(RGDyK) to nanocarriers. For example, c(RGDyK) was employed to modify paclitaxel-loaded micelles, which showed significantly enhanced anti-glioblastoma effect compared with nontargeting micelles (Zhan et al. 2010). The c(RGDyK) decorated liposome was also used to encapsulate peptide for treatment of glioblastoma, and it exerted potent antitumor effect with low toxicity (Li et al. 2012). Here we present a protocol for preparation and evaluation of c(RGDyK)-mediated targeting drug liposomes (Fig. 1). To modify liposomes with c(RGDyK), c(RGDyK) has to be conjugated with a PEGylated lipid (PEG-lipid) material. Firstly, liner RGD peptides are synthesized by solid-phase peptide synthesis strategy, and the liner peptide will be cyclized to obtain the c(RGDyK) peptide. Then c(RGDyK) is thiolated (RGD-SH) and reacted with maleimide (Mal)-derivatized PEG-lipids. The thiol of the RGD-SH can conjugate with the maleimide quickly and effectively, and the RGD-PEG-lipids will then be produced. The scheme of the reactions is shown in Fig. 2. Then the RGD-modified lipids are integrated with other lipid materials to prepare liposomes. The lipid segments of the RGD-PEG-lipids will be spontaneously inserted in the lipid bilayer of the liposomes, and the hydrophilic RGD peptides will be exposed both in the outside and inside water-phase, forming the active targeting liposomes. Polyethylene glycol (PEG) conjugated lipids are used as a portion of materials to facilitate a long circulation capability of the liposomes. Liposomes can be prepared using the thin film hydration or reverse evaporation method. To get a smaller size and more uniform
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Fig. 1 Schematic representation of doxorubicin (DOX)-loaded liposomes modified with RGD for targeting drug delivery (Deng et al. 2016). The RGD ligands on the liposomes interact with the integrin receptor on the cancer cell membrane and lead to the formation of endosomes. The liposomes are then degraded by enzymes in lysosomes, and DOX is released and then functions in nucleus. (The schematic is cited and modified from Deng et al. 2016.)
formulation, extrusion procedure can be employed. Here DOX and a D-conformation antitumor peptide (pDP) are used as the model of antitumor drugs. DOX is an anthracycline antitumor antibiotic derived from cultures of Streptomyces peucetius var. caesius. It presumably works by forming complexes with nucleic acids through intercalation between base pairs, then inhibiting topoisomerase II activity and preventing the DNA and RNA biosynthesis (Tewey et al. 1984). DOX is generally used for the treatment of broad-spectrum cancers, including acute lymphoblastic leukemia, neuroblastoma, breast carcinoma, and so on. DOX can be loaded into liposomes using the classic pH-gradient method. D-PMIβ is an antitumor D-peptide. It can effectively inhibit the interaction between tumor suppressor protein p53 and its negative regulator murine double minute 2 (MDM2) by binding to MDM2 with high affinity and unsurpassed specificity (Li et al. 2012). As it was reported that peptide derivatives modified with lipophilic moieties could achieve high peptide encapsulation within liposomes (Liang et al. 2005), the palmitylated derivate of DPMIβ (pDP) is used as loading drug. Lipophilic pDP peptide can be loaded into liposomes by the reverse evaporation method. Subsequently, the methods to characterize RGD-modified liposomes loaded with DOX (RGD-LS/DOX) and pDP (RGDLS/pDP) are described, including how to verify the conjugation of RGD to liposomes, how to proceed the physicochemical characterization of liposomes, and how to detect the drug encapsulation efficacy. The in vitro and in vivo tumor targeting ability of RGD-modified liposomes can be evaluated using fluorescence-loaded liposomes. Finally, the in vitro and in vivo antitumor effects of RGD-LS/pDP are illustrated (Li et al. 2012), which proves that RGD modification can significantly improve the treatment efficacy of the liposomes.
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Fig. 2 Schematic representation of synthesis of RGD-PEG-DSPE
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2
Materials
2.1
Synthesis of RGD Peptide
1. Protected Fmoc-amino acid derivatives Asp(OtBu), D-Tyr(OtBu), Lys(Boc), and Arg(Pbf) 2. H-Gly-2-Chlorotrityl resin 3. Trifluoroacetic acid (TFA) 4. Piperidine, N,N-Dimethylformamide (DMF), and ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) 5. O-Benzotriazole-N,N,N0 ,N0 -tetramethyl-uronium-hexafluorophosphate (HBTU) 6. Diisopropylethylamine (DIEA) 7. N-Hydroxybenzotriazole (HOBt) and Benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) 8. N-Succinimudyl-S-acetylthiopropionat (SATP) 9. Hydroxylamine hydrochloride (NH2OH∙HCL) 10. A peptide synthesis vessel which can be used for pumping filtration separation 11. A high performance liquid chromatograph (HPLC) system equipped with a reversed-phase column 12. Preparative HPLC 13. A vacuum pump 14. A freeze dryer 15. Phosphate-buffered saline (PBS): pH 7.4, 0.1 M
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Preparation of RGD Conjugated Polyethylene Glycol-Lipid (RGD-PEG-DSPE)
1. RGD-SH 2. Mal-derivatized polyethylene glycol 3400-phosphatidylethanolamine distearoyl (Mal-PEG3400-DSPE) 3. PBS: pH 7.4, 0.1 M 4. DMF 5. Distilled water 6. A 30-mL glass reactor 7. Magnetic stirrers 8. A magnetic stirring apparatus 9. A dialysis bag (cutoff molecular weight 3, 500 Da) 10. A 3-L glass reactor 11. A freeze dryer
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Characterization of RGD-PEG-DSPE
1. RGD-PEG-DSPE 2. Mal-PEG-DSPE
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3. Chloroform-d, 99.8 atom % D 4. Borosilicate glass tubes for nuclear magnetic resonance 5. Nuclear magnetic resonance (NMR) spectrometer
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Preparation of RGD-Modified Liposome
2.4.1 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Preparation of RGD-Modified Liposome Loaded with Doxorubicin (RGD-LS/DOX) PEG2000-DSPE Hydrogenated soybean phosphatidylcholine (HSPC) Cholesterol (Chol) RGD-PEG-DSPE Chloroform A 50-mL round-bottomed spherical Quick-fit flask A rotary evaporator equipped with water bath Vacuum desiccators to remove solvent from the dried film Doxorubicin (DOX) Double distilled water 0.155 M ammonium sulfate solution Normal saline (NS) Sephadex G-50 A vortex mixer A shaking water bath Mini-extruder
2.4.2 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
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Preparation of RGD-Modified Liposome Loaded with pDP (RGDLS/pDP) PEG2000-DSPE Hydrogenated soybean phosphatidylcholine (HSPC) Cholesterol (Chol) RGD-PEG-DSPE Chloroform pDP was in-house produced by solid-phase synthesis method NS A 50-mL round-bottomed spherical Quick-fit flask An ultrasonic apparatus A rotary evaporator equipped with water bath Sephadex G-50 Mini-extruder
Characterization of RGD-Modified Liposomes
1. RGD-LS/DOX, RGD-LS/pDP 2. NS
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3. 4. 5. 6.
2% (w/v) phosphotungstic acid RGD-LS Unmodified liposome (LS) Human integrin αvβ3 kit, including recombinant human integrin αvβ3 and reaction buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150 mM NaCl, 1 mM MgCl2, and 1 mM MnCl2, pH 7.4) N-Hydroxysuccinimide (NHS)-biotin PBS: pH 7.4, 0.1 M Streptavidin-gold conjugate Double distilled water 5% (v/v) triton DOX standard solutions: 0.125, 0.25, 0.5, 1.0, and 2.0 mg/ml DOX in 5% triton Ultrafiltration tubes (cutoff molecular weight 10, 000 Da) The dynamic light scattering particle size apparatus A transmission electron microscope A centrifuge An ultraviolet spectrophotometer HPLC
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
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Tumor Targeting Ability of RGD-Modified Liposomes
2.6.1
Cellular Uptake
Preparation of RGD-Modified Liposome Loaded with 5-Carboxyfluorescein (RGD-LS/FAM) 1. PEG2000-DSPE 2. Hydrogenated soybean phosphatidylcholine (HSPC) 3. Cholesterol (Chol) 4. RGD-PEG-DSPE 5. Chloroform 6. A 50-mL round-bottomed spherical Quick-fit flask 7. A rotary evaporator equipped with water bath 8. Vacuum desiccators to remove solvent from the dried film 9. 5-Carboxyfluorescein (FAM) 10. Double distilled water 11. 0.1-M sodium hydroxide solution (NaOH) 12. Normal saline (NS) 13. Sephadex G-50 14. A vortex mixer 15. A shaking water bath 16. Mini-extruder 17. A fluorescence spectrophotometer
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Uptake by Glioblastoma Cells 1. Human glioblastoma U87 cells 2. Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS) Penicillin-Streptomycin solution 100, PBS (pH 7.4, 1) and Trypsin EDTA 3. T75 cell culture flasks 4. Pipettes (5 mL and 1 mL) 5. A cell counter 6. A CO2 incubator 7. A 12-well plate 8. 40 ,6-diamidino-2-phenylindole (DAPI) 9. A confocal laser microscope 10. LS/FAM and RGD-LS/FAM
2.6.2
In Vivo Tumor Targeting Ability
Preparation of RGD-Modified Liposome Loaded with Near-Infrared Fluorescent Dyes (RGD-LS/Dir) 1. PEG2000-DSPE 2. Hydrogenated soybean phosphatidylcholine (HSPC) 3. Cholesterol (Chol) 4. RGD-PEG-DSPE 5. Chloroform 6. A 50-mL round-bottomed spherical Quick-fit flask 7. A rotary evaporator equipped with water bath 8. Vacuum desiccators to remove solvent from the dried film 9. 1,10 -Dioctadecyl-3,3,30 ,30 -tetramethyl indotricarbocyanine iodide (Dir) 10. Acetonitrile 11. Double distilled water 12. Normal saline (NS) 13. Sephadex G-50 14. A vortex mixer 15. A shaking water bath 16. Mini-extruder 17. A fluorescence spectrophotometer In Vivo Distribution 1. U87 cells 2. Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), Penicillin-Streptomycin solution 100, PBS (pH 7.4, 1), and Trypsin EDTA 3. T75 cell culture flasks, 96-well microplates 4. Pipettes (5 mL, 1 mL, 200 μL) 5. 1.7 mL, 5 mL centrifuge tubes 6. A cell counter 7. A CO2 incubator 8. Medical cottons
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Microinjectors Stereotaxic apparatus with mouse adaptor A scissor and a tweezer A centrifuge Male Balb/c nude mice, 6–8 weeks old Chloral hydrate LS/Dir and RGD-LS/Dir 1-mL and 20-mL syringe NS An In Vivo Imaging System (IVIS)
Antitumor Activity of RGD-Modified Liposomes
2.7.1 In Vitro Antitumor Activity 1. U87 cells 2. Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), Penicillin-Streptomycin solution 100, PBS (pH 7.4, 1), and Trypsin EDTA 3. T75 cell culture flasks, 96-well microplates 4. Pipettes (5 mL, 1 mL, 200 μL), Multichannel pipette (300 μL) 5. 1.7-mL, 5-mL centrifuge tubes 6. A cell counter 7. A CO2 incubator 8. 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazolium romide (MTT) 9. Dimethyl sulfoxide (DMSO) 10. A microplate reader 11. pDP and D-PMIβ peptides, Nutin-3, RGD-LS/DOX, LS/pDP, and RGDLS/pDP 2.7.2 In Vivo Antiglioblastoma Efficacy 1. U87 cells 2. Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), Penicillin-Streptomycin solution 100, PBS (pH 7.4, 1), and Trypsin EDTA 3. T75 cell culture flasks, 96-well microplates 4. Pipettes (5 mL, 1 mL, 200 μL), Multichannel pipette (300 μL) 5. 1.7-mL, 5-mL centrifuge tubes 6. A cell counter 7. A CO2 incubator 8. Medical cottons 9. Microinjectors 10. Stereotaxic apparatus with mouse adaptor 11. 1-mL syringes 12. A centrifuge 13. Male Balb/c nude mice, 6–8 weeks old
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14. Chloral hydrate 15. Empty RGD-LS, LS/pDP, LS/DOX, and RGD-LS/pDP
3
Methods
3.1
Synthesis of RGD Peptide
Firstly, synthesize the liner RGD peptide (Arg-Gly-Asp-Tyr-Lys) via Fmoc-protected solid-phase peptide synthesis strategy, then cyclize the liner peptide by PyBOP to get the c(RGDyK) peptide, finally thiolate c(RGDyK) using SATP to get c(RGDyK)-SH, which can be used to conjugate with PEGylated lipids. 1. Weigh out 0.33 g of Gly-2-Cl-Trt resin (0.2 mmol) and put it into a peptide synthesis vessel (see Note 1). 2. To remove the protective group on the amino of Gly on resin, add 5 mL of 20% (v/v) piperidine in DMF solution to deprotect the resin. 3. Remove the present solution 30 min later using a vacuum pump and wash the deprotected resin using 10 mL of DMF for three times. 4. Weight out 0.65 g of Arg(Pbf) amino acid (1 mmol), dissolve it using 5 mL of 0.5-M HOBt/HBTU in DMF solution and 0.8 mL of DIEA, and add it into the vessel to allow the carboxyl group of Arg to react with the amino group of Gly which is on the resin (see Note 2). 5. Remove the unreacted amino acid 45 min later and wash the resin with DMF for three times. Now Arg(Pbf) should have been conjugated to Gly-2-Cl-Trt resin. 6. Repeat the “2–5” steps to conjugate the following amino acid to the resin, using Lys(Boc), D-Tyr(OtBu), or Asp(OtBu) instead of Arg(Pbf) for the step “4.” 7. Cleave the peptide-resin bond using 20 mL of 1% (v/v) TFA in H2O for 30 min, purify the resultant liner peptide using a preparative HPLC, and use a freeze dryer to get frozen-dried liner RGD. 8. To obtain cyclic RGD peptide, mix 22.0 mg of liner RGD (0.01 mmol), 5.20 mg of PyBOP (0.01 mmol), and 2 μL of DIEA (0.015 mmol), and allow them to react for 6 h (see Note 3). 9. To deprotect the protective group on the side chains of the peptide, add 20 mL of 95% TFA in H2O into the cyclic RGD and let them react for 2 h. Purify the resultant cyclic peptide using the preparative HPLC, and froze dry the c(RGDyK) peptide. 10. To thiolate the c(RGDyK) peptide, dissolve 15 mg of c(RGDyK) with 1 mL of PBS and 12 mg of SATP by 20 μL of DMF, mix them together, let them react for 1 h, purify the resultant acetyl-protected c(RGDyK)-SH peptide using the preparative HPLC, and froze dry it. 11. Prepare a deprotection solution: 0.5 M NH2OH∙HCL and 25 mM EDTA-2Na in PBS.
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12. Dissolve the dried acetyl-protected c(RGDyK)-SH peptide by 2 mL of PBS and add 0.2 mL of deprotection solution into the peptide to remove the acetyl group. Purify the rude c(RGDyK)-SH 1 h later using the preparative HPLC and froze dry it.
3.2
Preparation of RGD-PEG-DSPE
1. Weigh out 5.3 mg of RGD-SH (7.2 μmol), place into a 30 ml glass reactor vessel, and dissolve it using 3 ml of PBS (pH = 7.4). 2. Dissolve 20.4 mg of Mal-PEG-DSPE (6 μmol) in 1 ml of DMF. 3. Add the 1 ml of Mal-PEG-DSPE in DMF into the 3 ml of RGD-SH in PBS dropwise and keep the reaction solution stirred for 1 h (see Note 4). 4. Purify the resultant RGD-PEG-DSPE using the dialysis method: add the resulting reaction solutions into a dialysis bag (3, 500 MW), dialyze it in a 3-L glass reactor with 3 L of distilled water, and change the outside water every 4 h for three times, so the unreacted RGD-SH and DMF will be removed (see Note 5). 5. Freeze-dry the purified RGD-PEG-DSPE using a freeze dryer (see Note 6).
3.3
Characterization of RGD-PEG-DSPE
1. Add 2 mg of RGD-PEG-DSPE into a borosilicate glass tube which is specially used for nuclear magnetic resonance. 2. Add 1 ml of Chloroform-d into the tube to dissolve the materials. 3. Characterize the materials by 1H-NMR, using a NMR spectrometer. Mal-PEG-DSPE can be used here as a negative control. An example of 1H-NMR characterization result is shown in Fig. 3. The characteristic peak of the maleimide group of Mal-PEG-DSPE, which occurs at 6.7 ppm, disappeared in the 1H-NMR spectra of c(RGDyK)-PEG-DSPE, indicating that the Mal group of Mal-PEG-DSPE had reacted with the thiol group of c(RGDyK) (Li et al. 2012).
3.4
Preparation of RGD-Modified Liposomes
3.4.1 Preparation of RGD-LS/DOX Prepare RGD-LS using thin film hydration and extrusion method, and then load the RGD-LS with DOX via pH-gradient method. 1. Weight out 10.4 mg of HSPC, 3.74 mg of Chol, 1.93 mg of PEG-DSPE, and 1 mg of RGD-PEG-DSPE (HSPC/Chol/PEG-DSPE/RGD-PEG-DSPE = 55:40:4:1, molar ratio) into a 50-ml round-bottom flask (see Note 7). 2. Dissolve all lipids using 5 mL of chloroform. 3. Remove the chloroform by rotary evaporation under the reduced pressure at 40 C, and then a thin film of lipids will form on the round-bottom flask (see Note 8).
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1
H-NMR spectrum of Mal-PEG-DSPE and c(RGDyK)-PEG-DSPE
4. Put the flask into a vacuum chamber overnight, to remove the residual chloroform. 5. Add 1 ml of 155 mM ammonium sulfate solution and suspend the film using vortex mixer (see Note 9). 6. Rotate the lipid solution in a 60 C incubator for 2 h (see Note 10). 7. Extrude repeatedly the resulting lipid solution through 400 nm, 200 nm, 100 nm, and 50 nm polycarbonate membranes successively, using a mini extruder (see Note 11). 8. Filter the liposomes over a Sephadex G-50 column, eluted with NS. 9. Add 2.8 mg of DOX into the liposomes (Drug/Lipid = 5:1, molar ratio) and incubate the solution at 60 C for 15 min (see Note 12). 10. To remove the unloaded DOX, the obtained DOX-loaded liposomes were purified over a Sephadex G-50 column, eluted with NS.
3.4.2 Preparation of RGD-LS/pDP Prepare RGD-LS/pDP using reverse evaporation method followed with extrusion method. 1. Weight out 10.4 mg of HSPC, 3.74 mg of Chol, 1.93 mg of PEG-DSPE, and 1 mg of RGD-PEG-DSPE (HSPC/Chol/PEG-DSPE/RGD-PEG-DSPE = 55:40:4:1, molar ratio) into a 50-ml round-bottom flask. 2. Dissolve all lipids (17.07 mg) using 6 mL of chloroform. 3. Dissolve 2.56 mg of pDP peptide into 1 mL of NS.
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4. Add the pDP solution into the lipid solution, and sonicate the resultant suspension until it turns to a W/O emulsion. 5. Evaporate the emulsion vertically under the reduced pressure at 40 C, until the emulsion turns into gel. 6. Add 2 mL of NS into the gel to dilute the liposomes and continue the rotary evaporation to remove the residual chloroform. 7. Extrude repeatedly the resulting liposomal pDP through 400 nm, 200 nm, 100 nm, and 50 nm polycarbonate membranes successively, using a mini extruder. 8. To remove the unloaded pDP, filter the liposomes over a Sephadex G-50 column, eluted with NS.
3.5
Characterization of RGD-Modified Liposomes
3.5.1 Physicochemical Characterization of RGD-Modified Liposomes The size, zeta potential, and morphology of RGD-LS were detected. 1. Determine the particle size and zeta potential of RGD-LS using a dynamic light scattering instrument. The typical particle size and zeta potential of RGD-LS is around 100 nm and 0 mV, respectively. 2. Detect the morphology of the liposomes using a transmission electron microscope. Dilute the liposomes using double distilled water for 50 times (see Note 13) and then stain the resulting liposomes using 2% (w/v) phosphotungstic acid. Observe the stained liposomes under the TEM. A representative image of DOXloaded liposomes is shown in Fig. 4 (Yan et al. 2011).
3.5.2
Characterization of Conjugated RGD on the Surface of Liposomes The conjugation of RGD peptide to liposomes can be detected using an immunogold staining method. To get a clear view of gold, the liposomes without drug were used here. 1. Weight out 50 μg of recombinant human integrin αvβ3 and 1 μg of NHS-biotin (NHS-biotin/integrin >10, molar ratio). 2. Incubate the integrin and NHS-biotin in 100 μL of PBS (pH 7.4) for 30 min. 3. Put the obtained solution into an ultrafiltration tube (cutoff MW 10, 000 Da). 4. Centrifuge the tube at 3,000 g for 15 min. Add 100 μL of PBS into the protein solution. 5. Repeat the step “4” two times. 6. Resuspend the purified biotinylated integrin in 75 μL of reaction buffer. 7. Add 125 μL of reaction buffer into 50 μL of RGD-LS (about 1 mg). Incubate the liposomes with 25 μL of biotinylated integrin for 4 h. 8. To remove the unbound biotinylated protein, the obtained integrin-labeled liposomes were purified over a Sephadex G-50 column, eluted with NS.
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Fig. 4 A TEM image showed nearly spherical morphology of LS/DOX
9. Incubate the integrin-labeled liposomes with streptavidin-gold conjugate (protein/streptavidin-gold = 50:1, volume ratio) for 1 h. 10. To remove the unbound gold, the resulting gold-labeled liposomes were purified over a Sephadex G-50 column, eluted with NS. 11. Gold-labeled liposomes were diluted using double distilled water for 10 times and then stained using 2% (w/v) phosphotungstic acid. 12. Observe the stained liposomes under the TEM. An example of characterization result is shown in Fig. 5 (Li et al. 2012). Two negative controls were used here. They were LS incubated with biotinylated integrin αvβ3 and streptavidin-labeled colloidal gold sequentially and RGD-LS without incubation with biotinylated integrin, respectively. From the TEM images, it could be clearly seen that the liposome bound with integrin αvβ3 via RGD modification.
3.5.3 DOX Encapsulation Efficacy The concentrations (Conc.) of DOX in the liposomal samples can be detected using an ultraviolet spectrophotometer. 1. Prepare the liposomes according to the 1–9 steps in the Sect. 3.4.1. 2. After incubating DOX with RGD-LS, add 0.5 ml of the obtained liposomes into 4.5 ml of 5% triton (solution 1), so the lipids would be dissolved by triton and all DOX be released into the solution. 3. Purify 0.5 ml of the obtained RGD-LS/DOX liposomes above through a Sephadex G-50 column and collect all of the liposomes loaded with DOX. 4. Dissolve all of the resulting purified RGD-LS/DOX in 5% triton with a final volume of 5 mL (solution 2) (see Note 14).
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Fig. 5 Transmission electron micrograph of (a) RGD-LS incubated with biotinylated integrin αvβ3 and streptavidin labeled colloidal gold sequentially, (b) RGD-LS only incubated with streptavidin labeled colloidal gold, and (c) LS incubated with biotinylated integrin αvβ3 and streptavidin-labeled colloidal gold sequentially. Scale bar: 10 nm
5. Prepare a DOX stock solution (4 mg/ml DOX and 24 mg/ml lipids) in 5% triton (see Note 15) and dilute it using 5% triton to get a series of DOX standard solutions: 0.125, 0.25, 0.5, 1.0, and 2.0 mg/ml DOX in 5% (v/v) triton. 6. Measure the absorbance of the DOX standard solutions at 480 nm using an ultraviolet spectrophotometer and generate a standard curve to establish the relationship between the absorbance value and the Conc. of DOX solution. 7. Measure the absorbance of the solution 1 and solution 2, and calculate the Conc. of DOX in solution 1 and solution 2 by the standard curve above. 8. The encapsulation efficacy of DOX in the liposomes is calculated according to the following equation: Encapsulation efficacy% ¼
Conc:of DOX in solution 2=Conc:of DOX in solution 1 100%
3.5.4 pDP Encapsulation Efficacy The protocol for assessment of pDP encapsulation efficacy is similar with that described in the Sect. 3.5.3, except that the Conc. of pDP standard solutions are 1.954, 3.908, 7.815, 15.63, 31.25, 62.50, and 125.0 μg/mL, and the Conc. of pDP in each sample is detected using HPLC. The HPLC conditions for detecting pDP are as follows: Column: C4 column (4.6 150 mm, 5 μm) Mobile phase: elution buffer A-0.1% TFA in water, elution buffer B-0.1% TFA in acetonitrile, gradient elution mode, 30~90% B, 30 min Column temperature: 40 C Detection wavelength: diode array detector, λ = 214 and 280 nm Flow rate: 0.7 mL/min
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Tumor Targeting Ability of RGD-Modified Liposomes
3.6.1 Cellular Uptake The cellular targeting abilities of RGD-LS/FAM and LS/FAM to U87 cells were evaluated using a confocal laser microscope.
Preparation of RGD-Modified Liposome Loaded with 5-Carboxyfluorescein (RGD-LS/FAM) Prepare RGD-LS/FAM using thin film hydration and extrusion method. 1. Weight out 10.4 mg of HSPC, 3.74 mg of Chol, 1.93 mg of PEG-DSPE, and 1 mg of RGD-PEG-DSPE (HSPC/Chol/PEG-DSPE/RGD-PEG-DSPE = 55:40:4:1, molar ratio) into a 50-ml round-bottom flask. 2. Dissolve all lipids using 5 mL of chloroform. 3. Remove the chloroform by rotary evaporation under the reduced pressure at 40 C, and then a thin film of lipids will form on the round-bottom flask. 4. Put the flask into a vacuum chamber overnight, to remove the residual chloroform. 5. Suspend 2 mg FAM using 1 mL NS and add 40 μL of 1 M NaOH solution to dissolve the FAM (see Note 16). 6. Add the FAM solution into the flask and suspend the film using vortex mixer. 7. Rotate the lipid solution in a 60 C incubator for 2 h. 8. Extrude repeatedly the resulting lipid solution through 400 nm, 200 nm, 100 nm, and 50 nm polycarbonate membranes successively, using a mini extruder. 9. Filter the liposomes over a Sephadex G-50 column to remove the unloaded FAM, eluted with NS. 10. The Conc. of FAM in liposomes is detected using a fluorescence spectrophotometer (Excitation/emission wavelength = 494/522 nm).
Uptake by Glioblastoma Cells Cell Culture
1. Prepare the cell culture medium by adding 5 mL of Penicillin-Streptomycin solution and 50 mL of FBS into 445 mL of DMEM. 2. Place about 5 105 U87 cells into a T75 flask with 10 mL of the prepared medium inside (see Note 17). 3. To cultivate the cells, put the flask with cells into the incubator at 37 C and 5% CO2. 4. Subculture the cells when 80% cells are confluent. Drain the present culture medium, wash the flask with 2 mL of PBS for one time, add 1 mL of trypsin to dissociate the cells, remove the trypsin 30 s later, add 2 mL of medium into the flask to stop trypsinization, suspend the cells using pipette, divide the cells equally into two T75 flasks and cultivate the cells in the incubator (see Note 18).
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Fig. 6 Cellular uptake of LS/FAM and RGD-LS/FAM by U87 cells. Cells were incubated with LS/ FAM or RGD-LS/FAM for 4 h, then the cell nucleus was stained with DAPI. Green and blue colors in the image represent FAM and DAPI, respectively. (Scale bar = 10 μm.)
Observation Using Confocal Laser Microscope
1. 2. 3. 4. 5. 6. 7. 8.
9.
Trypsinize one T75 flask of U87 cells and suspend the cells in 10 mL of medium. Use a cell counter to obtain the Conc. of the cell suspensions. Add 240, 000 cells into 12 mL of culture medium. Place 1 mL of the obtained cell suspensions into each well of a 12-well plate. Incubate the plate overnight. Use culture medium to prepare diluted RGD-LS/FAM and LS/FAM solutions (the final Conc. of FAM in each sample is 5 μM). Remove the present medium in the 12-well plate and treat the cells with 1 mL of each liposomal solution (n = 3) for 4 h in the incubator. Prepare 10 μg/mL DAPI solution using PBS. Rinse each well using PBS for three times. Add 1 mL of DAPI solution into each well to dye the cell nucleus. Remove the DAPI solution 10 min later and rinse each well by PBS for three times. Capture the fluorescent intensity using a confocal laser microscope. The excitation/emission wavelength of FAM and DAPI is at 494/522 nm and 358/461 nm, respectively.
A representative result of RGD-LS/FAM cellular uptake study is shown in Fig. 6 (Wei et al. 2015). While there is no significant fluorescence of FAM found in the cells treated with unmodified liposomes, abundant uptake was observed with the cells treated by liposomes modified with c(RGDyK). It indicates that RGD modification can facilitate the cellular uptake of liposomes by integrin mediation.
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3.6.2 In Vivo Tumor Targeting Ability The tumor tissue targeting ability of RGD-LS/Dir in the intracranial U87 glioma model was examined, using LS/Dir as a negative control. Preparation of RGD-Modified Liposome Loaded with Near-Infrared Fluorescent Dyes (RGD-LS/Dir) Prepare RGD-LS/Dir using thin-film hydration and extrusion method. 1. Weight out 10.4 mg of HSPC, 3.74 mg of Chol, 1.93 mg of PEG-DSPE, and 1 mg of RGD-PEG-DSPE (HSPC/Chol/PEG-DSPE/RGD-PEG-DSPE = 55:40:4:1, molar ratio) into a 50-ml round-bottom flask. 2. Dissolve all lipids using 5 mL of chloroform. 3. Prepare 5 mg/mL Dir solution using acetonitrile, and then add 10 μL of Dir solution into the lipid solution (see Note 19). 4. Remove the chloroform and acetonitrile by rotary evaporation under the reduced pressure at 40 C, and then a thin film of lipids will form on the round-bottom flask. 5. Put the flask into a vacuum chamber overnight, to remove the residual chloroform. 6. Add 1 mL NS into the flask and suspend the film using vortex mixer. 7. Rotate the lipid solution in a 60 C incubator for 2 h. 8. Extrude repeatedly the resulting lipid solution through 400 nm, 200 nm, 100 nm, and 50 nm polycarbonate membranes successively, using a mini extruder. 9. Filter the liposomes over a Sephadex G-50 column to remove the unloaded Dir, eluted with NS. 10. The Conc. of Dir in liposomes is detected using a fluorescence spectrophotometer (Excitation/emission wavelength = 748/780 nm). In Vivo Distribution Establishment of Intracranial Glioma Model
1. Prepare the cells for establishing the nude mice intracranial U87 glioma model. Culture two T75 flasks of U87 cells, trypsinize all the cells, and suspend them in 4 mL of culture medium, centrifuge the cells at 800 g for 5 min, remove the supernatant and resuspend the cells in 4 mL of PBS, then centrifuge the cells at 800 g for another 5 min, remove the supernatant and resuspend the cells in 0.9 mL of PBS. 2. Prepare 7% (w/w) chloral hydrate solution in PBS. 3. Anesthetize 6 mice using 0.1 mL of chloral hydrate solution. 4. Inject 5 μL of the obtained cell suspensions slowly into the right striatum (1.8 mm lateral, 0.6 mm anterior to the bregma, and 3 mm of depth) of each mouse, using a stereotactic fixation device with mouse adaptor. 5. Divide the intracranial tumor-bearing nude mice randomly into two groups (n = 3). On day 15 postinoculation, treat the two groups intravenously with RGD-LS/Dir or LS/Dir (0.25 mg/kg Dir).
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Fig. 7 The ex vivo images of brains from glioma-bearing nude mice treated with LS/Dir or RGDLS/Dir
6. After 12 h, euthanatize the mice, perfuse the mice with NS, dissect and collect the brain tissues. 7. Take images of the brain tissues using an In Vivo Imaging System (excitation/ emission wavelength at 748/780 nm). Figure 7 shows the result of in vivo distribution of RGD-modified liposomes in the excised mice brain (Wei et al. 2015). While the unmodified liposomes barely distributed in the brain, RGD-LS/Dir can accumulate in the brain tumor. This suggests the effective tumor targeting ability of RGD-modified liposomes.
3.7
Antitumor Activity of RGD-Modified Liposomes
3.7.1 In Vitro Antitumor Activity The in vitro antitumor activities of RGD-LS/pDP, RGD-LS/DOX, LS/pDP, and Nutin-3 (a small molecule antagonist of MDM2) against U87 cells were evaluated. Two D-peptides, D-PMIβ, and pDP were used as controls. 1. Culture one flask of U87 cells using the protocol mentioned in Uptake by Glioblastoma Cells, Step 1. 2. Trypsinize one T75 flask of U87 cells and suspend the cells in 10 mL of medium. 3. Use a cell counter to obtain the Conc. of the cell suspensions. 4. Add 500, 000 cells into 20 mL of culture medium. 5. Place 0.2 mL of the obtained cell suspensions into each well of a 96-well plate, leaving 3 wells to fill PBS instead of cell suspensions as blank control. Incubate the plate overnight.
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Fig. 8 In vitro antitumor activity of the RGD-liposomal pDP against U87 cells. U87 cells were seeded in 96-well plates, cultured overnight, treated with the liposomes, and then cell viabilities were tested after 3 days. Relative cell viability was expressed as a percentage of control cells treated with the medium
6. Use culture medium to prepare successively diluted pDP, D-PMIβ, Nutin-3, RGD-LS/DOX, LS/pDP, and RGD-LS/pDP solutions, with a final Conc. of each drug ranged from 0.1 μM to 25 μM. 7. Remove the present medium in the 96-well plate and treat the cells with 0.2 mL of each drug solution (n = 3) for 72 h in the incubator, leaving one set of wells without drug as control. 8. Prepare 5 mg/mL MTT solution using PBS and add 20 μL of MTT solution into each well. 9. Incubate the plate for another 4 h and then remove the culture medium (see Note 20). 10. Add 0.15 mL of DMSO into each well to dissolve the MTT formazan crystals and incubate at 37 C for 10 min. 11. Measure the absorbance at a test wavelength of 595 nm and a reference wavelength of 650 nm, using a microplate reader. 12. Calculate the cell viability of each sample and the IC50 value of each formulation. A representative result of RGD-LS/pDP cytotoxicity study is shown in Fig. 8 (Li et al. 2012). The free D-PMIβ cannot be internalized by U87 cells, therefore it does not affect the cell viability under the tested concentrations. In contrast, pDP, the palmitylated derivate of D-PMIβ, possesses improved lipophilicity and decreased cell viability under high concentrations. The IC50 value of LS/pDP and RGD-LS/ pDP is 1.30 μM and 550 nM, respectively. It shows that RGD modification can improve the in vitro antitumor activity of the pDP liposomes due to efficient internalization mediated by integrin receptor.
3.7.2 In Vivo Antiglioblastoma Efficacy 1. Prepare 32 glioma-bear nude mice using the protocol mentioned in In Vivo Distribution, Step 1.
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Fig. 9 Kaplan-Meier survival curve of intracranial U87 glioma-bearing nude mice model. The RGD-liposomal pDP has significantly improved the survival time of the treated mice compared to the unmodified liposomal group ( p < 0.001)
2. Divide the intracranial tumor-bearing nude mice randomly into four groups (n = 8). On day 8, 12, 16, and 20 postinoculation, treat the four groups intravenously with empty RGD-LS, LS/pDP, LS/DOX (5 mg/kg each time), and RGDLS/pDP (10 mg/kg each time), respectively. 3. Record the survival time of each mouse. Figure 9 shows the result of antiglioblastoma efficacy study on the RGD-modified liposome loaded with pDP (Li et al. 2012). The mice treated with RGD-LS/pDP have significantly longer survival time than the mice treated with LS/pDP, demonstrating that the RGD-mediated active targeting liposomes have much better antitumor efficacy than the unmodified liposomes.
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Notes
There are some common mistakes when following the above procedure. Here are some notes which can be used to troubleshoot possible problems. Note: 1. Avoid water during the progress of solid phase synthesis, as amino acids cannot react with other reactant when they are dissolved in water. 2. To get a high productive rate of the peptide, five times of excessive free amino acid was used here to react with the amino acid on the resin. HOBt, HBTU, and DIEA work as catalysts. 3. Here DIEA are used as catalysts. 4. In order to get a stable emulsion, add the 1 mL of organic phase into the 3 mL of aqueous phase. Do not operate in reverse.
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5. Keep the reaction temperature under 25 C, as RGD-PEG-DSPE is not stable at high temperature. 6. Keep the dried RGD-PEG-DSPE in a 20 C freezer. 7. Dry the flask thoroughly in a drying oven to make sure there is no water in the flask. 8. Keep the vacuum degree of the vacuum pump around 0.07 MPa to avoid forming bubbles during the rotary evaporation progress. 9. If the vortex mixer cannot shake the lipid film down from the flask, a water-bath ultrasound equipment could be applied here. 10. The temperature for the incubation progress should be above the phase-transition temperature of the lipids, which is 60 C here. Use a glass stopper and parafilm to seal the flask during the incubation progress. 11. Operate the extrusion progress on a heater and set the temperature at 60 C. 12. The procedures involving DOX should be performed away from light. 13. The liposomes should be properly diluted for TEM imaging. As salts would be shown as tiny black dots on the TEM image, double distilled water was used here to dilute the liposomes. 14. A 5-mL volumetric flask could be applied here to guarantee the accuracy of the final volume of the dissolved liposomes. 15. According to the previous research, the lipids used in the formulation would not influence the absorbance of DOX at 480 nm. For the drugs whose absorbance would be interrupted by lipids, first the drug should be extracted out of the lipids, and then its concentration is detected. 16. The procedures involving FAM should be performed away from light. 17. Warm up the culture medium to 37 C before using it. 18. When subculturing the U87 cells, a subcultivation ratio of 1:2 to 1:3 is recommended. 19. The procedures involving Dir should be performed away from light. 20. To avoid loss of MTT formazan crystals, remove the culture medium gently.
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Conclusion
Integrins are an important family of receptors overexpressed on several tumor cells and endothelial cells of tumor angiogenesis. RGD peptide and its derivatives having an amino acid sequence of Arg-Gly-Asp could specifically bind to integrins with high affinity. Therefore, RGD peptide-modified liposomes became one of the most popular targeting drug delivery nanocarriers. In this chapter, the knowledge about preparation and evaluation of RGD peptide-mediated targeting drug liposomes is introduced in detail. Classic methods about peptide modification, liposome preparation, and drug loading are described here, including monothiol-specific conjugation, thin-film hydration, reverse evaporation, and extrusion methods. Some experimental results are given to show the physiochemical characterization, tumor targeting ability, and antitumor ability of RGD-modified liposomes. Taken together,
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integrin receptor-mediated targeting drug liposomes are potential and promising therapeutics for cancer treatment.
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Li C, Shen J, Wei X, Xie C, Lu W (2012) Targeted delivery of a novel palmitylated D-peptide for antiglioblastoma molecular therapy. J Drug Target 20(3):264–271 Liang MT, Davies NM, Toth I (2005) Encapsulation of lipopeptides within liposomes: effect of number of lipid chains, chain length and method of liposome preparation. Int J Pharm 301(1–2):247–254 Maeda H, Wu J, Sawa T, Matsumura Y, Hori K (2000) Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J Control Release 65(1–2):271–284 Manzoni L, Belvisi L, Arosio D, Civera M, Pilkington-Miksa M, Potenza D, Caprini A, Araldi EM, Monferini E, Mancino M, Podestà F, Scolastico C (2009) Cyclic RGD-containing functionalized azabicycloalkane peptides as potent integrin antagonists for tumor targeting. ChemMedChem 4(4):615–632 Murphy EA, Majeti BK, Barnes LA, Makale M, Weis SM, Lutu-Fuga K, Wrasidlo W, Cheresh DA (2008) Nanoparticle-mediated drug delivery to tumor vasculature suppresses metastasis. Proc Natl Acad Sci U S A 105(27):9343–9348 Oba M, Aoyagi K, Miyata K, Matsumoto Y, Itaka K, Nishiyama N, Yamasaki Y, Koyama H, Kataoka K (2008) Polyplex micelles with cyclic RGD peptide ligands and disulfide cross-links directing to the enhanced transfection via controlled intracellular trafficking. Mol Pharm 5(6):1080–1092 Rodriguez MA, Pytlik R, Kozak T, Chhanabhai M, Gascoyne R, Lu B, Deitcher SR, Winter JN (2009) Vincristine sulfate liposomes injection (Marqibo) in heavily pretreated patients with refractory aggressive non-Hodgkin lymphoma: report of the pivotal phase 2 study. Cancer 115(15):3475–3482 Ruoslahti E (2003) The RGD story: a personal account. Matrix Biol 22(6):459–465 Samanen J, Ali F, Romoff T, Calvo R, Sorenson E, Vasko J, Storer B, Berry D, Bennett D, Strohsacker M (1991) Development of a small RGD peptide fibrinogen receptor antagonist with potent antiaggregatory activity in vitro. J Med Chem 34(10):3114–3125 Sudimack J, Lee RJ (2000) Targeted drug delivery via the folate receptor. Adv Drug Deliv Rev 41(2):147–162 Tewey KM, Rowe TC, Yang L, Halligan BD, Liu LF (1984) Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II. Science 226(4673):466–468 Vachutinsky Y, Oba M, Miyata K, Hiki S, Kano MR, Nishiyama N, Koyama H, Miyazono K, Kataoka K (2011) Antiangiogenic gene therapy of experimental pancreatic tumor by sFlt-1 plasmid DNA carried by RGD-modified crosslinked polyplex micelles. J Control Release 149(1):51–57 Wei X, Gao J, Zhan C, Xie C, Chai Z, Ran D, Ying M, Zheng P, Lu W (2015) Liposome-based glioma targeted drug delivery enabled by stable peptide ligands. J Control Release 218:13–21 Yan Z, Zhan C, Wen Z, Feng L, Wang F, Liu Y, Yang X, Dong Q, Liu M, Lu W (2011) LyP-1conjugated doxorubicin-loaded liposomes suppress lymphatic metastasis by inhibiting lymph node metastases and destroying tumor lymphatics. Nanotechnology 22(41):415103 Yang X, Hong H, Grailer JJ, Rowland IJ, Javadi A, Hurley SA, Xiao Y, Yang Y, Zhang Y, Nickles RJ, Cai W, Steeber DA, Gong S (2011) cRGD-functionalized, DOX-conjugated, and 64Cu-labeled superparamagnetic iron oxide nanoparticles for targeted anticancer drug delivery and PET/MR imaging. Biomaterials 32(17):4151–4160 Zhan C, Gu B, Xie C, Li J, Liu Y, Lu W (2010) Cyclic RGD conjugated poly(ethylene glycol)-copoly(lactic acid) micelle enhances paclitaxel anti-glioblastoma effect. J Control Release 143(1):136–142 Zhan C, Li B, Hu L, Wei X, Feng L, Fu W, Lu W (2011) Micelle-based brain-targeted drug delivery enabled by a nicotine acetylcholine receptor ligand. Angew Chem Int Ed Engl 50(24):5482–5485 Zhan C, Wei X, Qian J, Feng L, Zhu J, Lu W (2012) Co-delivery of TRAIL gene enhances the antiglioblastoma effect of paclitaxel in vitro and in vivo. J Control Release 160(3):630–636 Zhan C, Li C, Wei X, Lu W, Lu W (2015) Toxins and derivatives in molecular pharmaceutics: drug delivery and targeted therapy. Adv Drug Deliv Rev 90:101–118
Preparation of Functional Vincristine Liposomes for Treatment of Invasive Breast Cancer
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Fan Zeng
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Synthesis of Targeting Molecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Preparation of the Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Determination of Vincristine and Dasatinib . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Characterization of the Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Cell Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Evaluations on Invasive Breast Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7 Evaluations on Invasive Breast Cancer Spheroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8 Evaluations on Breast Cancer-Bearing Nude Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Synthesis of Targeting Molecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Preparation of the Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Determination of Vincristine and Dasatinib . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Characterization of the Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Cell Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Evaluations on Invasive Breast Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Evaluations on Invasive Breast Cancer Spheroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 Evaluations on Breast Cancer-Bearing Nude Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9 Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
240 242 242 242 242 243 243 243 244 244 244 244 244 247 248 250 250 251 252 254 254 256 256
F. Zeng (*) Beijing Neurosurgical Institute, Capital Medical University, Beijing, China e-mail: [email protected] © Springer-Verlag GmbH Germany, part of Springer Nature 2021 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49320-5_10
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Abstract
Clinical treatment of cancers usually adopts a comprehensive strategy, which consists of surgical treatment, radiotherapy, chemotherapy, and biotherapy. Among which, chemotherapy plays a crucial role. However, conventional chemotherapy fails to obtain a satisfactory efficacy, including unfavorable pharmacokinetic properties and systemic toxicities, which are caused by the direct delivery of free drugs into the circulatory system. This chapter focuses on the liposomal formulations of encapsulated vinca alkaloid and mainly introduces the preparation and characterization of functional vincristine liposomes, which were designed to treat the invasive breast cancer along with eliminating the vasculogenic mimicry channels. In this liposomal drug delivery system, vincristine was used as an anticancer agent, dasatinib was used as a promising regulator, and the targeting molecular c(RGDyK) was modified on the surface of the liposomes. The functional liposomes were round in shape and displayed a smooth surface with approximately 100 nm in diameter. The investigations were performed on invasive breast cancer MDA-MB-231 cells and MDA-MB-231 xenografts in nude mice. The functional vincristine plus dasatinib liposomes displayed a prolonged circulation time in blood system and an increased accumulation in tumor tissue, resulting in a robust overall anticancer efficacy. Keywords
Liposomes · Vincristine · Dasatinib · Breast cancer · Drug delivery
1
Introduction
Cancer has become the most common malignant disease and a major cause of mortality among people around the world. Clinical treatments of cancers usually adopt a comprehensive strategy, including surgical treatment, radiotherapy, chemotherapy, and biotherapy. Among which, chemotherapy plays a crucial role in eliminating cancer cells. However, the major problem of chemotherapy is the lack of selective toxicity, which results in a narrow therapeutic index and severe side effects (Li et al. 2006). The injected free drugs in the circulatory system are not able to penetrate into the vasculature in the tumor site and accumulate higher in normal tissues. These phenomenon often leads to incomplete cancer response, multiple drug resistance, and, ultimately, therapeutic failure (Wang et al. 2012). Vincristine sulfate, as an effective chemotherapeutic agent, is widely used to treat various cancers, including malignant lymphoma, acute leukemia, and breast cancer (Kassem et al. 2011). It is a cell cycle-specific anticancer agent. The cytotoxic activity of vincristine is based on the ability to inhibit microtubule, altering the tubulin polymerization equilibrium and resulting in altered microtubule structure and function, which then causes the arrest of cell division in metaphase (Chen et al. 2012). However, its clinical application exhibits low absorption and fast elimination
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attributed to P-gp-mediated efflux and causes side effects based on dose-limiting systemic toxicity (Wang et al. 2014; Barthomeuf et al. 2005). Therefore, designed nanocarriers for achieving precise delivery to cancer cells are expected to overcome the shortcomings of vincristine. Currently, many kinds of nanocarriers have been developed for the delivery of vincristine, such as liposomes and nanoparticles (Chen et al. 2012; Wang et al. 2014; Ling et al. 2010; Zucker and Barenholz 2010). Liposomes are round-shaped vesicles with bilayer structure, which are mainly made up of phospholipids and cholesterol, and have been widely used for drug delivery in the past several decades (Vijayakumar et al. 2016). The liposomal formulations exhibit significant advantages in anticancer therapy. The pegylated liposomes in suitable particle size had been characterized for prolonged circulation through avoiding the rapid clearance of reticuloendothelial system (RES) and were capable of making more accumulation in tumors by EPR effect (Matsumura and Maeda 1986; Qiao et al. n.d.; Woodle and Lasic 1992). The varying particle size of the liposomes changes the distribution of drugs in tissues caused by a physical retention due to the difference in compact degrees of each tissue. Therefore, the liposomes are able to reduce the toxicity of drugs through this mechanism (Forssen and Tökès 1981). More importantly, liposomes are biocompatible and can entrap both hydrophilic (in the internal water compartment) and hydrophobic (in the bilayers) pharmaceutical agents, protecting them from external damage. And also, the use of liposomes can maintain the desirable drug ratios and coordinate the release of the encapsulated drugs to achieve increased antitumor activity (Tardi et al. 2007, 2009; Mayer et al. 2006). The effect of vincristine in invasive cancer cells is restricted. To improve therapeutic efficiency, a kind of functional vincristine liposomes was fabricated in this chapter. Dasatinib, as an inhibitor of sarcoma gene family kinases, exhibits multiple effects on cancer, including antiproliferative activity, induction of apoptosis, and inhibition of invasion (Finn et al. 2007; Shor et al. 2007). Because of these properties, dasatinib represents a promising inhibitor in invasive breast cancer treatment and was also encapsulated in the functional liposomes. c(RGDyK) is a cyclic peptide that has a specific affinity for the integrin receptor, which is overexpressed on many malignant cancer cells (Chen et al. 2005; Nie et al. 2011). This cyclic peptide offers a potential avenue for liposomal drug delivery system to access target cancer cells. The liposomal formulations containing vincristine were prepared by ammonium sulfate gradient method. The method is designed according to the principle of chemical equilibrium and active encapsulation. The general procedures could be described as follows: firstly, blank liposomes are prepared by film dispersion method and hydrated with ammonium sulfate solution; secondly, the ammonium sulfate outside the liposomes was removed by dialysis against phosphatebuffered saline, thus forming an ammonium sulfate gradient between two sides of liposome vesicles, namely, the inside has a high concentration of ammonium sulfate, while the outside has a lower one. Finally, blank liposome suspensions were incubated with vincristine solution in water bath at 60 C with gentle shaking for 20 min (Men et al. 2011). The specific procedure of liposome preparation is described step by step in this protocol.
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2
Materials
2.1
Synthesis of Targeting Molecule
1. Cyclic RGD peptide (c(RGDyK)). 2. N-hydroxysuccinimidyl-polyethylene glycol distearoylphosphatidylethanolamine (DSPE-PEG2000-NHS) and polyethylene glycol distearoylphosphatidylethanolamine (DSPE-PEG2000). 3. Anhydrous dimethylformamide (DMF). 4. N-methyl morphine. 5. Regenerated cellulose dialysis tubing (molecular weight cutoff point, 3000 Da). 6. Magnetic stirrer. 7. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).
2.2 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
2.3
Preparation of the Liposomes Egg phosphatidylcholine (EPC). Cholesterol. Polyethylene glycol distearoylphosphatidylethanolamine (DSPE-PEG2000). Vincristine sulfate. Dasatinib. Coumarin. 1,10 -Dioctadecyl-3,3,30 ,30 -tetramethyl indotricarbocyanine iodide (DiR). Chloroform and methanol were of chromatographic grade. HEPES. Phosphate-buffered saline (PBS). Polycarbonate membranes (400 nm and 200 nm). 250 mM ammonium sulfate solution. 100 ml pear-shaped flask. Rotary vacuum evaporator. Ultrasonic cleaner. Ultrasonic cell crusher. Dialysis bags.
Determination of Vincristine and Dasatinib
1. Hundred-thousandth analytical balance. 2. High-performance liquid chromatography (HPLC) system with UV detector. 3. Extend-C18 column (5 μM, 4.6 250 mm).
Preparation of Functional Vincristine Liposomes for Treatment of Invasive. . .
15
4. 5. 6. 7.
Centrifuge. Methanol. Distilled water. Potassium dihydrogen phosphate (KH2PO4).
2.4 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
2.5 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
2.6
Characterization of the Liposomes Sephadex G-50. A vortex instrument. Methanol. Centrifuge. HBS solution. Malvern Zetasizer 3000HS for dynamic light scattering analysis. Atomic force microscope (AFM). Micropore filter membrane (200 nm). Polished silicon wafer. Fetal bovine serum (FBS). Constant temperature culture oscillator. Dialysis bags (molecular weight cutoff point, 12,000–14,000 Da). Phosphate-buffered saline. Distilled water.
Cell Cultures Invasive human breast cancer MDA-MB-231 cells. Leibovitz’s L-15 Medium. Fetal bovine serum. Trypsin EDTA. Penicillin-streptomycin solution 100 . Phosphate-buffered saline. Sterile centrifuge tube. Cell culture flask. Incubator. Clean bench. Optical microscope.
Evaluations on Invasive Breast Cancer Cells
1. Flow cytometer. 2. Coumarin. 3. Phosphate-buffered saline.
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2.7 1. 2. 3. 4. 5.
Evaluations on Invasive Breast Cancer Spheroids
Agarose. Coumarin. Phosphate-buffered saline. Confocal laser scanning fluorescent microscopy. Scanning electron microscope (SEM).
2.8 1. 2. 3. 4. 5.
F. Zeng
Evaluations on Breast Cancer-Bearing Nude Mice
Female BALB/c nude mice (16–18 g). Kodak multimodal imaging system. A weighing scale. Electronic digital vernier caliper. Isoflurane.
3
Methods
3.1
Synthesis of Targeting Molecule
1. Dissolve c(RGDyK) peptide and DSPE-PEG2000-NHS at a ratio of 2:1 (mol/mol) in anhydrous DMF (see Notes 1 and 2). 2. Adjust the pH value of the reaction solution to 9.0 using N-methyl morphine. 3. Continuously stir the solution at room temperature for 24 h. 4. Transfer the reaction mixture into a dialysis bag and dialyze them against deionized water for another 48 h. 5. Freeze-dry the resultant to obtain the targeting molecule, and store them at 20 C. 6. Identify and characterize the product using a MALDI-TOF mass spectrometer (Fig. 1).
3.2
Preparation of the Liposomes
3.2.1 Preparation of Functional Vincristine Plus Dasatinib Liposomes 1. Dissolve EPC, cholesterol, DSPE-PEG2000, DSPE-PEG2000-c(RGDyK) conjugate, and dasatinib in chloroform and methanol (3:1, v/v) at a ratio of 66:26:2.5:3:3.5 (mol/mol) in a pear-shaped bottle (see Notes 3 and 4). 2. Remove the solvent by a rotary vacuum evaporator to form the lipid film (see Notes 5 and 6). 3. Hydrate the formed lipid film with 250 mM ammonium sulfate by sonication in the water bath for 5 min (see Note 7). 4. Treat the obtained suspensions using an ultrasonic cell disruptor for 10 min, and successively extrude them through polycarbonate membranes with pore sizes of 400 and 200 nm, three times each (see Notes 8 and 9).
Preparation of Functional Vincristine Liposomes for Treatment of Invasive. . .
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2836.66 2968.87 3101.04
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Fig. 1 MALDTOF-MS spectra of targeting molecule. (a) DSPE-PEG2000-NHS; (b) DSPE-PEG2000-c (RGDyK) (Zeng et al. 2015)
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5. Dialyze the suspensions in HEPES-buffered saline for 24 h (see Note 10). 6. Incubate the dialyzed suspensions with vincristine solution in a water bath at 40 C with continual shaking for 30 min (lipids/drug = 20:1, w/w). 7. Store the obtained functional vincristine plus dasatinib liposomes (Fig. 2) at 4 C in a refrigerator.
3.2.2 Preparation of Vincristine Plus Dasatinib Liposomes Prepare vincristine plus dasatinib liposomes using the same procedures as functional vincristine plus dasatinib liposomes, but in the film formation process, replace the DSPE-PEG2000-c(RGDyK) to an equimolar amount of DSPE-PEG2000.
246 Fig. 2 Schematic drawing of functional vincristine plus dasatinib liposomes
F. Zeng DSPE-PEG2000-C(RGDyK)
Dasatinib
Vincristine
DSPE-PEG2000
3.2.3 Preparation of Vincristine Liposomes Prepare vincristine liposomes using the same procedures as functional vincristine plus dasatinib liposomes, but in the film formation process, replace the DSPEPEG2000-c(RGDyK) to an equimolar amount of DSPE-PEG2000 and without the addition of dasatinib. 3.2.4 Preparation of Dasatinib Liposomes Prepare dasatinib liposomes using the same procedures as functional vincristine plus dasatinib liposomes, but in the film formation process, replace the DSPE-PEG2000-c (RGDyK) to an equimolar amount of DSPE-PEG2000. Moreover, do not perform dialysis and drug loading operations after liposome extrusion. 3.2.5 Preparation of Functional Liposomes Prepare functional liposomes using the same procedures as functional vincristine plus dasatinib liposomes, but without the addition of dasatinib in the film formation process. Also, do not perform the dialysis and drug loading operations after liposome extrusion. 3.2.6 Preparation of Fluorescence Labeling Liposomes 1. Conduct all the preparation procedures in dark light. 2. By the same proportion, dissolve EPC, cholesterol, DSPE-PEG2000, DSPEPEG2000-c(RGDyK), and appropriate coumarin (or DiR) in chloroform into a pear-shaped flask to prepare coumarin liposomes (lipids/coumarin = 500:1, w/w), functional coumarin liposomes, DiR liposomes (lipids:DiR = 200:1, w/w), and functional DiR liposomes (lipids:DiR = 200:1, w/w). 3. Remove the chloroform by a rotary vacuum evaporator to form the lipid film. 4. Hydrate the newly formed lipid film with HBS buffer solution (25 mM HEPES/ 150 mM NaCl) by sonication in the water bath for 5 min. 5. Treat the suspensions using an ultrasonic cell disruptor for 10 min. 6. Extrude the obtained suspensions through polycarbonate membranes with pore sizes of 400 and 200 nm, three times each. 7. Store the liposomes in darkness.
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3.3
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Determination of Vincristine and Dasatinib
3.3.1 Linearity of Vincristine For vincristine, prepare the mobile phase with methanol and water (60:40, v/v), containing 0.02 M KH2PO4 in the aqueous phase. Set flow rate at 1.0 mL/min and detection wavelength at 297 nm. 1. Precisely weigh and dissolve vincristine in methanol. Prepare the vincristine solution in serial concentrations of 0.61, 1.22, 3.05, 6.1, 12.2, 30.5, 61.0, and 122.0 μg/mL to generate an external calibration curve. 2. Measure the samples using the above HPLC condition. 3. Evaluate the linearity by standard calibration curve. Calculate the lope, intercept, and linear correlation coefficient (r2), and carry out linear regression analysis. 4. After data acquisition, quantify the concentrations of vincristine via linear regression analysis of the standard curve from the relative peak area.
3.3.2 Linearity of Dasatinib For dasatinib, prepare the mobile phase with methanol and water (60:40, v/v), containing 0.02 M KH2PO4 in the aqueous phase. Set flow rate at 1.0 ml/min and detection wavelength at 320 nm. 1. Precisely weigh and dissolve dasatinib in methanol. Prepare the dasatinib solution in serial concentrations of 0.54, 1.08, 2.69, 5.38, 10.75, 26.88, 53.75, and 107.50 μg/mL to generate an external calibration curve. 2. Measure the samples using the above HPLC condition. 3. After data acquisition, quantify the concentrations of dasatinib via linear regression analysis of the standard curve from the relative peak area.
3.3.3 Precision of Vincristine or Dasatinib 1. Dilute the vincristine solution at concentrations of 2.52, 10.05, and 50.33 μg/mL. 2. Dilute the dasatinib solution at concentrations of 2.15, 10.75, and 51.60 μg/mL. 3. Measure the three different concentrations of vincristine or dasatinib solution by HPLC. Evaluate the intra- and inter-day coefficients of variation for five times on the same day and inter-day. 4. Calculate RSD to evaluate the intra- and inter-day coefficients of variation. 5. The RSD value of inter-day and intra-day precision was, respectively, less than 2%, which indicated the proposed method was quite precise and reproducible. 3.3.4 Detection Limits of Vincristine or Dasatinib 1. Dilute and measure vincristine or dasatinib solution by HPLC following the same method. 2. The detection limit for vincristine or dasatinib represented the concentrations that rendered signal to noise ratio reach 3:1.
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Characterization of the Liposomes
3.4.1 1. 2. 3. 4. 5. 6. 7. 8.
Encapsulation Efficiency Determination of Drug-Loaded Liposomes Add distilled water in Sephadex G-50 before loading the column. Saturate the Sephadex G-50 column with blank liposomes (see Note 11). Load liposomes on the column, and elute the column with HBS solution to remove unencapsulated vincristine or dasatinib. Collect the drug-loaded liposomal suspension, and add nine times volume of methanol to disrupt the liposomes. Shake for 1 min by a vortex instrument. Collect the supernatant fluid containing drugs after another 5 min centrifugation at a speed of 10,000 rpm. Measure vincristine and/or dasatinib in liposomes with HPLC. Calculate the EE (encapsulation efficiency) of vincristine or dasatinib according to the formula, EE% = (Wencap/Wtotal) 100%, where Wencap was the measured amount of vincristine or dasatinib in the liposomal suspensions after passing over the column and Wtotal was the measured amount of vincristine or dasatinib in the equal volume of liposomal suspensions before passing over the column (Table 1).
3.4.2 Characterization with Dynamic Light Scatter 1. Dilute the liposomes with distilled water to an appropriate concentration (100 μg/ mL lipid). 2. Measure the particle sizes, zeta potential values, and polydispersity indexes (PDI) of all liposomes using a dynamic light scatter (Table 1). Table 1 Characterization of liposomes Liposomes Blank functional liposomes Vincristine liposomes Vincristine plus dasatinib liposomes Functional vincristine plus dasatinib liposomes
Particle size (nm) 103.10 1.54
PDI 0.17 0.01
Zeta potential (mV) 5.48 0.35
EE(%) Vincristine –
Dasatinib –
103.37 2.27
0.17 0.01
8.61 0.83
97.92 0.85
–
104.73 2.02
0.16 0.03
8.57 0.09
98.21 0.78
93.29 0.75
104.50 1.97
0.17 0.02
6.21 0.41
97.72 0.87
91.79 0.42
Data are presented as mean SD (n = 3)
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3.4.3 Morphology Characterization with AFM 1. Dilute the liposome suspensions with distilled water. 2. Filter them through a 200 nm micropore filter membrane. 3. Spread a 10 μL volume of liposome suspension on a silicon slice, and dry it at room temperature. 4. Observe the liposomes using AFM (Fig. 3).
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Fig. 3 AFM images of liposomal formulations. A1 Two-dimensional image of vincristine liposomes. A2 Three-dimensional image of vincristine liposomes. B1 Two-dimensional image of functional vincristine plus dasatinib liposomes. B2 Three-dimensional image of functional vincristine plus dasatinib liposomes
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2. Mix 2 ml liposomes with 2 ml release medium, and seal the mixture in dialysis bags. 3. Immerse the dialysis bags in 16.0 ml of the release medium, and consecutively shake the vials in a constant temperature oscillator (100 rpm, 37 C). 4. Take a volume of 0.5 ml release medium at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 20, and 24 h, respectively, and immediately replace them with the same volume of fresh release medium after each sampling. 5. Precipitate the taken samples with methanol. Mixed the mixture for 3 min with a vortex, and centrifuge at 10,000 rpm for 5 min. 6. Inject 20 μL supernatant into the HPLC system, and record the signal peaks for vincristine and dasatinib. 7. Calculate the release rate (Fig. 4) with the formula RR = (Wi/Wtotal) 100%, where RR is the drug release rate (%), Wi is the measured amount of vincristine or dasatinib at the time point of i in release medium, and Wtotal is the total amount of vincristine or dasatinib in the equal volume of liposome suspensions prior to dialysis.
3.5
Cell Cultures
Grow the invasive human breast cancer MDA-MB-231 cells in Leibovitz’s L-15 Medium supplemented with 10% FBS in a 37 C humidified incubator.
3.6
Evaluations on Invasive Breast Cancer Cells
3.6.1 Cellular Uptakes in Breast Cancer Cells 1. Use coumarin as a fluorescent probe to determine the cellular uptake. Prepare the coumarin-labeled liposomes according to the method described above. 2. Seed MDA-MB-231 cells at a density of 4 105 cells/well in 6-well culture plates for 24 h. 20 16
Vincristine liposomes Vincristine plus dasatinib liposomes Functional vincristine plus dasatinib liposomes
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Fig. 4 In vitro release rates of drugs from varying liposomes in the simulated body fluids. (a) vincristine; (b) dasatinib. Data are presented as mean SD (n = 3)
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Fig. 5 Flow cytometric measurement of cellular uptake by breast cancer MDA-MB-231 cells. Notes: 1 blank control; 2 coumarin liposomes; 3 functional coumarin liposomes; 4 free coumarin
3. Treat the cells with free coumarin, coumarin liposomes, and functional coumarin liposomes at a final concentration of 0.5 μM coumarin for another 4 h. Use culture medium as a blank control (see Note 12). 4. After incubation, collect the cells and measure the fluorescence intensity using a flow cytometer according to the manufacturer’s instructions (Fig. 5).
3.7
Evaluations on Invasive Breast Cancer Spheroids
3.7.1 Construction of Multicellular Cancer Spheroids 1. Add agarose into serum-free culture medium, and heat them to 80 C for 30 min to form a 2% (w/v) solution. 2. Add 50 μL agarose solution into each well of a 96-well culture plate. 3. After cooling to ambient temperature, seed MDA-MB-231 cells at a density of 1 103 cells/well with 100 μL growth medium. 4. Gently shake the culture plates for 5 min, and incubate the cells to form cancer spheroids.
3.7.2 Penetrating Effect in Breast Cancer Spheroids 1. Use coumarin as a fluorescent probe to monitor the penetration ability of varying formulations. Prepare the coumarin-labeled liposomes according to the method described above. 2. Treat the MDA-MB-231 tumor spheroids with free coumarin, coumarin liposomes, and functional coumarin liposomes for 12 h, respectively. The final concentration of coumarin was 10.0 μM.
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3. After incubation, wash the spheroids with PBS and subsequently scan them at different layers from the top of the spheroids to the inside using the confocal laser scanning fluorescent microscope (Fig. 6).
3.7.3 Destructing Effect on the Breast Cancer Spheroids 1. Collect the MDA-MB-231 cancer spheroids in 6-well culture plates. 2. Treat the cancer spheroids with varying formulations at a concentration of 1 μM vincristine or dasatinib. Use culture medium as a blank control. 3. After incubation for 48 h, fix the spheroids by 2.5% glutaraldehyde for 60 min. 4. Rinse the spheroids three times with PBS, and then dehydrate and embed them. 5. Observe the spheroids under a scanning electron microscope.
3.8
Evaluations on Breast Cancer-Bearing Nude Mice
3.8.1 Establishing Breast Cancer-Bearing Nude Mice Models 1. Use female BALB/c nude mice (16–18 g) for the studies (see Note 13). 2. Re-suspend approximately 1 107 MDA-MB-231 cells in 200 μL serum-free Leibovitz’s L-15 Medium. 3. Subcutaneously inject the cancer cell suspensions into the right armpits of BALB/ c female nude mice. 4. Following growth to a volume of approximately 1000 mm3, extract the tumors, and cut them into pieces of approximately 2 2 2 mm3. 5. Seed the tumors into the right armpits of fresh nude mice with a tumor inoculation needle. 3.8.2 1. 2.
3. 4. 5.
Anticancer Evaluations of the Functional Liposomes on Nude Mice When the tumors reached approximately 100 mm3 in volume, randomly divide the mice into five treatment groups. Then treat the mice with physiological saline, free vincristine, vincristine liposomes, vincristine plus dasatinib liposomes, or functional vincristine plus dasatinib liposomes via the tail vein at days 12, 14, 16, 18, 20, and 22 after inoculation. The dosage of vincristine was 1 mg/kg (vincristine/dasatinib = 1:1, mol/mol). Weigh the mice, and measure the tumors with calipers. Calculate the tumor volumes (V ) with the formula V = (length width2) / 2 (mm3), and evaluate the tumor volume ratios with the formula R (%) = Vday i / V day 11 100%, where Vday i is the tumor volume at day 1 and Vday 11 is the tumor volume at day 11 (Fig. 7).
3.8.3 In Vivo Imaging in Mice 1. To monitor the distribution of liposomes, use DiR as a fluorescent probe. Prepare the DiR-labeled liposomes according to the method described above.
Free coumarin
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Fig. 6 Confocal images of breast cancer MDA-MB-231 spheroids incubated with varying coumarin formulations
Functional coumarin liposomes
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Fig. 7 Antitumor efficacy in the tumor-bearing nude mice xenografted with breast cancer MDA-MB-231 cells after treatment with varying formulations. p < 0.05, a, versus saline; b, versus free vincristine; c, versus vincristine liposomes; d, vincristine plus dasatinib liposomes. Data are presented as the mean SD (n = 6)
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2. Randomly divide the mice into four groups when the tumors reached approximately 600 mm3 in volume. 3. Subsequently administer the mice with physiological saline, free DiR, DiR liposomes, or functional DiR liposomes via the tail vein. 4. Anesthetize the mice with isoflurane. 5. Scan the mice at 1, 3, 6, 9, 12, 24, and 48 h using a Kodak multimodal imaging system (Fig. 8a). 6. Scarify the mice by cervical dislocation at 48 h. 7. Dissect tumor masses, hearts, livers, spleens, lungs, and kidneys from the mice. 8. Capture the fluorescent images and X-ray images for all ex vivo tissues (Fig. 8b).
3.9
Statistics
1. Present the data as mean standard deviation (SD). 2. Use one-way analysis of variance (ANOVA) to determine the significance among groups, and then use a Bonferroni correction for post hoc tests. 3. Set the value of P < 0.05 as significant.
4
Notes
1. The weighing operation should be as quick as possible, because the frozen materials could easily get stuck, especially for the c(RGDyK) peptide. 2. The DMF must be anhydrous so as to make sure the success of the reaction. 3. Add and dissolve these ingredients in accordance with the order. 4. The ingredients of liposomes showed better solubility in a mixed solution than that in a single methanol or chloroform solution. All the above materials must be dissolved in the solvent, and ultrasonic operation can be used in this procedure if necessary.
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Tumor Heart Liver Spleen Lung Kidney
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Fig. 8 In vivo real-time imaging observation in the tumor-bearing nude mice xenografted with breast cancer MDA-MB-231 cells after treatment with varying formulations. (a) In vivo real-time imaging of the tumor-bearing nude mice; (b) Ex vivo optical images of the tumor and normal tissues after the tumor-bearing mice sacrificed at 48 h
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5. The speed and temperature of evaporating chloroform and methanol (3:1, v/v) solution should be proper, or the ingredients of liposomes will precipitate. 6. The mixed solvent must be removed completely to create a uniform lipid film. Followed by a quick transfer to a vacuum pump, the sample is kept for several moments to ensure the removal of residual solvent. 7. After water-bath sonication, the lipid film needs to be completely dissolved from the wall of the bottle. And then, the milky solution was obtained. 8. The sonication time depends on the sample volume and concentration, and start sonication until the solution turns from milky to opalescent. 9. Particle size implies the stability of liposomes. If the particle size distribution is less than expected, the sample may be re-extruded to obtain the desired size range. 10. During dialysis, the dialysis fluid needs to be refreshed twice. 11. Sephadex G-50 was saturated with blank liposomes to prevent the drug-loaded liposomes from being adsorbed in the glucan pores. 12. All experiments must be conducted under dark conditions. 13. All procedures were performed according to the guidelines of the Institutional Authority for Laboratory Animal Care.
5
Conclusion
A type of nanostructured functional vincristine plus dasatinib liposomes was developed by thin-film hydration method and modified with the targeting molecule DSPE-PEG2000-c(RGDyK). The liposomes exhibited a significant efficacy in MDAMB-231 cells and cancer-bearing nude mice because of the following reasons: (i) the pegylated liposomes and the nanostructured particle size resulted in prolonged circulation in the blood and more accumulation in the tumor sites of drugs and (ii) the targeting molecule promoted cellular drug uptake and finally promoted the overall anticancer efficacy. It is concluded that the functional drug-loaded liposomal system may provide an alternative strategy for the treatment of invasive breast cancer.
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inhibits growth of basal-type/“triple-negative” breast cancer cell lines growing in vitro. Breast Cancer Res Treat 105(3):319–326 Forssen EA, Tökès ZA (1981) Use of anionic liposomes for the reduction of chronic doxorubicininduced cardiotoxicity. Proc Natl Acad Sci U S A 78(3):1873–1877 Kassem LA, Gamal El-Din MM, Yassin NA (2011) Mechanisms of vincristine-induced neurotoxicity: possible reversal by erythropoietin. Drug Discov Ther 5(3):136–143 Li X, Ruan GR, Lu WL, Hong HY, Liang GW, Zhang YT, Liu Y, Long C, Ma X, Yuan L, Wang JC, Zhang X, Zhang Q (2006) A novel stealth liposomal topotecan with amlodipine: apoptotic effect is associated with deletion of intracellular Ca2+ by amlodipine thus leading to an enhanced antitumor activity in leukemia. J Control Release 112(2):186–198 Ling G, Zhang P, Zhang W, Sun J, Meng X, Qin Y, Deng Y, He Z (2010) Development of novel selfassembled DS-PLGA hybrid nanoparticles for improving oral bioavailability of vincristine sulfate by P-gp inhibition. J Control Release 148(2):241–248 Matsumura Y, Maeda H (1986) A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res 46(12 Pt 1):6387–6392 Mayer LD, Harasym TO, Tardi PG, Harasym NL, Shew CR, Johnstone SA, Ramsay EC, Bally MB, Janoff AS (2006) Ratiometric dosing of anticancer drug combinations: controlling drug ratios after systemic administration regulates therapeutic activity in tumor-bearing mice. Mol Cancer Ther 5(7):1854–1863 Men Y, Wang XX, Li RJ, Zhang Y, Tian W, Yao HJ, RJ J, Ying X, Zhou J, Li N, Zhang L, Yu Y, WL L (2011) The efficacy of mitochondrial targeting antiresistant epirubicin liposomes in treating resistant leukemia in animals. Int J Nanomedicine 6:3125–3137 Nie Y, Schaffert D, Rödl W, Ogris M, Wagner E, Günther M (2011) Dual-targeted polyplexes: one step towards a synthetic virus for cancer gene therapy. J Control Release 152(1):127–134 Qiao Y, Huang X, Nimmagadda S, Bai R, Staedtke V, Foss CA, Cheong I, Holdhoff M, Kato Y, Pomper MG, Riggins GJ, Kinzler KW, Diaz LA Jr, Vogelstein B, Zhou S (2011) A robust approach to enhance tumor-selective accumulation of nanoparticles. Oncotarget 2(1–2):59–68 Shor AC, Keschman EA, Lee FY, Muro-Cacho C, Letson GD, Trent JC, Pledger WJ, Jove R (2007) Dasatinib inhibits migration and invasion in diverse human sarcoma cell lines and induces apoptosis in bone sarcoma cells dependent on SRC kinase for survival. Cancer Res 67(6):2800–2808 Tardi PG, Gallagher RC, Johnstone S, Harasym N, Webb M, Bally MB, Mayer LD (2007) Coencapsulation of irinotecan and floxuridine into low cholesterol-containing liposomes that coordinate drug release in vivo. Biochim Biophys Acta 1768(3):678–687 Tardi P, Johnstone S, Harasym N, Xie S, Harasym T, Zisman N, Harvie P, Bermudes D, Mayer L (2009) In vivo maintenance of synergistic cytarabine: daunorubicin ratios greatly enhances therapeutic efficacy. Leuk Res 33(1):129–139 Vijayakumar MR, Vajanthri KY, Balavigneswaran CK, Mahto SK, Mishra N, Muthu MS, Singh S (2016) Pharmacokinetics, biodistribution, in vitro cytotoxicity and biocompatibility of vitamin E TPGS coated trans resveratrol liposomes. Colloids Surf B: Biointerfaces 145:479–491 Wang Z, Yu Y, Dai W, Lu J, Cui J, Wu H, Yuan L, Zhang H, Wang X, Wang J, Zhang X, Zhang Q (2012) The use of a tumor metastasis targeting peptide to deliver doxorubicin-containing liposomes to highly metastatic cancer. Biomaterials 33(33):8451–8460 Wang Y, Dou L, He H, Zhang Y, Shen Q (2014) Multifunctional nanoparticles as nanocarrier for vincristine sulfate delivery to overcome tumor multidrug resistance. Mol Pharm 11(3):885–894 Woodle MC, Lasic DD (1992) Sterically stabilized liposomes. Biochim Biophys Acta 1113(2):171–199 Zeng F, Ju RJ, Liu L, Xie HJ, Mu LM, Zhao Y, Yan Y, Hu YJ, Wu JS, Lu WL (2015) Application of functional vincristine plus dasatinib liposomes to deletion of vasculogenic mimicry channels in triple-negative breast cancer. Oncotarget 6(34):36625–36642 Zucker D, Barenholz Y (2010) Optimization of vincristine-topotecan combination – paving the way for improved chemotherapy regimens by nanoliposomes. J Control Release 146(3):326–333
Preparation and Characterization of DNA Liposomes Vaccine
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Ya-Fei Du, Ming Chen, Jia-Rui Xu, Qian Luo, and Wan-Liang Lu
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Preparation of Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Preparation of Plasmid DNA Liposome Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Encapsulation Efficiency of DNA Liposome Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Size Reduction of DNA Liposome Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Methods (Gregoriadis et al. 1999) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Preparation of MLV and SUV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Encapsulation of Plasmid DNA by Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Encapsulation Efficiency of DNA in the Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Reduction of DRV Liposomes Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Genetic immunization is a process that uses plasmid DNA encoding antigens from bacteria, viruses, protozoa, and cancers to establish protective humoral and cell-mediated immunity system. The introducing antigen-encoding naked plasmid DNA in vivo by intramuscular injection can trigger humoral and cellmediated protective immunity against infection. However, the major limitation of naked DNA vaccines is the degradation of naked DNA by deoxyribonuclease so that the DNA is unable to act on antigen presenting cells (APCs) effectively.
Y.-F. Du · M. Chen · J.-R. Xu · Q. Luo · W.-L. Lu (*) State Key Laboratory of Natural and Biomimetic Drugs, Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System, School of Pharmaceutical Sciences, Peking University, Beijing, China e-mail: [email protected] © Springer-Verlag GmbH Germany, part of Springer Nature 2021 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49320-5_20
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This is because the muscle cells can absorb and degrade DNA when the naked DNA is administered by intramuscular injection. To avoid DNA degradation and enable DNA vaccine to reach APCs, the liposomes can be used as DNA vaccine carriers, referring to as DNA liposome vaccines. For this purpose, the plasmid DNA is encapsulated into liposomes by the dehydration-rehydration procedure. The entrapped DNA by liposomes can protect the DNA content from local nucleases at injection site and deliver the DNA liposome vaccine to APCs in the lymph nodes. Usually, the neutral, anionic, and cationic liposomes can quantitatively encapsulate a DNA vaccine and are capable of transfecting APC cells in vitro with varying efficiency, but the vesicle surface charge, size, and lipid composition of the liposomes influence the transfection efficiency of the DNA vaccine into APC cells, thereby affecting the expressions of cytokines or immune-stimulating effects (Gregoriadis et al., J Drug Target 3(6):469–475, 1996). This chapter mainly deals with a simple and high-yield approach for producing DNA liposome vaccines using a dehydration-rehydration procedure, followed by describing their characterization. Keywords
Dehydration-rehydration · DNA liposome vaccines · Encapsulation · Cationic lipid · Characterization
1
Introduction
The vaccine is a biological preparation that provides active acquired immunity to a particular disease. The first generation vaccines are the classical vaccines, which rely on the use of wholly killed or attenuated pathogens (Schwendener 2014). The second generation vaccines are the innovative vaccines, which mainly use the synthetic peptides, carbohydrates, or antigens. Recent investigation focuses on the development of the second generation subunit vaccines because they are safer and easier to produce. Subunits are antigens that are well-characterized recombinant proteins and peptides. For example, subunit vaccines are usually manufactured using protein subunits. However, their immune response is often weak because the artificial synthesis protein antigens may be incapable of inducing maturation of dendritic cells (DCs) and further decrease the activity of primary antigen-presenting cells (APCs), which can react to foreign pathogens and trigger the immune response (Schwendener 2014). Since the discovery of DNA vaccine in 1990, it has attracted more and more attention because of its better safety, wider range of humoral and cellular immunity. DNA vaccines are actually the third generation of vaccines. This kind of genetic immunization uses plasmid DNA encoding antigens from bacteria, viruses, protozoa, and cancers to provide humoral and cell-mediated immunity (Gregoriadis et al. 2002). Nowadays, a number of new DNA vaccines, also known as the nucleic acid vaccines or gene vaccines, have been developed aiming at building the specific
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antigen encoded genes into a plasmid so that the plasmid DNA vaccines are able to be utilized by the host cell’s expression system. The plasmid DNA vaccine is injected into the body via a certain method and can induce the immune response for prevention and treatment of disease. The DNA vaccines have potential advantages over other vaccines. They are able to induce a wider range of immune responses and have a longer persistence of immunity. After injection, cells can directly produce the antigens for a protective immunological response. DNA vaccines play important roles against bacterial, viral, parasitic, and tumor diseases. At the early stage, intramuscular injection (Davis et al. 2008) of antigenencoding naked plasmid DNA is used and tested. The experimental results demonstrate that they can trigger humoral and cell-mediated protective immune response against infection. After the muscle cellular uptake of DNA, the expression and extracellular release of antigen result in enhancement of the immunity. However, there are several potential disadvantages with the naked DNA vaccination: (i) DNA is only absorbed by a minor fraction of muscle cells; (ii) DNA is directly exposed to the nucleases in the interstitial fluid. These lead to the use of a large dose DNA, which meets the demand for providing an effective immune-response (Gregoriadis et al. 1997; Gregoriadis 1998). Plasmid DNA vaccines are vulnerable to deoxyribonuclease, and after administration, naked DNA is usually to lose most of their ability to target antigen presenting cells (APCs). To overcome the potential problem in the development of DNA vaccines, there is an urgent need for developing effective and safe DNA vaccines in the human immunization program. DNA liposome vaccine offers a promising solution to circumvent the problem above (Gregoriadis et al. 2002). Therefore, a number of injectable liposome-based DNA vaccines have been licensed in the USA and Europe for clinical treatment against hepatitis A and influenza (Gregoriadis et al. 2002; Gregoriadis 1998). When administration by local injection, liposome DNA vaccine can be taken up by APCs at the injection site and in the lymphatics (Gregoriadis et al. 1997; Gregoriadis 1990, 1995) to activate the immune activity of APCs. The immune activity of DNA is thus significantly enhanced by the liposome encapsulation by preventing the degradation of DNA from deoxyribonuclease attack (Velinova et al. 1996). In the preparation process, the transfection efficiency of DNA liposome vaccine can further be improved by choosing a suitable vesicle surface charge, size, and lipid composition of the liposomes. Besides, co-encapsulation of cytokine genes or other adjuvants (e.g., immune -stimulatory sequences) together into the DNA liposome vaccine is also able to evidently improve the transfection efficiency (Fig. 1). There is also a report that the plasmid DNA liposome vaccine is able to effectively transfect immune cells and results in immunization result regardless of the vesicle surface charge (Gregoriadis et al. 1997; Perrie et al. 2001) by using a plasmid (pRc/ CMV HBS) encoding the S region of the hepatitis B surface antigen (HBsAg; subtype ayw). Intramuscular injection of this DNA liposome vaccine, the immune outcome is 100-folds higher that of naked DNA vaccine via the same administration (Fig. 2). Usually, the cationic material-based DNA liposome vaccines can provide beneficial immunization as compared to naked or DNA liposome complexed vaccine, in
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Fig. 1 Schematic representation of a DNA liposome vaccine (Heegaard et al. 2011). Notes: Depending on the lipophilic property of various compounds, the incorporation can be either by encapsulation in the aqueous inner space or by integration in the bilayer or surface attachment on the lipid bilayer membrane. CpG, cytosine phosphorothioate guanine oligodeoxynucleotide; PEG, polyethylene glycol
view of the results of humoral (splenic interleukin-4) and cell-mediated (splenic interferon γ) immune responses in mice (Fig. 3) (Gregoriadis et al. 1997, 1999; Gregoriadis 1998; Perrie et al. 2002; Perrie and Gregoriadis 2000). Liposomes are one of the widely used delivery vehicles in the pharmaceutical applications and have the advantages of biodegradation, low toxicity, simple preparation, and adjustability of the components. They have nano-sized spherical vesicles, consisting of one or more lipid bilayers surrounding a central aqueous core. Hydrophilic substances can be incorporated into the aqueous compartment of liposomes and hydrophobic substances can be incorporated into the lipid bilayers. Several drug liposomes have been used for treatment of cancer and other diseases, such as Doxil and Caelyx (PEGylated liposomal doxorubicin), and DaunoXome (non-PEGylated liposomal daunorubicin) (Gaitanis and Staal 2010; Abraham et al. 2005). To prepare DNA liposome vaccines, the vesicles of liposomes can encapsulate the DNA antigen and its immune adjuvants. Liposomes can be divided into the large multilamellar vesicles (LMVs) and small unilamellar vesicles (SUVs), according to the size and structure of the liposomes (Fig. 4). Based on the surface electric charges, the liposomes can be divided into cationic liposomes, anionic liposomes, and neutral liposomes. Positively charged cationic liposomes are useful carriers for negatively charged plasmid DNA antigen. They also suitable vectors for immune adjuvants of the DNA. In addition to protect DNA vaccines from degradation of deoxyribonuclease, the cationic material-based liposomes are able to promote cell transfection efficiency by enhancing the cellular uptake (Wang et al. 2010). In analyzing the cellular
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Fig. 2 A comparison of immune responses between naked DNA vaccine and DNA liposome vaccine (Gregoriadis et al. 1997). Notes: The formulation was injected with naked, or liposomeencapsulated pRc/CMV HBS. BALB/c mice were injected intramuscularly on days 0, 10, 20, 27, and 37 with 5 μg of DNA encapsulated in cationic liposomes composed of PC, DOPE and DOTAP (a); DC-Chol (b); or SA (c) (molar ratios 1:0.5:0.25); or in the naked form (d). Animals were bled 7, 15, 26, 34, and 44 days after the first injection and sera tested by ELISA for IgG1 (black bars), IgG2a (white bars) or IgG2b (gray bars) responses against the encoded hepatitis B surface antigen (HBsAg; S region, ayw subtype). Values are means S.D. of log10 of reciprocal end point serum dilutions required for OD to reach readings of about 0.2. Similar values (all groups) were obtained in mice injected as above with 10 μg DNA in a separate experiment. Sera from untreated mice gave log10 values of less than 2.0. IgG1 responses were mounted by all mice injected with liposomal DNA but became measurable only at 26 days. Differences in log10 values (all IgG subclasses at alltime intervals) in mice immunized with liposomal DNA and mice immunized with naked DNA were statistically significant (P < 0.0001–0.002)
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Fig. 3 A comparison of the produced cytokine levels in mice spleen by immunization among naked DNA, cationic material based DNA liposome vaccine or liposome complexed DNA vaccine (pRc/CMV HBS) (Gregoriadis et al. 1997). Notes: BALB/c mice were immunized as in Fig. 2 with DNA encapsulated into either cationic (DOTAP) (a) or uncharged liposomes (b), mixed with cationic (DOTAP) liposomes (c), or in the naked form (d). “Control” denotes cytokine levels in normal unimmunized mice. Three weeks after the final injection, mice were killed and their spleens subjected to IL-4 and IFN-γ analysis. Each bar represents the mean S.E. of a group of four mice. Cytokine values in mice immunized with cationic liposomes were significantly higher than those in the other groups (P < 0.001–0.05)
transfection, the mechanism of the cationic material-based DNA liposome vaccine is different from that of naked DNA vaccine (Fig. 5). This is because, although cationic liposomes could bind to and be taken up by the negatively charged myocytes, the protein in the interstitial fluid would neutralize (Gregoriadis 1995) the liposomal surface and thus preventing such a binding. Moreover, the vesicle size (about 600–700 nm average diameter (Perrie and Gregoriadis 2000)) would render access of the liposomes into the cells. However, the cationic liposomes can be endocytosed by APCs (dendritic cells possibly included) in the lymphatics (Velinova et al. 1996). This phenomenon is supported by the experiments in which the mice are intramuscularly or subcutaneously injected with naked plasmid DNA or cationic DNA liposome vaccine (pCMV4.EFGP) (Fig. 6). This chapter describes a typical method for preparation and characterization of cationic DNA liposome vaccine as the following.
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Fig. 4 Scanning electron micrograph of a mixture of small unilamellar and multilamellar liposomes of various sizes (Gregoriadis et al. 1999)
Fig. 5 A comparison on the transfection mechanism between cationic DNA liposome vaccine and naked DNA vaccine (Gregoriadis et al. 2002). Notes: Cationic DNA liposome vaccine may be taken up directly by APCs such as dendritic cells, while the naked DNA vaccine is firstly taken up by muscle cells and then transfects the APC cells
2
Materials
2.1
Preparation of Liposomes
1. 2. 3. 4.
Phosphatidylcholine (PC) Distearoylphosphatidylcholine (DSPC) Phosphatidyl ethanolamine (PE) Phosphatidic acid (PA)
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Fig. 6 Fluorescence images indicating the cellular uptake by muscle cells and APC cell in lymph node of mice after intramuscularly administration of cationic DNA liposome vaccine or naked DNA vaccine (pCMV4.EGFP) (Perrie et al. 2001)
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Phosphatidylglycerol (PG) Phosphatidylserine (PS) Dioleoylphosphatidylcholine (DOPE) Stearylamine (SA) 1,2-Bis(hexadecylcycloxy)-3-trimethylaminopropane (BisHOP) l,2-Dioleoyloxy-3-(N,N,N-trimethylammonium) propane (DOTAP) 1,2-Dioleoyl-3-dimethylammoniumpropane (DODAP) 3ß-(N,N-dimethylaminoethane)carbamyl cholesterol (DC-Chol) Chloroform (CHCl3) Nitrogen (N2) Round-bottomed spherical quick-fit flask Ultrasonic apparatus Rotary evaporator Glass beads Centrifugal machine
2.2
Preparation of Plasmid DNA Liposome Vaccine
1. 2. 3. 4. 5.
Plasmid DNA Liquid nitrogen Phosphate buffered saline (PBS pH 7.2) Centrifugal machine Freeze dryer
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1. Isotope labeling DNA 2. Triton X-100 3. 2-Propanol
2.4 1. 2. 3. 4. 5. 6. 7. 8.
Size Reduction of DNA Liposome Vaccine
Sucrose Polyethylene glycol 6000 (PEG 6000) Microfluidizer 110S Sepharose CL-4B Dialysis devices Freeze dryer Vortex oscillator Centrifugal machine
3
Methods (Gregoriadis et al. 1999)
To prepare DNA liposome vaccine, the plasmid DNA should be encapsulated into the liposomes. The procedure consists of the following steps: (i) preparation of the lipid membrane of liposomes; (ii) hydration for preparing multilamellar vesicles (MLVs)/bigger liposomes, followed by producing small unilamellar vesicles (SUVs)/smaller liposomes; (iii) encapsulation of plasmid DNA into SUVs; (iv) dehydration of the product and crush of the dry lumps; and (v) finally rehydration of the product for obtaining unilamellar “dehydration-rehydration” liposomes (DRVs) in which the plasmid DNA is encapsulated. To separate the DNA loaded liposomes from free DNA, a high speed centrifuge is used. To reduce the size of the particles when necessary, DNA loaded liposomes can be milled by going through a microfluidizer.
3.1
Preparation of MLV and SUV
3.1.1 Preparation of Phospholipid Membranes 1. Dissolve 16 μmol PC and 8 μmol DOPE (or PE) in 2 ~ 5 ml chloroform. Add 4 μmol PA, PS, or PG for negatively charged liposomes; Add 3.2 ~ 8 μmol SA, BisHOP, DOTMA, DOTAP, or DC-CHOL for positively charged liposomes. 2. Place the solution of lipids in a 50 ml round-bottomed spherical quick-fit flask and remove the solvent by rotary evaporator at 37 C. Flush the thin lipid film formed on the walls of the flask for about 60 s with oxygen-free nitrogen (N2) to replace air and ensure complete solvent removal.
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3.1.2 Preparation of MLV 1. Add 2 ml distilled H2O (solution 3.1.1-2 instead if step 3.1.3 below is not detrimental to the vaccine) into the flask, together with a few glass beads if needed. Shake the mixture vigorously by hand or mechanically until totally transform the lipid film into a milky suspension. 2. Warm up the flask and H2O or solution 3.1.2-1, carry out this process in a water bath endowed with a shaking facility above the Tc of the phospholipid (>Tc). 3. Place the emulsion at >Tc for 1–2 h to form multilamellar vesicles (MLV) of diverse sizes. 3.1.3 Preparation of SUV 1. Remove the glass beads. Slightly immerse the titanium probe in the emulsions. Sonicate the milky suspension at >Tc (with frequent intervals of rest). Deliver a gentle stream of N2 continuously through thin plastic tube to carry out the sonication. The product is a slightly opaque to clear suspension of small unilamellar vesicles (SUV) about 30–80 nm in diameter. 2. The ultrasonic time depends on the used amount of lipid and the diameter of the probe. For the amounts of lipid in solution 3.1.1-2, usually obtain a clear or slightly opaque suspension within up to four sonication cycles, each lasting 30 s with 30 s rest intervals in-between using a probe 0.75 inch in diameter. 3. Adjust the ultrasonic setting to make the suspension oscillate violently. 4. After ultrasound, centrifuge SUV suspensions for 2 min at 1700 g to remove titanium fragment and place the supernatant for 1 ~ 2 h at >Tc.
3.2
Encapsulation of Plasmid DNA by Liposomes
3.2.1 Dehydration of SUV After Encapsulating Plasmid DNA 1. Dissolve up to 500 μg of the water-soluble vaccine (plasmid DNA) in 2 ml distilled water (H2O) or 10 ml PBS (PBS pH 7.2), if needed. The nature of buffers with respect to composition, pH, and molarity is variable as long as it does not interfere with liposome formation or the yield of encapsulation. 2. Rest the sonicated suspensions of SUV at >Tc for about 1–2 h and mix it with above solution 3.2.1 (when water is used in the step 3.2.2). Rapidly freeze it in liquid nitrogen, and freeze-dry it overnight under vacuum (Tc. Add H2O (0.1 ml per 16 μmol of phospholipid) pre-heated at >Tc to it. Keep the volume of H2O at a minimum while enough to ensure complete dissolution of the powder. 2. Keep the sample at >Tc for about 30 min. 3. Repeat process 1 and 2. Add 0.8 ml PBS (pre-heated at >Tc) and keep the sample at >Tc for 30 min.
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3.2.3 Depletion of Free DNA 1. Centrifuge the suspensions containing multilamellar liposomes (dehydration–rehydration vesicles, DRVs) with embedded and un-encapsulated vaccine at 40,000 g for 60 min (4 C). 2. Suspend the obtained (vaccine containing DRVs) pellets in H2O or PBS. Centrifuge it again under the same conditions. Repeat the process once again to remove the remaining un-encapsulated material. 3. Suspend the final pellets in 2 ml H2O or PBS. Add NaCl to a final concentration of 0.9% when the liposome suspension is used for intravenous injection. 4. To measure the z-average size of the mixed suspension liposomes (600 ~ 700 nm), use the photon correlation spectroscopy (PCS).
3.3
Encapsulation Efficiency of DNA in the Liposomes
1. Monitor the extent of drug encapsulation in DRV liposomes by measuring the vaccine in the suspended pellets and combined supernatants. Use the radiolabeled vaccine to measure the encapsulation efficiency of DNA. 2. Employ appropriate quantitative techniques if a radiolabel is not available or cannot be used. Mix a sample of the liposome suspensions with Triton X-100 (up to 5% final concentration) or 2-propanol (1:1 volume ratio) to release the embedded DNA, then determine the amounts. 3. Extract the lipids or DNA by appropriate techniques if Triton X-100 or the solubilized liposomal lipids interfere with the assay of DNA. Encapsulation efficiency range between about 29% and 99% (Fig. 6), depending on the amounts of lipid and the vaccine (DNA) used. It can achieve the highest values by encapsulating DNA with positively charged DRV (Table 1). However, as part of the liposome-associated solute may interact with the liposome surface during the encapsulation procedure, it is essential to determine actual encapsulation of the solute (as opposed to surface-bound solute). In the case of DNA or proteins, use deoxyribonuclease and proteinase (which will degrade most of the external material) to measure the encapsulation efficiency of DNA in liposomes.
3.4
Reduction of DRV Liposomes Size
The following step for vaccine containing DRV liposomes must be converted to smaller vesicles (down to about 100 nm z-average in diameter).
3.4.1 Reduction of DRV Liposomes Size by a Microfluidizer 1. Dilute the liposomal suspensions obtained in step 3.2.2 (prior to separation of the encapsulated from the un-encapsulated vaccine in step 3.2.3; unwashed liposomes) to 10 ml with H2O and pass the suspension for a number of full cycles by microfluidizer 110S (Microfluidics); set pressure gauge at 60 psi to give a flow rate of 35 ml/min.
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Table 1 Encapsulation of plasmid DNA into liposomes by dehydration-rehydration method (Gregoriadis et al. 1999) Liposomes PC, DOPEa PC, DOPEb PC, DOPE, PSa PC, DOPE, PSb PC, DOPE, PGa PC, DOPE, PGb PC, DOPE, SAa PC, DOPE, SAb PC, DOPE, BisHOPa PC, DOPE, DOTMAa PC, DOPE, DC-Chola PC, DOPE, DC-Cholb PC, DOPE, DOTAPa PC, DOPE, DOTAPb PC, DOPE, DOTAPa PC, DOPE, DOTAPb
Incorporated plasmid DNA (%) pGL2 pRc/CMV HBS pRSVGH 44.2 55.4 45.6 12.1 11.3 57.3 12.6 53.5 10.2 74.8 48.3 69.3 86.8 87.1 76.9 77.2 80.1 79.8 88.6 80.6 57.4 64.8
pCMV4.65 pCMV4.EGEP VR1020 28.6
52.7 67.7
71.9
89.6 81.6
Notes: S-Labeled plasmid DNA (10–500 μg) was (a) incorporated into or (b) mixed with neutral (PC, DOPE), anionic (PC, DOPE, PS, or PG), or cationic (PC, DOPE, SA, BisHOP, DOTMA, DCChol, DOTAP, or DODAP) dehydration-rehydration vesicles (DRVs). Incorporation values for the different amounts of DNA used for each of the liposomal formulations did not differ significantly and were therefore pooled (values shown are means of values obtained from three to five experiments). PC (16 μmol) was used in molar ratios of 1:0.5 (neutral) and 1:0.5:0:25 anionic and cationic liposomes). PC, Egg phosphatidylcholine; DOPE, dioleoylphosphatidylethanolamine; PS, phosphatidylserine; PG, phosphatidylglycerol; SA, stearylamine; BisHOP, 1,2-bis(hexadecylcycloxy)-3-trimethylaminopropane; DOTMA, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-triethylammonium; DC-Chol, 3β-(N,N-dimethylaminoethane) carbonylcholesterol; DOTAP, 1,2dioleoy1-3-(trimethylammonium)-propane; DODAP, 1,2-dioleoy1-3-dimethylammoniumpropane. Plasmid DNAs used encoded luciferase (pGL2), hepatitis B surface antigen (S region) (pRc/CMV HBS), human growth hormone (pRSVGH), mycobacterium leprae protein (pCMV 4.65), fluorescent green protein (pCMV4.EGFP), and schistosoma protein (VR1020)
2. The number of cycles used depends on the vesicle size required (Table 2) or the sensitivity of the encapsulated vaccine (plasmid DNA). The greater the number of cycles is, the smaller the amount of drug is retained by the vesicles, and multiple cycles may destroy the DNA and influence transfection. After 10 cycles of microfluidization, minimum vesicle diameters are about 100–160 nm, depending on whether the microfluidization is carried out in H2O or PBS. 3. Carry out microfluidization of the sample after removal of un-encapsulated vaccine as in step 3.2.3 (washed liposomes) is also practicable. However, it reduces drug retention in this case. It appears that the presence of embedded drug during microfluidization diminishes solute leakage, probably by reducing the osmotic rupture of vesicles (Kirby and Gregoriadis 1984) and/or the initial concentration gradient across the bilayer membranes.
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Table 2 Z-average mean size of microfluidized DRVs (Gregoriadis et al. 1999) Medium Washed DRVs Water PBS Unwashed DRVs Water PBS
Number of cycles 1.8 3.5
5.2
7.1
10.6
463.5 447.4
149.9 198.6
115.0 168.1
121.9 159.5
114.7 155.7
473.9 456.3
132.9 186.2
116.9 186.7
116.6 169.8
101.9 159.9
Notes: Maltose-containing washed or unwashed DRVs (32 μmol PC) were microfluidized in the presence of water or PBS for up to 10.6 cycles, and samples were measured for vesicle size (diameter in nanometers) by dynamic light scattering (photon correlation spectroscopy). Polydispersity indexes ranged from 0.503 to 0.653 (water) and from 0.517 to 0.653 (PBS)
3.4.2 Preparation of Small DNA Liposomes DNA liposomes can be prepared using the next method by encapsulating DNA into small liposomes (approximately 200 nm) without microfluidization (Zadi and Gregoriadis 2000). 1. Prepare SUV according to 3.1.3, then mix liposomes, sucrose (ratio of quality as 1:1, e.g. 21 mg total lipid and 21 mg sucrose), and proper amount of plasmid DNA (10–500 μg). 2. Refrigerate and dehydrate the mixture by freeze-dryer. Rehydrate the frozen dry block according to 3.2.2. 3. Centrifuge suspensions to separate the encapsulated DNA by liposomes from free DNA according to method 3.2.3.
3.4.3 Separation of Free DNA 1. Place the microfluidized sample (in dialysis tubing) in PEG 6000 within a flat container to concentrate the sample (about 10 ml) to about 1–2 ml if needed. The removal of excess H2O from the tubing is relatively rapid (within 30–60 min). Therefore, it is essential to inspect the sample regularly. 2. Separate the un-encapsulated DNA when the required volume has been reached. Carry it out by molecular sieve chromatography using a sepharose CL-4B column, in which case vaccine-containing liposomes are eluted at the end of the void volume. 3.4.4
Characterization of DNA Content and Properties of DRVs After Microfluidization 1. The content of vaccine within liposomes is estimated as in step 3.3 and is expressed as percentage of vaccine in the original preparation obtained in step 3.2.3. 2. Because the sample is microfluidized by following step 3.2.2, i.e., before the estimation of encapsulation, it is necessary that a small portion of the sample to be microfluidized is kept aside for estimation of encapsulation according to step 3.3.
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3. Vesicle size measurements are carried out by photon correlation spectroscopy or as described elsewhere (Perrie and Gregoriadis 2000; Skalko et al. 1996). 4. The liposomes encapsulated DNA can also be determined by micro electrophoresis in the Zetasizer instrument. In order to determine the surface net charge of the cationic liposomes, the zeta potential is usually determined. 5. The morphology of liposomes was studied by transmission electron microscope (TEM). 6. Evaluate DNA vaccines effects for the related cell and animal experiments.
3.4.5 An Example of DNA Liposomes Vaccine Intramuscular immunization of mice with pRc/CMV HBS (encoding the S region of hepatitis B surface antigen; ayw subtype) encapsulated into such liposomes leads to improved humoral and cell-mediated immunity. For example, IgGi antibody isotype produced by cationic liposome-encapsulated DNA vaccines has 100 fold higher titers compared with naked DNA or DNA complexed with preformed similar (cationic) liposomes. It also has increased levels of IFN-γ and IL-4. It is likely that the immunization with liposome-encapsulated plasmid DNA needs antigen-presenting cells (APC) locally or in the regional draining lymph nodes (Gregoriadis et al. 1997). When comparing the encapsulated (charged and uncharged liposomes) and complexed (with similar cationic liposomes) pRc/CMV-HBS, the results from immune responses (Figs. 2 and 7) showed that the cationic DNA liposome vaccine (by DOTAP material) produced greater (over 80-fold) IgG1 titers than the complexed DNA (10 μg dose; 28 days). Interestingly, the electric charge neutral DNA liposomes are also effectively capable of transfecting encapsulated DNA although having a reduced efficiency (Gregoriadis et al. 1997) (Fig. 8).
4
Notes
1. For the liposome extruded through membranes, it is critical to extrude slowly to avoid rupture of the membrane. Extrusion should be done above the phase transition temperature (Tc) of lipids. 2. All the equipment related to the preparation of liposomes should warm up above the phase transition temperature. 3. For the preparation of phospholipid membranes, it is critical to produce a membrane as thin as possible. Thick films are difficult to hydrate. 4. For encapsulating plasmid DNA into liposomes, DNA and lipids should be dissolved together to prepare drug/lipid film for hydration and the following extrusion or sonication. For encapsulating a hydrophilic molecule into liposomes, the hydrophilic molecule should be dissolved in hydration buffer for extrusion or sonication. 5. Vigorous sonication may destroy some agents of the liposomes. Liposomes prepared by using sonication technique are usually not too stable. The liposomes tend to fuse together to form larger vesicles due to the high curvature energy of the lipid bilayer. It is recommended to keep the sample for a few hours at room temperature in the dark and then remove the fused MLV by centrifugation.
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Reciprocal end point serum dilution(log10)
5
273
a
4
3
2 4
b
3
2 3
c
2 3
d
2 21
28
Days after first injection Fig. 7 A comparison of immune responses in mice between the complexed or liposome-encapsulated plasmid DNA. (Reproduced with permission from reference Gregoriadis et al. 1997). Notes: Balb/c mice were injected intramuscularly on days 0. 7, 14, 21, and 28 with 1 (white bars) or 10 ug (black bars) of pRc/CMV HBS encapsulated in positively charged liposomes composed of PC, DOPE, and DOTAP (a), uncharged liposomes composed of PC and DOPE (b), complexed with similar preformed cationic DOTAP liposomes (c) or in naked form (d). Sera from animals bled at 7, 14, 21, and 28 days after the first injection were analyzed for anti-HBsAg IgG1 by ELISA. Immune responses were mounted by all mice injected with liposomal DNA but became measurable only at 21–28 days. For other details, see legend to Fig. 2. Differences in log10 values (10 ug dose, 21 and 28 days) between mice immunized with cationic liposomal DNA and mice immunized with neutral liposomal, complexed and naked DNA as well as differences between neutral or liposomal or complexed and naked DNA were statistically significant (P < 0.0001–0.005)
5
Conclusions
Liposomes have enormous versatility which makes them be a highly valuable carrier system for vaccines. Owing to their specific properties including composition, size, and surface property, liposomes not only improve antigen stability and presentation to APCs but also possess the ability to overcome biological barriers, such as skin and mucosa, and provide controlled and slow release of antigens. Bind with
274 6
IFN- (ng/spleen)
5 4 3 2 1 0 2
IL-4 (ng/spleen)
Fig. 8 Cytokine levels in the spleens of mice immunized with naked, complexed, or liposome-encapsulated plasmid DNA (Gregoriadis et al. 1997). Notes: Mice were immunized as in Figure 7 with pRc/CMV HBS encapsulated into either positively charged (a) or uncharged liposomes (b), complexed with positively charged liposomes (c), or in naked form (d). “Control” represents cytokine levels in normal unimmunized mice. Three weeks after the final injection, mice were killed and their spleens subjected to cytokine analysis. Each bar represents the mean S.D. of a group of 4 mice. Cytokine values in mice immunized with cationic liposomes were significantly higher than those in the other groups (P < 0.001–0.05)
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1.6
1.2
0.8
0.4
0
a
control
b
c
d
DNA vaccine
co-formulated adjuvants, liposomes can induce strong immune responses. Therefore, liposomes can provide a platform for development of modern vaccine formulations. Also, DNA immunization is a promising way to mediate immune for situations where antigens are either ineffective or unavailable. Immunization studies have shown that cationic liposomes promote much greater humoral and cytotoxic T lymphocyte immune responses against the antigen encoded by the encapsulated DNA vaccine. In conclusion, as the third generation of vaccines, DNA liposome vaccine demonstrates a broad range of applications in treatment of severe diseases.
References Abraham SA, Waterhouse DN, Mayer LD, Cullis PR, Madden TD, Bally MB (2005) The liposomal formulation of doxorubicin. Methods Enzymol 391:71–97
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Davis HL, Whalen RG, Demeneix BA (2008) Direct gene transfer into skeletal muscle in vivo: factors affecting efficiency of transfer and stability of expression. Hum Gene Ther 4(2):151–159 Gaitanis A, Staal S (2010) Liposomal doxorubicin and nab-paclitaxel: nanoparticle cancer chemotherapy in current clinical use. Cancer Nanotechnol Methods Mol Biol 624:385–392 Gregoriadis G (1990) Immunological adjutants: a role for liposomes. Immunol Today 11(3):89–97 Gregoriadis G (1995) Engineering liposomes for drug delivery: progress and problems. Trends Biotechnol 13(12):527–537 Gregoriadis G (1998) Genetic vaccines: strategies for optimization. Pharm Res 15(5):661–670 Gregoriadis G, Saffie R, de Souza JB (1997) Liposome-mediated DNA vaccination. FEBS Lett 402 (2–3):107–110 Gregoriadis G, McCormack B, Obrenovic M, Saffie R, Zadi B, Perrie Y (1999) Vaccine encapsulation in liposomes. Methods 19(1):156–162 Gregoriadis G, Bacon A, Caparros-Wanderley W, McCormack B (2002) A role for liposomes in genetic vaccination. Vaccine 20(Supplement 5):B1–B9 Heegaard PMH, Dedieu L, Johnson N, Le Potier MF, Mockey M, Mutinelli F, Vahlenkamp T, Vascellari M, Sørensen NS (2011) Adjuvants and delivery systems in veterinary vaccinology: current state and future developments. Arch Virol 156(2):183–202 Kirby C, Gregoriadis G (1984) Dehydration-rehydration vesicles: a simple method for high yield drug encapsulation in liposomes. Nat Biotechnol 2(11):979–984 Perrie Y, Gregoriadis G (2000) Liposome-encapsulated plasmid DNA: characterisation studies. Biochim Biophys Acta Gen Subj 1475(2):125–132 Perrie Y, Frederik PM, Gregoriadis G (2001) Liposome-mediated DNA vaccination: the effect of vesicle composition. Vaccine 19(23):3301–3310 Perrie Y, Obrenovic M, McCarthy D, Gregoriadis G (2002) Liposome (liposome™)-mediated DNA vaccination by the oral route. J Liposome Res 12(1–2):185–197 Schwendener RA (2014) Liposomes as vaccine delivery systems: a review of the recent advances. Ther Adv Vaccin 2(6):159–182 Skalko N, Bouwstra J, Spies F, Gregoriadis G (1996) The effect of microfluidization of proteincoated liposomes on protein distribution on the surface of generated small vesicles. Biochim Biophys Acta 1301:249–254 Velinova M, Read N, Kirby C, Gregoriadis G (1996) Morphological observations on the fate of liposomes in the regional lymph nodes after footpad injection into rats. Biochim Biophys Acta, Lipids Lipid Metab 1299(2):207–215 Wang D, Xu J, Feng Y, Liu Y, McHenga SSS, Shan F, Sasaki J-i, Lu C (2010) Liposomal oral DNA vaccine (mycobacterium DNA) elicits immune response. Vaccine 28(18):3134–3142 Zadi B, Gregoriadis G (2000) A novel method for high-yield encapsulation of solutes into small liposomes. J Liposome Res 10(1):73–80
Preparation and Evaluation of BiomineralBinding Antibiotic Liposomes
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Xin-Ming Liu, Ke Ren, Geoffrey Wu, and Dong Wang
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Preparation of Biomineral-Binding Lipid ALN-TEG-Chol . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Preparation and Characterization of Biomineral-Binding Liposomes (BBL) . . . . . . . . 2.3 Binding Kinetics of Biomineral-Binding Liposomes (BBL) on HA . . . . . . . . . . . . . . . . 2.4 In Vitro Oxacillin Release from Biomineral-Binding Liposomes (BBL) . . . . . . . . . . . . 2.5 In Vitro Inhibition of S. aureus Biofilm Growth Using Oxacillin-Loaded Biomineral-Binding Liposomes (BBL) Staphylococcus aureus (S. aureus) . . . . . . . . . 3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Preparation of Biomineral-Binding Lipid ALN-TEG-Chol (Fig. 2, Note 1) . . . . . . . . 3.2 Preparation and Characterization of Biomineral-Binding Liposomes (BBL) and Non-Binding Liposome (NBL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Binding Kinetics of Biomineral-Binding Liposomes (BBL) on HA . . . . . . . . . . . . . . . . 3.4 In Vitro Oxacillin Release from Biomineral-Binding Liposomes (BBL) . . . . . . . . . . . . 3.5 In Vitro Inhibition of S. aureus Biofilm Growth Using Oxacillin-Loaded Biomineral Binding Liposomes (BBL) (Note 11) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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X.-M. Liu · K. Ren (*) · D. Wang Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE, USA e-mail: [email protected]; [email protected]; [email protected] G. Wu Office of Lifecycle Drug Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA e-mail: [email protected] © Springer-Verlag GmbH Germany, part of Springer Nature 2021 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49320-5_17
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Abstract
The prevention and treatment of bacterial contamination induced bone infection is challenging due to the lack of osteotropicity of available antimicrobials. This protocol describes a novel biomineral-binding liposomal (BBL) platform for the efficient delivery of antimicrobials to the skeletal tissues. The biomineral-binding lipid, alendronate-tri(ethylene-glycol)-cholesterol conjugate (ALN-TEG-Chol), was synthesized through Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition, a versatile “click” reaction. Using ALN-TEG-Chol, BBL-containing antimicrobial oxacillin was then successfully developed using extrusion and sonication methods. Oxacillin-loaded BBL showed fast and strong binding to hydroxyapatite particles (model bone surface), and demonstrated significantly better preventive efficacy against bacterial colonization when challenged with Staphylococcus aureus when compared to that of free oxacillin and non-BBL. This suggests that the development of biomineral-binding liposomal formulations of antimicrobials may be used in conjunction with orthopedic implants or bone-grafting materials to prevent osteomyelitis associated with orthopedic surgery. Keywords
Liposomes · Biomineral-binding · Bone infection · Osteomyelitis · Implant · Antimicrobials · Oxacillin
1
Introduction
Even with the extensive use of antibiotics, osteomyelitis or bone infection is still a substantial clinical challenge in orthopedic practice (Wu and Grainger 2006; Lew and Waldvogel 1997). The maintenance of very high antimicrobial level at the infection sites is necessary for bacterial pathogen eradication. However, the limited blood supply to the hard tissue and the lack of osteotropicity of available antimicrobials are major obstacles to achieve high local antimicrobial concentration at the infected sites (Wu and Grainger 2006; Diaz-Rodriguez et al. 2011). While systemic administration of high doses of antimicrobials may help achieve a high antimicrobial level at the infection sites, its ubiquitous systemic distribution can lead to serious toxicities (Wu and Grainger 2006). Antimicrobial-loaded implantable devices and osteotropic antimicrobial prodrugs have been developed to maintain the local drug concentration and to improve the antimicrobial efficacy with limited benefit (El-Husseiny et al. 2011; Tanaka et al. 2008; Takahashi et al. 2008). Considering the development of an antibiotic delivery platform for the better treatment of osteomyelitis, we believed the maintenance of high antimicrobial concentration at the potential infection sites and at the time of initial surgery would be most effective as the bacterial colonization is still very limited at this stage. Liposomes are one of the most well-studied and widely used delivery vehicles for pharmaceutical applications. They are nano-size spherical vesicles consisting of one or more lipid bilayers surrounding a central aqueous core. Hydrophilic substances
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can be incorporated into the aqueous compartment of liposomes, and hydrophobic substances can be incorporated into the lipid bilayers. Several liposome formulations are currently on the market for the treatment of cancer and other diseases, such as Doxil and Caelyx (PEGylated liposomal doxorubicin) and DaunoXome (nonPEGylated liposomal daunorubicin) (Gaitanis and Staal 2010; Abraham et al. 2005). The circulation time of liposomes can be significantly improved by grafting polyethylene glycol (PEG) onto the liposome surface to reduce elimination by MPS (Immordino et al. 2006). To improve drug delivery to the disease site, PEGylated liposomes can be further decorated with the specific ligands (Torchilin 2005). In this protocol, we will present the design and development of a biomineral-binding liposomal formulation (Liu et al. 2012), which may be utilized to help prevent/ treat osteomyelitis. This novel platform may be loaded with a variety of antimicrobials, quickly (within a few minutes) binds onto the bone, the surface of grafting materials (e.g., hydroxyapatite, or HA), and HA-coated implant surface, to efficiently concentrate the drugs locally to deter bacterial colonization and therefore significantly improve clinical management of skeletal infection (Fig. 1). One central feature of this platform is the design and synthesis of a biomineralbinding lipid for the liposome preparation. Cholesterol, as a major component of liposomes, has unique properties in regulating phase transition of liposomal bilayers and improving the stability of liposomes (Ohvo-Rekila et al. 2002). Bisphosphonates (BP) are clinically used antiresorptive drugs for the treatment of osteoporosis. A unique feature of BP is their strong affinity to the mineral component of the hard tissue (Zhang et al. 2007; Wang et al. 2005). As a member of BP, alendronate (ALN) has been used extensively as an osteotropic moiety for the development of bone-targeted drug delivery systems (Liu et al. 2007; Pan et al. 2008; Wang et al. 2003; Liu et al. 2008). Due to the availability of chemical handles for modification, cholesterol (OH) and ALN (NH2) were selected for the development of a new
Fig. 1 Proposed working mechanism of biomineral-binding liposomes (BBL) on the orthopedic implant surface. A representative hydroxyapatite (HA)-coated implant surface is presented. (Adapted from Liu et al. 2012, with permission)
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lipid derivative with osteotropicity. Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition of azides and alkynes is one of the most frequently used “click” reactions for bioconjugation, polymer synthesis, and nanoparticle functionalization (Hein et al. 2008). It is characterized by mild reaction conditions (room temperature), good functional group compatibility, high reaction yields, simple workup, strong reliability, and working very well both in organic and aqueous solvent systems (Kolb et al. 2001; Rostovtsev et al. 2002). This versatile “click” reaction was used to synthesize novel alendronate-tri(ethylene glycol)-cholesterol conjugate (ALN-TEG-Chol, Fig. 2; Liu et al. 2012). After inserting into the lipid bilayer of liposomes, ALNTEG-Chol enables the nanocarrier with osteotropicity, permitting its anchoring to implant surface (e.g., HA coated). When used to deliver antimicrobials, the drug-loaded liposomes will be accumulated onto the bone and orthopedic implant surface and gradually release the drug to prevent local bacterial colonization. In this protocol, Staphylococcus aureus (S. aureus) biofilm assay was chosen for antimicrobial activity evaluation because it is one of the major pathogens of deviceassociated osteomyelitis. Oxacillin was chosen as a model drug because it is widely used clinically to treat penicillin-resistant S. aureus (Kumar et al. 2007).
Fig. 2 Synthetic route of biomineral-binding lipid ALN-TEG-Chol. (Adapted from Liu et al. 2012, with permission)
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The biomineral-binding liposomes were prepared using different methods and evaluated in vitro for their biofilm inhibition efficacy (Liu et al. 2012). This delivery platform was intended for the effective prevention of orthopedic infections caused by a variety of bacterial pathogens. Due to the versatile design of the system and the outstanding in vitro biofilm inhibition efficacy, the same liposome can be used to load different or even a combination of antibiotics; and it can also be easily applied to other implant surfaces (e.g., titanium, stainless steel) because specific-binding moieties to these surfaces have been developed and can be incorporated into the liposomes using similar strategy (Khoo et al. 2010; Yoshinari et al. 2010).
2
Materials
2.1
Preparation of Biomineral-Binding Lipid ALN-TEG-Chol
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
Cholesterol Alendronate (ALN) Triethylene glycol Pentynoic acid p-Toluenesulfonyl chloride Sodium azide 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) N-Hydroxysuccinimide (NHS) 4-Dimethylaminopyridine (DMAP) Tris-(hydroxypropyltriazolylmethyl)amine (THPTA) Sodium ascorbate Copper(II) sulfate pentahydrate (CuSO4.5H2O) Triethylamine Hydrochloride (HCl) Sodium Hydroxide (NaOH) Sodium bicarbonate (NaHCO3) Silica-supported triamine tetracetate sodium salt Dichloromethane 1,4-Dioxane Ethanol Acetonitrile Tetrahydrofuran Methanol Hexane Chloroform Ethyl acetate Silica gel chromatography Varian Inova Unity 500 NMR Spectrometer
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Preparation and Characterization of Biomineral-Binding Liposomes (BBL) L-α- phosphatidylcholine (LPC) 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) Cholesterol Oxacillin sodium salt Rotary evaporator Glass flask Vacuum desiccator Mini-Extruder (Avanti Polar Lipid, Inc.) Cole Parmer Ultrasonic processor Sephadex G50 column Ultracentrifuge PD-10 columns (Sephadex G-25 M) Dynamic light scattering (DLS) analyzer (Malvern Nano-Zetasizer) Agilent 1100 HPLC system Monopotassium phosphate (KH2PO4) Chloroform Methanol Acetonitrile
Binding Kinetics of Biomineral-Binding Liposomes (BBL) on HA
1. Hydroxyapatite (HA) particles 2. Benchtop centrifuge
2.4
In Vitro Oxacillin Release from Biomineral-Binding Liposomes (BBL)
1. Dialysis membrane with molecular weight cutoff (MWCO) size of 25 kDa of globular protein 2. Phosphate buffered saline (PBS, 10 mM)
2.5
In Vitro Inhibition of S. aureus Biofilm Growth Using OxacillinLoaded Biomineral-Binding Liposomes (BBL) Staphylococcus aureus (S. aureus)
1. Trypticase Soy Broth (TSB) medium 2. HA discs (0.50 diameter 0.04–0.060 thick)
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3. 24-Well cell culture plate 4. Teflon cell spatula 5. Vortex mixer
3
Methods
3.1
Preparation of Biomineral-Binding Lipid ALN-TEG-Chol (Fig. 2, Note 1)
3.1.1 1.
2.
3.
4.
5.
6.
7. 8.
9.
Preparation of Azido-Terminated Cholesterol (Azido-TEG-Chol, Compound 5) (Note 2) Add p-toluenesulfonyl chloride (1.43 g, 7.5 mmol) slowly into an ice-cooled solution of cholesterol (Compound 1, 1.933 g, 5 mmol) and triethylamine (1.125 mL, 7.5 mmol) in 40 mL dry dichloromethane at 0 C with 4dimethylaminopyridine (DMAP, 60 mg, 0.5 mmol) as the catalyst, and stir the reaction mixture first in an ice-bath and then at room temperature overnight. Extract the reaction mixture with 1 N HCl and brine, evaporate the organic solvent, and purify the crude product with silica gel chromatography (Hexane/ Chloroform 4:1) to yield Cholest-5-en-3β-yloxy-tosylate (Tos-Chol, Compound 2). Yield: 60%. Add anhydrous tri(ethyleneglycol) (10 mL, 74 mmol) to a solution of Tos-Chol (2.16 g, 4 mmol) in dry 1,4-dioxane (30 mL) and reflux the reaction mixture for about 6 h under an argon atmosphere (TLC to confirm the end of the reaction). Evaporate 1,4-dioxane, extract the crude product with chloroform/brine, evaporate the organic solvent, and purify the crude product with silica gel chromatography (Hexane/Ethyl acetate 2:1) to yield 8-(cholest-5-en-3β-yloxy)3,6-dioxaoctan-1-ol (TEG-Chol, Compound 3) as a white oily solid. Yield: 78.5%. Add p-toluenesulfonylchloride (248 mg, 1.3 mmol) slowly into an ice-cooled solution of TEG-Chol (518 mg, 1 mmol) and triethylamine (0.2 mL, 1.3 mmol) in 10 mL dry dichloromethane at 0 C with DMAP (12 mg, 0.1 mmol) as the catalyst, and stir the reaction mixture firstly in an ice-bath and then at room temperature overnight. Extract the reaction mixture with 1 N HCl and brine, evaporate the organic solvent, and purify the crude product with silica gel chromatography (Hexane/ Chloroform 4:1) to yield 8-(cholest-5-en-3β-yloxy)-3,6-dioxaoctanyl tosylate (Tos-TEG-Chol, Compound 4). Yield: 75%. Add sodium azide (325 mg, 5 mmol) in Tos-TEG-Chol (337 mg, 0.5 mmol) in ethanol (5 mL); reflux the reaction mixture for 6 h. Concentrate the reaction mixture, extract the crude product with chloroform/ saturated NaHCO3 and chloroform/brine, and evaporate the organic solvent to yield 8-(cholest-5-en-3β-yloxy)-3,6-dioxaoctanyl azide (Azido-TEG-Chol, Compound 5) as an oily product. Yield: 95%. Confirm the structure of the product with 1H NMR.
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3.1.2 1.
2.
3.
4.
5.
Preparation of Acetylene-Terminated Alendronate (Acetylene ALN, Compound 7) (Note 3) Add EDC (2.11 g, 11 mmol) in an ice-cooled solution of 4-pentynoic acid (1.0 g, 10 mmol) and NHS (1.27 g, 11 mmol) in dichloromethane (50 mL), and stir the reaction mixture for 6 h firstly in an ice-bath and then at room temperature overnight. Extract the product with dichloromethane/brine and evaporate the organic solvent to yield pentynoic acid 2,5-dioxo-pyrrolidin-1-yl ester (Compound 6). Yield: 80%. Add the acetonitrile solution of pentynoic acid 2,5-dioxo-pyrrolidin-1-yl ester (2.38 g, 6 mmol) dropwise into the aqueous alendronate solution (1.58 g, 5 mmol in 30 mL) with three batches and 4 h reaction apart, and control the pH to around 7.4 with NaOH solution before the addition of next batch of pentynoic acid 2,5dioxo-pyrrolidin-1-yl ester. Precipitate the reaction mixture in ethanol for 3 times to give the pure 1-hydroxy4-pent-4-ynamidobutane-1,1-diyldiphosphonic acid (Acetylene ALN, Compound 7). Yield: 70%. Confirm the structure of the product with 1H NMR.
3.1.3 1.
2. 3.
4.
5.
Preparation of Biomineral-Binding Alendronate-Cholesterol Lipid (ALN-TEG-Chol, Compound 8) Using Click Reaction (Note 4) Add the 0.5 mL tetrahydrofuran (THF)/methanol (MeOH)/water (1:1:1, v/v) solution of sodium ascorbate (40 mg, 0.2 mmol) slowly into the 0.5 ml THF/ MeOH/water (1:1:1, v/v) solution of CuSO4.5H2O (5 mg, 0.02 mmol), and stabilizing agent (THPTA, 8.7 mg, 0.02 mmol) under argon to produce the catalyst. Stir the catalyst solution for 2–5 min under argon. Add the catalyst solution dropwise into the THF/(MeOH)/water (1:1:1, v/v, 2 mL) solution of Azido-TEG-Chol (120 mg, 0.22 mmol) and Acetylene ALN (80 mg, 0.2 mmol) under argon and stir the reaction mixture for 3 days at room temperature. Precipitate the reaction mixture into methanol for three times to get the crude product, add a large excess of silica-supported triamine tetracetate sodium salt into the THF/water (1:1, v/v) solution of the crude product to remove copper, and filter the solution to yield 8-(cholest-5-en-3β-yloxy)-3,6-dioxaoctanyl alendronate (ALN-TEG-Chol, Compound 8). Yield: 67%. Confirm the structure of the product with 1H NMR.
3.2 3.2.1
Preparation and Characterization of Biomineral-Binding Liposomes (BBL) and Non-Binding Liposome (NBL)
Preparation of Oxacillin-Loaded Liposomes by Extrusion Method (Gabizon et al. 2003; Note 5–8) 1. Dissolve 100 mg of lipid mixture of 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC)/Cholesterol/ALN-TEG-Chol (70:25:5, molar ratios) in 5 mL chloroform/ methanol (1:1 v/v) solution in a round bottom flask.
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2. Evaporate the organic solvents under vacuum in a rotary evaporator to form a thin lipid film in flask and then remove the traces of the solvents in a vacuum desiccator for 4 h. 3. Add 1 ml of oxacillin sodium solution (100 mg/mL) into the flask containing the thin lipid film and hydrate the lipid film in a rotary evaporator with a rotation speed of 120 rpm at 65 C for 15–20 min. 4. Extrude the solution through 400, 200, and 100 nm polycarbonate membranes (11 times for each of the membranes) at 65 C using a Mini-Extruder (Avanti Polar Lipids, USA), and keep the sample at least 1 h at 65 C and 1 h at room temperature in the dark under nitrogen atmosphere to form stable liposomes. 5. Reserve an aliquot (50 μl) of the initial lipid-drug mixture for the determination of oxacillin loading using HPLC. 6. Equilibrate a Sephadex G50 column with phosphate-buffered saline (PBS), load the liposomal dispersion on the column, and elute the column with PBS at a flow rate of 1 ml/min to remove unencapsulated oxacillin. 7. Quantify oxacillin in the final and initial formulation by an Agilent 1100 HPLC system: Agilent C18 reverse-phase column (4.6 250 mm, 5 μm); mobile phase, 0.02 M KH2PO4/acetonitrile/methanol (70:25:5, v/v) at a flow rate of 1 mL/min; UV detection at 225 nm. 8. Determine the size, particle size distribution, and zeta-potential of resulting biomineral-binding liposomes (BBL) using dynamic light scattering (DLS) ZetaPlus analyzer (Nano-Zetasizer Malvern, Table 1). 9. Repeat step 1–8 of this section to prepare non-binding liposomes (NBL) using 100 mg of lipid mixture of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/ Cholesterol (70:30, molar ratios).
3.2.2 1. 2. 3. 4. 5.
Preparation of Oxacillin-Loaded Liposomes by Sonication Method (Note 9, 10) Prepare multilamellar lipid vesicles (MLV) using the step 1–3 of Sect. 3.2.1. Transfer the MLV dispersion into glass vial and place it in a 65 C water bath. Immerse the sonicator probe into the sample and adjust it to at least 1 cm (0.4 inch) above the bottom of vial. Flush the vial with nitrogen during the whole sonication process. Switch on the Cole Parmer Ultrasonic processor (Vernon Hills, IL) and set the sonication power at about 50% amplitude output.
Table 1 Composition and physicochemical characterization of liposomes. From the data generated by the DLS analysis, BBL and NBL were found to be similar in particle size and size distribution (~150 nm, PDI 0.1), suggesting the incorporation of ALN-TEG-Chol instead of cholesterol did not induce any significant change in the physicochemical properties of the liposome formulation (Adapted from Liu et al. 2012, with permission) Liposomes NBL BBL
Molar ratios/% (LPC/Chol/ ALN-TEG-Chol) 70/30/0 70/20/10
Particles size (nm) 152.1 0.8 164.5 1.3
PDI 0.12 0.03 0.13 0.02
Zeta potential (mV) 30.4 0.6 58.4 0.2
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6. Set the sonication time to about 10 min with 30 s on and 30 s off (depending on the sample volume and concentration), and start sonication until the dispersion turn from milky to opalescent. 7. Keep the sample at least 1 h at 65 C and 1 h at room temperature in the dark under a nitrogen atmosphere. 8. Centrifuge the sample at 12,000 rpm for 10–15 min to remove large liposomes and potential titanium particle shaded from sonicator. 9. Repeat the step 5–8 of Sect. 3.2.1 to prepare and characterize oxacillin-loaded biomineral-binding liposomes (BBL). 10. Repeat step 1–9 of this section to prepare non-binding liposomes (NBL) using 100 mg of lipid mixture of 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC)/Cholesterol (70:30, molar ratios).
3.3
Binding Kinetics of Biomineral-Binding Liposomes (BBL) on HA
1. Add hydroxyapatite (HA, 100 mg) into 1 mL of oxacillin-loaded biomineralbinding and non-binding liposomal solutions, respectively; stir the mixtures at room temperature. 2. Take 100 μL HA and liposome mixture after stirring for 1, 3, 5, 10, and 30 min separately and remove HA by centrifugation (7000 rpm, 1 min). 3. Collect 50 μL of the supernatant, and analyze the oxacillin concentration by an Agilent 1100 HPLC system using the method described in Sect. 3.2.1. 4. Calculate the amount of oxacillin bound to HA particles by subtracting the amount of oxacillin left in the liposomal supernatant after binding from the initial amount of oxacillin in liposomes before binding (Fig. 3).
3.4
In Vitro Oxacillin Release from Biomineral-Binding Liposomes (BBL)
1. Add 1 mL of oxacillin-loaded liposomal solutions into a dialysis bag (MWCO 25,000 kDa) and put the dialysis bag into 30 mL of release medium (10 mM PBS, pH 0 7.4) with gentle stirring at 37 C for in vitro oxacillin release. 2. Take 1 mL of the release medium form the dialysis bag and replace with 1 mL of fresh release medium at predetermined time intervals. 3. Analyze oxacillin concentration in the release medium by an Agilent 1100 HPLC system using the method described in Sect. 3.2.1. (Fig. 4)
3.5
In Vitro Inhibition of S. aureus Biofilm Growth Using OxacillinLoaded Biomineral Binding Liposomes (BBL) (Note 11)
1. Incubate autoclaved HA discs with different liposome formulations and saline in a 24-well plate for 10 min.
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100
Liposome binding to HA (%)
0% mol ALN-TEG-Chol 10% mol ALN-TEG-Chol
80
60
40
20
0
0
5
10 15 20 Incubation time (min)
25
30
Fig. 3 In vitro binding kinetics of biomineral-binding liposomes (BBL) and non-binding liposomes (NBL) on hydroxyapatite (HA) surface. The data from the binding kinetics studies reveal that BBL exhibits a strong affinity and rapid binding rate to biomineral material. All data are means standard deviations (n = 3). (Adapted from Liu et al. 2012, with permission) 100 0% mol ALN-TEG-Chol 10% mol ALN-TEG-Chol
Drug Released (%)
80
60
40
20
0
0
6
12 Incubation time (min)
18
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Fig. 4 In vitro oxacillin release study of biomineral-binding liposomes (BBL) and non-binding liposomes (NBL). The oxacillin containing liposome was observed with a sustained release profile. All data are means standard deviations (n = 3). (Adapted from Liu et al. 2012, with permission)
2. Wash the HA discs with saline to remove unbound liposome or/and free drug. 3. Transfer liposome-bound HA discs to the wells of 24-well plate containing 1 mL of S. aureus suspension UAMS-1 (OD 0.05 at 600 nm) in Trypticase Soy Broth (TSB) medium.
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4. Culture S. aureus suspension statically for 24 h at 37 C to allow biofilm development on the HA discs. 5. Transfer the HA discs to the wells of 24-well plate containing 1 mL of TSB medium. 6. Gently scrape the surface of each HA disc with a sterile spatula to harvest the adherent cells on HA discs. 7. Vortex mixing the removed biofilms for 10 s, serially dilute the cells in TSB medium at a 1:10 ratio using the track dilution method, and incubate the cells for 24 h at 37 C (Jett et al. 1997). 8. Determine the CFUs recovered per biofilm using to quantify bacterial growth (Fig. 5).
3.6
Statistical Analysis
Data were expressed as mean SD. In the bacterial study, specific differences between the log-CFU/biofilm of each experimental group were analyzed using the Student t-test (Microsoft Office 2007, Excel). A P-value of 98% purity). SPC is used in solid state (powder). 2. Cholesterol (Chol, pure). Chol is used in solid state (powder). 3. 3β(N-(N0 , N0 -dimethylaminoethane) carbamoyl) cholesterol (DC-Chol). 4. N-(carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoyl-sn-glycero-3phosphoethanolamine, sodium salt (DSPE-mPEG2000). 5. DSPE-PEG2000-PRP was synthesized as described in Sect. 3.1. 6. DSPE-PEG5000-Folate was synthesized as described in Sect. 3.2. 7. Negative control siRNA (siN.C.), 6-carboxyfluorescein-labeled negative control siRNA (FAM-siRNA), and siRNA targeting polo-like kinase 1 (PLK-1) mRNA (siPLK-1) were synthesized and stored at 20 C. 8. Glucose.
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Dual-Modified siRNA-Loaded Liposomes for Prostate Cancer Therapy
9. 10. 11. 12. 13. 14.
d.d. H2O (see Note 1). d.d. H2O pretreated with diethyl pyrocarbonate (DEPC) (0.1%, v/v) (see Note 1). Chloroform. Methanol. Polycarbonate membranes(0.4-μm and 0.2-μm). Hand-extruder (Avestin, Canada).
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Gel Electrophoresis
1. Powdered agarose. 2. d.d. H2O (see Note 1). 3. Tris-Borate-EDTA (TBE) buffer (0.5): 5.4 g Tris, 2.75 g Boric acid, 1 mM EDTA-Na2, pH 8.0 (see Note 1). 4. Ethidium bromide solution (500 mg/mL in water). Store at 2–8 C and keep away from light owing to its sensitivity (see Note 1). 5. Loading buffer (6) (see Note 1).
2.5
Cell Culture
1. 22Rv1 xenograft prostate tumor cell line. 2. Complete medium: folate-deficient RPMI 1640 medium, fetal bovine serum (FBS), Penicillin-Streptomycin solution 100 (see Note 1). 3. Phosphate-buffered saline (PBS; 50 mM potassium phosphate monobasic, pH 7.4) (see Note 1). 4. Trypsin-EDTA, 0.05% trypsin plus 0.53 mM EDTA-Na2 in PBS (pH 7.4) (see Note 1). 5. T75 cell culture flasks. 6. A CO2 incubator.
2.6
Cellular Uptake and Flow Cytometric Analysis
1. 2. 3. 4. 5.
22Rv1 cells. Complete medium (same as in Sect. 2.5). 6-well plates. PBS (1, pH 7.4) (see Note 1). FAM-siRNA (same as in Sect. 2.7) and various liposomes encapsulating FAM-siRNA were prepared as described in Sect. 3.3. 6. Heparin (125 U/mg).
2.7
Cell Apoptosis Assay
1. 22Rv1 cells. 2. Complete medium (same as in Sect. 2.5).
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3. 25 cm2 tissue culture flasks. 4. Various samples loading siPLK-1 or siN.C. were prepared as described in Sect. 3.3. 5. Annexin V-FITC apoptosis detection kit.
2.8
Analytical Instruments
1. Matrix-assisted laser desorption-ionization time of flight mass spectrometer (MALDI-TOF MS, Bruker Daltonics, Germany) was employed to confirm the MW of DSPE-PEG2000-PRP and DSPE-PEG5000-Folate. 2. Dynamic light scattering measurement was utilized to record size, polydispersity index (PDI), and zeta potential of liposomes using the Malvern Zetasizer Nano ZS (Malvern, UK) (Table 2). 3. Ultraviolet (UV) illuminator was used to visualize and photograph the agarose gel using GK-330C plus Transilluminator 2020D gel imaging analysis system (United-Bio, Tokyo, Japan). 4. Flow cytometric analysis was performed via FACSCalibur flow cytometer (BD, USA).
3
Methods
3.1
Synthesis of DSPE-PEG2000-PRP (See Note 2)
1. Synthesize DSPE-PEG2000-PRP using literature method (Zhu et al. 2012). Place DSPE-PEG2000-mal (20 mg, 6.91 μmol) into a 100 mL round-bottom flask, and dissolve it with 4 mL of chloroform-methanol (3:1,v/v). 2. Remove the solvent on an evaporator under 40 C to form a thin lipid film on the flask wall, and keep the flask in a vacuum chamber. 3. Add 2 mL of HEPES buffer (pH 6.5) to hydrate the dried lipid film, and form a lipid suspension in the round-bottom flask. 4. Dissolve the PRP (20 mg, 6.38 μmol) in 4 mL of HEPES buffer (pH 6.5). 5. Add the DSPE-PEG2000-Mal suspension dropwise to the PRP solution, and mix the system with magnetic agitation for 48 h at room temperature under nitrogen atmosphere. 6. Incubate the resulting solution with L-cysteine (8 mg, 65.7 μmol) for 4 h (see Note 3). 7. Dialyze the reaction mixture with a MW cutoff of 25,000 Da against d.d. H2O for 48 h to remove excess peptides and L-cysteine. 8. Lyophilize the final solution and store the product at 20 C. 9. Determine the MW distribution of the resulting DSPE-PEG2000-PRP on a MALDI-TOF MS (see Note 4).
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Synthesis of DSPE-PEG5000-Folate (See Note 5)
1. Synthesize the DSPE-PEG5000-Folate following the procedure in previous report (Gao et al. 2013). Dissolve folate (100 mg, 226.55 μmol) into 4 mL of anhydrous DMSO (see Note 6). 2. Add the DCC (93.5 mg, 453.10 μmol) and NHS (21 mg, 182.46 μmol) to the folate solution, and stir the system with magnetic agitation for 6 h at room temperature. 3. Remove the emerging white precipitate from the suspension by centrifuging, and add 30 mL of cold acetone-diethyl ether (3:7, v/v) into the resulting supernatant with stirring. 4. Collect the resulting precipitate by centrifuging, and perform at least three rinses with cold acetone and anhydrous diethyl ether, respectively. 5. Dry the solid under vacuum, and obtain a dark yellow powder (folate-NHS ester). 6. Dissolve folate-NHS (11.5 mg, 21.33 μmol) in 1 mL of DMSO, and add the DSPE-PEG5000-NH2 (57 mg, 10.41 μmol) and TEA (7 μL, 50.54 μmol) into the folate-NHS solution. 7. Stir the system at room temperature for 48 h. 8. Remove the TEA on an evaporator under 40 C. 9. Dialyze the resulting system with an MW cutoff of 25,000 Da against d.d. H2O for 48 h. 10. Lyophilize the dialysate and store the product at 20 C. 11. Determine the MW of the resulting DSPE-PEG5000-Folate using MALDI-TOF MS (see Note 7).
3.3
Preparation and Characterization of siRNA-Loaded Liposomes (See Note 8)
1. Prepare the normal liposomes (N-L) and folate-modified liposomes (F-L) with a lipid composition (molar ratio see Table 1) of SPC, cholesterol, DC-chol, DSPEmPEG2000, and DSPE-PEG5000-Folate (only for F-L). Dissolve the lipids in chloroform-methanol (3:1, v/v), and remove the solvent in a rotary evaporator for 15 min at 40 C. 2. Continue vacuum drying for 1 h at room temperature. 3. Prepare the 5% glucose solution (w/v) using the DEPC-pretreated d.d. H2O (see Note 9), and dissolve the lyophilized siRNA (siN.C., siPLK-1 or FAM-siRNA) in the glucose solution (10 mM). 4. Hydrate the lipid film using resulting siRNA solution at N/P ratio 5:1(see Note 10), and extrude the lipid dispersion sequentially through polycarbonate membranes with 0.4-μm (five times) and 0.2-μm (five times) pore sizes using a handextruder.
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Table 1 Various liposome formulations containing siRNA
N-L F-L P-L PF-L
Liposome components (mol ratio of total lipid) SPC/Chol/DC-chol/DSPE-mPEG2000 (48.5: 8.1: 40.4: 3.0) SPC/Chol/DC-chol/DSPE-mPEG2000/DSPE-PEG5000-Folate (48.0: 8.0: 40.0: 3.0: 1.0) SPC/Chol/DC-chol/DSPE-mPEG2000/DSPE-PEG2000-PRP (47.1: 7.8: 39.2: 2.9: 2.9) SPC/Chol/DC-chol/DSPE-mPEG2000/DSPE-PEG5000-Folate/DSPE-PEG2000-PRP (46.6: 7.8: 38.8: 2.9: 1.0: 2.9)
Table 2 Size distribution and zeta potential of siRNA-loaded liposomes (n = 3)
N-L F-L P-L PF-L
Diameter (nm) 177.6 5.9 181.2 8.5 195.2 10.2 202.1 10.4
PDI 0.085 0.019 0.079 0.011 0.239 0.039 0.221 0.047
Zeta-potential (mV) +9.06 0.88 +0.19 0.21 +10.19 1.39 +2.71 1.20
The data are expressed as the mean SD value for at least three different preparations
5. Form PRP-modified liposomes (P-L) and PRP/folate comodified liposomes (PF-L) by the postinsertion method. Prepare a lipid film of DSPE-PEG2000PRP by rotary evaporation, and keep further drying under vacuum for 1 h. 6. Hydrate the dried lipid film with the DEPC-pretreated glucose solution (5%, w/v), and incubate the system for 15 min at 60 C. 7. Add 0.25 mL of suspension (DSPE-PEG2000-PRP micelles) into 1 mL of the above N-L at the required molar ratio (3% DSPE-PEG-peptides of total lipid). 8. Incubate the mixture for 4 h in a water bath at 37 C, and form the P-L preparation. 9. Mix the micelle of DSPE-PEG2000-PRP with preformed F-L at the above molar ratio and condition, and obtain the PF-L preparation. 10. Permit all of the resulting liposomes to cool to room temperature before use. 11. Analyze the particle size and zeta potential of liposome suspension using a dynamic light scattering instrument.
3.4
Gel Electrophoresis
1. Disperse the required amount of agarose in TBE buffer to prepare 3% gel in a glass flask, and heat the agarose system in water bath to help dissolution.
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2. Cool down the solution below 50 C, and add ethidium bromide to the agarose solution with a final concentration of 0.5 μg/mL. 3. Pour the gel in the acrylic plate to form a mold. Place a comb of 0.5–1.0 mm above the plate to obtain complete wells as the agarose solidifies (see Note 11). 4. Stiffen the gel for 20–40 min at room temperature, and remove the comb carefully. 5. Put the gel in the electrophoresis tank, and add TBE buffer enough to cover the gel with the top of the wells immerged in TBE buffer. 6. Mix the 6 loading buffer with F-L liposome sample at N/P ratios of 1/1, 2/1, 3/1, 4/1, and 5/1, respectively, and then load the mixture into the wells slowly with a micropipette (see Note 12). 7. Cover the tank and electrophore the loaded samples for 15–30 min at 100 V in TBE buffer (see Note 13). 8. Visualize the electrophoretic mobility of the siRNA on a UV illuminator at 254 nm (see Note 14).
3.5
Cell Culture
1. To prepare the complete medium, add 5 mL of penicillin-streptomycin solution and 50 mL of FBS into 445 mL of folate-deficient RPMI 1640 medium (see Note 15). 2. Transfer about 3 106 22Rv1 xenograft prostate tumor cells into a T75 flask with 10 mL of the complete medium inside (see Note 16). 3. Grow the cells to ~80% confluency by maintaining the flask with cells in a 37 C humidified incubator with a 5% CO2 atmosphere. 4. To subculture the cells, remove the present culture medium, rinse the cells twice with 2 mL of PBS (pH 7.4), add 1 mL of trypsin to dissociate the cells, put 2 mL of complete medium into the flask to stop trypsinization, collect the cells by centrifuging, divide the cells equally into several T75 flasks, and culture the cells in the incubator (see Note 17).
3.6
Cellular Uptake and Flow Cytometric Analysis
1. Seed the 22Rv1 cells onto 6-well plates at a density of 3 105 cells per well in 2 mL of complete folate-deficient RPMI 1640 medium, and culture the cells at 37 C in a 5% CO2 humidified atmosphere for 24 h. 2. Remove the present complete medium, and rinse the cells with PBS (pH 7.4) for three times. 3. Displace the PBS (pH 7.4) in each well with medium containing free FAM-siRNA or various liposomes encapsulating FAM-siRNA (see Note 18). 4. Incubate the cells with the above medium containing FAM-siRNA (75 nM) for 2 h at 37 C. 5. Trypsinize the cells and perform at least two washes with cold ( 4 C) PBS (pH 7.4) containing heparin (125 U/mL).
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6. Rinse the cells for two times with cold ( 4 C) PBS (pH 7.4), and suspend the cells (each well) in 0.5 mL of PBS (pH 7.4). 7. Filter the resulting suspension through a 300 mesh, and subject the cell samples to flow cytometric analysis utilizing a FACSCalibur flow cytometer (see Note 19).
3.7
Cell Apoptosis Assay
1. Plate 22Rv1 cells on 25 cm2 tissue culture flasks at a density of 1.5 106 cells per flask in 5 mL of complete folate-deficient RPMI 1640 medium. 2. Keep the flasks at 37 C in 5% CO2 humidified atmosphere for 24 h, and wash the cells for two times with PBS (pH 7.4). 3. Incubate the cells with fresh serum-free medium containing siRNA-loaded samples with a final concentration of siRNA (siPLK-1 or siN.C.) being 100 nM (see Note 20). 4. Perform a 5-h exposition, and replace the medium with complete medium for an additional culture of 72 h at 37 C. 5. Remove the complete medium in the culture flasks and rinse the cells for two times with PBS (pH 7.4). 6. Collect the adherent cells by trypsinization and two washes with PBS (pH 7.4), and suspend the cells (each well) in 0.5 mL of PBS (pH 7.4). 7. Stain the cells with the Annexin V-FITC apoptosis detection kit according to the manufacturer’s instructions, and analyze the resulting cell samples via the FACSCalibur flow cytometer with 10,000 events collected (e.g., 488 nm; Em: 530 nm) (see Note 21).
4
Notes
1. Aqueous solutions (including d.d. H2O, buffer, and culture medium) should be sterile and DNase- and RNase-free grade. 2. By Michael addition reaction, the terminal sulfhydryl group of PRP was conjugated with maleimide group of DSPE-PEG2000-Mal (Fig.1a). 3. To block the unreacted maleimide groups, L-cysteine was introduced at a molar ratio of 10:1 (L-cysteine/maleimide). 4. The MALDI-TOF-MS spectra verified the MW of the major multi-peaks centered at 6024.83 which was in agreement with the theoretical MW of the corresponding conjugates, DSPE-PEG2000-PRP (6024 Da) (Fig. 2a). 5. The incorporation of various biological ligands into drug delivery systems facilitates the selective delivery of drugs to tumor cells. Such ligands are recognized and matched by specific receptors on certain types of cancer cell surfaces, which induce the subsequent internalization of the ligand-decorated carriers via receptor-mediated endocytosis (Allen 2002). In view of the high affinity between folate and the extra membrane fraction of PSMA (Hattori and Maitani 2005), DSPE-PEG5000-Folate was introduced to enhance targeted
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Fig. 1 Synthesis diagram of DSPE-PEG2000-PRP (a) and DSPE-PEG5000-Folate (b)
6. 7.
8. 9. 10.
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delivery to PSMA-positive PC cells. To acquire the folate-linked conjugates, carbodiimide-mediated coupling of folate was performed to readily attainable DSPE-PEG5000-NH2 (Fig. 1b). In order to obtain anhydrous DMSO, the HPLC-grade solvent was dried over 4 Å molecular sieve at room temperature. As presented in Fig. 2b, the major multi-peaks centered at a 5910 mass-charge ratio demonstrated that the mean MW of DSPE-PEG5000-Folate was consistent with the calculated data (5897). To avoid the degradation of siRNA in all the procedure, all glassware and plasticware are DNase- and RNase-free grade. The d.d. H2O was pretreated with DEPC (0.1%, v/v) and then sterilized. The N/P ratio represents the relative value of moles of the amine groups of cationic DC-chol to those of the phosphate ones of siRNA. In general, 1 molar ratio of DC-chol is equivalent to 1 molar ratio of N. 1 molar ratio of siRNA corresponds 42 molar ratio of phosphorus because the double-strand siRNA usually has 21 base pairs and single-base pair contains double phosphate domains. The molten gel should be added carefully onto a slab gel apparatus to avoid air bubbles.
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Fig. 2 MALDI-TOF mass spectra of DSPE-PEG2000-PRP (a) and DSPE-PEG5000-Folate (b)
12. The 10 μL of liposome sample should be premixed with 2 μL 6 loading buffer prior to the loading into the wells of gels. The loading buffer acts as dye so as to indicate the running and the ending point. 13. The voltage of the electrophoresis and running time need to be set according to the nature of the sample loaded. 14. The siRNA encapsulated in liposome should remain inside the well, while the free one should migrate in the gel. The result of gel electrophoresis showed that
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Fig. 3 Gel electrophoresis assay of liposomes encapsulating siRNA at N/P ratio of 1/1, 2/1, 3/1, 4/1, and 5/1, respectively
15.
16. 17. 18. 19.
20.
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siRNA could be sufficiently packaged in the liposome at an N/P ratio of 5:1 or greater (Fig. 3). Folate-deficient RPMI 1640 medium was introduced to offer folate-deficient culture conditions, under which PSMA, as a folate transporter, has been confirmed to significantly promote folate uptake into PSMA-expressing cells (Yao et al. 2010). 22Rv1 was identified as a PSMA-positive prostate cell line (Yamamichi et al. 2012; Regino et al. 2009). When subculturing the 22Rv1 cells, a subcultivation ratio of 1:2 to 1:3 is recommended. Various liposomal formulations containing FAM-siRNA should undergo 16 h pretreatment with a 5 mg/mL PSA solution prior to the exposure. A representative result of internalization is shown in Fig. 4. Following the pretreatment with exogenous PSA, only two groups, P-L and PF-L, obtained a significant promotion in cellular uptake, suggesting the expected activation of PSA-responsive peptide. Additionally, a significantly increased (1.7-fold) mean fluorescence intensity was observed in cells incubated with F-L compared with N-L, indicating the mediation of PSMA. PLK-1, as a key regulator of mitotic progression, is overexpressed in many human solid tumors, including PC (Dassie et al. 2009). This gene’s downregulation induces apoptosis in proliferating tumor cell cultures (Strebhardt and Ullrich 2006). Accordingly, the gene is selected as a therapeutic target against PC. Following exposure to different formulations, the cell apoptotic activity was determined, and the data was presented in Fig. 5. Consequently, the exposure to every type of liposome loading siPLK-1 led to a great quantity of apoptotic (both Annexin V-positive and propidium iodide-positive) cells (38–55%). By comparison, only a slight effect ( Lp > rivastigmine solution after IN administration of three rivastigmine formulations. The result is in accordance with the drug distribution in the cortex and hippocampus (Fig. 6), indicating that liposomes, as a drug delivery system, and the modification of CPP can be used to enhance the pharmacodynamic efficacy of rivastigmine, and this is also consistent with the results of transport across the BBB model in vitro (Fig. 5).
3
Discussion
There are common mistakes when following the above procedure. Here are some notes which can be used to solve possible problems. Note 1. Rivastigmine is a weakly alkaline drug, and its tartrate form is generally used with the molecular weight of 400.43. Select ammonium sulfate gradient method which is considered suitable for encapsulation of weakly alkaline drug to prepare rivastigmine liposomes (Haran et al. 1993). 2. The membrane for extrusion should be used in order to obtain the uniform size without destroying the structure of liposomes. Preheating the solutions at 50 C is recommended before extrusion especially when the extrusion is difficult to perform. 3. Select PBS with pH 6.5 as a solvent of rivastigmine liposomes since the nasal cavity has the pH of 5.5–6.5 (Dahl and Mygind 1998). 4. Avoid water during the progress of reaction. Add trimethylamine to remove the free ammonia during the reaction and adjust the pH value. 5. Nitrogen should be filled before covering, or a balloon full of nitrogen is recommended to cover on top of the flask. 6. During dialysis, the deionized water should be changed every 3–4 h to remove the impurities sufficiently. 7. Perform the same procedure as the rivastigmine liposomes in Subheading 2.2.1. 8. Normally, soak the Sephadex G-50 in deionized water for over 24 h to ensure fully swelling before use. 9. Solution with light blue opalescence can be found while separating by the Sephadex G-50 column. The elution curve could be used to refer the volumes of separated liposomes to ensure picking out of all liposomes diluent. 10. To ensure the detected accuracy of entrapment efficiency, the recovery of rivastigmine measured by HPLC should be previously determined through adding the drug into the blank liposomes which are prepared as the same process. 11. Suitable beaker is needed to ensure the dialysis bag can be immersed by the 100 mL release medium. 12. Rinse with 2% gelatin to coat the inside of flask for attaching cells more easily. 13. Add endothelial cell growth factor (ECGF) into the medium in order to promote the growth of BMVECs.
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14. 15. 16. 17.
Warm up the culture medium and trypsin to 37 C before using. A subcultivation ratio of 1:2 to 1:3 is recommended. The generation is suggested to subculture within 40 since primary culture. Put the electrode of the TEER instrument into the out slot and inner slot, respectively, to measure the TEER value. Add less culture medium slightly into the out slot of the 12-well incubator than that of the inner slot to observe the change between bilateral liquid surface of outside and inside slot. If the differential liquid level can keep over 4 h, it can also infer the cell monolayer have formed the tight junction (Xie et al. 2004). Warm up the formulations at 37 C before using. Add 30 μL formulations and 970 μL culture medium into each well to make the final 150 μg/mL of rivastigmine. House the male Sprague-Dawley rats under standard conditions with free access to food and water. Fast the rats for at least 12 h prior to the experiment and give water freely. Store all homogenates at 80 C prior to analysis. Prepare calibration curves with known amounts of rivastigmine in the range of 1–1000 ng/ml for tissue samples, utilizing the peak area ratio of rivastigmine to internal standard (antipyrine) versus the rivastigmine concentrations. The retention time is ~1.6 min for rivastigmine by HPLC/MS method. The correlation coefficients of regression equations are all above 0.99. The limit of quantification is 0.5 ng/ml for tissue samples. Use poloxamer 188 here to accommodate viscosity similar to liposomes and enhance penetration of rivastigmine to abate the discrepancies between liposomes and solution in vivo, since it has good permeation enhancing effect during intranasal delivery (Chen et al. 2013). The color-substrate solution should be performed away from light while using. The reaction time should be strictly controlled according to the instruction of the assay kit.
18.
19.
20.
21. 22.
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Dequalinium-Mediated MitochondriaTargeting Drug Liposomes for the Treatment of Drug-Resistant Lung Cancer
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Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Synthesis of DQA-PEG2000-DSPE Conjugate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Preparation of Targeting Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Characterization of Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Targeting Mechanism and Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7 Efficacy in Drug-Resistant Lung Cancer Xenografts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8 In Vivo Imaging Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Synthesis of DQA-PEG2000-DSPE Conjugate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Preparation of Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Characterization of Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Targeting Mechanism and Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Efficacy in Drug-Resistant Lung Cancer Xenografts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 In Vivo Imaging Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9 Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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X. Ying (*) School of Pharmaceutical Sciences/Key Laboratory of Sichuan Province for Specific Structure of Small Molecule Drugs, Chengdu Medical College, Chengdu, China e-mail: [email protected] © Springer-Verlag GmbH Germany, part of Springer Nature 2021 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49320-5_26
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Abstract
The liposomes are useful drug carriers that are able to improve the physicochemical property and targeted ability of drugs. Lonidamine has shown a promising adjuvant anticancer drug for treatment of metastatic breast cancer and non-small cell lung carcinoma (NSCLC). However, its anticancer efficacy is hindered by its low water solubility and therapeutic index. This protocol introduces the fabrications of dequalinium-mediated mitochondria-targeting liposomes containing lonidamine or epirubicin and further describes the efficacy and action mechanism by targeting mitochondria after their combined use for treatment of drug-resistant lung cancer. In the targeting liposome constructs, lonidamine, epirubicin, and dequalinium lipid conjugate are used as an apoptotic inducer of resistant cancer cells, an anticancer agent, and a targeting molecule, respectively. This kind of drug liposomes provides a useful formulation to treat drug-resistant lung cancer. Keywords
Liposomes · Mitochondrial targeting · Lonidamine · Drug-resistant lung cancer · Signaling pathways
1
Introduction
Non-small cell lung cancer (NSCLC) is one of the malignant tumors of lung cancer, accounting for 85% of all lung cancer diagnosis (Molina et al. 2008). According to the statistics, the stage IV NSCLC patients have an overall 5-year survival rate of 16% globally (Siegel et al. 2016), and most of the patients are relapsed due to drug resistance. Substantial investigations reveal that the drug resistance of NSCLC is derived from the intrinsic and extrinsic mechanisms. The intrinsic drug resistance is mostly caused by DNA quick repair mechanism (Bouwman and Jonkers 2012) or gene tolerance, while the extrinsic is mainly caused by the overexpressions of ATPbinding cassette transporters (ABC transporter) (Efferth and Volm 2017), causing the efflux of anticancer drugs from the cancer cells and thereby reducing the treatment efficacy. Targeting drug liposomes emerge to be powerful carriers for troubleshooting both intrinsic and extrinsic drug resistance. Firstly, polyethylene glycol-modified targeting liposomes are able to deliver drugs more into solid tumors via long circulation in blood system, evading the rapid uptake of reticuloendothelial system after drug administration (Luo et al. 2017; Aoyama et al. 2017). Secondly, a suitable particle size of the targeting drug liposomes enables drug carriers to be accumulated into the solid tumor tissues through the enhanced permeability and retention (EPR) effect. Finally, specifically modified targeting liposomes are able to inhibit drug efflux from cancer cells by blocking the ABC transporters (Mamot et al. 2003) and by attracting onto the subcellular organelles. Mitochondria-targeting drug liposomes are just these cases constructed for the purposes aimed at killing both intrinsic and extrinsic resistant cancers.
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Mitochondria are the intracellular “power house”, which make ATP molecules to provide energy for all cell activities. Meanwhile, mitochondria also regulate the process of cellular apoptosis by two families of genes that are expressed on the mitochondrial membrane, as anti-apoptotic and apoptotic genes, respectively. Besides, the mitochondrial membrane permeability is involved in the induction of apoptotic signaling (McArthur et al. 2018). During apoptosis, apoptosis-inducing factor (AIF) and cytochrome C (Cyt C) release and activate apoptotic enzymes caspases to produce a cascade of reaction by various signal transduction pathways (Adachi et al. 2015; Zhou et al. 2013). Additionally, mitochondria also mediate cell growth and cell cycle (Harbauer et al. 2014; Abdul and Hoosein 2002). Hence, in addition to targeting cancer cells, mitochondria-targeting drug liposomes can further target mitochondria of cancer cells, thereby significantly enhancing the killing effect in drug-resistant cancer cells by inducing apoptosis via activating apoptotic enzymes and pro-apoptotic genes while inhibiting anti-apoptotic genes. This protocol is aimed at describing a kind of mitochondria-targeting drug liposomes that enable to treat drug-resistant NSCLC. The liposomes are composed of lonidamine, epirubicin, egg phosphatidylcholine, D-α-tocopherol polyethylene glycol succinate (TPGS1000), and dequalinium lipid conjugate (DQA-PEG2000DSPE). In the constructs, lonidamine is inserted into the bilayer of liposome vesicles as the apoptotic inducer of resistant cancer cells, epirubicin is entrapped into liposome inner vesicles as an anticancer agent, and DQA-PEG2000-DSPE conjugate is incorporated onto the membrane of the liposomes as the mitochondria-targeting molecule. Dequalinium (DQA) is a quaternary ammonium cation used to treat malaria (Rodrigues and Gamboa 2009; Abdul and Hoosein 2002). In the latest years, it is found that DQA is able to penetrate across the lipid bilayers and accumulate in the mitochondria of cancer cells (Weiss et al. 1987), and thus used to modify drug nanoparticles for targeting drug-resistant cancers (Bae et al. 2018). Lonidamine is a kind of indole derivatives. Studies demonstrate that lonidamine interfere the energy metabolism of cancer cells by inhibiting glycolysis and respiratory chain, causing apoptosis of cancer cells (Nath et al. 2016; Le Bras et al. 2006). However, the low water solubility of lonidamine makes it hard to exert anticancer effect and to be delivered to mitochondria (Liu et al. 2017). Epirubicin is one of anthracyclines and extensively used for clinical treatment of various cancers. It acts as anticancer efficacy by binding with DNA and directly inserting into the double-helix base pairs of DNA, damaging the function of DNA, interfering with the transcription, and preventing the formation of mRNA (Olinski et al. 1997). The major safety concerns of epirubicin are cardio and hematologic toxicity (Vici et al. 2011). The construction of dequalinium-mediated mitochondria-targeting liposomes not only reaches a mitochondria-targeting purpose for eliminating drug-resistant cancer but also reduces the systemic toxicity of anticancer drug, including cardiotoxicity due to the altered distribution after drug administration. The following text will mainly describe the preparation and characterization of the mitochondria-targeting drug liposomes by incorporating either lonidamine or epirubicin.
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2
Materials
2.1
Synthesis of DQA-PEG2000-DSPE Conjugate
1. Dequalinium (DQA) 2. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (COOH-PEG2000-DSPE) 3. 4-dimethylaminopyridine (DMAP) 4. N, N0 -dicyclohexylcarbodiimide (DCC) 5. Acetonitrile 6. 1-hydroxy-1H-benzotriazole (HoBt) 7. Regenerated cellulose dialysis tubing (molecular weight cutoff point, 2000) 8. Nuclear magnetic resonance spectroscopy 9. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
2.2 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
2.3 1. 2. 3. 4. 5. 6. 7. 8. 9.
Preparation of Targeting Liposomes Egg phosphatidylcholine D-α-tocopherol polyethylene glycol 1000 succinate (TPGS1000) DQA-PEG2000-DSPE Lonidamine and epirubicin Methanol Ammonium sulfate (250 mM) Phosphate buffered saline Coumarin Polyethylene glycol-distearoyl phosphatidyl ethanol-amine (PEG2000-DSPE) Pear-shaped flasks Rotary vacuum evaporator Ultrasonic cell disruptor (200 W) Polycarbonate membranes (400 nm and 200 nm) Dialyze bag (12,000 to 14,000 molecular mass cutoff)
Characterization of Liposomes Sephadex G-50 Phosphate buffered saline Acetonitrile Acetate (0.1 M) NaH2PO4 (0.5 M) Trimethylamine Distilled water Micropore filter membrane (200 nm) ODS C18 column (Phenomenex, i.e., 5 mm; 250 mm 4.6 mm)
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10. High-performance liquid chromatography (HPLC) system with a UV detector 11. Zetasizer Nano Series Zen 4003 12. Atomic force microscopy (AFM)
2.4
Cell Culture
1. Carcinomic human alveolar basal epithelial A549 cells and resistant A549 cells (A549cDDP) 2. F-12K medium (American Type Culture Collection, ATCC) 3. Fetal bovine serum 4. 1% Antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) 5. RPMI-1640 6. Cisplatin (4 mM) 7. CO2 incubator
2.5 1. 2. 3. 4. 5. 6. 7. 8. 9.
Cytotoxicity
A549 cells A549cDDP cells Lonidamine Sulforhodamine B (SRB) Acetic acid (1%, v/v) Trichloroacetic acid Tris base solution (10 mM) 96-well culture plates 96-well plate reader
2.6
Targeting Mechanism and Effect
2.6.1 Mitochondrial Co-localization 1. A549 cells 2. A549cDDP cells 3. MitoTracker Deep Red FM (0.5 mM and 2 mM) 4. MitoTracker Green FM (200 nM) 5. Chambered coverslips 6. Leica confocal software 2.6.2 Drug Content in Mitochondria 1. A549 cells 2. A549cDDP cells 3. Coumarin 4. Cold PBS
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Mitochondria extraction reagent Cell Mitochondria Isolation Kit Homogenizer FACScan flow cytometer
2.6.3 Mitochondrial Depolarization 1. A549 cells 2. A549cDDP cells 3. Cationic lipophilic fluorochrome rhodamine 123 4. Phosphate buffered saline 5. MPTP blocker cyclosporin A (CsA, 1 μM) 6. 6-well, flat-bottom tissue culture plates 2.6.4 Release of Cytochrome C from Mitochondria 1. A549 cells 2. A549cDDP cells 3. Lonidamine 4. Epirubicin 5. Cold PBS 6. Cytosol extraction buffer 7. Mitochondrial dissolution buffer 8. Horseradish peroxidase (HRP)-conjugated reagent 9. Chromogen solution A and chromogen solution B 10. Stop solution 11. Human cytochrome C enzyme-linked immunosorbent assay (ELISA) kit 12. Cytosol and mitochondrial protein extraction kit 13. Vortex mixer 14. Glass homogenizer 15. Centrifuge 16. Microplate reader 2.6.5 Caspase Activation 1. A549 cells 2. A549cDDP cells 3. Caspase 3 and caspase 9 substrates 4. Lonidamine 5. Epirubicin 6. Cell lysate 7. Centrifuge 8. Microplate reader 2.6.6 Effects on Pro-apoptotic Bax and Anti-apoptotic Mcl-1 1. ELISA kit 2. Bicinchoninic acid
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Cell lysate Bax monoclonal antibody Mcl-1 monoclonal antibody HRP-tagged protein of Bax antibody HRP-tagged protein of Mcl-1 antibody Chromogen solutions A and B Centrifuge Microplate reader
2.6.7 ROS Assay 1. A549 cells 2. A549cDDP cells 3. 20 ,70 -Dichlorofluorescein diacetate (H2DCFDA) 4. H2DCFDA stock solution (200 mM) 5. FACScan flow cytometer 2.6.8 ATP Assay 1. A549 cells 2. A549cDDP cells 3. ATP detection working dilution 4. 96-well plates 5. Firefly luciferase-based ATP assay kit 6. Centrifuge 7. Monochromator microplate reader
2.7 1. 2. 3. 4. 5. 6.
A549cDDP cells Female BALB/c nude mice (initial weight 16–18 g) Serum-free RPMI-1640 culture medium Physiological saline Epirubicin Caliper
2.8 1. 2. 3. 4. 5.
Efficacy in Drug-Resistant Lung Cancer Xenografts
In Vivo Imaging Observation
Female BALB/c nude mice 1,10 -dioctadecyl-3,3,30 ,30 tetramethyl indotricarbocyanine iodide (DiR) 0.25% trypsin Physiological saline Kodak multimodal imaging system
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3
Methods
3.1
Synthesis of DQA-PEG2000-DSPE Conjugate
1. Synthesize the DQA-PEG2000-DSPE conjugate from COOH-PEG2000-DSPE and DQA by acylation reaction (see Note 1). 2. Transfer crude product to regenerate cellulose dialysis tubing. 3. Dialyze against deionized water for 48 h to remove uncoupled DQA, DMAP, DCC, HoBt, and acetonitrile. 4. Lyophilize the residue to obtain powder. 5. Use nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to characterize the mixture. 6. Add serial known aliquots of pure DQA to the same sample of the above reaction product. 7. Add pure DQA to yield a linear in the signal from the aromatic hydrogen (~6.8 ppm). 8. Use slope to determine the initial mass of DQA-PEG2000-DSPE in the sample. 9. Semi-quantify the DQA-PEG2000-DSPE content by the signal peak of DQA remained on DQA-PEG2000-DSPE conjugate. 10. Calculate the yield of DQA-PEG2000-DSPE.
3.2
Preparation of Liposomes
3.2.1 Preparation of Targeting Lonidamine Liposomes 1. Add egg phosphatidylcholine, TPGS1000 and DQA-PEG2000-DSPE, and lonidamine into methanol in a pear-shaped flask (see Note 2). 2. Remove the methanol by rotary vacuum evaporator. 3. Hydrate the lipid film by sonication in a water bath for 10 min. 4. Treat suspensions with an ultrasonic cell disruptor for 10 min (200 W). 5. Extrude the suspension through polycarbonate membranes (400 nm thrice and 200 nm thrice) to obtain targeting lonidamine liposomes in Fig. 1.
3.2.2 Preparation of Lonidamine Liposomes 1. Add egg phosphatidylcholine, TPGS1000 and PEG2000-DSPE, and lonidamine into methanol in a pear-shaped flask. 2. Remove the methanol by rotary vacuum evaporator. 3. Hydrate the lipid film by sonication in a water bath for 10 min. 4. Treat suspensions with an ultrasonic cell disruptor for 10 min (200 W). 5. Extrude the suspension through polycarbonate membranes (400 nm thrice and 200 nm thrice) to obtain lonidamine liposomes.
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DQA-PEG2000-DSPE Cholesterol Lipid and TPGS1000 Epirubicin* COOH-PEG2000-DSPE Lonidamine* *not encapsulated simultaneously
Fig. 1 Schematic representation of targeting lonidamine liposomes or targeting epirubicin liposomes. (Adapted from Li et al. (2013), with permission)
3.2.3 Preparation of Targeting Coumarin Liposomes (See Note 3) 1. Dissolve egg phosphatidylcholine, TPGS1000 and DQA-PEG2000-DSPE, and coumarin in methanol in a pear-shaped flask (lipids/coumarin = 200:1, w/w). 2. Remove the methanol by rotary vacuum evaporator. 3. Hydrate the lipid film by sonication in a water bath for 10 min. 4. Treat suspensions with an ultrasonic cell disruptor for 10 min (200 W). 5. Extrude the suspension through polycarbonate membranes (400 nm thrice and 200 nm thrice) to obtain targeting coumarin liposomes. 3.2.4 Preparation of Coumarin Liposomes 1. Dissolve egg phosphatidylcholine, TPGS1000 and PEG2000-DSPE, and coumarin in methanol in a pear-shaped flask (lipids/coumarin = 200:1, w/w). 2. Remove the methanol by rotary vacuum evaporator. 3. Hydrate the lipid film by sonication in a water bath for 10 min. 4. Treat suspensions with an ultrasonic cell disruptor for 10 min (200 W). 5. Extrude the suspension through polycarbonate membranes (400 nm thrice and 200 nm thrice) to obtain coumarin liposomes. 3.2.5 Preparation of Targeting Epirubicin Liposomes 1. Dissolve egg phosphatidylcholine, TPGS1000, and DQA-PEG2000-DSPE (90/5/5, mmol/mmol) in methanol in a pear-shaped flask. 2. Remove the methanol by rotary vacuum evaporator. 3. Hydrate the lipid film by sonication in a water bath for 10 min. 4. Treat suspensions with an ultrasonic cell disruptor for 10 min (200 W). 5. Extrude the suspension through polycarbonate membranes (400 nm thrice and 200 nm thrice).
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6. Dialyze the suspension in PBS for three times (each for 12 h). 7. Add epirubicin and an appropriate amount of ammonium sulfate. 8. Incubate the mixture at 60 C in a water bath, and shake intermittently for 25 min to obtain targeting epirubicin liposomes.
3.2.6 Preparation of Epirubicin Liposomes 1. Dissolve egg phosphatidylcholine, TPGS1000, and PEG2000-DSPE (90/5/5, mmol/mmol) in methanol in a pear-shaped flask. 2. Remove the methanol by rotary vacuum evaporator. 3. Hydrate the lipid film by sonication in a water bath for 10 min. 4. Treat suspensions with an ultrasonic cell disruptor for 10 min (200 W). 5. Extrude the suspension through polycarbonate membranes (400 nm thrice and 200 nm thrice). 6. Dialyze the suspension in PBS for three times (each for 12 h). 7. Add epirubicin. 8. Incubate the mixture at 60 C in a water bath, and shake intermittently for 25 min to obtain epirubicin liposomes.
3.3
Characterization of Liposomes
1. Pass over targeting lonidamine liposomes, lonidamine liposomes, targeting coumarin liposomes, coumarin liposomes, targeting epirubicin liposomes, and epirubicin liposomes, respectively, through a Sephadex G-50 column with PBS as equilibrium liquid. 2. Measure the content of lonidamine and epirubicin by HPLC system with UV detector (see Notes 4 and 5). 3. Calculate the encapsulation efficiencies (EE) of lonidamine and epirubicin (see Note 6). 4. Measure the particle sizes and zeta potential values using a Zetasizer Nano Series Zen 4003. 5. Observe the morphology of liposomes by AFM. 1. Dilute liposomes with distilled water. 2. Filter through a 0.2 μm micropore filter membrane. 3. Spread a 10 μl volume of liposome suspension on a silicon slice. 4. Dry at room temperature. 5. Observe using AFM in Fig. 2.
3.4
Cell Culture
1. Culture A549 cells in F-12K medium supplemented with 10% FBS and 1% antibiotics. 2. Culture MDR lung cancer A549cDDP cells in RPMI-1640 supplemented with 10% FBS and 1% antibiotics.
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Fig. 2 (A) Atomic force microscopic images of lonidamine liposomes (A1, plane surface image; A2, three-dimensional image). (B) Atomic force microscopic images of targeting lonidamine liposomes (B1, plane surface image; B2, three-dimensional image). (Adapted from Li et al. (2013), with permission)
3. Culture A549cDDP cells in the presence of 4 mM cisplatin. 4. Passage A549cDDP cells in a drug-free medium for 1 week. 5. Maintain cells under 5% CO2 at 37 C.
3.5
Cytotoxicity
1. Seed A549 cells or A549cDDP cells in 96-well culture plates. 2. Culture the cells for 24 h under 5% CO2 at 37 C.
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3. Replace the medium with fresh culture media containing varying concentrations of free lonidamine, lonidamine liposomes, and targeting lonidamine liposomes, respectively (see Note 7). 4. Remove the medium at 48 h. 5. Fix the cells with trichloroacetic acid. 6. Wash the plates four times with water. 7. Dry the plates in air at room temperature (25 C). 8. Stain cells with 100 μl SRB. 9. Remain the plates at room temperature for 30 min. 10. Quickly rinse the plates four times with 1% (v/v) acetic acid. 11. Dry the plates in air at room temperature (25 C). 12. Add 200 μl of 10 mM Tris base solution to each well. 13. Shake the plates for 5 min. 14. Measure the OD at 540 nm using a 96-well plate reader. 15. Calculate the cell survival rates using the formula: survival % = (A540 nm for the treated cells/A540 nm for the control cells) 100%, where A540 nm is the absorbance value. 16. Set five determinations for each dose level in each assay. 17. Repeat each assay thrice. 18. Create dose-effect curves. 19. Incubate A549 cells or A549cDDP cells with fresh culture media containing various concentrations of the targeting epirubicin liposomes (0–20 μM) plus targeting lonidamine liposomes (2.5 μM, 5 μM, and 10 μM). 20. Follow step 5 to 19 after 48 h of incubation (see Note 8).
3.6
Targeting Mechanism and Effect
3.6.1 Mitochondrial Co-localization 1. Seed A549 cells or A549cDDP cells into chambered coverslips (3 104/well). 2. Incubate for 24 h. 3. Add coumarin liposomes (10 μM) or targeting coumarin liposomes (10 μM). 4. Incubate for 4 h. 5. Wash the cells with PBS. 6. Stain the cells with MitoTracker Deep Red FM (see Note 9). 7. Incubate the cells for 30 min. 8. Create images from the individual channels. 9. Overlap the images. 10. Composite images. 11. Observe the co-localization of targeting epirubicin liposomes into the mitochondria by the same procedures (see Note 10). 3.6.2 Drug Content in Mitochondria 1. Incubate A549 cells or A549cDDP cells.
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2. Add free coumarin, coumarin liposomes, or targeting coumarin liposomes (10 μM coumarin). 3. Incubate for 4 h. 4. Harvest and wash with cold PBS (pH 7.4) twice. 5. Isolate mitochondria by the Cell Mitochondria Isolation kit. 6. Measure the amount of mitochondria uptake by FACScan flow cytometer with the events collected 1 104. 7. Represent by fluorescent intensity.
3.6.3
Mitochondrial Depolarization
Measure the Mitochondria Membrane Potential (ΔΨm) 1. Seed A549 cells or A549cDDP cells (4 105 cells/well) in a 6-well, flat-bottom tissue culture plate. 2. Incubate for 24 h. 3. Treat the cells with lonidamine liposomes, epirubicin liposomes, targeting lonidamine liposomes, targeting epirubicin liposomes, and targeting epirubicin liposomes plus targeting lonidamine liposomes (20 μM lonidamine and 10 μM epirubicin), respectively. 4. Incubate for 6 h. 5. Wash the cells with PBS once. 6. Dye with the cationic lipophilic fluorochrome rhodamine 123. 7. Analyze the cells (1 104) by a FACScan flow cytometer immediately to measure the mitochondria membrane potential (ΔΨm). Confirm the Specificity of Induced Mitochondrial Depolarization by Targeting Lonidamine Liposomes or Targeting Epirubicin Liposomes 1. Seed A549 cells or A549cDDP cells (4 105 cells/well) in a 6-well, flat-bottom tissue culture plate. 2. Inhibit with the MPTP blocker cyclosporin A for 30 min. 3. Remove the MPTP blocker. 4. Add a new medium containing the lonidamine liposomes (20 mM), epirubicin liposomes (10 mM), targeting lonidamine liposomes (20 mM), targeting epirubicin liposomes (10 mM), and targeting epirubicin liposomes (10 mM) plus targeting lonidamine liposomes (20 mM). 5. Add blank medium to perform control experiments. 6. Wash the cells by PBS once. 7. Dye with the cationic lipophilic fluorochrome rhodamine 123. 8. Analyze the cells (1 104) by a FACScan flow cytometer immediately to measure the mitochondria membrane potential (ΔΨm).
3.6.4 Release of Cytochrome C from the Mitochondria 1. Incubate A549 cells or A549cDDP cells for 24 h. 2. Add free lonidamine, lonidamine liposomes, targeting lonidamine liposomes, free epirubicin, epirubicin liposomes, targeting epirubicin liposomes, and
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targeting lonidamine liposomes plus targeting epirubicin liposomes (20 μM lonidamine or 5 μM epirubicin). Incubate the cells for 24 h. Harvest the cells and wash with cold PBS twice. Incubate the cells in cytosol extraction buffer (see Note 11). Vortex for 15 s. Homogenize with a glass homogenizer 30–35 times. Centrifuge the homogenization buffer at 3000 revolutions per min (rpm) for 10 min at 4 C. Centrifuge the supernatant at 11,000 rpm for 30 min at 4 C. Store the supernatant from the centrifugation of step 9 as the cytosol fraction. Treat the precipitates with mitochondrial dissolution buffer (see Note 12). Vortex and centrifuge at 13,000 rpm for 10 min at 4 C. Store the supernatant from the centrifugation of step 12 as the mitochondrial fraction. Add protein samples to the sample testing wells. Incubate the samples for 30 min at 37 C, and then wash with washing buffer five times. Add horseradish peroxidase (HRP)-conjugated reagent (provided in the kit) to each well. Add chromogen solution A and chromogen solution B (provided in the kit) to the wells. Incubate for 15 min at 37 C in the dark. Treat the wells with the stop solution (provided in the kit). Measure at 450 nm on a microplate reader within 10 min. Calculate the content ratio of cytochrome C by comparison with the content of the blank control in Fig. 3 (see Note 13).
3.6.5 Caspase Activation 1. Culture A549 or A549cDDP cells for 24 h. 2. Add free lonidamine, lonidamine liposomes, targeting lonidamine liposomes, free epirubicin, epirubicin liposomes, targeting epirubicin liposomes, and targeting lonidamine liposomes plus targeting epirubicin liposomes (20 μM lonidamine or 5 μM epirubicin). 3. Incubate for 24 h. 4. Harvest the cells. 5. Lyse the cells. 6. Centrifuge the cell lysates at 10,000 rpm for 1 min at 4 C. 7. Store the supernatants. 8. Treat the supernatants with caspase 3 and caspase 9 substrates. 9. Measure caspase 3 and caspase 9 activities by a microplate reader (405 nm). 10. Calculate the activity ratio according to kit instructions. 3.6.6 Effects on Pro-apoptotic Bax and Anti-apoptotic Mcl-1 1. Culture the cells with medium at 37 C in 5% CO2 for 24 h.
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Mitochondria Cytosol
d,e,f,g
Protein ratio
2.5 2.0 a,b 1.5 1.0 0.5 0.0
1
2
3
4
b
5
6
7
Mitochondria
3.0
Cytosol
d,e,f,g
2.5
Protein ratio
8
2.0
a,b,c
1.5 1.0 0.5 0.0
1
2
3
4
5
6
7
8
Fig. 3 Cytochrome C protein ratios in the cytosol and mitochondria of A549 (a) and A549cDDP (b) cells after incubation with varying drug formulations for 12 h. Each assay is repeated in triplicate. Data are presented as the mean standard derivation (n = 3). (Adapted from Li et al. (2013), with permission). (1) Blank control, (2) free epirubicin, (3) epirubicin liposomes, (4) targeting epirubicin liposomes, (5) free lonidamine, (6) lonidamine liposomes, (7) targeting lonidamine liposomes, (8) targeting epirubicin liposomes plus targeting lonidamine liposomes. p < 0.05; a, vs. 1; b, vs. 1; c, vs. 3; d, vs. 4; e, vs. 5; f, vs. 6; g, vs. 7
2. Add epirubicin liposomes (5 mM), lonidamine liposomes (20 mM), targeting epirubicin liposomes (5 mM), targeting lonidamine liposomes (20 mM), and targeting epirubicin liposomes (5 mM) plus targeting lonidamine liposomes (20 mM). 3. Incubate the cells for 12 h.
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4. 5. 6. 7.
Lyse the cells. Centrifuge the cell lysates at 10,000 rpm at 4 C for 1 min. Collect the total protein (containing Bax or Mcl-1) from the supernatants. Add samples containing Bax or Mcl-1 (50 ml) to wells pre-coated with Bax or Mcl-1 monoclonal antibodies. Incubate at 37 C for 30 min. Wash with the kit washing buffer. Add HRP-tagged protein of a Bax or Mcl-1 antibody (50 ml). Protect the samples (50 ml) from light. Incubate at 37 C for 30 min. Remove the unbound enzyme by washing with the kit washing buffer. Add chromogen solutions A and B (each 50 ml) (see Note 14). Add the stop solution (50 ml) (see Note 15). Measure the absorbance of the yellow product at 450 nm with a microplate reader. Measure the concentration of total protein (see Note 16).
8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
3.6.7 ROS Assay 1. Culture A549 or A549cDDP cells for 24 h. 2. Add epirubicin liposomes, lonidamine liposomes, targeting epirubicin liposomes, targeting lonidamine liposomes, and targeting epirubicin liposomes plus targeting lonidamine liposomes (20 μM lonidamine or 10 μM epirubicin). 3. Incubate for 6 h. 4. Add 25 μl H2DCFDA (200 μM) to the medium. 5. Incubate the cells for 30 min in the dark. 6. Measure the fluorescence intensity (1 104 cells) by FACScan flow cytometer. 3.6.8 ATP Assay 1. Culture A549 or A549cDDP cells for 24 h. 2. Add epirubicin liposomes, lonidamine liposomes, targeting epirubicin liposomes, targeting lonidamine liposomes, or targeting epirubicin liposomes plus targeting lonidamine liposomes. 3. Incubate for 6 h. 4. Centrifuge A549 or A549cDDP cells at 12,000 rpm for 5 min. 5. Measure the cellular ATP levels by a firefly luciferase-based ATP assay kit. 6. Mix 100 ml of each supernatant with 100 ml ATP detection working dilution in 96-well plates. 7. Measure luminance (relative luminescence units, RLU) by a monochromator microplate reader.
3.7
Efficacy in Drug-Resistant Lung Cancer Xenografts
1. Re-suspend approximately 1 107 A549cDDP cells in 200 μl serum-free RPMI1640 culture medium.
Dequalinium-Mediated Mitochondria-Targeting Drug Liposomes for the. . .
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2. Inject the cells into the right flanks of the BALB/c nude mice. 3. Randomly divide the mice (tumor volume, 200–220 mm3) into six treatment groups (six mice each). 4. Administrate physiological saline, free epirubicin (3 mg/kg), epirubicin liposomes (3 mg/kg), targeting epirubicin liposomes (3 mg/kg), lonidamine liposomes (20 mg/kg), and targeting epirubicin liposomes (3 mg/kg) plus targeting lonidamine liposomes (20 mg/kg) via tail vein injection on the 22th, 24th, 26th, and 28th days after inoculation. 5. Monitor the tumor progression with a caliper every other day. 6. Weigh the mice and measure the tumors with a caliper every day or every 2 days. 7. Observe the mice before sacrifice. 8. Confirm each tumor mass by necropsy on the 31th day after inoculation. 9. Calculate the tumor volumes as length width2/2 (mm3) in Fig. 4.
3.8 1. 2. 3. 4. 5. 6. 7. 8. 9.
In Vivo Imaging Observation
Divide female BALB/c mice into three groups (three mice each). Dissociate A549cDDP cells by 0.25% trypsin (g/100 ml). Inoculate A549cDDP cells. Administrate the mice with physiological saline, free DiR, and targeting DiR liposomes via tail vein injection on the 26th day. Scan the mice at 1, 2, 6, 12, and 24 h by a Kodak multimodal imaging system. Administrate the mice with targeting DiR liposomes, free DiR, and physiological saline for 1 h. Sacrifice the tumor-bearing mice. Remove the tumor masses, hearts, livers, spleens, lungs, and kidneys. Photograph the fluorescence signal intensities in different tissues.
3.9
Statistical Analysis
1. Present data as the mean standard deviation (SD). 2. Use one-way analysis of variance (ANOVA) to determine the significance among groups. 3. Use post hoc tests with the Bonferroni correction for multiple comparisons between individual groups.
4
Notes
1. The DQA-PEG2000-DSPE conjugate is synthesized by acylation reaction. The mixture should be detected by nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
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2500
Blank Lonidmaine liposomes
Volume (mm3)
2000
Epirubicin liposomes Targeting epirubicin liposomes plus targeting lonidamine liposomes
1500
1000
500
0
a, b. c 0
5
10
15
20
25
30
35
Days post inoculation (day)
Fig. 4 Antitumor activity in the A549cDDP xenografts in BALB/c nude mice after treatment with 0.9% NaCl (blank); 20 mg/kg lonidamine liposomes; 3 mg/kg epirubicin liposomes; 3 mg/kg targeting epirubicin liposomes plus 20 mg/kg targeting lonidamine liposomes. Data are presented as mean standard deviation (n = 6). (Adapted from Li et al. (2013), with permission). a, p < 0.05, targeting epirubicin liposomes plus targeting lonidamine liposomes versus physiological saline; b, p < 0.05, targeting epirubicin liposomes plus targeting lonidamine liposomes versus lonidamine liposomes; c, p < 0.05, targeting epirubicin liposomes plus targeting lonidamine liposomes versus epirubicin liposomes
2. During the preparation process of targeting lonidamine liposomes, the ratio of egg phosphatidylcholine, TPGS1000, and DQA-PEG2000-DSPE should be 90:5:5 (mmol/mmol) and the ratio of lipids and lonidamine 10:1 (w/w). 3. The targeting coumarin liposomes are also prepared and used as controls, potent cytotoxic agents, or fluorescent probes to lonidamine liposomes, epirubicin liposomes, targeting epirubicin liposomes, and coumarin liposomes. 4. Mobile phase of lonidamine should be consisted of acetonitrile and 0.1 M acetate buffer (50:50, v/v). The detected wavelength is set at 298 nm. 5. Mobile phase of epirubicin should be consisted of acetonitrile, 0.5 M NaH2PO4 and triethylamine (34:66:0.3, v/v). The detected wavelength is set at 254 nm. 6. The encapsulation efficiencies (EE) of lonidamine and epirubicin is calculated using the formula EE% = (Wi/Wtotal) 100%. Wi is the measured amount of lonidamine, coumarin, or epirubicin in the liposome suspensions after passing over the Sephadex G-50 column, and Wtotal is the measured amount of lonidamine, coumarin, or epirubicin in the liposome suspensions before passing over the Sephadex G-50 column. 7. The volume of lonidamine liposome preparations that need to be added to the cells is 10 μl, and the final concentration of lonidamine should be 20 times more than the theoretical concentration.
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8. In cytotoxicity experiment, the culture medium is used as a blank control. The cells are further incubated for 48 h under 5% CO2 at 37 C and detected by the SRB assay. 9. A549 cells and A549cDDP cells should be separately stained with 0.5 mM and 2 mM of MitoTracker Deep Red FM. 10. The co-localized time of targeting epirubicin liposomes into the mitochondria should be 1 h. And the cells should be stained with MitoTracker Green FM (200 nM). 11. The contents in cytosol extraction buffer should be consisted of 0.1% (v/v) protease inhibitor and 0.1% (v/v) DL-Dithiothreitol [DTT]. 12. The mitochondrial dissolution buffer contains 20 mmol/L Tris-Cl (pH 7.5), 2 mmol/L EGTA, 1% (v/v) Triton X-100, 0.1% (v/v) protease inhibitor and 1 mM DTT. 13. Cytochrome C protein content should be determined by human cytochrome C ELISA kit. 14. The color of sample liquids should change to blue after adding chromogen solutions A and B. 15. After adding the stop solution, the liquids color finally turns to yellow because of the effect of the acid. The depth of the color is positively correlated with the concentration of Bax or Mcl-1 protein in a sample. 16. The concentration of total protein should be measured using the bicinchoninic acid method at 540 nm.
5
Conclusion
This protocol dealt with the preparation and characterization of targeting lonidamine liposomes and targeting epirubicin liposomes, both of which are developed to treat resistant lung cancer by a combinational use. For this purpose, functional liposome materials and apoptotic inducing agent are crucial to the final efficacy for treating the resistant cancer. The liposome membrane materials, DQA-PEG2000-DSPE, PEG2000DSPE, and TPGS1000, play important roles to extend the long circulation of the targeting drug liposomes in blood, to enhance the permeability and retention in tumor tissue, and to target the mitochondria of resistant lung cancer cells. Lonidamine entrapped in the targeting liposomes significantly contribute to enhance treatment efficacy of epirubicin by inducing apoptosis of drug-resistant cancer.
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Aoyama K, Kuroda S, Morihiro T, Kanaya N, Kubota T, Kakiuchi Y, Kikuchi S, Nishizaki M, Kagawa S, Tazawa H, Fujiwara T (2017) Liposome-encapsulated plasmid DNA of telomerasespecific oncolytic adenovirus with stealth effect on the immune system. Sci Rep 7(1):14177 Bae Y, Jung MK, Lee S, Song SJ, Mun JY, Green ES, Han J, Ko KS, Choi JS (2018) Dequaliniumbased functional nanosomes show increased mitochondria targeting and anticancer effect. Eur J Pharm Biopharm 124:104–115 Bouwman P, Jonkers J (2012) Genomic instability in cancer: the effects of deregulated DNA damage signalling on cancer chemotherapy response and resistance. Nat Rev Cancer 12(9):587–598 Efferth T, Volm M (2017) Multiple resistance to carcinogens and xenobiotics: P-glycoproteins as universal detoxifiers. Arch Toxicol 91(7):2515–2538 Harbauer AB, Opalińska M, Gerbeth C, Herman JS, Rao S, Schönfisch B, Guiard B, Schmidt O, Pfanner N, Meisinger C (2014) Cell cycle-dependent regulation of mitochondrial preprotein translocase. Science 346(6213):1109–1113 Le Bras M, Borgne-Sanchez A, Touat Z, El Dein OS, Deniaud A, Maillier E, Lecellier G, Rebouillat D, Lemaire C, Kroemer G, Jacotot E, Brenner C (2006) Chemosensitization by knockdown of adenine nucleotide translocase-2. Cancer Res 66(18):9143–9152 Li N, Zhang CX, Wang XX, Zhang L, Ma X, Zhou J, Ju RJ, Li XY, Zhao WY, Lu WL (2013) Development of targeting lonidamine liposomes that circumvent drug-resistant cancer by acting on mitochondrial signaling pathways. Biomaterials 34(13):3366–3380 Liu Y, Zhang X, Zhou M, Nan X, Chen X, Zhang X (2017) Mitochondrial-targeting lonidaminedoxorubicin nanoparticles for synergistic chemotherapy to conquer drug resistance. ACS Appl Mater Interfaces 9(50):43498–43507 Luo R, Li Y, He M, Zhang H, Yuan H, Johnson M, Palmisano M, Zhou S, Sun D (2017) Distinct biodistribution of doxorubicin and the altered dispositions mediated by different liposomal formulations. Int J Pharm 519(1–2):1–10 Mamot C, Drummond DC, Hong K, Kirpotin DB, Park JW (2003) Liposome-based approaches to overcome anticancer drug resistance. Drug Resist Updat 6(5):271–279 McArthur K, Whitehead LW, Heddleston JM, Li L, Padman BS, Oorschot V, Geoghegan ND, Chappaz S, Davidson S, San Chin H, Lane RM, Dramicanin M, Saunders TL, Sugiana C, Lessene R, Osellame LD, Chew TL, Dewson G, Lazarou M, Ramm G, Lessene G, Ryan MT, Rogers KL, van Delft MF, Kile BT (2018) BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis. Science 359(6378):eaao6047 Molina JR, Yang P, Cassivi SD, Schild SE, Adjei AA (2008) Non-small cell lung cancer: epidemiology, risk factors, treatment, and survivorship. Mayo Clin Proc 83(5):584–594 Nath K, Guo L, Nancolas B, Nelson DS, Shestov AA, Lee SC, Roman J, Zhou R, Leeper DB, Halestrap AP, Blair IA, Glickson JD (2016) Mechanism of antineoplastic activity of lonidamine. Biochim Biophys Acta 1866(2):151–162 Olinski R, Jaruga P, Foksinski M, Bialkowski K, Tujakowski J (1997) Epirubicin-induced oxidative DNA damage and evidence for its repair in lymphocytes of cancer patients who are undergoing chemotherapy. Mol Pharmacol 52(5):882–885 Rodrigues JR, Gamboa ND (2009) Effect of dequalinium on the oxidative stress in Plasmodium berghei-infected erythrocytes. Parasitol Res 104(6):1491–1496 Siegel RL, Miller KD, Jemal A (2016) Cancer statistics, 2016. CA Cancer J Clin 66(1):7–30 Vici P, Colucci G, Giotta F, Sergi D, Filippelli G, Perri P, Botti C, Vizza E, Carpino A, Pizzuti L, Latorre A, Giannarelli D, Lopez M, Di Lauro L (2011) A multicenter prospective phase II randomized trial of epirubicin/vinorelbine versus pegylated liposomal doxorubicin/vinorelbine as first-line treatment in advanced breast cancer. A GOIM study. J Exp Clin Cancer Res 30:39 Weiss MJ, Wong JR, Ha CS, Bleday R, Salem RR, Steele GD Jr, Chen LB (1987) Dequalinium, a topical antimicrobial agent, displays anticarcinoma activity based on selective mitochondrial accumulation. Proc Natl Acad Sci U S A 84(15):5444–5448 Zhou J, Zhao WY, Ma X, Ju RJ, Li XY, Li N, Sun MG, Shi JF, Zhang CX, Lu WL (2013) The anticancer efficacy of paclitaxel liposomes modified with mitochondrial targeting conjugate in resistant lung cancer. Biomaterials 34(14):3626–3638
Preparation of Anthracyclines Liposomes for Tumor-Targeting Drug Delivery
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Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Preparation of Liposomes by pH Gradient Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Preparation of Liposomes Modified with MAN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Daunorubicin Liposomes Modified with MAN and TF . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Determination of Encapsulation Efficiency, Drug Release, and Particle Size . . . . . . 2.5 Determination of Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7 BBB Model In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8 Transport Across the BBB and Competition Assay of MAN . . . . . . . . . . . . . . . . . . . . . . 2.9 C6 Glioma Cellular Uptake and Competition Assay of TF . . . . . . . . . . . . . . . . . . . . . . . . . 2.10 Antiproliferative Activity Against C6 Glioma Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.11 Dual-Targeting Effects In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.12 Effect on the Avascular C6 Glioma Spheroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.13 Effects on the Survival of Brain Tumor-Bearing Animals . . . . . . . . . . . . . . . . . . . . . . . . . . 2.14 Measurement of Daunorubicin in Plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.15 Measurement of Daunorubicin in Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.16 Pharmacokinetics and Biodistribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Preparation and Characterization of the Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 BBB Model In Vitro (Note 7) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Transport Across the BBB and Competition Assay of MAN . . . . . . . . . . . . . . . . . . . . . . 3.5 C6 Glioma Cellular Uptake Assay of TF (Note 8) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Competition Assay of TF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Antiproliferative Activity Against C6 Glioma Cells (Note 9) . . . . . . . . . . . . . . . . . . . . . . 3.8 Dual-Targeting Effects In Vitro (Note 10) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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X. Ying (*) School of Pharmaceutical Sciences/Key Laboratory of Sichuan Province for Specific Structure of Small Molecule Drugs, Chengdu Medical College, Chengdu, China e-mail: [email protected] © Springer-Verlag GmbH Germany, part of Springer Nature 2021 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49320-5_8
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3.9 Effect on the Avascular C6 Glioma Spheroids (Note 11) . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.10 Effects on the Survival of Brain Tumor-Bearing Animals (Note 12) . . . . . . . . . . . . . . 3.11 Measurement of Daunorubicin in Plasma (Note 13) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.12 Measurement of Daunorubicin in Tissues (Note 14) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.13 Pharmacokinetics and Biodistribution (Note 15) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.14 Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
378 379 380 380 381 382 382 383 384
Abstract
Anthracyclines are the most effective anticancer drugs with broad cancer spectrum. They demonstrate strong anticancer efficacy in vitro but are less effective in vivo during treatment of brain cancer due to the hindrance of the blood-brain barrier (BBB). In this regard, this protocol focuses on the fabrication of dual-ligand modified anthracycline liposomes for treatment of brain cancer through transferring drug across the BBB and then for targeting brain cancer. Here, the preparation and characterization techniques of dual-targeting daunorubicin liposomes are described. In this construct, daunorubicin is used as a model drug, while all anthracyclines could be loaded into the liposomes with the same procedures. The present results demonstrate that the dual-targeting daunorubicin liposomes exhibit a high drug encapsulation efficiency (>90%), an increased transport of the drug liposomes across the BBB, and then a targeted effect in killing brain glioma cells, thereby improving the therapeutic efficacy of brain glioma in vitro and in animals. Keywords
Anthracyclines · Dual`-targeting · Liposomes · Blood-brain barrier · Brain glioma-bearing rats · Survival
1
Introduction
Anthracyclines are a class of anticancer drugs, which are extracted from streptomyces bacterium (McGowan et al. 2017). They are used to treat a variety of cancers, including leukemias, lymphomas, breast, stomach, uterine, ovarian, bladder cancer, and lung cancers (Minotti et al. 2004; Peng et al. 2005; Bouma et al. 1986). Laboratory studies demonstrate that anthracyclines are also effective in killing brain cancer cells in vitro but less effective in vivo. This decreased efficacy is due to the hindrance of the blood-brain barrier (BBB), and consequently, anthracyclines are less often used to treat brain cancer. The adverse effects of anthracyclines, like any other chemotherapeutic agent, are linked to their cytotoxicity to non-differentiated and proliferating normal cells. The major adverse effects of anthracyclines are cardiotoxicity including cardiomyopathy and congestive heart failure, which obviously restrict their clinical applications
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Preparation of Anthracyclines Liposomes for Tumor-Targeting Drug Delivery
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(Lyman et al. 2011). In addition, anthracyclines have also been evidenced having severe or febrile neutropenia and other adverse effects, such as vomiting, nausea, and alopecia. The first anthracycline discovered was daunorubicin (trade name Daunomycin), which is produced naturally by Streptomyces peucetius. Doxorubicin (trade name Adriamycin) was developed shortly thereafter, and many other related compounds have followed, such as epirubicin, idarubicin, and valrubicin (Weiss 1992). Epirubicin is less cardiotoxic than doxorubicin but may not totally eliminate the risk of chronic cardiotoxicity (Cortés-Funes and Coronado 2007). Thus, clinical utility of anthracyclines is hampered by cardiotoxicity, myelosuppression, and resistance. Accordingly, various strategies are employed to prevent the cardiotoxicity of anthracyclines, including change of drug administration method, limit of the overall dosage, encapsulation of drug into liposomes, combinational use of cardioprotectors, and modification of anthracyclines. During the latest decades, a remarkable progress has been made in the development of drug liposomes (Tila et al. 2015). Liposomes are spherical vesicles consisting of one or more lipid bilayers surrounding a central aqueous core. They can encapsulate hydrophilic substances into their aqueous compartment and incorporate hydrophobic substances into the lipid bilayers. Liposomal formulations have characteristics of good biocompatibility, biodegradability, lower toxicity, and so on (Simone et al. 2008; Coune 1988). The liposomes have demonstrated an improved pharmacokinetic property of drug by altering drug distribution in the human body, hence reducing systemic toxicity or local toxicity of anticancer drug. The liposomal formulations of daunorubicin and doxorubicin are less toxic to cardiac tissue than the non-liposomal form because a lower proportion of drug liposomes is delivered to heart tissue (Forssen and Tökes 1979). However, conventional liposomes have demonstrated a rapid uptake by the reticuloendothelial system [RES] after intravenous administration (Huwyler et al. 2008). In the 1990s, a kind of PEGylation modified drug liposomes had been developed for preventing the rapid clearance of conventional liposomes by RES (Moghimi and Patel 1992), reaching a long circulation in blood system after administration. This kind of long-circulation drug liposomes, such as doxorubicin liposomes, had been successfully approved by regulatory authority and used in clinical treatments. Nevertheless, long-circulatory anthracycline liposomes are still ineffective to treat brain cancer. In order to deliver anthracyclines to the brain cancer, two main obstacles are to transfer drug across the BBB and to target brain cancer cells. Consequently, ligand-modified targeting liposomes are developed for treatment of brain cancer. In the dual-targeting fabrication, ligand-modified liposomes are designed to bind with overexpressing receptors on the BBB and then on brain cancer cells (Du et al. 2009). Here, the protocol describes a kind of dual-targeting daunorubicin liposomes for reaching above purposes. In this fabrication, daunorubicin is used as a model drug, which is able to kill cancer cells by DNA intercalation, reactive oxygen species (ROS) production, interaction with DNA topoisomerase I and II, and induction of apoptosis (Simeonova et al. 2009; Lotfi et al. 2002). In order to transport drug across BBB and to improve the therapeutic efficacy, daunorubicin is incorporated into the aqueous compartment
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of liposomes using ammonium sulfate gradient approach, and p-aminophenyl-α-Dmannopyranoside (MAN) and transferrin (TF) are conjugated on the surface of liposomes. The kind of dual-targeting daunorubicin liposomes can transport across the BBB by MAN and then target brain glioma by TF. The evaluations of therapeutic efficacy are performed on the BBB model in vitro and brain cancerbearing animals (Ying et al. 2010, 2011).
2
Materials
2.1
Preparation of Liposomes by pH Gradient Method
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
2.2 1. 2. 3. 4. 5. 6. 7. 8. 9.
Egg phosphatidylcholine (EPC) Cholesterol Polyethylene glycol distearoylphosphosphatidylethanolamine 1,2-Distearoyl-sn-glycero-3-phosphoethamolamine-N-[carboxy(polyethylene glycol) 2000] (COOH-PEG2000-DSPE) 1,2-Distearoyl-sn-glycero-3-phosphoethanola-mine-N-[amino(polyethylene glycol) 2000] (NH2-PEG2000-DSPE) Polycarbonate membranes (400 nm and 200 nm) Phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4) 250 mM ammonium sulfate solution Chloroform (analytical grade) Methanol (analytical grade) 100 ml pear-shaped flask Rotary vacuum evaporator Bath-type sonicator Circulating water multipurpose vacuum pump Ultrasonic cell disruptor
Preparation of Liposomes Modified with MAN Egg phosphatidylcholine (EPC) Cholesterol Polyethylene glycol distearoylphosphosphatidylethanolamine 1,2-Distearoyl-sn-glycero-3-phosphoethamolamine-N-[carboxy(polyethylene glycol) 2000] (COOH-PEG2000-DSPE) 1,2-Distearoyl-sn-glycero-3-phosphoethanola-mine-N-[amino(polyethylene glycol) 2000] (NH2-PEG2000-DSPE) Polycarbonate membranes (400 nm and 200 nm) Phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4) 250 mM ammonium sulfate solution Chloroform
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Preparation of Anthracyclines Liposomes for Tumor-Targeting Drug Delivery
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Methanol (2:1, v/v) p-aminophenyl-α-D-manno-pyranoside (MAN) Trinitrobenzene sulfonic acid (TNBS) 4% NaHCO3 10% Sodium dodecyl sulfate (SDS) 100 ml pear-shaped flask Rotary vacuum evaporator Bath-type sonicator Circulating water multipurpose vacuum pump Ultrasonic cell disruptor Magnetic stirrer Ultraviolet-visible spectrophotometer
2.3 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
369
Daunorubicin Liposomes Modified with MAN and TF Egg phosphatidylcholine (EPC) Cholesterol Polyethylene glycol distearoylphosphosphatidylethanolamine 1,2-Distearoyl-sn-glycero-3-phosphoethamolamine-N-[carboxy(polyethylene glycol) 2000] (COOH-PEG2000-DSPE) 1,2-Distearoyl-sn-glycero-3-phosphoethanola-mine-N-[amino(polyethylene glycol) 2000] (NH2-PEG2000-DSPE) Polycarbonate membranes (400 nm and 200 nm) Phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4) Chloroform (analytical grade) Methanol (analytical grade) 250 mM ammonium sulfate solution Transferrin (TF) p-Aminophenyl-α-D-manno-pyranoside (MAN) Trinitrobenzene sulfonic acid (TNBS) 4% NaHCO3 10% sodium dodecyl sulfate (SDS) 1-(3-Dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDCI) N-hydroxysuccinimide (NHS) Sephadex G-100 Bicinchoninic acid (BCA) kit N-hydroxysuccinimide (NHS) 100 ml pear-shaped flask Rotary vacuum evaporator Bath-type sonicator Circulating water multipurpose vacuum pump Ultrasonic cell disruptor Magnetic stirrer Ultraviolet-visible spectrophotometer
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2.4
Determination of Encapsulation Efficiency, Drug Release, and Particle Size
1. 2. 3. 4. 5. 6. 7.
Daunorubicin hydrochloride ODS column (Phenomenex, 250 4.6 mm) HPLC system with ultraviolet detector Acetonitrile (chromatographic grade) Sodium dihydrogen phosphate (NaH2PO4) (analytical grade) Triethylamine (analytical grade) Phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4) Methanol (chromatographic grade) Distilled water Centrifuge Dynamic light scattering (DLS) analyzer (Malvern Nano Series Zen 4003 Zeta Sizer)
8. 9. 10. 11.
2.5 1. 2. 3. 4. 5. 6. 7.
Determination of Morphology
Transmission electron microscopy Distilled water Micropore filter membrane (0.2 μm) Copper grids coated with carbon films 1% uranyl acetate solution Atomic force microscopy (NSK, SPI3800N series SPA-400) Silicon slice
2.6 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Cell Culture Murine brain microvascular endothelial cells (BMVECs) Murine C6 glioma cell lines Dulbecco’s modification of Eagle’s medium (DMEM) Fetal calf serum Penicillin Streptomycin l-Glutamine Endothelial cell growth factor (ECGF) Heparin F10 medium Heat-inactivated fetal bovine serum (FBS) Heat-inactivated horse serum and antibiotics Cell culture flask (T-25, T-75) 24-well tissue culture plates Trypsin EDTA
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371
16. CO2 incubator 17. Vertical flow clean bench 18. Optical microscope
2.7
BBB Model In Vitro
1. 2. 3. 4.
Murine brain microvascular endothelial cells (BMVECs) 2% gelatin solution Culture insert Transendothelial electrical resistance (TEER) instrument (World Precision Instruments) 5. Scan electron microscope 6. Horseradish peroxidase (HRP) (RZ = 3.0–3.5, enzyme activity >265 U/mg, Mw = 44 kDa)
2.8 1. 2. 3. 4. 5. 6. 7.
Transport Across the BBB and Competition Assay of MAN
Murine brain microvascular endothelial cells (BMVECs) Dulbecco’s modification of Eagle’s medium (DMEM) p-Aminophenyl-α-D-manno-pyranoside (MAN) Daunorubicin hydrochloride ODS column (Phenomenex, 250 4.6 mm) High-performance liquid chromatography (HPLC) system with UV detector Six-well cell culture plates
2.9 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
C6 Glioma Cellular Uptake and Competition Assay of TF Murine C6 glioma cells Six-well plates Daunorubicin hydrochloride F10 culture medium FAScan flow cytometer Transferrin (TF) Laser scanning confocal microscope Chambered coverslips Paraformaldehyde (4% (v/v)) Hoechst 33,258
2.10
Antiproliferative Activity Against C6 Glioma Cells
1. Murine C6 glioma cells 2. 96-well culture plates
372
3. 4. 5. 6. 7. 8.
Daunorubicin hydrochloride Sulforhodamine B (SRB) Trichloroacetic acid Acetic acid (analytical grade) Tris base Microplate reader
2.11 1. 2. 3. 4. 5. 6.
Effects on the Survival of Brain Tumor-Bearing Animals
Male Sprague-Dawley rats(240–250 g) Stereotaxic apparatus Murine C6 glioma cells Serum-free F10 medium Formalin (4%) Vernier caliper Bone wax Penicillin injection
2.14 1. 2. 3. 4.
Effect on the Avascular C6 Glioma Spheroids
C6 glioma cells Agarose 96-cell culture plates Serum-free F10 culture medium Inverted microscope
2.13 1. 2. 3. 4. 5. 6. 7. 8.
Dual-Targeting Effects In Vitro
Murine brain microvascular endothelial cells Murine C6 glioma cells Dulbecco’s modification of Eagle’s medium (DMEM) Apical compartment Basolateral compartment Sulforhodamine B (SRB)
2.12 1. 2. 3. 4. 5.
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Measurement of Daunorubicin in Plasma
HPLC system with ultraviolet detector ODS column (Phenomenex ODS-C18, 250 4.6 mm, 5 μm) Methanol (chromatographic grade) Distilled water
22
5. 6. 7. 8.
Preparation of Anthracyclines Liposomes for Tumor-Targeting Drug Delivery
Glacial acetic acid Plasma N2 gas stream Centrifuge
2.15 1. 2. 3. 4. 5. 6. 7. 8.
373
Measurement of Daunorubicin in Tissues
Male Kunming mice (23–25 g) HPLC with ultraviolet detector Homogenizer Physiological saline (137 mmol/l NaCl, 2.7 mmol/l KCl, 8 mmol/l Na2HPO4, and 2 mmol/l KH2PO4, pH 7.4) Vortex Methanol (chromatographic grade) Centrifuge N2 gas stream
2.16 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Pharmacokinetics and Biodistribution
Kunming mice (23–25 g) Heparin Microcentrifuge tube Centrifuge Freezer Homogenizer Physiological saline (137 mmol/l NaCl, 2.7 mmol/l KCl, 8 mmol/l Na2HPO4, and 2 mmol/l KH2PO4, pH 7.4) Vortex Methanol (chromatographic grade) Centrifuge N2 gas stream HPLC with ultraviolet detector
3
Methods
3.1
Preparation and Characterization of the Liposomes
3.1.1 Blank Liposomes 1. Dissolve EPC, cholesterol, PEG2000-DSPE, COOH-PEG2000-DSPE, and NH2PEG2000-DSPE (52:43:4:0.5:0.5, mmol/mmol) in chloroform and methanol (2:1, v/v) in a pear-shaped flask.
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2. Evaporate the chloroform and methanol to dryness under vacuum with a rotary evaporator. 3. Hydrate the lipid film with 250 mM ammonium sulfate by sonication in the water bath for 5 min. 4. Extrude through polycarbonate membranes thrice using 400 nm pore sizes and then thrice using those of 200 nm pore sizes. 5. Dialyze the suspensions against the PBS for three times (each time 12 h) to obtain the blank liposomes.
3.1.2 Daunorubicin Liposomes (Note 1) 1. Add daunorubicin into the blank liposomes. 2. Mix the suspensions. 3. Incubate the suspensions at 40 C in water bath, and intermittently shake for 20 min to produce the daunorubicin liposomes. 4. Remove unloaded daunorubicin by dialysis against tenfold PBS (v/v) for three times (each 12 h). 3.1.3 Daunorubicin Liposomes Modified with MAN (Note 2) 1. Mix a volume of 8 ml blank liposome suspensions (18.6 mg lipid/ml) with 14.75 μmol (4 mg) of MAN. 2. Add excessive amount of glutaraldehyde (200 μmol, 20 mg) to the liposome suspensions. 3. Incubate the suspensions for 5 min at room temperature. 4. Remove uncoupled MAN and glutaraldehyde by dialysis against PBS. 5. Estimate the coupling efficiency of MAN on the liposomes using the following formula: CEMAN = (1 Auncoupled/Atotal) 100%, where CEMAN is the coupling efficiency of MAN, Auncoupled is the absorbance of uncoupled amino groups of NH2-PEG2000-DSPE on the liposomes after modifying with MAN, and Atotal is the absorbance of amino groups of NH2-PEG2000-DSPE on the liposomes prior to conjugation with MAN. 6. Load daunorubicin as item 3.1.2 to obtain the daunorubicin liposomes modified with MAN basing on the blank liposomes modified with MAN. 3.1.4 Daunorubicin Liposomes Modified with MAN and TF (Note 3) 1. Add 5.22 μmol (1.0 mg) of EDCI and 12.51 μmol (1.44 mg) of NHS into 1 ml of the blank daunorubicin liposomes modified with MAN (COOH-PEG2000-DSPE: EDCI:NHS = 0.063:2.5:6.3, μmol/μmol). 2. Stir the above liposome suspensions for 10 min at room temperature using a magnetic stirrer. 3. Add 0.063 μmol (5 mg) of TF into the suspensions. 4. React for 3 h at room temperature using the same stirring. 5. Apply a Sephadex G-100 column equilibrated with PBS to remove the uncoupled TF in the liposome suspensions. 6. Collect liposome fractions from Sephadex G-100 column.
22
Preparation of Anthracyclines Liposomes for Tumor-Targeting Drug Delivery
COOH-PEG2000-DSPE
NH2-PEG2000-DSPE
375
p-aminophenyl--Dmanno-pyranoside (
)
Transferrin (
)
Lipid bilayer membrance Daunorubicin Daunorubicin liposomes
Dual-targeting daunorubicin liposomes
Fig. 1 Overall schematic representation for the preparation of daunorubicin liposomes modified with p-aminophenyl-α-D-manno-pyranoside (MAN) and transferrin (TF). (Adapted from reference Ying et al. (2010), with permission)
7. Determine the content of TF on the liposomes by BCA kit. 8. Load daunorubicin as item 3.1.2 to obtain daunorubicin liposomes modified with MAN and TF in Fig. 1.
3.1.5 Daunorubicin Liposomes Modified with TF (Note 3) 1. Add 0.063 μmol (5 mg) of TF into 1 ml of the blank liposomes. 2. React for 3 h at room temperature using the same stirring. 3. Apply a Sephadex G-100 column equilibrated with PBS for removing the uncoupled TF in the liposome suspensions. 4. Collect liposome fractions from Sephadex G-100 column. 5. Determine the content of TF on the liposomes by BCA kit. 6. Load daunorubicin as item 3.1.2 to obtain daunorubicin liposomes modified with TF. 3.1.6 Encapsulation Efficiency (Note 4) 1. Destroy the liposome suspensions by adding methanol to measure the content of daunorubicin. 2. Measure daunorubicin with ODS column at 1.0 ml/min of flow rate by HPLC system with UV detector. 3. Calculate the encapsulation efficiency of daunorubicin with the following formula: EE = (Wdialysis/Wtotal) 100%, where EE is the encapsulation efficiency of daunorubicin, Wdialysis is the measured amount of daunorubicin in the liposome suspensions after dialysis, and Wtotal is the measured amount of daunorubicin in the equal volume of liposome suspensions before dialysis. 3.1.7 Drug Release (Note 5) 1. Measure daunorubicin by HPLC system with UV detector after destroying through adding methanol. 2. Perform in vitro release ability of daunorubicin-loaded liposomes by the dialysis against the release medium containing serum protein. 3. Calculate the release rate.
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3.1.8 Particle Size (Note 6) 1. Detect the particle sizes and zeta potential values of daunorubicin formulations. 3.1.9 Determination of the Morphology Under TEM (Note 6) 1. Resuspend the liposomes in distilled water. 2. Filtered through a micropore filter membrane (0.2 μm). 3. Use copper grids coated with carbon films to absorb the liposome particles for 1 min in the liposome suspension. 4. Take out copper grids, and blot with filter paper. 5. Float these grids in 1% uranyl acetate solution for 1 min, remove, blot with filter paper, and dry at room temperature overnight. 6. Observe the specimens by TEM at 120 kV. 3.1.10 Determination of the Morphology Under AFM (Note 6) 1. Dilute the liposomes with distilled water. 2. Filter through a micropore filter membrane. 3. Spread a volume of 10 μl of the liposome suspension on a silicon slice. 4. Dry at room temperature. 5. Observe by AFM.
3.2
Cell Culture
1. Passage BMVECs in the DMEM(20% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mmol/L l-glutamine, 100 μg/ml endothelial cell growth factor (ECGF), 40 U/ml heparin). 2. Culture C6 glioma cells in F10 medium supplemented by 5% heat-inactivated fetal bovine serum (FBS), 15% heat-inactivated horse serum, and antibiotics (penicillin 100 U/ml and streptomycin 100 μg/ml). 3. Split the cells within 36 h after reaching full monolayer confluency in order to keep the cells in a healthy condition. 4. Maintain cells at 37 C in the presence of 5% CO2.
3.3
BBB Model In Vitro (Note 7)
1. Seed BMVECs on 2% gelatin-coated culture insert at a density of 37,500 cells/ insert and cultured 6 days. 2. Change the culture medium every 2 days. 3. Assess the tightness of the monolayer by measuring TEER using a TEER instrument. 4. View the tightness of the monolayer with scan electron microscope at instrumental magnification of 10,000-fold in Fig. 2. 5. Select HRP as an indicator to evaluate the permeability of the BBB model.
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377
Fig. 2 Morphology of the blood-brain barrier (BBB) model in vitro. Scan electron microscopic microphotograph showed that there was no aperture between the adjacent cells. (Adapted from reference Ying et al. (2010), with permission)
3.4
Transport Across the BBB and Competition Assay of MAN
1. Use DMEM as a transport medium. 2. Add excessive amount of MAN (0.74 μmol, 200 μg) into the apical compartment in advance for the competition assay. 3. Add daunorubicin formulations at a concentration of 8.87 μM (5 μg/ml daunorubicin) after 30 min. 4. Take a volume of 500 μl sample from the basolateral compartment at 0, 2, 4, 8, and 24 h. 5. Replace with 500 μl fresh DMEM immediately after each sampling. 6. Determine the samples by HPLC. 7. Calculate the transport ratio (%).
3.5
C6 Glioma Cellular Uptake Assay of TF (Note 8)
Seed C6 glioma cells into six-well plates and culture overnight at 37 C. Apply 8.87 μM (5 μg/ml daunorubicin) of daunorubicin formulations into cells. Use the drug-free F10 culture medium as a blank control. Culture for 2 h. Trypsinize cells and harvest. Determine the fluorescence intensities of daunorubicin in cells by FAScan flow cytometer. 7. Seed cells in chambered coverslips and treat with 8.87 μM (5 μg/ml daunorubicin) of daunorubicin liposomal formulations, respectively. 8. Fix cells with 4% (v/v) paraformaldehyde, and finally stain with Hoechst 33,258 after 2 h incubation.
1. 2. 3. 4. 5. 6.
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9. Observe the intracellular localization of daunorubicin in C6 glioma cells with laser scanning confocal microscope.
3.6
Competition Assay of TF
1. Seed cells into six-well plates and culture overnight at 37 C. 2. Incubate the C6 glioma cells with excessive amount of TF (0.013 μmol, 1000 μg) for 30 min in advance. 3. Add daunorubicin liposomes modified with TF and daunorubicin liposomes modified with MAN and TF into C6 glioma cells at a concentration of 8.87 μM (5 μg/ml daunorubicin), respectively. 4. Further incubate the cells for 2 h. 5. Determine the fluorescence intensities of daunorubicin in different formulations by FAScan flow cytometer.
3.7
Antiproliferative Activity Against C6 Glioma Cells (Note 9)
1. Seed C6 glioma cells into 96-well culture plates at a density of 2.5 103 cells/ well and grow at 37 C in the presence of 5% CO2 for 24 h. 2. Add free daunorubicin and daunorubicin liposomal formulations into 96-well culture plates. 3. Measure the cytotoxicity at 48 h later by SRB staining assay. 4. Read the absorbance on a microplate reader at 540 nm. 5. Calculate the survival percentages using the following formula: Survival % = (A540nm for the treated cells/A540 nm for the control cells) 100%, where A540 nm is the absorbance value. 6. Draw dose-effect curves. 7. Calculate IC50 values.
3.8
Dual-Targeting Effects In Vitro (Note 10)
1. Establish a BMVEC/C6 glioma co-culture model. 2. Add free daunorubicin and daunorubicin liposomal formulations into the apical compartment at a concentration of 8.87 μM (5 μg/ml daunorubicin). 3. Determine the survival percentage of C6 glioma cells in the basolateral compartment by the SRB staining assay after 48 h in Fig. 3.
3.9
Effect on the Avascular C6 Glioma Spheroids (Note 11)
1. Prepare agarose solution in serum-free F10 (2%, w/v) by heating at 80 C for 30 min.
Preparation of Anthracyclines Liposomes for Tumor-Targeting Drug Delivery
Inhibitory rate (%)
22
379
1. Free daunorubicin 2. Daunorubicin liposomes 3. Daunorubicin liposomes modified with MAN 4. Daunorubicin liposomes modified with TF 80 5. Daunorubicin liposomes modified with MAN and TF a,b c 60 40 20 0
1
2
3
4
5
Fig. 3 The dual-targeting effect: inhibitory rate of daunorubicin liposomes modified with MAN and TF against C6 glioma cells following transport across the BBB in vitro. Data are presented as the mean standard deviation (SD) (Adapted from reference Ying et al. (2010), with permission). a: P < 0.01, daunorubicin liposomes modified with MAN and TF versus free daunorubicin. b: P < 0.01, daunorubicin liposomes modified with MAN and TF versus daunorubicin liposomes. c: P < 0.01, daunorubicin liposomes modified with MAN versus daunorubicin liposomes modified with TF
2. Coat each well of 96-cell culture plates with a thin layer of this sterilized solution. 3. Seed C6 glioma cells at a density of 2000 cells/well. 4. Gently agitate the plates for 5 min on the first day. 5. Allow C6 glioma spheroids to grow for 24 h at 37 C in the presence of 5% CO2. 6. Incubate C6 glioma spheroids with serum-free F10 culture medium, free daunorubicin, daunorubicin liposomal formulations with 17.73 μM (10 μg/ml) of daunorubicin concentration. 7. Monitor growth inhibition by measuring the size of C6 glioma spheroids using an inverted microscope at days 0, 1, 2, 3, and 5, respectively. 8. Measure the major (dmax) and minor (dmin) diameters of each spheroid. 9. Calculate the spheroid volume using the following formula: V = (π dmax dmin)/6. 10. Estimate the C6 glioma spheroid volume ratio with the formula: R = (Vday i/Vday 0) 100%, where Vday i is the C6 glioma spheroid volume at the ith day after applying the drug and Vday 0 is the C6 glioma spheroid volume prior to administration.
3.10
Effects on the Survival of Brain Tumor-Bearing Animals (Note 12)
1. House male Sprague-Dawley rats under standard conditions with free access to food and water.
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2. Operate a sagittal incision through the skin to expose the cranium. 3. Stereotaxically implant approximate 5 105 C6 glioma cells/10 μl in serumfree F10 medium into the right forebrain of each rat. 4. Randomly divide the rats into six groups (nine rats per group) at day 8 after tumor inoculation. 5. Administer animals with physiological saline in blank control group. 6. Treat animals in other five groups with free daunorubicin, daunorubicin liposomal formulations via tail vein at a dose of 5 mg/kg daunorubicin. 7. Administrate drugs every 2 days with total three doses per rat. 8. Sacrifice three rats of each group for measuring the tumor size at day 14. 9. Apply other six rats for monitoring the survival curves. 10. Dissect and fix the brain tissues of the sacrificed rats in formalin. 11. Determine glioblastoma volume with a vernier caliper. 12. Estimate the tumor volume using the formula: Volume = length width height. 13. Calculate the tumor inhibitory rate with the formula: Rv = (1 Vdrug/ Vsaline) 100%, where Vdrug is the C6 glioma volume after treating with drug and Vsaline is the C6 glioma volume after treating with physiological saline. 14. Calculate the survival time from day 0 since tumor inoculation to the day of death. 15. Plot Kaplan-Meier survival curves for each group in Fig. 4.
3.11
Measurement of Daunorubicin in Plasma (Note 13)
1. Analyze the plasma samples by HPLC with an ultraviolet detector. 2. Dilute daunorubicin in blank plasma to a concentration between 0.25 and 20 μg/ ml, including 0.25, 0.5, 1.0, 2.5, 5.0, 10.0, and 20.0 μg/ml in the final sample solutions after processing. 3. Prepare the samples for recovery and precision measurements at 0.25, 5.0, and 20.0 μg/ml. 4. Add a 3.0 ml volume of methanol to the above mixture and mix for 1 min with a vortex. 5. Centrifuge the mixture at 10,000 rpm for 5 min. 6. Transfer the liquid phase to a clean tube and dry under a gentle N2 gas stream to obtain residues containing the drugs. 7. Reconstitute the drug residue with 100 μl mobile phase and centrifuge at 10,000 rpm for 5 min. 8. Inject a 20 μl volume of supernatant into the HPLC system. 9. Calculate concentrations of daunorubicin.
3.12
Measurement of Daunorubicin in Tissues (Note 14)
1. House male Kunming mice under standard conditions with free access to food and water.
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381
1. Physiological saline 2. Free daunorubicin 3. Daunorubicin liposomes 4. Daunorubicin liposomes modified with MAN 5. Daunorubicin liposomes modified with TF 6. Daunorubicin liposomes modified with MAN and TF 100
1
Survival (%)
80
2
60 3
40
4
20 0
5 6
10
14 18 Post innoculation days
6 22
26
Fig. 4 Kaplan-Meier survival curves of C6 glioma-bearing rats treated with daunorubicin liposomes modified with MAN and TF at days 8, 10, and 12 (1-week treatment) after inoculation. Results indicated that the daunorubicin liposomes modified with MAN and TF (22 days) significantly improved the survival rate of animals as compared with physiological saline (13 days, P = 0.001), free daunorubicin (17 days, P = 0.001), daunorubicin liposomes (18 days, P = 0.005), daunorubicin liposomes modified with MAN (19 days, P = 0.038), and daunorubicin liposomes modified with TF (18 days, P = 0.034), respectively. (Adapted from reference Ying et al. (2010), with permission)
2. Construct calibration curves by daunorubicin samples prepared in blank tissue homogenate. 3. Prepare the samples for recovery and precision measurements at 0.25, 5.0, and 20.0 μg/g. 4. Add 50 μl of a daunorubicin standard solution and 75 μl of the 20 μg/ml doxorubicin internal standard solution to 100 μl of blank tissue homogenate (0.1 g of tissue). 5. Mix for 30 s with a vortex. 6. Add a 3.0 ml volume of methanol to the above mixture and mix for 1 min with a vortex. 7. Centrifuge at 10,000 rpm for 5 min. 8. Transfer the liquid phase to a clean tube and dry under a gentle N2 gas stream. 9. Reconstitute the drug residue with 100 μl mobile phase and centrifuge at 10,000 rpm for 5 min. 10. Inject a 20 μl volume of supernatant into the HPLC system and detect the concentrations of daunorubicin.
3.13
Pharmacokinetics and Biodistribution (Note 15)
1. Divided Kunming mice into five groups (36 mice per group).
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2. Intravenously inject the drugs into the tail vein at a dose of 10 mg daunorubicin/ kg body weight. 3. Collect 1 ml volume of plasma in a heparinized microcentrifuge tube (six mice for each time point) after animal sacrifice by decapitation at the following time points: 0, 1, 3, 6, 24, and 48 h. 4. Separate the plasma by centrifugation at 10,000 rpm for 5 min. 5. Store at 20 C until detection. 6. Collect tissues, including heart, liver, spleen, lung, kidney, and brain at these time points. 7. Wash the tissues with physiological saline. 8. Dry with filter paper. 9. Weigh accurately and store at 20 C until analysis. 10. Detect the drug concentrations in the plasma and tissues under HPLC.
3.14
Statistical Analysis
1. Present data as the mean standard deviation (SD). 2. Apply one way analysis of variance (ANOVA) to determine significance among groups, after which post hoc tests with the Bonferroni correction were used for comparison between individual groups. 3. Present survival data as Kaplan-Meier plots and analyze with a log-rank test. 4. Consider a value of P < 0.05 to be significant. 5. Use the SOLVER software (Microsoft Office 2003, Excel) to calculate the pharmacokinetic parameters of daunorubicin administered as various formulations.
4
Notes
1. During the procedure of loading drugs, a volume of 8 ml blank liposome suspensions (18.6 mg lipid/ml) should be mixed with 10 mg of daunorubicin hydrochloride (daunorubicin hydrochloride: phospholipids = 1:15, w/w). 2. After completing the reaction, the absorbance of the final solution is read on ultraviolet-visible spectrophotometer at 320 nm against a blank prepared as above with 1 ml of water instead of the liposome suspensions. 3. A volume of 1 ml liposome suspensions is applied to a Sephadex G-100 column to remove the uncoupled TF. The coupling efficiency of TF should be defined to be the amount (μmol) of TF coupled on the liposome surface in 1 ml suspensions. 4. Mobile phase is consisted of acetonitrile, 0.02 M NaH2PO4, and triethylamine (34:66:0.3, v/v). The detected wavelength is set at 233 nm. All encapsulation efficiencies should be 92%. 5. A volume of 4 ml liposome suspensions in dialysis tubing is immersed in 20.0 ml of the release medium and oscillated with a shaker at a rate of
22
6. 7.
8. 9. 10.
11. 12.
13.
14.
15.
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Preparation of Anthracyclines Liposomes for Tumor-Targeting Drug Delivery
383
100 times per minute at 37 C. After each sampling, the same volume of fresh release medium must be immediately replaced with a volume of 0.5 ml release medium. The liposome suspension should be fresh and diluted enough before characterization using Nano Series Zen, TEM, and AFM. The cells must be mixed well when seeding cells into the wells. Cells would settle down to the bottom of the cell suspension tubes. In order to have consistent cell number added into the plates, it is essential to have cells uniformly suspended. The wavelength of daunorubicin emission is set at 560 nm, and FL2-H filter is in the range 564–606 nm for the collection of fluorescence intensity. Edge wells in plates must be not used in experiment since the cells in those wells usually grows slower than inner wells. C6 glioma cells should be seeded on the basolateral compartment of the insert at a density of 1000 cells/ compartment. After 5 days of incubation, the model can be used for the experiments. After seeding the C6 glioma cells, 96-well culture plates must be gently agitated for 5 min on the first day, which contributes to form the C6 glioma spheroids. Brain tumor-bearing animals should be constructed on the specific location (coordinates, 0.4 mm anterior and 3.0 mm lateral from the bregma, at a depth of 5.0 mm from the brain surface on rat’s head) where is inoculated with tumor cells. The mobile phase consists of methanol and water containing 5% glacial acetic acid (46: 54, v/v) with a flow rate of 1.0 ml/min under isocratic conditions. The detector is set at a wavelength of 254 nm. Doxorubicin hydrochloride is used as an internal standard. A 50 μl volume of daunorubicin standard solution and 50 μl of a 20 μg/ml doxorubicin internal standard solution are added to 100 μl blank plasma and mix for 30 s with a vortex. Daunorubicin samples are prepared in blank tissue homogenate (100 μl, containing 0.1 g of tissues) for calibration curves within the range of 0.25–20 μg daunorubicin/g wet tissue, including 0.25, 0.5, 1.0, 2.5, 5.0, 10.0, and 20.0 μg/g in the final samples after processing. A tissue sample should be homogenized with a homogenizer in physiological saline. Concentrations of daunorubicin in the heart, liver, spleen, lung, kidney, and brain are measured with the same HPLC method. The injection volume should be adjusted to 0.8 ml/100 g of mouse weight.
Conclusion
This protocol presents a kind of dual-targeting daunorubicin liposomes modified with p-aminophenyl-α-D-mannopyranoside (MAN) and transferrin (TF), which are designed to transport drug across the BBB, to enhance the targeting effect to brain tumor, and to reduce the side effect of anthracyclines. The liposomes are prepared by film dispersion method and ammonium sulfate gradient method. Encapsulation
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efficiency, drug release, particle size, and morphology of the liposomes are characterized. Results demonstrate that the liposomes are able to transport across the BBB and then target on brain glioma cells, thereby improving the therapeutic efficacy of brain glioma in vitro and in animals.
References Bouma J, Beijnen JH, Bult A, Underberg WJ (1986) Anthracycline antitumour agents. A review of physicochemical, analytical and stability properties. Pharm Weekbl Sci 8(2):109–133 Cortés-Funes H, Coronado C (2007) Role of anthracyclines in the era of targeted therapy. Cardiovasc Toxicol 7(2):56–60 Coune A (1988) Liposomes as drug delivery system in treatment of infectious diseases. Potential applications and clinical experience. Infection 16(3):141–147 Du J, Lu WL, Ying X, Liu Y, Du P, Tian W et al (2009) Dual-targeting topotecan liposomes modified with tamoxifen and wheat germ agglutinin significantly improve drug transport across the blood-brain barrier and survival of brain tumor-bearing animals. Mol Pharm 6(3):905–917 Forssen EA, Tökes ZA (1979) In vitro and in vivo studies with adriamycin liposomes. Biochem Biophys Res Commun 91(4):1295–1301 Huwyler J, Drewe J, Krähenbuhl S (2008) Tumor targeting using liposomal antineoplastic drugs. Int J Nanomedicine 3(1):21–29 Lotfi K, Zackrisson AL, Peterson C (2002) Comparison of idarubicin and daunorubicin regarding intracellular uptake, induction of apoptosis, and resistance. Cancer Lett 178(2):141–149 Lyman GH, Kuderer NM, Crawford J, Wolff DA, Culakova E, Poniewierski MS, Dale DC (2011) Predicting individual risk of neutropenic complications in patients receiving cancer chemotherapy. Cancer 117(9):1917–1927 McGowan JV, Chung R, Maulik A, Piotrowska I, Walker JM, Yellon DM (2017) Anthracycline chemotherapy and cardiotoxicity. Cardiovasc Drugs Ther 31(1):63–75 Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L (2004) Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev 56 (2):185–229 Moghimi SM, Patel HM (1992) Opsonophagocytosis of liposomes by peritoneal macrophages and bone marrow reticuloendothelial cells. Biochim Biophys Acta 1135(3):269–274 Peng X, Chen B, Lim CC, Sawyer DB (2005) The cardiotoxicology of anthracycline chemotherapeutics: translating molecular mechanism into preventative medicine. Mol Interv 5(3):163–171 Simeonova M, Ivanova G, Enchev V, Markova N, Kamburov M, Petkov C, Devery A, O'Connor R, Brougham D (2009) Physicochemical characterization and in vitro behavior of daunorubicinloaded poly(butylcyanoacrylate) nanoparticles. Acta Biomater 5(6):2109–2121 Simone EA, Dziubla TD, Muzykantov VR (2008) Polymeric carriers: role of geometry in drug delivery. Expert Opin Drug Deliv 5(12):1283–1300 Tila D, Ghasemi S, Yazdani-Arazi SN, Ghanbarzadeh S (2015) Functional liposomes in the cancertargeted drug delivery. J Biomater Appl 30(1):3–16 Weiss RB (1992) The anthracyclines: will we ever find a better doxorubicin? Semin Oncol 19(6):670–686 Ying X, Wen H, Lu WL, Du J, Guo J, Tian W, Men Y, Zhang Y, Li RJ, Yang TY, Shang DW, Lou JN, Zhang LR, Zhang Q (2010) Dual-targeting daunorubicin liposomes improve the therapeutic efficacy of brain glioma in animals. J Control Release 141(2):183–192 Ying X, Wen H, Yao HJ, Zhang Y, Tian W, Zhang L, Ju RJ, Wang XX, Yu Y, Lu WL (2011) Pharmacokinetics and tissue distribution of dual-targeting daunorubicin liposomes in mice. Pharmacology 87(1–2):105–114
Preparation and Characterization of pH Sensitive Drug Liposomes
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Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Synthesis of Zwitterionic Oligopeptide Lipid (e.g., 1,5-Dioctadecyl-L-Glutamyl 2-Histidyl-Hexahydrobenzoic Acid, HHG2C18) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Preparation of pH-Sensitive Drug Liposomes by Thin-Film Dispersion Methods (HHG2C18-L) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Preparation of Conventional Drug Liposomes by Thin-Film Dispersion Methods (SPC-L) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Determination of Particle Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Determination of Zeta Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Determination of the Buffering Capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7 Determination of the Degradation of Hexahydrobenzoic Amide from the HHG2C18-L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8 Determination of Zeta Potential Variation of the HHG2C18-L with the Degradation of the Amide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.10 Cellular Uptake of the HHG2C18-L Under Different pH Values . . . . . . . . . . . . . . . . . . . 2.11 Qualitatively Evaluation of Endolysosomal Escape and Mitochondrial Targeting of the HHG2C18-L by Confocal Laser Scanning Microscopy (CLSM) . . . . . . . . . . . . 3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Synthesis of Zwitterionic Oligopeptide Lipid (e.g., HHG2C18) . . . . . . . . . . . . . . . . . . . . 3.2 Preparation of pH-Sensitive Drug Liposomes by Thin-Film Dispersion Methods (HHG2C18-L) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Preparation of Conventional Drug Liposomes by Thin-Film Dispersion Methods (SPC-L) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Determination of Particle Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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C. Ju · C. Zhang (*) Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Center of Drug Discovery, Academic Institute of Pharmaceutical Science, China Pharmaceutical University, Nanjing, China e-mail: [email protected] © Springer-Verlag GmbH Germany, part of Springer Nature 2021 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49320-5_14
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3.5 3.6 3.7
Determination of Zeta Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Determination of the Buffering Capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Determination of the Degradation of Hexahydrobenzoic Amide from the HHG2C18-L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 Determination of Zeta Potential Variation of the HHG2C18-L with the Degradation of the Amide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.10 Cellular Uptake of the HHG2C18-L Under Different pH Values . . . . . . . . . . . . . . . . . . . 3.11 Qualitatively Evaluation of Endolysosomal Escape and Mitochondrial Targeting of the HHG2C18-L by CLSM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
There are multiple physiological and pathological barriers in cancer therapy faced by anticancer drug on the route from the injection site to the final antitumor target. In order to improve the targeting efficiency and antitumor efficacy, pH-sensitive nanocarriers have attracted much interest, owing to the abnormal pH values (pH 6.0–7.0) of tumor tissues compared with normal tissues and the stronger acidic endolysosomal lumens (pH 4.5–5.5). Recently, a novel multistage responsive drug liposome based on pH variation is prepared to vanquish the series of barriers in the whole-process delivery. The zwitterionic oligopeptide liposomes (HHG2C18-L) prepared by thin-film dispersion method contain a synthetic zwitterionic oligopeptide lipid (HHG2C18), soy phosphatidylcholine (SPC), and cholesterol. HHG2C18 is an intelligent lipid with two stearyl alkane chains as a hydrophobic block, and two amino acid groups (glutamic acid and histidine) and one pH-cleavable group (hexahydrobenzoic amide) as a hydrophilic block, which simulates natural phospholipids in the structure. In this chapter, besides the synthesis of HHG2C18 and the preparation of the HHG2C18-L, multistage pH response of HHG2C18-L to the mildly acidic tumor microenvironment and the acidic intracellular compartment successively is evaluated by the determination of particle size and zeta potential according to the pH change, buffering capacity, hydrolysis of hexahydrobenzoic amide, as well as the cellular uptake and intracellular delivery. Consequently, it has been found that this smart pH sensitive drug liposome has the ability to enhance tumor cellular uptake, improve cytosol distribution, and well target mitochondria, providing a meaningful nanoplatform for cancer therapy.
Keywords
pH sensitive liposome · Multistage responsive · Charge conversion · pH-cleavable hexahydrobenzoic amide · Mitochondrial targeting
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Introduction
Liposomes, lipid bilayer vesicles, have been referred as one of the most fantastic nanocarriers for widespread application in chemical drug and gene delivery, especially for the delivery of tumor-targeted drugs (Allen and Cullis 2013). In addition to the preclinical employment of liposomes for cancer therapy, it has been demonstrated that liposomes are beneficial for clinically deliver anticancer drugs due to the increase of antitumor effects and decrease of undesired side effects (Wibroe et al. 2016). For example, Doxil ®, a liposomal injection of doxorubicin hydrochloride (DOX HCL) approved by the US Food and Drug Administration (FDA), has the ability to prolong the retention time of DOX HCl and weaken the congestive heart failure compared with Doxorubicin Hydrochloride Injection (Barenholz 2012). The advantages of liposomes are basically on their characteristics including the protection of packaged anticancer drugs to improve the plasma stability and considerable retention of anticancer drugs in the blood, the nanoscale size to enlarge the accumulation of anticancer drugs in tumor compartments by the enhanced permeability and retention (EPR) effect (as passively target effect), and potential multi-functionalization to realize the active target effect and stimuli-responsive release (Deshpande et al. 2013). Although a myriad of well-fabricated liposomes have been prepared for cancer therapy, the liposomal-based delivery systems still face tremendous challenge in substantial increasing the drug accumulation in tumor tissue, a prerequisite of well anticancer efficacy (Kanamala et al. 2016). In other words, the anticancer efficacy of liposomes depends on their ability to reach the target sites of action. The unfortunate reality is that the delivery of liposomes from the injection site to the final antitumor targets should go through multiple physiological and pathological barriers, resulting in the limited therapeutically effective amount of drugs (Blanco et al. 2015). There might be four levels of barriers that liposomes face stepwise during the in vivo transport process. Firstly, it is the “organ level” that liposomes transport via blood to the tumor extracellular matrix. As it is known, the abundance of plasma protein would absorb onto the surface of liposomes by the specific or non-specific interaction after liposomes enter the systemic circulation (Tenzer et al. 2013). Once labeling by opsonin, the accelerated elimination of liposomes may occur by the reticuloendothelial system (RES) in liver, spleen, and lung (Nel et al. 2009). In addition, the liposomes may undergo the degradation of phospholipase, which would lead to the burst release of drug before reaching the target organs (Moghimi and Patel 1998). Secondly, the “tissue level” is referred to the transport from the tumor extracellular matrix to the tumor cell membrane that binding the cell membrane is essential for the internalization of liposomes. The pathological characteristics of tumor include leaky tumor vessels, (Maeda et al. 2013) dense extracellular matrix, (Zhang et al. 2016) high tumor interstitial pressure, (Heldin et al. 2004) acidic (Li et al. 2016) and hypoxic microenvironment, (Luo et al. 2016) which restrict the deep penetration and uniform distribution of liposomes in the tumor tissue, thus impeding the approach of
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liposomes to the cell membrane. The third level is “cellular level” which means the barriers of cellular uptake. The internalization of liposomes also plays an important role in the antitumor efficacy since most targets of drugs are within the cells. The particle size and zeta potential of liposomes might influence the uptake of liposomes by tumor cells (Nel et al. 2009; Black et al. 2014). The intracellular delivery referred as “subcellular level” is the eventual barrier of liposomes reaching the action site. Clathrin-mediated endocytosis is the major pathway of nanoparticles to enter the cells, resulting in the subsequent location in endolysosomes which is able to degrade the loaded drug of liposomes (Banerjee et al. 2016; Le Roy and Wrana 2005). To avoid this undesired degradation and improve the drug effect, it is necessary for the liposomes to escape from the endolysosomes into the cytoplasm and subsequently arrive at the cell organelles where the drugs work. In a word, these physiological and pathological barriers greatly cut down the therapeutically effective amount of drugs, which means strategies overcoming these barriers are highly desired. In order to improve the anticancer efficacy of liposomes, several passive targeting, (Cabral et al. 2011) active targeting methods, (Cheng et al. 2012) as well as stimulus-responsive targeting methods (Karimi et al. 2016) have been developed (Deshpande et al. 2013). For example, PEGylation on the surface of liposomes has been demonstrated as the most efficient method to sustain the drug retention time in circulation system, thus increasing the drug accumulation in tumor tissues through passive targeting (Barenholz 2012; Bunker et al. 2016). The active targeting methods involve the introduction of antibody or ligand to the surface of liposomes, which further boost the arrival of liposomes to the tumor apartment (Veneti et al. 2016; Arranja et al. 2016). In addition, with the purpose of improving the specific action of antitumor drugs against tumor cells, a large number of tumor microenvironmental stimulus-responsive strategies have been under study, including pH, temperature, enzymes, redox, light, magnetic field, and so on (Karimi et al. 2016; Torchilin 2014). Among these stimulus-responsive systems, pH-sensitive liposomes have attracted much interest for two quite different reasons (Kanamala et al. 2016). One is that mildly acidic tumor microenvironment (pH 6.0–7.0) (Cardone et al. 2005) due to the glycolytic metabolism of glucose to lactate in tumor tissues may be exploitable for selective targeting of tumors relative to normal tissues (pH 7.4), complementing and extending the selectivity achievable by the EPR effect. Of note, the basic prerequisites of EPR for liposomes to have pharmaceutical applications are the safety and stability in the blood resulting from the surface chemistry of liposomes, such as surface charge. As is well known, the positively charged liposomes may lead to severe cytotoxicity, rapid clearance from the plasma compartment, and instability with opsonin (Ozpolat et al. 2014). However, liposomes with positive charge are liable cellular uptake on account of the binding through electrostatic interaction with negatively charged cell membrane (Chen et al. 2011). In an attempt to improve the retention of nanoparticles, much research has focused on nanoparticles functionalized to change their surface charge from neutral/negative to positive when triggered by the tumor extracellular pH, for enhanced tumor cellular uptake (Mo et al. 2013). The other reason is that endosomes into which most of nanocarriers
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are incorporated via endocytosis develop markedly acidified lumens (pH 4.5–5.5). The endolysomal entrapment of liposomes can be intracellular barrier that may significantly reduce efficacy, as a result of the degradation of liposomes and their cargo through endolysosomal acid and hydrolase. For this reason, endolysomal pH sensitive liposomes have been formulated for endolysosomal escape and payload release. These designs include nanovectors with (a) pH-cleavable linkers bonded to drugs (Aryal et al. 2010); (b) pH-induced charge conversion (Ju et al. 2014; Lee et al. 2008; Yuan et al. 2012); (c) pH stimulant membrane fusion and disruption (Obata et al. 2010); (d) pH-triggered swelling (Ke et al. 2011); and (e) chemical agents for endosomal escape (Yu et al. 2011). Besides, one other thing to note is that cytoplasmic pH values (pH 7.2–8.0) are higher than the endolysosomal pH values (pH 4.5–5.5). When liposomes with pH-induced charge conversion escaped from the endolysosomes into the cytosol, a charge reconversion of liposomes from positive to negative takes place, resulting in the loss of subcellular targeting, including nuclear and mitochondrial targeting, which requires highly positive charge. Despite these extraordinary advances of pH sensitive liposomes, the majority of previous reports have only paid attention to overcome one or two barriers, such as charge conversion or endolysosomal escape. Consequently, an easy-to-fabricate liposome that can break down successive physiological and pathological barriers from blood to organelles for anticancer drug delivery is still very much in demand. It has recently reported a novel multistage pH sensitive drug liposome referred as zwitterionic oligopeptide liposomes with mitochondrial targeting to vanquish the series of barriers mentioned above in the whole-process delivery (Mo et al. 2012). To prepare the zwitterionic oligopeptide liposomes, the zwitterionic oligopeptide lipid (HHG2C18) was firstly synthesized using esterification reaction and acylation reaction. HHG2C18 is a smart lipid with two amino acid groups (glutamic acid and histidine) and one pH-cleavable group (hexahydrobenzoic amide) (Xu et al. 2007) as a hydrophilic block, and two stearyl alkane chains as a hydrophobic block, which simulates natural phospholipids in the structure. The histidine groups endow the oligopeptide lipid with proton sponge effect and the potential of charge reversion, while the hexahydrobenzoic amide has the ability to respond to the lower pH values followed by the exposure of histidine group to enhance the positive charge. Afterwards, a thin-film dispersion method was applied to prepare the zwitterionic oligopeptide liposomes (HHG2C18-L). The optimized formulation of HHG2C18-L consisted of HHG2C18, the synthetic zwitterionic oligopeptide lipid, soy phosphatidylcholine (SPC), and cholesterol. To study the capacity of HHG2C18-L breaking down successive physiological and pathological barriers from blood to organelles, the multistage pH response of HHG2C18-L to the mildly acidic tumor microenvironment, and the acidic intracellular compartment successively was set as an evaluation criterion. The surface charge of HHG2C18-L in the blood circulation is strongly negative to pursue the safety and stability for potential pharmaceutical applications. When HHG2C18-L extravasates from the blood into tumor tissue by means of the EPR effect, the first-stage pH response happens on account of the mildly acidic environment in tumor tissues. The surface charge of HHG2C18-L reverses to positive and increases tumor cellular uptake due to the electrostatic
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attraction to the negatively charged tumor plasmalemma. Accompanied by endocytosis into the endosomes and lysosomes, the second-stage pH response occurs since the strongly acidic environment of endolysosomes, in which the imidazole group of histidine in HHG2C18 facilitates proton influx (proton sponge effect) to endolysosomes, leading to endolysosomal bursting. The third-stage pH response takes place in the endolysosomes based on the intracellular compartmental acidity, as well in which the degradation of pH-cleavable hexahydrobenzoic amide gives rise to a higher positive surface charge of HHG2C18 through the loss of carboxy groups in HHG2C18, impeding a charge reconversion from positive to negative in the back of escaping to the cytoplasm as the elevated pH values. Ultimately, the strong positive charge of HHG2C18-L promotes the accumulation of HHG2C18-L at the mitochondria by electrostatic interaction for mitochondrial targeting. Hence, the intelligent pH sensitive drug liposome has the ability to maintain the plasma stability, enhance effective entry of the tumor cells, improve escape from the endolysosomes, and well accumulate at the mitochondria. It suggests that this strategy will provide a safe and meaningful carrier platform for cascade mitochondrial drug delivery and bring the opportunities to explore more intelligent drug delivery systems for efficient cancer therapy. In this chapter, the synthesis of HHG2C18, the preparation of HHG2C18-L, and the evaluation of multistage pH response of HHG2C18-L including particle size and zeta potential according to the pH change, buffering capacity, as well as the hydrolysis of hexahydrobenzoic amides are shown. Furthermore, the cellular uptake of liposomes under the different pH values, the endolysosomes escape, and the mitochondrial targeting are involved in this chapter as well.
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Materials
2.1
Synthesis of Zwitterionic Oligopeptide Lipid (e.g., 1,5-Dioctadecyl-L-Glutamyl 2-Histidyl-Hexahydrobenzoic Acid, HHG2C18)
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
A 500-mL round-bottomed spherical flask A 250-mL round-bottomed spherical flask A rotary evaporator, water bath L-Glutamic acid (Glu) Aliphatic chain (e.g., stearyl alcohol) p-toluene sulfonic acid (p-Tos) Methylbenzene Dichloromethane (DCM) 5% (w:v) sodium bicarbonate solution Sodium chloride saturated solution Anhydrous sodium sulfate Methanol Triethylamine (TEA)
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14. N-tert-butoxycarbonyl-N-(imidazole)-(4-toluenesulfonyl)-L-histidine (Boc-LHis(Tos)) 15. N, N-dicyclohexylcarbodiimide (DCC) 16. N-hydroxysuccinimide (NHS) 17. N, N-dimethylformamide (DMF) 18. Distilled water 19. Trifluoroacetic acid (TFA) 20. Hexahydrophthalic anhydride (HHPA) 21. Chloroform (CHCl3) 22. Deprotection solution for Tos group: 0.41 M 1-Hydroxybenzotrizole (HOBt) in tetrahydrofuran (THF) 23. Acetone
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Preparation of pH-Sensitive Drug Liposomes by Thin-Film Dispersion Methods (HHG2C18-L)
1. Lipids: soy phosphatidylcholine (SPC), HHG2C18 and cholesterol (7.5:2.5:1, w: w:w) 2. Model drug (e.g., CCI-779) 3. A 50-mL round-bottomed spherical flask 4. Organic solvents: mixture of chloroform and methanol (2:1, v:v) 5. A rotary evaporator, water bath 6. Vacuum desiccators to remove any traces of remaining solvents from the dried film 7. Milli-Q ® water 8. Ultrasonic cell disruptor 9. 0.22 μm polycarbonate membrane filters 10. Sephadex G-50 column
2.3 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Preparation of Conventional Drug Liposomes by Thin-Film Dispersion Methods (SPC-L) Lipids: SPC and cholesterol (10:1, w:w) Model drug (e.g., CCI-779) A 50-mL round-bottomed spherical flask Organic solvents: mixture of chloroform and methanol (2:1, v:v) A rotary evaporator, water bath Vacuum desiccators to remove any traces of remaining solvents from the dried film Milli-Q ® water Ultrasonic cell disruptor 0.22 μm polycarbonate membrane filters Sephadex G-50 column
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Determination of Particle Size
1. Dilution of liposome dispersion to 3 mL with 20 mM Hepes buffer (pH 7.4) or 20 mM acetate buffer (pH 4.5, 5.5, 6.5) 2. A Dynamic Light Scattering Analyzer
2.5
Determination of Zeta Potential
1. Dilution of liposome dispersion to 3 mL with 20 mM Hepes buffer (pH 7.4) or 20 mM acetate buffer (pH 4.5, 5.5, 6.5) 2. A ZetaPlus Zeta Potential Analyzer
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Determination of the Buffering Capacity
1. A 20 mL of each liposome (the total lipid concentration of 2.64 mg/mL) adjusted to pH 10 with 0.3 M NaOH 2. Titrant: 0.01 M or 0.05 M HCl 3. A pH meter
2.7 1. 2. 3. 4. 5. 6.
Determination of the Degradation of Hexahydrobenzoic Amide from the HHG2C18-L
Fluorescamine acetone solution (2 mg/mL) 20 mM Hepes buffer (pH 7.4) 20 mM acetate buffer (pH 6.5, 5.5, 4.5) Methanol 20 mM acetic acid (HAc) A fluorospectrophotometer
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Determination of Zeta Potential Variation of the HHG2C18-L with the Degradation of the Amide
1. 20 mM Hepes buffer (pH 7.4) 2. 20 mM acetate buffer (pH 6.5, 5.5, 4.5) 3. A ZetaPlus Zeta Potential Analyzer
2.9
Cell Culture
1. Human renal carcinoma (A498) cells 2. Human lung carcinoma (A549) cells
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Human breast carcinoma (MCF-7) cells RPMI-1640 medium (1640) Dulbecco modified eagle medium (DMEM) 0.25% (w:v) trypsin Fetal bovine serum (FBS) Penicillin-streptomycin solution Phosphate buffered saline (PBS) Cell incubator
2.10 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Cellular Uptake of the HHG2C18-L Under Different pH Values
24-well plates Human renal carcinoma (A498) cells Human lung carcinoma (A549) cells Human breast carcinoma (MCF-7) cells Coumarin 6-loaded HHG2C18-L (C6/HHG2C18-L, equal to 100 ng/mL concentration of C6) C6-loaded SPC-L (C6/SPC-L, equal to 100 ng/mL concentration of C6) RPMI-1640 medium (1640) Dulbecco modified eagle medium (DMEM) PBS High performance liquid chromatography (HPLC) Pierce BCA Protein Assay Kit
2.11
1. 2. 3. 4. 5. 6. 7.
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35-mm TC-Treated Culture Dish (35 mm) C6/HHG2C18-L (equal to 100 ng/mL concentration of C6) C6/SPC-L (equal to 100 ng/mL concentration of C6) 50 nM LysoTracker Red 200 nM MitoTracker Red PBS CLSM
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Methods
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Synthesis of Zwitterionic Oligopeptide Lipid (e.g., HHG2C18)
The zwitterionic oligopeptide lipid (HHG2C18) was synthesized as shown in Fig. 1.
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Fig. 1 Synthesis of the zwitterionic oligopeptide lipid (1,5-dioctadecyl-L-glutamyl 2-histidylhexahydrobenzoic acid, HHG2C18)
1. Place 11.8 g (80.2 mmol) of L-Glutamic acid and 18.3 g (96.2 mmol) of p-Tos into a 500-mL round-bottomed spherical flask and dissolve them in 350 mL of methylbenzene. 2. Reflux the solution (Step 1) for 1 h at 110 C. 3. Add 47.8 g (176.7 mmol) of stearyl alcohol to the solution in step 2, followed by stirring for 12 h under reflux at 110 C (see Note 1). 4. Evaporate the reaction mixture in step 3 with vacuum distillation to remove methylbenzene. 5. Dissolve the concentrated solution (Step 4) in 150 mL of DCM. 6. Wash the DCM solution in step 5 with 100 mL of 5% (w:v) sodium bicarbonate solution twice and with 100 mL of distilled water successively. 7. Dry the organic layer in step 6 with anhydrous sodium sulfate. 8. Evaporate the organic layer (Step 7) with rotary evaporation to obtain a 1,5dioctadecyl-L-glutamate (G2C18) concentrated solution. 9. Recrystallize the G2C18 (Step 8) from 100 mL of methanol and obtain a white powder with a yield of 55.4%. Analyze the purified G2C18 using 1H nuclear magnetic resonance spectroscopy (1H NMR) and mass spectrometry (MS). Show the data in step 27. 10. Place 11.0 g (16.9 mmol) of G2C18 and 5.1 g (50.4 mmol) of TEA into a 500mL round-bottomed spherical flask and dissolve them in 300 mL of DMF with stirring for 1 h at room temperature. 11. Place 6.9 g (16.8 mmol) of Boc-L-His(Tos), 10.3 g (49.9 mmol) of DCC, and 2.9 g (25.2 mmol) of NHS into a 250-mL round-bottomed spherical flask and dissolve them in 100 mL of DMF with stirring for 3 h at room temperature.
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12. Mix the two solutions in step 10 and step 11 with stirring for 12 h at room temperature. 13. Transfer the reaction mixture (Step 12) into 500 mL of distilled water and filtrate. 14. Dissolve the filter residue (Step 13) in 300 mL of DCM and further filtrate. 15. Collect and evaporate the filter liquor (Step 14) with rotary evaporation to obtain an amino group-protected intermediate ((Boc)H(Tos)G2C18) concentrated solution. 16. Recrystallize (Boc)H(Tos)G2C18 from 180 mL of methanol and obtain a white powder with a yield of 50.8%. Analyze the purified (Boc)H(Tos)G2C18 using 1H NMR and MS. Show the data in step 27. 17. Dissolve the obtained (Boc)H(Tos)G2C18 in 15 mL of DCM, followed by the addition of 15 mL of TFA, and react for 4 h at room temperature to remove the protected Boc group (see Note 2). 18. Adjust the pH value of the reaction solution in step 17 to neutral with 5% (w:v) sodium bicarbonate solution. 19. Collect the organic layer from the solution in step 18 and dry with anhydrous sodium sulfate. 20. Evaporate the organic layer (Step 19) with rotary evaporation to obtain a faint yellow powder (H(Tos)G2C18) with a yield of 47.3%. Analyze the purified H (Tos)G2C18 using 1H NMR and MS. Show the data in step 27. 21. Dissolve 2.0 g (2.1 mmol) of H(Tos)G2C18, 1.64 g (10.6 mmol) of HHPA and 0.43 g (4.2 mmol) of TEA in 30 mL of CHCl3 and stir for 15 h at room temperature. 22. Wash the reaction mixture in step 21 with 10 mL of 5% (w:v) sodium chloride saturated solution twice and 10 mL of distilled water twice successively. 23. Dry the organic layer obtained in step 22 with anhydrous sodium sulfate, followed by the rotary evaporation to get an amino group-protected intermediate (HH(Tos)G2C18) concentrated solution. 24. Recrystallize HH(Tos)G2C18 from 70 mL of acetone and obtain a white powder with a yield of 64.4%. 25. Dissolve 1.5 g (1.4 mmol) of HH(Tos)G2C18 in 40 mL of THF with 2.2 g (16.4 mmol) of HOBt by stirring for 5 h at room temperature to remove the protected Tos group. 26. Purify the HHG2C18 solution by chromatography separation with the eluent of DCM/ methanol (15:1, v:v) and evaporate with the rotary evaporation to obtain a white powder with a yield of 5.1%. Analyze the purified HHG2C18 using 1H NMR and MS. Show the data in step 27. 27. G2C18: 1H NMR (CDCl3, 300 MHz, δ ppm): 0.88 (t, 6H, CH2CH3), 1.26–1.55 (m, 60H, CH2 (octadecyl)), 2.17 (m, 2H, NH2CHCH2), 2.45 (t, 2H, CH2CO), 3.99 (t, 1H, NH2CH), 4.05 (t, 4H, COOCH2); HRMS (ESI) m/z: [M þ H]+ calcd for C33H65NO4, 651.62; found, 651.6233. (Boc)H(Tos)G2C18: 1H NMR (CDCl3, 300 MHz, δ ppm): 0.88 (t, 6H, CH2CH3), 1.26–1.55 (m, 60H, CH2 (octadecyl)), 1.43 (s, 9H, CH3(Boc)), 2.17 (m, 2H, NH2CHCH2), 2.45 (t, 2H, CH2CO), 2.43 (s, 3H, CH3(Tos)), 2.87–3.06 (q, 2H, NHCHCH2), 4.04–4.07 (t, 4H, COOCH2), 4.48 (q, 1H, NHCH), 4.52 (q, 1H, NHCH), 7.08 (d, 1H, CH2C = CH), 7.33–7.35 (d, 2H, CH = CH), 7.7–7.81(d, 2H, CH = CH), 7.91 (d, 1H, N = CH); HRMS (ESI) m/ z: [M þ H]+ calcd for C59H102N4O9S, 1042.74; found, 1042.74.
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H(Tos)G2C18: 1H NMR (CDCl3, 300 MHz, δ ppm): 0.88 (t, 6H, CH2CH3), 1.26–1.55 (m, 60H, CH2 (octadecyl)), 2.17 (m, 2H, NH2CHCH2), 2.45 (t, 2H, CH2CO), 2.43 (s, 3H, CH3(Tos)), 2.87–3.06 (q, 2H, NHCHCH2), 4.04–4.07 (t, 4H, COOCH2), 4.48 (q, 1H, NHCH), 4.52 (q, 1H, NHCH), 7.08 (d, 1H, CH2C = CH), 7.33–7.35 (d, 2H, CH = CH), 7.7–7.81 (d, 2H, CH = CH), 7.91 (d, 1H, N = CH); HRMS (ESI) m/z: [M þ H]+ calcd for C54H94N4O7S, 942.69; found, 942.69. HHG2C18: 1H NMR (CDCl3, 500 MHz, δ ppm): 0.85–0.89 (t, 6H, CH2CH3), 1.25–1.60 (m, 64H, CH2 (octadecyl)), 1.77–3.18 (m, 10H, CH2 (HHPA)), 2.03 (m, 2H, NHCHCH2), 2.18(m, 2H, NHCHCH2CH2), 3.02–3.18 (q, 2H, NHCHCH2), 4.02–4.12 (t, 4H, COOCH2), 4.49 (q, 1H, NHCH), 4.94 (q, 1H, NHCH), 7.87 (d, 2H,CH2C = CH, N = CH); HRMS (ESI) m/z: [M þ H]+ calcd for C55H98N4O8, 942.74; found, 942.7497.
3.2
Preparation of pH-Sensitive Drug Liposomes by Thin-Film Dispersion Methods (HHG2C18-L)
1. Dissolve all lipids (SPC, HHG2C18, and cholesterol (7.5:2.5:1, w:w:w)) in 5 mL of the mixture of chloroform and methanol (2:1, v:v) in a 50-mL round-bottomed spherical flask (see Note 3). 2. Add CCI-779 (2.5% (w:w) of total lipids weight) to the lipids solution in step1. 3. Dry the solution (Step 3) with the rotary evaporation at 40 C and form a thin lipid film. 4. Further desiccate the thin lipid film under vacuum overnight. 5. Rehydrate the thin lipid film in 5 mL of Milli-Q ® water with the rotary evaporation for 30 min at 37 C (see Note 4). 6. Sonicate the rehydrated solution with an ultrasonic cell disruptor at 10% ultrasonic intensity for 15 min on ice bath (see Note 5). 7. Extrude the liposome suspension through polycarbonate membrane filters with a pore size of 0.22 μm. 8. Place 0.2 mL of CCI-779/HHG2C18-L on the top of Sephadex G-50 column, and elute with Milli-Q ® water at the speed of 1 mL/min. Collect the eluent containing the CCI-779/HHG2C18-L and determinate the amount of drugs. 9. Calculate the encapsulation efficiencies (EE) by the following formula: EE = W/W0 100%, where W and W0 are the amount of drugs in the liposomes after and before passing over Sephadex G-50 column. The EE of CCI-779 in HHG2C18-L was approximately 96.75%.
3.3
Preparation of Conventional Drug Liposomes by Thin-Film Dispersion Methods (SPC-L)
The preparation method of SPC-L was the same as the preparation method of HHG2C18-L except the lipids employed. And SPC-L was prepared as a control.
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1. Dissolve all lipids (SPC and cholesterol (10:1, w:w)) in 5 mL of the mixture of chloroform and methanol (2:1, v:v) in a 50-mL round-bottomed spherical flask (see Note 6). 2. Add CCI-779 (2.5% (w:w) of total lipids weight) to the lipids solution in step1. 3. Dry the solution (Step 2) with the rotary evaporation at 40 C and form a thin lipid film. 4. Further desiccate the thin lipid film under vacuum overnight. 5. Rehydrate the thin lipid film in 5 mL of Milli-Q ® water with the rotary evaporation for 30 min at 37 C (see Note 4). 6. Sonicate the rehydrated solution with an ultrasonic cell disruptor at 10% ultrasonic intensity for 15 min on ice bath (see Note 5). 7. Extrude the liposome suspension through polycarbonate membrane filters with a pore size of 0.22 μm. 8. Place 0.2 mL of CCI-779/SPC-L on the top of Sephadex G-50 column, and elute with Milli-Q ® water at the speed of 1 mL/min. Collect the eluent containing the CCI-779/SPC-L and determinate the amount of drugs. 9. Calculate the encapsulation efficiencies (EE) by the following formula: EE = W/ W0 100%, where W and W0 are the amount of drugs in the liposomes after and before passing over Sephadex G-50 column. The EE of CCI-779 in SPC-L was approximately 95.24%.
3.4
Determination of Particle Size
1. Dilute 200 μL of CCI-779/HHG2C18-L or CCI-779/SPC-L in 3 mL of 20 mM Hepes buffer (pH 7.4) or 20 mM acetate buffer (pH 4.5, 5.5, 6.5). 2. Measure the mean particle diameters of the diluted liposomes by a dynamic light scattering analyzer (see Note 7). Figure 2 presents the mean particle diameters of CCI-779/HHG2C18-L and CCI-779/SPC-L in different dilute solutions.
3.5
Determination of Zeta Potential
To demonstrate the first-stage pH response of HHG2C18-L to the tumor extracellular acidity, the zeta potentials of HHG2C18-L dispersed in the buffer solutions at various pH values (pH 7.4, 6.5, 5.5, 4.5) were measured. The pH values of 7.4, 6.5, 5.5, and 4.5 simulated that of the physiological environment, tumor microenvironment, endosomal, and lysosomal compartments, respectively. 1. Dilute 150 μL of CCI-779/HHG2C18-L or CCI-779/SPC-L in 3 mL of 20 mM Hepes buffer (pH 7.4) or 20 mM acetate buffer (pH 4.5, 5.5, 6.5). 2. Measure the zeta potentials of the diluted liposomes at 37 C by a zetaplus zeta potential analyzer (see Note 8). Figure 3 presents the zeta potentials of CCI-779/ HHG2C18-L and CCI-779/SPC-L at different pH values.
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Fig. 2 Particle sizes of CCI779/SPC-L and CCI-779/ HHG2C18-L at different pH values
Fig. 3 Zeta potentials of CCI-779/SPC-L and CCI779/HHG2C18-L at different pH values
3.6
Determination of the Buffering Capacity
To demonstrate the second-stage pH response of HHG2C18-L to the endosomal and lysosomal acidity, the buffering capacity of HHG2C18-L reflecting the effect of HHG2C18-L on endolysosomal escape was measured. 1. Adjust the pH value of HHG2C18-L or SPC-L (20 mL) with the total lipid concentration of 2.64 mg/mL to pH 10 with 0.3 M NaOH. (The concentration of HHG2C18 in HHG2C18-L was about 0.6 mg/mL). 2. Add aliquots of 0.01 M or 0.05 M HCl into the liposomes (Step 1) sequentially to reduce the pH value from 10 to 3.5. 3. Measure the pH values of the solutions after each addition of HCl with a pH meter. 4. Draw the curve of pH value versus the amount of HCl as shown in Fig. 4, indicating the intrinsic buffering capacity of HHG2C18-L (see Note 9).
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Fig. 4 Acid titration profiles of aqueous solutions of SPC-L and HHG2C18-L. Solutions of each liposome were adjusted to pH 10 using 0.3 M NaOH and then titrated with 0.05 M or 0.01 M HCl
3.7
Determination of the Degradation of Hexahydrobenzoic Amide from the HHG2C18-L
To avoid the surface change reconversion from positive to negative due to the HHG2C18-L escaping from endolysosomes (pH 4.5–5.5) to the cytoplasm (pH 7.4), causing loss of interactions with the mitochondria, an acid-labile β-carboxylic acid amide presented pH-dependent hydrolysis was introduced to HHG2C18. The degradation of hexahydribenzoic amide was measured to demonstrate the third-stage pH response of HHG2C18-L by the fluorescamine method. Fluorescamine, an amine-reactive fluorescent dyn, reacts instantaneously with primary amines to bring about high fluorescence. In contrast, fluorescamine itself has no fluorescence. After incubating HHG2C18-L with the buffer solutions at the different pH values (pH 7.4, 6.5, 5.5, 4.5), the fluorescence intensity through fluorescamine reaction indicates the degrading level of hexahydribenzoic amide at different pH values, since the primary amine of histidine group was exposed after the degradation of hexahydribenzoic amide in HHG2C18. 1. Mix 1 mL of HHG2C18-L with HHG2C18 concentration of 5 mg/mL with 2 mL of 20 mM Hepes buffer (pH 7.4) or 20 mM acetate buffer (pH 6.5, 5.5, 4.5). 2. Incubate the solutions in step 1 at 37 C for specified time (0, 2, 4, 6, 8, 12, 24 h). 3. Withdrawn 150 μL of each sample (Step 2) and dilute them into 3 mL of methanol. 4. Add 100 μL of 2 mg/mL fluorescamine acetone solution into each sample in step3. 5. Incubate each sample (Step 4) in dark at room temperature for 10 min. 6. Mix 1 mL of HHG2C18-L with HHG2C18 concentration of 5 mg/mL with 2 mL of 20 mM HAc for 48 h as 100% of exposed amine. And repeat step 3, step 4, and step 5.
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7. Assay the fluorescence intensity of each sample at the excitation wavelength of 382 nm and the emission wavelength of 474 nm by fluorospectrophotometer. Employ the blank buffer solution as a negative control and 100% of exposed amine as a positive control (see Note 10). Figure 5 shows the data of the degradation of hexahydrobenzoic amide from the HHG2C18-L at different pH values.
3.8
Determination of Zeta Potential Variation of the HHG2C18-L with the Degradation of the Amide
To further confirm the removal of carboxy groups from HHG2C18, zeta potential variation of the HHG2C18-L after incubation with buffer solutions was measured at different pH values (pH 7.4, 6.5, 5.5, 4.5). 1. Mix 1 mL of HHG2C18-L with HHG2C18 concentration of 5 mg/mL with 2 mL of 20 mM Hepes buffer (pH 7.4) or 20 mM acetate buffer (pH 6.5, 5.5, 4.5). 2. Incubate the solutions in step 1 at 37 C for specified time (0, 2, 4, 6, 8, 12, 24 h). 3. Withdrawn 300 μL of each sample (Step 2) and dilute them into 2 mL of the buffer solution at the same pH value. 4. Measure the zeta potentials of the resulting samples in step 3 at 37 C by a zetaplus zeta potential analyzer (see Note 11). Figure 6 presents the zeta potential variation of the HHG2C18-L accompanying the degradation of the amide at different pH values.
3.9
Cell Culture
1. Incubate A498, A549, or MCF-7 cells in the 1640 or DMEM medium with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin, respectively. Fig. 5 Degradation of the hexahydrobenzoic amide in HHG2C18-L at different pH values
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Fig. 6 Zeta potential variation of HHG2C18-L accompanying the degradation of the hexahydrobenzoic amide at different pH values
2. Put these cells in an incubator with 5% of CO2 atmosphere and 90% of relative humidity at 37 C. 3. Subculture the cells after approximately 80% confluence of cells. 4. Remove the culture medium and wash the cells with 4 C PBS thrice. 5. Add 1 mL of 0.25% (w:v) trypsin to the culture dish and incubate the trypsin with cells for 1–2 min (Note 12). 6. Add another 5 mL of fresh medium with 10% FBS to terminate the effect of trypsin. 7. Dissociate and collect the cells using the centrifugal at 1000 rpm for 3 min. 8. Dissociate and count the collected cells, followed by seeding in plates or confocal microscopy dishes as required.
3.10
Cellular Uptake of the HHG2C18-L Under Different pH Values
To identify whether the pH-triggered charge conversion of HHG2C18-L was beneficial for the internalization of liposomes, the cellular uptake of the HHG2C18-L was operated at pH 7.4 or pH 6.5 simulating the normal or tumoral extracellular pH values, respectively. In addition, C6 was chosen as a fluorescent probe due to the hydrophobic property, nonspecific mitochondrial targeting, and the fluorescent visuality of C6. The amount of C6 was analyzed by high performance liquid chromatography (HPLC) that the detecting parameters of C6 were not shown in this chapter. 1. Seed A498, A549, or MCF-7 cells at a density of 1 105 cells per well in 24 well plates and culture these cells until 90% confluence of cells. 2. Dilute C6/HHG2C18-L and C6/SPC-L with FBS free culture medium (pH 7.4 or pH 6.5) to a final concentration as 100 ng/mL of C6, respectively. 3. Add 400 μL of C6/HHG2C18-L or C6/SPC-L in the wells and incubate the solutions with cells at 37 C for 2 h.
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4. Remove the culture medium and wash the cells with 4 C PBS thrice. 5. Assay the uptake amount of C6 by HPLC and BCA protein assay kit. 6. Calculate the uptake of C6 (ng/mg) as the following equation, UptakeC6 = QC6/ Qcells protein, where QC6 was the amount of C6 obtained by HPLC and Qcells protein was the amount of cells protein obtained by BCA protein assay kit (Note 13). Figure 7 presents the cellular uptake of C6/SPC-L and C6/HHG2C18-L on A549, MCF-7, and A498 cells for 2 h at different pH values (pH 7.4 and pH 6.5).
3.11
Qualitatively Evaluation of Endolysosomal Escape and Mitochondrial Targeting of the HHG2C18-L by CLSM
To proof that HHG2C18-L is capable of escaping from endolysomes and targeting the mitochondria, a qualitatively evaluation using CLSM was carried out. The cytoplasmic distribution of HHG2C18-L on A498 cells was visualized by labeling the cell organelles (lysosomes and mitochondria) with specific fluorescent probes (LysoTracker and MitoTracker) and observing the localization of HHG2C18-L with different cell organelles according to the time variation. 1. Seed A498 cells at a density of 1 105 cells per well in glass-bottom cell culture dish for 24 h at 37 C. 2. Dilute C6/HHG2C18-L and C6/SPC-L with FBS free culture medium to a final concentration as 100 ng/mL of C6, respectively. 3. Add 1 mL of C6/HHG2C18-L or C6/SPC-L in the wells and culture the solutions with cells for 1, 4, 8, or 12 h, respectively. 4. Remove the culture medium and wash the cells with 4 C PBS thrice. 5. Stain the lysosomes of the cells cultured with liposomes for 1, 4, or 8 h with 50 nM LysoTracker Red for 30 min at 37 C and follow by washing the cells with PBS thrice. Fig. 7 Cellular uptake of C6/ SPC-L and C6/HHG2C18-L on A549, MCF-7, and A498 cells for 2 h at different pH values (pH 7.4 and pH 6.5). *P < 0.05, **P < 0.01
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6. Stain the mitochondria of the cells cultured with liposomes for 12 h with 200 nM MitoTracker Red for 30 min at 37 C and follow by washing the cells with PBS thrice. 7. Observe the localization of C6/HHG2C18-L or C6/SPC-L with lysosomes and mitochondria using CLSM (Note 14). Figure 8 shows the intracellular delivery of C6/SPC-L and C6/HHG2C18-L on A498 cells at different time observed by CLSM.
4
Notes
1. Recrystallized stearyl alcohol was flaky and too light to mix uniformly with activated L-Glutamic acid solution, which meant that a charging hopper should be employed to avoid the flaky stearyl alcohol float on the solution. 2. 15 mL of TFA should be added drop by drop on the ice bath to counteract the heat generated and avoid the unexpected reaction, followed by transferring to room temperature.
Fig. 8 Intracellular delivery of C6/SPC-L and C6/HHG2C18-L on A498 cells at different time observed by CLSM. The late endosomes and lysosomes were stained by LysoTracker Red. White arrows signified the occasions of coincidence between the liposomes and endolysosomes. Green arrows indicated the liposomes escaping from endolysosomes into cytoplasm. The mitochondria were stained by MitoTracker Red. Scale bars are 10 μm
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3. The ratio of SPC and HHG2C18 employed in HHG2C18-L play an important role in the particle size and zeta potential of HHG2C18-L at different pH values due to the difference in charge that SPC was negative while HHG2C18 was pHsensitive. When the ratio of SPC and HHG2C18 was 5:1 (w:w), the charge conversion of HHG2C18-L according to the pH change was unobvious. In contrast, when the ratio of SPC and HHG2C18 decreased to 4:1 (w:w), 3:1 (w: w), or 2:1 (w:w), the HHG2C18-L held better charge conversion according to the pH change and had an isoelectric point around pH 6.8, which meant that more HHG2C18 was suitable for the pH sensitive liposome in need. However, more HHG2C18 would lead to larger size. To consider the particle size and the pH sensitivity of liposome, 3:1 (w:w) was chosen as the ratio of SPC and HHG2C18 employed in HHG2C18-L for further study. 4. Rehydration time was important to the encapsulation efficient of CCI-779 that the encapsulation efficient of CCI-779 obviously decreased as the extension of rehydration time. The temperature of rehydration affected the encapsulation efficient of CCI-779 as well due to the easily oxidation of phospholipid at higher temperature. 5. Longer sonication time or larger sonication intensity would cause the leakage of CCI-779, resulting in the decline of encapsulation efficient of CCI-779. In contrast, shorter sonication time could not disperse the liposome uniformly with a lot of larger drug liposomes. The ice bath was essential to avoid the oxidation of phospholipid and drug leakage under the hyperpyrexia during the sonication. 6. Cholesterol played a key role in the stability of liposomes. However, too much cholesterol would lead to the decrease of encapsulation efficient due to the competition of hydrophobic location in liposomes with hydrophobic drug. 7. The particle sizes of CCI-779/SPC-L and CCI-779/HHG2C18-L were 100.8 6.74 nm and 134.5 1.66 nm at pH 7.4, respectively. Furthermore, there were no significant differences of CCI-779/HHG2C18-L during the pH change (Fig. 2). 8. The zeta potential of HHG2C18-L changed sharply from negative (22.9 mV) to positive (þ6.3 mV) over the narrow pH range of 7.4–6.5, which confirmed that charge conversion of HHG2C18-L from negative to positive occurred in the mildly acidic tumor microenvironment. Furthermore, the zeta potential of HHG2C18-L continuously rose to þ15.3 mV at pH 5.5 and þ25.5 mV at pH 4.5 due to the increasing acidity, which endowed HHG2C18-L with stronger positive charge at the endolysosomal acidities. Accordingly, HHG2C18-L had the ability to change the zeta potential according to the environmental pH, which was attributed to the chemical structure of the zwitterionic lipid, which includes the carboxy group of hexahydrobenzoic acid and the amino group of histidine. By contrast, the pH-nonresponsive SPC-L showed a constant zeta potential at all pH values investigated (Fig. 3). 9. HHG2C18-L showed a remarkable pH buffering effect (proton sponge effect) for neutral to acidic conditions (Fig. 4). In other words, HHG2C18-L containing histidine with an imidazole ring were able to absorb protons at endolysosomal
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pH as the second-stage pH response, leading to an increase in osmotic pressure inside the endolysosomes, followed by plasma membrane disruption and HHG2C18-L release into the cytoplasm. The generated hexahydrobenzoic amide in HHG2C18-L exhibited great degradability at pH 5.5 and pH 4.5. Compared with the hydrolysis at pH 7.4, the amide hydrolyzed more than 5-fold and 7.5-fold at pH 5.5 and pH 4.5, respectively, after 24 h (Fig. 5). It confirmed the third-stage pH response of HHG2C18-L through a pH-cleavable linker, hexahydrobenzoic amide, which was prone to degrade in endolysosomal compartments with lower pH values, resulting in the removal of carboxy groups from HHG2C18. The positive charge of HHG2C18-L increased correspondingly with the hydrolysis of the hexahydrobenzoic amide. The zeta potential of HHG2C18-L gradually reached about þ22 mV at pH 5.5 and þ40 mV at pH 4.5 in 24 h. By comparison, HHG2C18-L showed a zeta potential of about 20 mV at pH 7.4 even after 24 h as a consequence of the presence of carboxyl groups (Fig. 6). It further validated the third-stage pH response of HHG2C18-L through hexahydrobenzoic amide, resulting in the removal of carboxy groups from HHG2C18, and laying a good foundation for subsequent mitochondrial targeting. The incubation time of trypsin with the cells was monitored by observing the cellular morphology. The short incubation time made the cells hard to departure from the culture dish, whereas the long incubation time might hurt the cells. Hence, it is important to control the incubation time of trypsin with the cells. The cellular uptake of HHG2C18-L by A549, MCF-7, and A498 cells were pH-dependent that significantly increased from 123.05 3.15 ng/mg, 216.95 10.96 ng/mg, 190.48 7.90 ng/mg at pH 7.4 to 187.64 20.05 ng/mg (P < 0.01), 415.00 54.31 ng/mg (P < 0.01), 243.83 24.87 ng/mg (P < 0.05) at pH 6.5, respectively. In contrast, the cellular uptake of SPC-L showed no significant difference due to the unchangeable zeta potential. It was demonstrated that the first-stage charge conversion of HHG2C18-L from negative to positive actually increased the cellular uptake by different tumor cells attributing in part to the stronger affinity between the positive HHG2C18-L at pH 6.5 and the negative cell membrane. The intracellular delivery of HHG2C18-L on A498 cells was shown in Fig. 8 that the red fluorescence indicated the lysosomes or mitochondria while the green fluorescence presented the C6/HHG2C18-L or C6/SPC-L. The yellow fluorescence happened when the liposomes colocalized with the specific organelle dyes. After incubation with A498 cells for 4 h, the green fluorescence of C6/ HHG2C18-L largely dissociated from the red fluorescence of lysosomes, while the noticeable yellow fluorescence presented at 1 h, displaying the successfully endolysosomal escape of HHG2C18-L. At 8 h, the stronger green fluorescence along with the weaker red fluorescence over time demonstrated more effective endolysosomal release of HHG2C18-L into the cytosol. In addition, a yellow fluorescence was displayed at 12 h suggesting the accumulation of C6/HHG2C18-L in mitochondria because of the stronger positive charge of
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HHG2C18-L following the departure of carboxy groups. In contrast, the pHnon-responsive C6/SPC-L showed no ability to escape from endolysosomes judging by the overwhelming majority of overlapped green and red fluorescence till 8 h, accordingly no ability to target mitochondria. The CLSM images further confirmed that HHG2C18-L was capable of penetrating through the endolysosomes and further achieving the mitochondrial targeting.
5
Conclusion
In summary, a novel multistage pH-responsive liposome based on zwitterionic oligopeptide lipid has been developed for cancer therapy. Charge conversion responded to the tumor extracellular environment, proton sponge effect, and pH degradable linker for enhancing positive charge based on endolysosomal acidity have been introduced to overcome the multiple physiological and pathological barriers. The strengths of the pH sensitive drug liposome contain enhanced tumor cellular uptake, improved cytoplasmic distribution, as well as good mitochondrial targeting, which has been confirmed by a series of characterizations. The results provide an inspired platform for cascade mitochondrial drug delivery and new opportunities in cancer therapy.
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HER2-Specific PEGylated Immunoliposomes Prepared by Lyophilization/Rehydration Method
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Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Preparation of Lyophilized PEGylated Immunoliposomes (LPIL) . . . . . . . . . . . . . . . . . . 2.3 Determination of Particle Size and Zeta Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 SiRNA Serum Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Preparation of LPIL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Encapsulation of siRNA into the Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Determination of Particle Size and Zeta Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Measurement of Transfection Efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Measurement of Gene Silencing Efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 SiRNA Serum Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
3b-[N-(N0 N0 –dimethylaminoethane) carbamoyl] cholesterol (DC-chol) liposomes are the first approved cationic liposomes by FDA for clinical trials, and their safety have been demonstrated in treating cystic fibrosis in vivo. Nowadays, the cationic liposomes composed of DC-Chol and dioleoylphosphatidylethanolamine (DC-Chol/DOPE) have been often used as nonviral gene delivery vectors, while their application is severely hampered by low transfection efficiency and poor J. Gao · Y. Zhong (*) Department of Pharmaceutical Science, College of Pharmacy, The Second Military Medical University, Shanghai, China e-mail: [email protected]; [email protected] © Springer-Verlag GmbH Germany, part of Springer Nature 2021 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49320-5_16
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serum stability. This protocol describes the preparation of lyophilized PEGylated DC-Chol/DOPE immunoliposomes (LPIL), which are conjugated with the Fab’ of humanized anti-HER2 monoclonal antibody for siRNA delivery through lyophilization/rehydration. Briefly, the cationic liposomes were prepared by a lipid film method, and the Fab’ of humanized anti-HER2 monoclonal antibody was mixed with cationic liposomes containing maleimide-terminated linker. Afterward, the HER2-specific PEGylated immunoliposomes (LPIL) were lyophilized and rehydrated with a certain amount of diluted siRNA solution for encapsulating siRNA. The resultant LPIL had a size of 150 nm and a zeta potential of 40 mv. The results showed that the lyophilization/rehydration method could significantly increase the transfection efficiency and gene silencing of immunoliposomes in HER2-overexpressing cells. In addition, PEGylation degree had evident effects on transfection efficiency and gene silencing activity of the liposomes. Results indicated that 2.5% PEG LPIL showed the best transfection efficiency and gene silencing activity. In view of the serum stability, siRNA in 2.5% PEG LPIL showed the lowest degrading rate, indicating that 2.5% PEG LPIL provided superior protection for siRNA. In conclusion, 2.5% PEG LPIL provided the best transfection efficiency, gene silencing efficiency, and siRNA protection, hence representing a promising approach for gene therapy. Keywords
Cationic liposomes · siRNA · DOPE · DC-Chol · Lyophilization/Rehydration · Gene silencing
1
Introduction
Cationic liposomes are the positively charged liposomes and usually used for gene therapy due to their favorable interactions with negatively charged DNA and cell membranes. As the gene delivery vectors, cationic liposomes have a variety of advantages. On the one hand, cationic liposomes are biodegradable and biocompatible (Oh and Park 2009) after administration in vivo. During biodegradation, the endogenous enzymes can break down the lipid components of the liposomes. On the other hand, the cationic liposomes can be assigned with specific functions. For example, the lipid composition-dependent modification of surface charge density is able to alter the interaction forces with negatively charged nucleic acids, while the inclusion of PEGylated lipids or specific lipids enables a variety of functionalized modifications of liposomes. In addition, the inclusion of lipophilic chemical drugs in the lipid bilayers of cationic liposomes can codeliver anticancer drug and therapeutic nucleic acids (Kang et al. 2011; Shim et al. 2011). Therefore, cationic liposomes have been used for delivery of various nucleic acids, such as plasmid DNA, antisense oligonucleotides, and siRNA (Bhavsar et al. 2012; Xiong et al. 2011).
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The first approved cationic liposomes by FDA for clinical trials are 3b-[N-(N0 , N -dimethylaminoethane) carbamoyl] cholesterol (DC-chol) liposomes, and the safety of DC-Chol liposomes has been demonstrated in the treatment of cystic fibrosis (Li et al. 1996). Currently, DC-Chol and dioleoylphosphatidyl ethanolamine (DOPE) (DC-Chol/DOPE) liposomes have demonstrated to be the efficient gene delivery vectors (Li et al. 1996; Maitani et al. 2007; Wasungu and Hoekstra 2006) with higher biocompatible nature and lower cytotoxicity (Choi et al. 2004). However, the clinical application of DC-chol/DOPE liposomes was severely hampered by their drawbacks, including low transfection efficiency, poor serum stability, and short circulation lifetime (Oh and Park 2009; Salvati et al. 2006; Esposito et al. 2006). To address the above issues, attaching polyethylene glycol (PEG) at the surface of cationic liposomes, i.e., PEGylation, could provide a useful approach to overcome the short circulation lifetime of cationic liposomes (Dass and Choong 2006). Nevertheless, PEGylation is also a barrier for nucleic acid internalization and endosomal escape, resulting in a reduction in transfection efficiency (Wasungu and Hoekstra 2006). To overcome the siRNA-releasing barrier, the PEGylation degree of cationic liposomes should be optimized. In addition, developing immunoliposomes by conjugating the targeting ligand is another approach to increase the transfection efficiency of PEGylated cationic liposomes (Puri et al. 2008). For this purpose, the recombinant humanized anti-HER2 monoclonal antibody (rhuMAbHER2) can be used as the targeting ligand. Furthermore, the Fab’ of rhuMAbHER2 (anti-HER2 Fab’) has been used as the optimal ligand for its reduced immunogenicity, improved pharmacokinetics, and better penetration into solid tumors, as compared with the intact whole monoclonal antibody (Sapra et al. 2005; Kirpotin et al. 1997). Loading method in the PEGylated cationic liposomes significantly influences the transfection efficiency of siRNA (Sapra et al. 2005). The direct mixing of siRNA with PEGylated cationic liposomes often leads to the exposure of siRNA on the outer surface of the liposomes, and hence results in the immediate release of siRNA, followed by degradation of siRNA by serum nucleases (Buyens et al. 2008, 2009). Accordingly, two strategies are used to improve the entrapment of siRNA into the core of PEGylated cationic liposomes. The first strategy is to hydrate the lipid film which contains PEG and cationic lipids using a concentrated solution of siRNA (Buyens et al. 2009). The second strategy, also named as a post-PEGylation approach, is to prepare liposomes/siRNA complexes using non-PEGylated liposomes, followed by a subsequent PEGylation of the liposomes/siRNA complexes (Remaut et al. 2007). However, if these two strategies were used to prepare PEGylated immunoliposomes/ siRNA complexes, the postpreparation environment should be nuclease-free after the formation of liposomes/siRNA complexes. Besides, the prepared liposomes/ siRNA complexes may lose the entrapped siRNA. Therefore, the lyophilization/ rehydration method is used as an improved approach to overcome the above drawbacks, consisting of two steps: (i) lyophilization of blank liposomes, and (ii) encapsulation of siRNA into the core of liposomes by reconstitution through rehydration in an aqueous solution of siRNA (Peer and Margalit 2000). This method 0
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is able to avoid the degradation of siRNA by nuclease and reduce the loss of siRNA during the liposome manufacturing (Peer and Margalit 2000). By using the lyophilization/rehydration method, Peer et al. successfully developed a kind of targeted stabilized liposomes entrapping siRNA (b7 I-tsNPs) for specifically silencing cyclin D1 expression in leukocytes (Peer et al. 2008). This protocol describes the preparation of PEGylated DC-Chol/DOPE immunoliposomes conjugated anti-HER2 Fab’ (PIL) and lyophilized PIL (LPIL), and illustrates the influence factors of PIL and LPIL containing siRNA on the silencing efficiency of cancer genes.
2
Materials
2.1
Reagents
1. Dioleoylphosphatidyl ethanolamine (DOPE) , 3b-[N-(N0 , N0 -dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine [methoxy (polyethyleneglycol)-2000](mPEG–DSPE), maleimide derivatized PEG2000-DSPE (Mal-PEG– DSPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-carboxyfluorescein (CFPE). 2. The siRNA targeting human HER1, negative control (NC) siRNA and fluorescein amidite (FAM) labeled NC siRNA. 3. RhuMAbHER2 and C225 (human mouse chimeric anti-HER1 monoclonal antibodies).
2.2 1. 2. 3. 4.
Preparation of Lyophilized PEGylated Immunoliposomes (LPIL)
Rotary evaporator and water bath Handheld extruder of liposomes Sephadex G-75 Free-drying machine
2.3
Determination of Particle Size and Zeta Potential
1. Deionized water 2. Nano sizer
2.4
Flow Cytometry
1. FACScan flow cytometer
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SiRNA Serum Stability
1. Electrophoresis apparatus
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Methods
3.1
Preparation of LPIL
1. Prepare multilamellar liposomes (MLL) by a lipid film method with a mixture of DC-Chol, DOPE, Mal-PEG–DSPE, and mPEG–DSPE at molar ratios of (49.5–0.5X): (49.5–0.5X): 1:X, where X% represents the molar ratio of mPEG–DSPE in total lipids. 2. Dissolve the mixture of lipids in 4 mL chloroform in a 500 mL eggplant flask, and then evaporate the organic liquid to form the lipid film in the flask by rotatory evaporation at 45 C. 3. Hydrate the lipid film with 1 mL 10 mM phosphate buffer saline (PBS, pH 7.4) to create MLL. 4. Extrude the resultant MLL into unilamellar nanoscale liposomes (ULL) with a handheld extruder at an ambient temperature in a stepwise manner using polycarbonate membranes (200, 100, and 80 nm, respectively) with 10 cycles for each pore size (see Note 1). 5. Thiolate anti-HER2 Fab’ with 2-iminothiolane at a molar ratio of 100:1 (2-iminothiolane: Fab’), as follows: incubate 1 mL of anti- HER2 Fab’ (1 mg/ mL in PBS containing 5 mM EDTA, pH 7.4) with 75 μg 2-iminothiolane for 2 h at room temperature, and then dialyze the resultant anti-HER2 Fab’ to PBS (containing 5 mM EDTA, pH 7.4) for triplicate to eliminate the excessive 2-iminothiolane. 6. Determine the sulfhydryl group of anti-HER2 Fab’ using the Ellman’s assay as follows: prepare DNTB (5,50 -dithiobis-2-nitrobenzoic acid) stock solution with a final concentration of 50 mM sodium acetate and 2 mM DTNB in molecular biology grade water; keep refrigerated; then prepare Tris solution with a final concentration of 1 M Tris and adjust the pH to 8.0; set a standard -SH (acetyl cysteine) calibration curve starting at 10 μM; prepare the DTNB working solution by adding 50 μL of the DTNB solution, 100 μL of Tris solution, and 840 μL of molecular grade biology water to reach 990 μL final volume; mix 10 μL sample with 990 μL DTNB working solution carefully, incubate at room temperature for 5 min, and then measured with UV spectrophotometer at 412 nm; and finally, calculate the molarity of (SH) groups in accordance with the measured absorbance and the extinction coefficient (13,600 M1 cm1) (see Note 2). 7. Mix the thiolated anti-HER2 Fab’ with the prepared liposomes containing maleimide-terminated linker at a molar ratio of 1/10 (anti-HER2 Fab’/ Mal-PEG-DSPE) and incubate the mixture at ambient temperature under
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nitrogen for 2 h. Thiolated anti-HER2 Fab’ reacted with maleimide group on the liposomes forming the PEGylated immunoliposomes, i.e., PIL or targeted liposomes, as the following illustration. o PEG
N
O +
s SH
PEG
o Liposome
Maleimide Group
N O
Thiolated anti-HER2 Fab’
Targeted liposome
8. Remove unconjugated anti-HER2 Fab’ by gel chromatography on Sephadex G-75 using PBS (pH 7.4) as the washing solution (see Note 3). 9. Collect the resultant PIL in the void volume fraction, sterilize PIL by passing through a 0.22 mm sterile filter, and store PIL at 4 C. 10. Mix PIL with 5.4 mg/mL of 9% sucrose (w/v) and lyophilize the liposomes for 16–18 h using free-drying machine to yield LPIL.
3.2
Encapsulation of siRNA into the Liposomes
1. Dissolve siRNA into the diethyl pyrocarbonate (DEPC) treated water to reach a final concentration of 20 mM siRNA, and then dilute siRNA solution (9 mL) with DEPC-treated water to a certain volume (9–90 mL) (see Note 4). 2. For entrapping siRNA into the lyophilized liposomes, rehydrate LPL or LPIL (containing 7.2–72 mg DC-chol) using a series of the diluted siRNA solutions (9–90 mL) separately, and incubate the mixture at ambient temperature for 20 min. 3. For entrapping siRNA into the nonlyophilized liposomes, mix PL or PIL (9–90 mL, containing 7.2–72 mg DC-chol) using a series of the diluted siRNA solutions (9–90 mL) separately, and incubate the mixture at ambient temperature for 20 min. Perform the entrapment procedure immediately before use. 4. As shown in Fig. 1, the size of LPIL containing siRNA became much larger. When the weight ratio of DC-chol to siRNA was 5, LPIL-containing siRNA had the largest particle size (Fig. 1a). When the above ratio increased to 10–30 or when the PEGylation degree increased, the particle size of LPIL containing siRNA decreased gradually. With the increased weight ratio of DC-chol to siRNA, the zeta potential of LPIL containing siRNA increased gradually, up to a plateau when the ratio was above 10 (Fig. 1b).
3.3
Determination of Particle Size and Zeta Potential
1. Add 10 μL sample to 90 μl PBS (pH 7.4) and sonicate the mixture for 1 min using a bath sonicator.
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Fig. 1 Influence of the PEGylation degrees and the weight ratio of DC-chol to siRNA on the particle sizes (a) and on the zeta potential values (b) of LPIL containing siRNA. Data are expressed as mean SD (n = 3)
2. Add 900 μL PBS (pH 7.4) to above mixture. 3. Rinse the sample cell with PBS (pH 7.4) using 50 mL syringe, and push air to exclude the PBS. 4. Use a 1 mL syringe to inject the sample (the first cell in the upper side while the second cell in the lower side). 5. Analyze the size and zeta potential values of the liposomes using the dynamic light scattering instrument. 6. Rinse the sample cell with the deionized H2O and keep the cell in the deionized H2O for the next measurement.
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Measurement of Transfection Efficiency
1. Inoculate human breast cancer SK-BR3 cells in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 C in a humidified 5% CO2 atmosphere. 2. Culture SK-BR3 cells in 48-well plates with a density of 60,000 cells per well overnight. 3. Incubate SK-BR3 cells with the above liposomes containing FAM labeled-siRNA at a final siRNA concentration of 100 nM siRNA (0.4 mg siRNA per well) for 6 h (see Note 5). 4. Trypsinize and then wash the cells, followed by analysis by flow cytometry (FCM). 5. As showed in Fig. 2, the transfection efficiency of LPIL was higher than that of PIL at each weight ratio of DC-chol to siRNA, indicating that the lyophilization/ rehydration method could significantly enhance the transfection efficiency of PIL. In addition, the transfection efficiencies of both LPIL and PIL were significantly higher than that of the controls (LPL and PL), implying that the conjugated antiHER2 Fab’ could enhance the binding affinity of the liposomes to HER2overexpressing cells.
3.5
Measurement of Gene Silencing Efficiency
1. For observing gene silencing efficiency, incubate SK-BR3 cells in 48-well plates with a density of 60,000 cells per well overnight (see Note 6, Note 7 and Note 8). 2. Incubate the cells with above liposomes containing NC or containing anti-HER2 siRNA at a final concentration of 100 nM siRNA (0.4 mg siRNA per well) for 6 h until changing fresh culture medium. 3. After 48 h incubation, trypsinize and then wash the cells, followed by incubating cells with C225 antibody at a final concentration of 5 mg/mL at 4 C for 45 min. 4. Then wash the cells, and further incubate with FITC-goat anti-human IgG (H + L) at 4 C for 30 min. 5. After washing, analyze the HER1 expression of the cells by FCM. In this assay, HER1 was used as the gene silencing target as HER1 was moderately expressed in SK-BR3 cells and hence was easily detected by FCM. 6. The results showed that the reduction in transfection efficiency of LPIL was dependent on PEGylation degree (Fig. 3a). A PEGylation degree at 3% PEG LPIL had the lowest transfection efficiency, while A PEGylation degree at 3.5% PEG or 4%PEG LPIL increased the transfection efficiency. In addition, the transfection efficiency of PIL was lower than that of LPIL. As shown in Fig. 3b, a PEGylation degree at 2.5%PEG LPIL demonstrated the best gene silencing effect on HER1.
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Fig. 2 Transfection efficiency of 2.5% PEG (a) and 4% PEG (b) LPIL, PIL, LPL, or PL at a series of the weight ratio of DC-chol to siRNA. The mean fluorescence intensity represented the intensity of FAM-siRNA fluorescence in SK-BR3 cells. Data are expressed as mean SD (n = 3)
3.6
SiRNA Serum Stability
1. Characterize the serum stability of free siRNA or liposomes encapsulated siRNA using agarose gel electrophoresis (see Note 9). 2. Mix samples of either free siRNA in aqueous solution or the liposomes encapsulated siRNA with fresh serum at a ratio of 1: 1 (v/v), and incubate the mixture at 37 C. 3. At varying incubation times, load free siRNA or the liposome-containing siRNA sample (0.25 mg siRNA) onto gel system. Perform the electrophoresis using an electrophoresis apparatus to detect the intact siRNA.
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Fig. 3 Transfection efficiency (a) and gene silencing effect on HER1 (b) of LPIL and PIL at different PEGylation degrees. The mean fluorescence intensity in transfection efficiency (a) represented the intensity of FAM-siRNA fluorescence in SK-BR3 cells, while the mean fluorescence intensity in gene silencing effect on HER1 (b) represented the HER1 expression in SK-BR3 cells. Data are expressed as mean SD (n = 3)
4. The results showed that free siRNA was fully degraded in serum after 12 h in view of the lost oligonucleotide band in the gel. In contrast, siRNA in the 2.5% PEG PIL or in the 2.5%PEG PL was entirely degraded in serum after 24 h (Fig. 4). Furthermore, siRNA in 2.5%PEG LPIL or 2.5% PEG LPL was only partially degraded in serum after 24 h, demonstrating a protection effect of siRNA from degradation.
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Fig. 4 siRNA serum stability assay. At varying incubation times with the serumcontaining medium, the degradation of free siRNA or the liposome-encapsulated siRNA was observed by electrophoresis apparatus
4
Notes
1. The size of liposomes can be adjusted by a handheld extruder. In case of difficult operation, the warming is beneficial for the extrusion of the liposomes passing through polycarbonate membrane in the extruder. 2. The -SH of anti-HER2 Fab’ should be more than 1 per Fab to ensure the conjugation efficiency of antibody with the liposomes. 3. When removing unconjugated anti-HER2 Fab’ from the liposomes, the resultant immunoliposomes should be concentrated with a suitable method for obtaining a desired targeting efficiency of the final product. 4. Unless stated otherwise, RNase-free water should be used during preparing siRNA solution for preventing the degradation of siRNA. 5. The incubation of FAM-labeled siRNA liposomes should be kept in dark to avoid fluorescence quenching. 6. The cancer cells should be passaged within 24 h after reaching full monolayer confluency in order to keep the cells growing in a healthy condition. 7. The cancer cell suspensions should be gently but thoroughly mixed to avoid cell clumping. In case of difficulty in obtaining single cells, a trypsin-EDTA treatment could be used for this purpose. 8. The cancer cells should always be kept in a good growth condition to guarantee the HER2 and HER1 expressing quality. 9. In the electrophoresis, the running time should not be too long to produce the obscure bands of siRNA.
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Conclusion
This protocol describes the preparation of lyophilized HER2-specific PEGylated DC-chol/DOPE immunoliposomes (LPIL) for siRNA delivery by lyophilization/ rehydration method. The results showed that the LPIL prepared by lyophilization/ rehydration method could significantly improve gene silencing effect on HER1 gene in the human breast cancer SK-BR3 cells. Besides, the PEGylation degree of LPIL had a significant effect on the gene silencing effect, and 2.5% PEG LPIL exhibited the optimal gene silencing effect on HER1 gene. In conclusion, 2.5% PEG LPIL provided the best transfection efficiency, gene silencing efficiency, and siRNA protection, hence representing a promising vector for gene therapy.
References Bhavsar D, Subramanian K, Sethuraman S, Krishnan UM (2012) Translational siRNA therapeutics using liposomal carriers: prospects & challenges. Curr Gene Ther 12:315–332 Buyens K, Lucas B, Raemdonck K, Braeckmans K, Vercammen J, Hendrix J, Engelborghs Y, De Smedt SC, Sanders NN (2008) A fast and sensitive method for measuring the integrity of siRNA-carrier complexes in full human serum. J Control Release 126:67–76 Buyens K, Demeester J, De Smedt SS, Sanders NN (2009) Elucidating the encapsulation of short interfering RNA in PEGylated cationic liposomes. Langmuir 25:4886–4891 Choi WJ, Kim JK, Choi SH, Park JS, Ahn WS, Kim CK (2004) Low toxicity of cationic lipid-based emulsion for gene transfer. Biomaterials 25:5893–5903 Dass CR, Choong PF (2006) Selective gene delivery for cancer therapy using cationic liposomes: in vivo proof of applicability. J Control Release 113:155–163 Esposito C, Generosi J, Mossa G, Masotti A, Castellano AC (2006) The analysis of serum effects on structure, size and toxicity of DDAB–DOPE and DC-Chol–DOPE lipoplexes contributes to explain their different transfection efficiency. Colloids Surf B Biointerfaces 53:187–192 Kang SH, Cho HJ, Shim G, Lee S, Kim SH, Choi HG, Kim CW, Oh YK (2011) Cationic liposomal co-delivery of small interfering RNA and a MEK inhibitor for enhanced anticancer efficacy. Pharm Res 28:3069–3078 Kirpotin DB, Park JW, Hong K, Shao Y, Shalaby R, Colbern G, Benz CC, Papahadjopoulos D (1997) Targeting of liposomes to solid tumors: the case of sterically stabilized anti-HER2 immunoliposomes. J Liposome Res 7:391–417 Li S, Gao X, Son K, Sorgi F, Hofland H, Huang L (1996) DC-Chol lipid system in gene transfer. J Control Release 39:273–281 Maitani Y, Igarashi S, Sato M, Hattori Y (2007) Cationic liposome (DC-Chol/DOPE ¼ 1:2) and a modified ethanol injection method to prepare liposomes, increased gene expression. Int J Pharm 342:33–39 Oh YK, Park TG (2009) siRNA delivery systems for cancer treatment. Adv Drug Deliv Rev 61: 850–862 Peer D, Margalit R (2000) Physicochemical evaluation of a stability-driven approach to drug entrapment in regular and in surface-modified liposomes. Arch Biochem Biophys 383:185–190 Peer D, Park EJ, Morishita Y, Carman CV, Shimaoka M (2008) Systemic leukocyte directed siRNA delivery revealing cyclin D1 as an anti-inflammatory target. Science 319:627–630 Puri A, Kramer-Marek G, Campbell-Massa R, Yavlovich A, Tele SC, Lee SB, Clogston JD, Patri AK, Blumenthal R, Capala J (2008) HER2-specific affibody-conjugated thermosensitive liposomes (affisomes) for improved delivery of anticancer agents. J Liposome Res 18:293–307 Remaut K, Lucas B, Braeckmans K, Demeester J, De Smedt SC (2007) Pegylation of liposomes favours the endosomal degradation of the delivered phosphodiester oligonucleotides. J Control Release 117:256–266
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Salvati A, Ciani L, Ristori S, Martini G, Masi A, Arcangeli A (2006) Physico–chemical characterization and transfection efficacy of cationic liposomes containing the pEGFP plasmid. Biophys Chem 121:21–29 Sapra P, Tyagi P, Allen TM (2005) Ligand-targeted liposomes for cancer treatment. Curr Drug Deliv 2:369–381 Shim G, Han SE, Yu YH, Lee S, Lee HY, Kim K, Kwon IC, Park TG, Kim YB, Choi YS, Kim CW, Oh YK (2011) Trilysinoyl oleylamide-based cationic liposomes for systemic co-delivery of siRNA and an anticancer drug. J Control Release 155:60–66 Wasungu L, Hoekstra D (2006) Cationic lipids, lipoplexes and intracellular delivery of genes. J Control Release 116:255–264 Xiong F, Mi Z, Gu N (2011) Cationic liposomes as gene delivery system: transfection efficiency and new application. Pharmazie 66:158–164
Preparation and Characterization of Drug Liposomes by Nigericin Ionophore
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Liang Zhang
Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Preparation of Ion Liposomes by Thin-Film Hydration . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Formation of Ion Gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Loading of Vincristine or Topotecan and Measurement of Encapsulation Efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Measurement of Particle Size, Zeta Potential, and Polydispersity Index (PDI) . . . . 2.5 Transmission Electron Microscopy (TEM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 In Vitro Release of Vincristine or Topotecan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8 Cytotoxicity to MDA-MB231or MCF-7 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9 In Vivo Inhibition of the Tumor Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.10 Pharmacokinetics and Tissue Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Preparation of Standard Curve of Vincristine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Preparation of Standard Curve of Topotecan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Linearity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 HPLC Method for Measurement of Vincristine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 HPLC Method for Measurement of Topotecan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Stability Testing of Vincristine or Topotecan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Precision of Vincristine or Topotecan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 Experiment Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9 Preparation of Vincristine Liposomes and Measurement of Encapsulation Efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.10 Preparation of Topotecan Liposomes and Measurement of Encapsulation Efficiency (Note 1, 2, 3, 4, 5, and 6) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
425 426 426 426 426 427 427 427 427 427 428 428 428 428 428 428 429 429 429 430 430 431 432
L. Zhang (*) Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences, Beijing, China e-mail: [email protected] © Springer-Verlag GmbH Germany, part of Springer Nature 2021 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49320-5_5
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3.11
Measurement of Particle Size, Zeta Potential, and Polydispersity Index (PDI, Note 6) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.12 Transmission Electron Microscopy (TEM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.13 In Vitro Release of Vincristine or Topotecan (Note 20) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.14 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.15 Cytotoxicity to MDA-MB231 or MCF-7 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.16 Animal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.17 In Vivo Inhibition of the Tumor Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.18 Pharmacokinetics (Note 21, 22, and 23) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.19 Tissue Distribution (Note 24) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.20 Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
434 434 434 435 435 436 436 436 437 437 437 440 441
Abstract
In the latest decades, the liposomes have been extensively employed as the drug delivery system to enhance the treatment efficacy and improve the safety of different pharmaceutical agents. In order to meet the requirements of industrial manufacture and clinical application, the liposomes should have high encapsulation efficiency of drug. This protocol reports a high encapsulation efficiency method of drug liposomes by nigericin ionophore. Nigericin ionophore is a polyether antibiotic which affects ion transport. Based on the ion-translocating property of nigericin ionophore, a fabrication approach is developed for loading the weakly basic drug into liposomes to get a high encapsulation efficiency. In this method, nigericin ionophore is utilized to prepare liposomes by facilitating the exchange metal ions for protons. Liposomes are prepared by encapsulating a solution of a metal ion, and composed of egg phosphatidylcholine (EPC), cholesterol, and polyethylene glycol-distearoylphosphatidylethanolamine (PEG2000-DSPE) to exhibit the ion gradient. Then, adding nigericin ionophore can acidify the liposomal interior by outward movement of the entrapped ions and inward movement of protons. Vincristine or topotecan can be loaded in response to this induced transmembrane pH gradient. The encapsulation efficiency will be obtained more than 90%. After intravenous injection administration in vivo, vincristine or topotecan liposomes exhibit long circulation time and strong inhibitory effect in the xenografted breast cancer cells in nude mice. In summary, the agents can be loaded into liposomes by nigericin ionophoremediated method and administered to mice with greater efficacy than possible with free agents.
Keywords
Liposomes · Nigericin ionophore · Vincristine · Topotecan · Breast cancer · Pharmacokinetics · Tissue distribution
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Preparation and Characterization of Drug Liposomes by Nigericin Ionophore
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Introduction
Liposomal delivery systems are nanoparticle lipoid vesicles which are being developed for loading a variety of drugs and represent an advanced technology for delivering anticancer agent to enhance permeability and retention (EPR) effects in tumor tissues and reduce systemic side effect (Langer 1998). A significant step in the development of liposomes came with inclusion of the PEGylated lipids in liposome composition. PEGylated lipids (polyethylene glycol-distearoylphosphosphatidyl ethanolamine, PEGDSPE) can inhibit opsonization by plasma proteins and contribute longer circulation for liposome because of the “steric stabilization” effect. In the presence of the surface hydrophilic protective layer of PEG chain, PEGylated liposomes show the characteristics of higher stability, the prolonged blood circulation time, and the reduced uptake by reticuloendothelial system. This technology has resulted in a number of liposomal formulations encapsulating anticancer agents. At present, several liposomal formulations are now available commercially or in the stage of clinical trials (Lasic and Papahadjopoulos 1995; Akashi et al. 1996; Abraham et al. 2002; Taggar et al. 2006). Vincristine is an anticancer chemotherapy drug and a member of the vinca alkaloids family. It is a useful agent in treatment of certain types of leukemia, Wilms tumor, neuroblastoma, and rhabdomyosarcoma. It works by causing depolymerization of microtubules leading to mitotic arrest and death. However, it also causes severe side effects, including a mixed sensory-motor neuropathy, seizures, mental changes, orthostatic hypotension, inappropriate secretion of antidiuretic hormone, and potentiation of preexisting neurological disease (Cui et al. 2010). Topotecan is a semisynthetic derivative of camptothecin, and it is effective against ovarian and small-cell lung cancer (SCLC) by inhibiting the activity of topoisomerase I, resulting in lethal DNA strand breaks, leading to the cancer cells’ death. However, after administration, topotecan may cause severe and sometimes fatal bone marrow suppression and blood problems (Fenske et al. 1998). The adverse effects of vincristine or topotecan have limited their uses and administration. The liposomal formulation could provide a good solution to this limitation, while how to improve the drug encapsulation efficiency of liposomes remains to be a key issue (Hamidinia et al. 2004; Melchior et al. 2016; Zhang et al. 2014, 2015). As the ability of transmembrane pH gradients can influence the equilibrium distributions of certain weak acidic and weak basic drugs, the pH gradient loading has been used in the preparation of drug liposomes, and has shown distinct advantages over other methods in view of higher drug encapsulation efficiency. Nigericin is an ionophore that establishes the inside liposome high and outside low proton gradient by exchanging ion inside liposomes for proton in the medium. This technology can be used to load liposomes with the vincristine or topotecan as high encapsulation efficiency, resulting in significantly increasing efficacy and reducing adverse effects (Zhang and Zhao 2013; Zhang et al. 2012; Ju et al. 2014; Ying et al. 2010).
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This chapter shows the optimized methods for loading liposomes with vincristine or topotecan by adding nigericin ionophore to liposomes containing ion. The outward movement of the ion and inward movement of H+ result in the acidification of the liposomal interior, driving the loading of vincristine or topotecan. This method shows a high drug encapsulation efficiency obtained for vincristine or topotecan liposomes by using nigericin ionophore. Then, the antitumor efficacy, pharmacokinetics, and distribution of vincristine or topotecan liposomes are evaluated after intravenous administration. This protocol contributes to the understanding of the preparation and the characterization of nigericin ionophore-mediated liposomes.
2
Materials
2.1
Preparation of Ion Liposomes by Thin-Film Hydration
1. Egg phosphatidylcholine (EPC) 2. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)2000] (PEG2000 -DSPE) 3. Cholesterol 4. K2SO4, KH2PO4, K2HPO4 5. Na2SO4, NaH2PO4, Na2HPO4 6. Chloroform 7. Pear-shaped flask 8. Rotary evaporator 9. N2 gas 10. Vacuum pump 11. Bath type sonicator 12. Vortex apparatus 13. Probe type sonicator 14. 400 nm and 200 nm polycarbonate membranes
2.2 1. 2. 3. 4. 5.
Formation of Ion Gradient
Nigericin sodium salt Sucrose Histidine buffer Quick Spin Sephadex G-50 column Centrifuge
2.3
Loading of Vincristine or Topotecan and Measurement of Encapsulation Efficiency
1. Vincristine sulfate 2. Topotecan
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Preparation and Characterization of Drug Liposomes by Nigericin Ionophore
3. Phenomenex C18 column (200 4.6 mm, 5 um) 4. HPLC system with UV detector
2.4
Measurement of Particle Size, Zeta Potential, and Polydispersity Index (PDI)
1. Phosphate buffered saline 2. Zetasizer 3000HSA for dynamic light scattering (DLS) analysis
2.5
Transmission Electron Microscopy (TEM)
1. Copper grids 2. JEM-1230 transmission electron microscope
2.6
In Vitro Release of Vincristine or Topotecan
1. Dialysis tube 2. Fetal bovine serum 3. Shaker
2.7 1. 2. 3. 4. 5. 6. 7.
Dulbecco’s minimum essential medium (DMEM) Fetal bovine serum Phosphate buffer saline (PBS) Trypsin EDTA Pen–Strep solution 100 T75 cell culture flask Incubator
2.8 1. 2. 3. 4. 5. 6.
Cell Culture
Cytotoxicity to MDA-MB231or MCF-7 Cells
96-well culture plates Trichloroacetic acid Sulforhodamine B Acetic acid Tris base Microplate reader
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2.9 1. 2. 3. 4.
In Vivo Inhibition of the Tumor Growth
Matrigel 1.0 ml syringes Caliper Alcohol cotton
2.10 1. 2. 3. 4. 5. 6.
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Pharmacokinetics and Tissue Distribution
2.0 ml microcentrifuge tubes Glass capillary tubes Heparin sodium salt Methanol Sodium chloride physiological solution Tissue homogenizer
3
Methods
3.1
Preparation of Standard Curve of Vincristine
1. Prepare the vincristine sulfate with 99% purity of concentrations of 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10, 25, 50 μg/ml in 100% methanol to generate an external calibration curve 2. Analyze the samples by HPLC 3. Quantify concentrations of vincristine sulfate via linear regression analysis of the standard curve from the relative peak area
3.2
Preparation of Standard Curve of Topotecan
1. Prepare the standard solution containing topotecan (50 μg/ml) in acetonitrile (ACN) and store it at 4 C 2. Make appropriate dilutions of the standard solution in ACN to produce solutions as 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10, 25, 50 μg/ml 3. Analyze the samples by HPLC 4. Quantify concentrations of topotecan via linear regression analysis of the standard curve from the relative peak area
3.3
Linearity
1. Prepare standard solutions of vincristine or topotecan at different concentration levels
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2. Generate the standard calibration curve of detector response (peak area) against vincristine or topotecan concentration 3. Calculate the slope, intercept, and linear correlation coefficient (r2) to carry out linear regression analysis
3.4
HPLC Method for Measurement of Vincristine
1. Develop the analytical technique of vincristine using HPLC equipment and fit it with a Phenomenex C18 column (200 4.6 mm) 2. 0.02 M sodium dihydrogen phosphate and methanol (36:64, v/v, pH = 4.7) compose the mobile phase 3. Keep the flow rate as 1.0 ml/min 4. Maintain the column temperature at 25 C and set the UV detection wavelength at 276 nm 5. Keep the injection volume as 20 μl 6. Validate the measurement methods after estimating the linearity, stability, interday and intra-day precision
3.5
HPLC Method for Measurement of Topotecan
1. Measure concentration of topotecan using HPLC equipment and fit it with a Phenomenex C18 column (200 4.6 mm). 2. In order to measure the amount of topotecan in the ring open (carboxy) and ring closed (lactone) form, separate the two forms of topotecan using 3% triethylamine as aqueous mobile phase and mixture of methanol/acetonitrile (30%:70%) as the organic phase. 3. Keep the sample temperature at 4 C and the column temperature at 40 C. 4. Run each sample for 15 min and increase the amount of organic phase from 10% to 25% over 9 min using a gradient method. 5. Detect the ring open and ring closed forms using an excitation wavelength of 380 nm and emission wavelength of 525 nm. 6. Validate the measurement methods after estimating the linearity, stability, interday and intra-day precision.
3.6
Stability Testing of Vincristine or Topotecan
Evaluate the stability and extent of degradation of the stock solution of vincristine or topotecan by stability testing. 1. 2. 3. 4.
Prepare topotecan stock solution (50 μg/ml) or vincristine stock solution (50 μg/ml) Dilute the solutions at three concentration levels (1, 10, and 50 μg/ml) Keep them at 4 C Detect solutions at regular time intervals for a period of 7 days in triplicate
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Table 1 Four independent variables and three levels in the optimization study Studied variable EPC: Cholesterol (mol:mol) EPC:Vincristine (mol:mol) or EPC:Topotecan (mol: mol) EPC: PEG2000-DSPE (mol:mol) Concentration of ion (mM)
Level 1 60:40 100:1 or 10:1 60:1 200
Level 2 55:45 50:1 or 5:1 55:1 300
Level 3 50:50 25:1 or 2.5:1 50:1 400
5. Run each solution by HPLC after filtering through a 0.2 μm filter 6. Compare the peak areas of the vincristine or topotecan at different time points to test the stability
3.7
Precision of Vincristine or Topotecan
1. Dilute topotecan stock solution or vincristine stock solution at concentrations of 1, 10, and 50 μg/ml. 2. Monitor the three different concentrations of vincristine solution or topotecan solution by HPLC to evaluate the intra- and inter-day coefficients of variation for three times on the same day and inter day. 3. Report the RSD for a statistically significant number of replicate measurements to evaluate the intra- and inter-day coefficients of variation. 4. Obtain an intra- or inter-day precision with RSD