Nanomaterials for Cancer Diagnosis (Nanotechnologies for the Life Sciences, Volume 7) [1 ed.] 3527313877, 9783527313877

Citation preview

VII

Contents Preface

XVII

List of Authors 1

1.1 1.2 1.3 1.3.1 1.3.1.1 1.3.1.2 1.3.1.3 1.3.2 1.4 1.4.1 1.4.1.1 1.4.1.2 1.4.2 1.4.2.1 1.4.2.2 1.4.3 1.4.3.1 1.4.3.2 1.5 1.5.1

XXI

Dendrimers in Cancer Treatment and Diagnosis 1 Srinivasa-Gopalan Sampathkumar, and Kevin J. Yarema Overview 1 Introduction 1

Basic Properties and Applications of Dendrimers 3 Structural Features and Chemical and Biological Properties 3 Basic Features of Dendritic Macromolecules are Inspired by Nature 3 Comparison of the Properties of Dendrimers and Conventional Synthetic Polymers 5 Comparison of the Properties of Dendrimers and Proteins (a Biological Polymer) 6 Dendritic Macromolecules Possess a Wealth of Possible Applications 8 Methods for Dendrimer Synthesis 10 History and Basic Strategies 10 Cascade Reactions are the Foundation of Dendrimer Synthesis 10 Dendrimer Synthesis has Expanded Dramatically in the Past Two Decades 12 Strategies, Cores, and Building Blocks for Dendritic Macromolecules 12 Dendrimers are Constructed from Simple ‘‘Building Blocks’’ 12 The Synthesis of Dendrimers Follows Either a Divergent or Convergent Approach 13 Heterogeneously-functionalized Dendrimers 13 Basic Description and Synthetic Considerations 13 Glycosylation is an Example of Surface Modification with Multiple Bioactivities 14 Dendrimers in Drug Delivery 15 Dendrimers are Versatile Nano-devices for the Delivery of Diverse Classes of Drugs 15

Nanotechnologies for the Life Sciences Vol. 7 Nanomaterials for Cancer Diagnosis. Edited by Challa S. S. R. Kumar Copyright 8 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31387-7

VIII

Contents

1.5.2 1.5.2.1 1.5.2.2 1.5.2.3 1.5.3 1.5.3.1 1.5.3.2 1.5.3.3 1.5.4 1.5.4.1 1.5.4.2 1.5.5 1.5.5.1 1.5.5.2 1.5.5.3 1.6 1.6.1 1.6.2 1.6.3 1.6.3.1 1.6.3.2 1.6.3.3 1.6.4 1.6.4.1 1.6.4.2 1.6.5 1.6.5.1 1.6.5.2 1.6.6 1.6.6.1 1.6.6.2 1.6.6.3 1.7

Dendritic Drug Delivery: Encapsulation of Guest Molecules 16 Dendrimers have Internal Cavities that can Host Encapsulated Guest Molecules 16 Using Dendrimers for Gene Delivery 16 Release of Encapsulated ‘‘Pro-drugs’’ 17 Covalent Conjugation Strategies 17 Dendrimers Overcome many Limitations Inherent in Polymeric Conjugation Strategies 17 Dendrimer Conjugates can be Used as Vaccines 19 Release of Covalently-delivered ‘‘Pro-drugs’’ 19 Fine-tuning Dendrimer Properties to Facilitate Delivery and Ensure Bioactivity 20 Delivery Requires Avoiding Non-specific Uptake 20 ‘‘Local’’ Considerations: Contact with, and Uptake by, the Target Cell 21 Drug Delivery: Ensuring the Biocompatibility of Dendritic Delivery Vehicles 22 Biocompatibility Entails Avoiding ‘‘Side Effects’’ such as Toxicity and Immunogenicity 22 Water Solubility and Immunogenicity 22 Inherent and Induced Toxicity 23 Dendrimers in Cancer Diagnosis and Treatment 24 Dendrimers have Attractive Properties for Cancer Treatment 24 Dendrimer-sized Particles Passively Accumulate at the Sites of Tumors 24 Multifunctional Dendrimers can Selectively Target Biomarkers found on Cancer Cells 25 Methods for Targeting Specific Biomarkers of Cancer 25 Targeting by Folate, a Small Molecule Ligand 26 Targeting by Monoclonal Antibodies 26 Dendrimers in Cancer Diagnosis and Imaging 28 Labeled Dendrimers are Important Research Tools for Biodistribution Studies 28 Towards Clinical Use: MRI Imaging Agents 28 Steps Towards the Clinical Realization of Dendrimer-based Cancer Therapies 29 The Stage is now set for Dendrimer-based Cancer Therapy 29 Boron Neutron Capture Therapy 29 Innovations Promise to Speed Progress 30 ‘‘Mix-and-Match’’ Strategy of Bifunctional Dendritic Clusters 30 Towards Therapeutic Exploitation of Glycosylation Abnormalities found in Cancer 31 Towards Targeting Metabolically-engineered Carbohydrate Epitopes 31 Concluding Remarks 33

Contents

Acknowledgments References 33 2

2.1 2.2 2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.3 2.3.1 2.3.2 2.3.3 2.4 2.4.1 2.4.2 2.4.2.1 2.4.2.2 2.4.2.3 2.5 2.5.1 2.5.2 2.5.3 2.5.4 2.5.5 2.6 2.7

3

3.1 3.2 3.3 3.3.1 3.3.1.1 3.3.1.2 3.3.1.3 3.3.1.4

33

Nanoparticles for Optical Imaging of Cancer Swadeshmukul Santra and Debamitra Dutta Introduction 44 Cancer Imaging Techniques 46

44

Computed Tomography (CT) Scanning 47 Magnetic Resonance (MR) 47 Positron Emission Tomography (PET) 47 Single-photon Emission CT (SPECT) 48 Ultrasonography (US) 48 Optical Imaging 48 Basics of Optical Imaging 48 Optical Imaging Techniques 49 Optical Contrast Agents 50 Nanoparticles for Optical Imaging 51 Why Nanoparticles for Optical Imaging? 51 Development of Nanoparticle-based Contrast Agents 53 Quantum Dots 53 Gold Nanoparticles 57 Dye-doped Silica Nanoparticles 61 Optical Imaging of Cancer with Nanoparticles 65 Active Targeting 65 Passive Targeting 66 Cancer Imaging with Quantum Dots 66 Cancer Imaging with Gold Nanoparticles 68 Cancer Imaging with Dye-doped Silica Nanoparticles 69 Other Nanoparticle-based Optical Contrast Agents 70 Conclusions and Perspectives 70 Acknowledgments 72 References 72 Nanogold in Cancer Therapy and Diagnosis 86 Priyabrata Mukherjee, Resham Bhattacharya, Chitta Ranjan Patra, and Debabrata Mukhopadhyay Introduction 86 Medicinal use of Gold: A Historical Perspective 87 Application of Gold Nanoparticles in Cancer 88 Angiogenesis and Cancer 88 Agents that Inhibit Endothelial Proliferation or Response 90 Agents that Block Activation of Angiogenesis 91 Agents that Block Extracellular Matrix Breakdown 91 Unique Anti-angiogenic Properties of Gold Nanoparticles 91

IX

X

Contents

3.3.1.5 3.3.1.6 3.3.1.7

Gold Nanoparticles Inactivate VEGF165 92 What is the Mechanism of Action? 93 Effect of Gold Nanoparticles on the Activity of VEGF165, VEGF121, bFGF and EGF 94 3.3.1.8 Effect of Gold Nanoparticles on Signaling Events of VEGF165 94 3.3.1.9 Effect of Nanogold on Downstream Signaling events of VEGF165 95 3.3.1.10 Effect of Gold Nanoparticles on Migration of HUVEC Cells 95 3.3.1.11 Effect of Gold Nanoparticles on Angiogenesis in vivo 95 3.3.1.12 Gold Radioisotopes in Cancer Treatment 96 3.3.2 Application of Gold Conjugates in the Treatment of Cancer 96 3.3.2.1 Gold–TNF Conjugate in Cancer Therapeutics 96 3.3.2.2 ‘‘2 in 1’’ System in Cancer Therapeutics 97 3.4 Biocompatibility of Gold Nanoparticles 99 3.4.1 Cellular Adhesion Effects 99 3.4.2 Local Biological Effects 99 3.4.3 Systemic and Remote Effects 99 3.4.4 Effects of the Host on the Implant 100 3.4.5 Addressing the Biocompatibility of Gold Nanoparticles using DNA Microarray Analysis 100 3.4.6 Internalization of Gold Nanoparticles by HUVECs 101 3.4.7 Nanogold Particles do not Alter Global Pattern of Transcription by HUVEC Cells under Serum-free Conditions 101 3.4.8 Nanogold Particles do not Alter the Global Pattern of Transcription by HUVECs in Near-normal Culture Conditions 103 3.5 Synthetic Approaches to Gold Nanoparticles 105 3.5.1 Chemical Methods 105 3.5.2 Physical Methods 105 3.5.3 Biological Methods 106 3.6 Nanotechnology in Detection and Diagnosis with Gold Nanoparticles 106 3.6.1 Cancer Detection 106 3.6.2 Detection in DNA 107 3.6.2.1 Single-mismatch Detection in DNA 107 3.7 Future Direction 109 Acknowledgments 110 References 110 4

4.1 4.2 4.3 4.3.1 4.4 4.4.1

Nanoparticles for Magnetic Resonance Imaging of Tumors 121 Tillmann Cyrus, Shelton D. Caruthers, Samuel A. Wickline, and Gregory M. Lanza Introduction 121 Magnetic Resonance Imaging (MRI) 121 Targeting Mechanisms 124 Passive versus Active Targeting 124 Superparamagnetic Nanoparticles 126 Ligand-directed Targeting of Iron Oxides 127

Contents

4.4.2 4.5 4.5.1 4.5.2 4.5.3 4.5.4 4.5.5 4.6 4.7 4.8

Cell Tracking of Iron Oxides 128 Paramagnetic Nanoparticles 128 Perfluorocarbon Nanoparticles 129 Liposomes 131 Fullerenes 132 Nanotubes 133 Dendrimers 133 Quantum Dots 133 Polymer Nanoparticles 134 Conclusion 135 References 138

5

Magnetic Resonance Nanoparticle Probes for Cancer Imaging 147 Young-wook Jun, Jung-tak Jang, and Jinwoo Cheon Introduction 147 Magnetic Nanoparticle Contrast Agents 150 Silica- or Dextran-coated Iron Oxide Contrast Agents 150 Magnetoferritin 152 Magnetodendrimers and Magnetoliposomes 152

5.1 5.2 5.2.1 5.2.2 5.2.3 5.2.4 5.3 5.3.1 5.3.2 5.3.3 5.3.4 5.3.5 5.4

6

6.1 6.2 6.2.1 6.2.2 6.2.2.1 6.2.2.2 6.3 6.3.1 6.3.1.1 6.3.1.2 6.3.2

New Type of Contrast Agent: Non-hydrolytically Synthesized High Quality Iron Oxide Nanoparticles 154 Iron Oxide Nanoparticles in Molecular MR Imaging 157 Infarct and Inflammation 158 Angiogenesis 159 Apoptosis 160 Gene Expression 161 Cancer Imaging 163 Summary and Outlook 166 References 169 LHRH Conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers 174 Carola Leuschner Introduction 174 Cancer 175 Conventional Approaches to Cancer/Metastases Detection 177

Current Chemotherapeutic Approaches and their Disadvantages in Cancer Treatments 179 Multidrug Resistance 179 Drug Delivery to Tumors 180 Nanoparticles as Vehicles for Drug Delivery and Diagnosis 181 Targeting Tumor Cells 183 Passive Targeting 183 Active Targeting 185 Detection of Tumors and Metastases using Nanoparticles 186

XI

XII

Contents

6.3.2.1 6.3.2.2 6.4 6.4.1 6.4.2 6.4.3 6.4.4 6.4.5 6.4.6 6.5 6.5.1 6.5.2 6.5.3 6.5.3.1 6.5.4 6.5.4.1 6.5.4.2 6.5.4.3 6.6

7

7.1 7.2 7.2.1 7.2.1.1 7.2.1.2 7.2.2 7.2.2.1 7.3 7.4 7.4.1 7.4.2 7.4.3 7.4.4 7.4.4.1 7.4.4.2 7.4.4.3 7.5 7.6

Nanoparticles for Magnetic Resonance Imaging 186 Targeted Delivery of Nanoparticles to Increase Cellular Uptake for Higher MRI Resolution 188 LHRH and its Receptors 189 The Ligand Luteinizing Hormone Releasing Hormone – LHRH 189 Analogs of LHRH 192 Receptors for LHRH 193 Function–Signal Transduction Pathways 194 LHRH Receptor-mediated Uptake 197 LHRH Receptor Type II 198 LHRH-bound Magnetic Nanoparticles 201 Synthesis and Characterization 201 Treatment using Hyperthermia 202 Treatment using Lytic Peptides 203 Destruction of Metastases through LHRH-SPION-Hecate 203 Detection of Tumors and Metastases 204 Targeted Delivery of SPION Contrast Agents for MRI 204 In Vitro Studies on Receptor-targeted LHRH-SPION Uptake 205 In Vivo Studies on Receptor-targeted LHRH-SPION Uptake 206 Future Outlook 210 Acknowledgments 212 Abbreviations 212 References 213 Carbon Nanotubes in Cancer Therapy and Diagnosis Pu Chun Ke and Lyndon L. Larcom Overview 232

232

SWNT Modification for Solubility and Biocompatibility 234 Chemical Modifications of SWNTs for Solubility 234 Functionalization of SWNTs through Oxidation 235 Functionalization of SWNTs through Covalent Modifications 236 Noncovalent Modifications of SWNTs for Solubility 237 Solubilization of SWNTs Using Lysophospholipids Enables Cellular Studies 239 Diffusion of SWNT–Biomolecular Complexes 247 Gene and Drug Delivery with SWNT Transporters 252 RNA Translocation with SWNT Transporters 253 Gene Transfection with SWNT Transporters 258 Gene Transfection with SWNT Transporters for RNA Interference 261 Drug Delivery with SWNT Transporters 263 Vaccine Delivery by SWNTs 263 Protein Delivery by SWNTs 266 Biosensing by SWNTs 269 Sensing and Treating Cancer Cells Utilizing SWNTs 269 Cytotoxicity of SWNTs 272

Contents

7.7 7.8

Cancers and SWNTs 275 Summary 277 Acknowledgments 278 References 278

8

Nanotubes, Nanowires, Nanocantilevers and Nanorods in Cancer Treatment and Diagnosis 285 Kiyotaka Shiba Introduction 285 Nanotubes, Nanowires and Nanorods 285 Carbon Nanotubes 286 Noncarbon Nanotubes 287 Single-wall Carbon Nanohorns 287 Nanorods and Nanowires 288 Self-assembled Nanotubes 289 Cancer Diagnosis 290 Carbon Nanotube-based Detection System 290 Non-carbon Nanotube-based Detection Systems 291 Microcantilevers 292 Nano-tag made of Nanorods 293 Cancer Treatment 293 Carriers for Drug Delivery Systems 294 Imaging Agents 294 Conclusions 295 References 296

8.1 8.2 8.2.1 8.2.2 8.2.3 8.2.4 8.2.5 8.3 8.3.1 8.3.2 8.3.3 8.3.4 8.4 8.4.1 8.4.2 8.5

9

9.1 9.2 9.2.1 9.2.2 9.3 9.3.1 9.3.2 9.3.3 9.4 9.4.1 9.4.2 9.5 9.5.1 9.5.2 9.5.3

Multifunctional Nanotubes and Nanowires for Cancer Diagnosis and Therapy 304 Sang Bok Lee and Sang Jun Son Introduction 304

Advanced Technologies in Magnetic Nanoparticles for Biomedical Applications 305 MRI and Therapeutic Application of Magnetic Nanoparticles 305 Biomedical Diagnostic Application of Magnetic Nanoparticles 307 Carbon Nanotubes 310 Carbon Nanotubes for Targeted Cancer Cell Death 310 Carbon Nanotubes for Detection of Cancer Cell 312 Carbon Nanotubes for Targeted Delivery 313 Nanotubes and Nanowires Composed of Artificial Peptides 315 Peptide Nanotubes 315 Peptide-Amphiphile Nanofibers 316 Template-synthesized Nanotubes and Nanorods 318 Differential Functionalization of Nanotube 319 Selective Extraction of Drug Molecules 320 Silica Nanotube Carrier for DNA Transfection 322

XIII

XIV

Contents

9.5.4 9.5.5 9.5.6 9.5.6.1 9.5.6.2 9.5.6.3 9.6

DNA Nanotubes 323 Nanobarcodes for Multiplexing Diagnosis 325 Magnetic Nanotubes 328 Synthesis and Characterization of Magnetic Nanotubes 328 Magnetic Field-assisted Chemical Separation and Biointeraction Drug Uptake and Controlled Release 330 Conclusion 332 References 333

10

Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling 338 Li-Shing Huang, Yuh-Yih Chien, Shu-Hua Chen, Po-Chiao Lin, Kai-Yi Wang, Po-Hung Chou, Chun-Cheng Lin, and Yu-Ju Chen Introduction 338 Fabrication and Biomedical Applications of Nanoparticles 339 Fabrications and Properties of Nanoparticles 339 Metal Nanoparticles in Cancer Diagnosis 343 Principles of Mass Spectrometry 348

10.1 10.2 10.2.1 10.2.2 10.3 10.3.1 10.3.2 10.4 10.4.1 10.4.2 10.4.3 10.4.4 10.5 10.5.1 10.5.2 10.5.3 10.5.4 10.5.5 10.6 10.6.1 10.6.2 10.7

329

Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry 348 Affinity Mass Spectrometry 349 Nanoprobe-based Affinity Mass Spectrometry (NBAMS) 350 Preparation of Nanoprobes and Workflow 351 Proof-of-principle Experiment 353 Kinetic Study of the Nanoscale Immunoreaction 356 Detection Limit and Concentration Effect of Nanoprobe-based Immunoassay 356 Human Plasma and Whole Blood Analysis by Nanoprobe-based Affinity Mass Spectrometry 359 Selected Protein Profiling from Human Plasma 359 Comparison of Nanoscale and Microscale Immunoassay 359 Suppression of Nonspecific Binding on Magnetic Nanoparticles 361 Enrichment of Target Antigen in Human Plasma 362 Plasma Protein Profiling in Normal Individuals and in Patients 364 Multiplex Assay 364 Workflow of Multiplexed Assay 366 Screening for Patient and Healthy Individuals 367 Future Outlook 369 Acknowledgments 369 References 369

Contents

11

11.1 11.2 11.2.1 11.2.1.1 11.2.1.2 11.2.1.3 11.2.1.4 11.2.1.5 11.2.1.6 11.2.1.7 11.2.2 11.2.2.1 11.2.2.2 11.2.2.3 11.2.2.4 11.2.2.5 11.2.3 11.2.3.1 11.2.3.2 11.2.3.3 11.3

Nanotechnological Approaches to Cancer Diagnosis: Imaging and Quantification of Pericellular Proteolytic Activity 377 Thomas Ludwig Introduction 377 Quantification of Local Proteolytic Activity – an Objective 380 Regulation of Protease Activity 382 Secretion 382 Activation 382 Inactivation 383 Endogenous Protease Inhibitors 383 Glycosylation 384 Oligomerization 384 Protein Trafficking 385 Mechanisms of Confining Proteolytic Activity 385 Membrane-type Matrix Metalloproteinases 386 Cell Surface Receptors for Protease Binding 387 ECM Binding of Proteases 387 Cellular Microdomains 388 The Tumor–Host Conspiracy 388

Local Proteolytic Activity Regulates Complex Cellular Functions 389 Local Proteolytic Activity in Cancer Cell Migration 389 Local Proteolytic Activity and Cell Signaling 389 Functional Insights from Matrix-metalloprotease Deficient Mice 390 Evaluation of Classical Methods for Quantification of Net Proteolytic Activity 391 11.3.1 Functional Detection of Local Proteolytic Activity by In Situ Zymography 392 11.3.2 Tumor Cell Invasion Assays 393 11.3.2.1 Electrical Resistance Breakdown Assay 394 11.3.3 In vivo Detection of Proteolytic Activity 394 11.3.4 Multiphoton Microscopy and Second-harmonic Generation 395 11.3.5 In vitro Detection of Local Proteolytic Activity by Labeled Substrates 396 11.4 Novel Approaches to Local Proteolytic Activity 396 11.5 Conclusions and Perspectives 400 Acknowledgments 401 Abbreviations 401 References 401 Index

413

XV

XVII

Preface Two volumes (6 & 7) in the ten-volume series Nanotechnologies for the Life Sciences have been dedicated to the application of nanotechnology in cancer. The first of these two has captured nanotechnological approaches for the treatment of cancer and is already published. I do hope you had a chance to read it. This is the second of the pair, bringing out the utility of nanotechnology in developing tools and materials for sensitive and early diagnosis of cancer. These two volumes are timely as it is projected that cancer will be the leading cause of death, overtaking heart diseases, in the near future. One of the major goals of the American Cancer Society is early and sensitive detection of cancer. It is astonishing to note that the five year survival rate for breast cancer patients, if the cancer is diagnosed at localized stage, is 97.5%. Currently only 63.5% of breast cancers are diagnosed at the localized stage and there is no sensitive diagnostic tool for detecting micro-metastases, where the survival rate is less than 30%. Having made only limited progress in early and sensitive diagnosis of cancer using traditional methods, a paradigm shift in our approach is required in order to develop imaging agents and diagnostics for detecting cancer in its earliest and pre-symptomatic stage when it can be treated most easily. Needless to say, nanotechnology is offering this new approach and investigations reported so far demonstrate its immense potential. Since these investigations are being published in a very broad range of journals, this book provides a unique collection of consolidated and up-to-date information on nanotechnological diagnostic tools for the detection of cancer. It is my pleasure to present, on behalf of the eminent contributors, the 7th volume in the series, entitled Nanomaterials for Cancer Diagnosis. The uniqueness of nanotechnological approaches in the battle against cancer is a distinct possibility to create technologies for simultaneous diagnosis and treatment of cancer. Therefore, you will find some of the chapters covering both these aspects while focusing primarily on diagnosis. I am grateful to all the contributors who made this book into a comprehensive source of information on the impact of a variety of nanomaterials on a number of diagnostic tools. A book of this magnitude is not possible but for their scholarly contributions. At this point, I also would like to gratefully acknowledge the support of a number of others, especially my employer, family, friends and Wiley VCH publishers for this timely publication.

Nanotechnologies for the Life Sciences Vol. 7 Nanomaterials for Cancer Diagnosis. Edited by Challa S. S. R. Kumar Copyright 8 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31387-7

XVIII

Preface

The book is divided into eleven chapters, encompassing a number of diagnostic approaches for cancer through use of a variety of nanomaterials such as dendrimers, quantum dots, gold nanoparticles, dye-doped silica nanoparticles, superparamagnetic iron oxide nanoparticles (SPIONs), liposomes, fullerenes, carbon nanotubes, nanowires, nanorods, and so on. The book begins with a chapter reviewing the progress that has been made to date on dendrimers – nano-sized, radially symmetric molecules with well-defined, homogeneous and monodisperse structure consisting of tree-like arms or branches – and dendrimer-based nanomaterials in the diagnosis and treatment of cancer. The authors, Srinivasa-Gopalan Sampathkumar and Kevin J. Yarema from the Johns Hopkins University in Baltimore, USA, have done an excellent job in collating pertinent information on how several different attributes of dendrimers are being utilized to fine-tune their biological activity in order to obtain effective solutions to long standing problems in diagnosis and treatment of cancer. The chapter, Dendrimers in Cancer Treatment and Diagnosis, is valuable for all those interested in acquiring knowledge on this exciting and most promising class of nanomaterials in cancer nanotechnology. The second chapter, Nanoparticles for Optical Imaging of Cancer, has been contributed from the laboratories of University of Central Florida in Orlando, USA. Authors Swadeshmukul Santra and Debamitra Dutta provide a broad overview on various existing cancer-imaging techniques followed by a detailed description on optical imaging in general and nanoparticle-based optical imaging in particular. This chapter will provide readers with all the necessary information that is required for them to obtain a grasp of this exciting and continuously evolving field of nanoparticle-based optical contrast agents. More specifically, readers will have an opportunity to obtain a broader perspective on applications of quantum dots, dye-doped nanoparticles, gold nanoparticles, phosphors & fluorescent polymer particles as potential contrast agents in optical imaging of cancer. Of all the nanomaterials with optical properties, gold nanoparticles are the most widely investigated materials and therefore, a complete chapter is dedicated to the utility of gold nanoparticles in cancer diagnosis. The third chapter, Nanogold in Cancer Therapy and Diagnosis, a contribution from the laboratories of Debabrata Mukhopadhyay from Mayo Clinic Cancer Center in Rochester, USA, brings out the importance of gold nanoparticles in cancer diagnosis and therapy. The authors also have reviewed relevant information on synthetic methods, biocompatibility and the mechanism of action of gold nanoparticles. Moving from nanomaterials-based optical imaging techniques for diagnosis of cancer, the next three chapters describe investigations into utilization of magnetic nanoparticles for enhancing the sensitivity of detection of tumors using magnetic resonance imaging (MRI). Of the existing non-invasive diagnostic tools such as Computed Tomography (CT), Positron Emission Tomography (PET), Single Photon Emission CT (SPECT), Ultrasound (US) and optical imaging, MRI appears to be the most promising and sensitive technique. It is gaining more importance with the discovery of nanomaterials-based contrast agents. Researchers from the Washington University Medical School in St. Louis, USA, contributed the fourth chapter wherein an overview of the MRI technology and principle targeting mechanisms

Preface

are described, followed by a detailed discussion on the application of superparamagnetic and paramagnetic nanoparticles. The chapter, Nanoparticles for Magnetic Resonance Imaging of Tumors, brings out clearly the differences between the superparamagnetic nanoparticles, which are mainly SPIONs and paramagnetic nanoparticles belonging to groups such as liposomes, perfluorocarbon nanoparticles, fullerenes, and others. The chapter contributed by Tillmann Cyrus, Shelton D. Caruthers, Samuel A. Wickline, and Gregory M. Lanza also provides up-to-date information on rapidly evolving hybrid technologies using quantum dots for non-invasive imaging with MRI and intraoperative direct visualization. While the fourth chapter provides an overview of nanomaterials being developed for MRI, the fifth chapter primarily focuses on superparamagnetic iron oxide nanoparticles. This chapter, Magnetic Resonance Nanoparticle Probes for Cancer Imaging, contributed by Young-Wook Jun, Jung-tak Jang, and Jinwoo Cheon from Yonsei University in Seoul, Korea, reviews recently developed biocompatible magnetic nanoparticles and their utilization in molecular MRI. The final chapter on the application of SPIONs in MRI, the sixth chapter in the book, is from the laboratories of Carola Leuschner, Pennington Biomedical Research Center at Baton Rouge, USA. Being one of my close collaborators and having closely associated with the development of Leutenizing Hormone and Releasing Hormone (LHRH)-conjugated SPIONs, I can confidently say that this chapter is very unique and provides readers with all the information that is available on LHRH-conjugated magnetic nanoparticles. The chapter, LHRH-conjugated Magnetic Nanoparticles for Diagnosis and Treatment of Cancers, provides an elaborate account of the role of LHRH and LHRH receptors in malignant tissue. The central theme of the chapter is a description of comparative advantages of LHRH-SPIONs with other targeting agents in targeting specifically primary tumors and metastases, thereby demonstrating the potential for LHRH-SPIONs in dramatically improving the sensitivity of MRI. Switching gears from nanoparticles, chapter seven written by Pu Chun Ke and Lyndon L. Larcom from Clemson University in Clemson, USA, is a testimony to the ever-increasing number of applications of carbon nanotubes. Not surprisingly, single-walled carbon nanotubes (SWNTs) have already shown promise in cancer diagnosis and therapy and have distinct advantages over multi-walled carbon nanotubes (MWNTs). The chapter, Carbon Nanotubes in Cancer Therapy and Diagnosis, captures up-to-date information available in the literature on functionalization for solubility and biocompatibility, cytotoxicity, gene and drug delivery, and sensing of cancer cells utilizing SWNTs. Following this chapter, Kiyotaka Shiba from the Cancer Institute of the Japanese Foundation for Cancer Research in Tokyo, Japan, has brought out a thorough review on the application of ‘‘non-spherical’’ nanomaterials in cancer treatment and diagnosis. The theme of this 8th chapter, truly reflected in the title Nanotubes, Nanowires, Nanocantilevers and Nanorods in Cancer Treatment and Diagnosis, is the current status of carbonaceous as well as non-carbonaceous nanotubes, nanowires, and nanorods with respect to their potential applications in cancer diagnosis and treatment. In the ninth chapter, Multifunctional Nanotubes and Nanowires for Cancer Diagnosis and Therapy, authors Sang Bok Lee and Sang Jun Son from the University of Maryland at College Park, USA, describe in detail

XIX

XX

Preface

the importance of carbon nanotubes, nanotubes and nanowires composed of artificial peptides, and template-synthesized silica and magnetic nanotubes in general for various biomedical applications and in particular related to cancer. The authors demonstrate that tubular nanomaterials have several advantages over spherical nanoparticles, especially when multifunctionality is needed as nanotubes have distinctive inner and outer surfaces that can be modified and utilized differently, enabling them to be equipped with the right function in the right position. The last two chapters of this volume are testimony to the breadth of nanotechnological approaches for cancer diagnosis. The penultimate chapter, Nanoprobe-based Affinity Mass Spectrometry for Cancer Marker Protein Profiling, provides an overview on a recently developed technique – nanoprobe-based affinity mass spectrometry (NBAMS) – for the screening of normal individual and cancer patients. The chapter is a contribution from the Institute of Chemistry and Genomic Research Center of the Academia Sinica in Taipei, Taiwan, authored by Yu-Ju Chen and co-workers. This chapter provides fundamental principles in mass spectrometry and the detection method in NBAMS, in addition to details of the design, workflow and performance of NBAMS. The final chapter is presented by Thomas Ludwig from Yale University at New Haven, USA. The author points out some of the challenges in cancer diagnosis by focusing on local, nanoscale processes for in vitro and in vivo diagnostics. The chapter, Nanotechnological Approaches to Cancer Diagnosis: Imaging and Quantification of Pericellular Proteolytic Activity, is an interesting chapter on how development of improved nanotechnology enabled methods that are likely to provide real-time high-resolution imaging and quantification of local enzymatic activities will play a major role in the design of new drugs and the understanding of basic principles in tumor cell invasion. This book concludes the two volumes dedicated to cancer nanotechnology. I do realize that there are some topics that have not been covered and also some topics that have been covered are not exhaustive enough. I am hoping that the next edition of this book series will fill these gaps and also will take into consideration suggestions from the readers. I am looking forward to hearing from you. As I end this preface, I am pleased to note that the first six volumes of the ten volume series have already been published and the next four volumes are in print. I am also pleased to know that the Nanotechnologies for the Life Sciences in general is being well received. September 2006 Baton Rouge, USA

Challa S. S. R. Kumar

1

1

Dendrimers in Cancer Treatment and Diagnosis Srinivasa-Gopalan Sampathkumar, and Kevin J. Yarema 1.1

Overview

Dendrimers are nano-sized, radially symmetric molecules with well-defined, homogeneous and monodisperse structure consisting of tree-like arms or branches. Over the past two decades since the term ‘‘dendrimer’’ was formally defined, research interest in these molecules has gradually evolved from a primary focus on overcoming purely synthetic challenges to include aesthetic and theoretical perspectives, and, more recently, with the ongoing flurry of ‘‘nanobiotechnology’’ advances, to develop practical and commercial applications for these elegant nanodevices. Today, a critical mass of knowledge exists to synthetically control the physicochemical properties of dendrimers and thereby govern their ensuing biological behaviors. These fundamental scientific advances, coupled with practical methods to covalently conjugate a wide range of bioactive molecules to the surface of a dendrimer or encapsulate them as guest molecules within void spaces, provide a highly versatile and potentially extremely powerful technological platform for drug delivery. This chapter recaps synthetic advances in dendrimer construction and summarizes the many features of these fascinating macromolecules that endow them with favorable properties for drug delivery applications. Finally, with this enticing technology having matured to the point where it is ready to confront ‘‘real-world’’ challenges, a synopsis is outlined of the prospects for exploiting dendrimer-based nanodevices for one of the most intractable medical challenges, the diagnosis and treatment of cancer.

1.2

Introduction

The discovery, design, and development of anticancer therapeutic agents have proven to be remarkably intractable despite intense efforts at the research and clinical levels over many decades. A brief consideration of the challenges facing an Nanotechnologies for the Life Sciences Vol. 7 Nanomaterials for Cancer Diagnosis. Edited by Challa S. S. R. Kumar Copyright 8 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31387-7

2

1 Dendrimers in Cancer Treatment and Diagnosis

anticancer drug illustrates some of the reasons for frustratingly-slow progress: first the drug must be able to seek out subtle changes that distinguish a transformed cell from the other 200 or so types of healthy cells found in the body and then provide a sufficiently high dose of a toxic agent to kill the cell. The difficulty of this task is amplified by the potential metastasis of cancer cells to widely-spread niches throughout the body, each with unique properties. Furthermore, to successfully cure a patient, each and every cancer cell must be eradicated because even one in a thousand – often harboring latent resistance – can re-grow into a second tumor refractory to therapeutic intervention. Readers of this chapter, contained within a volume devoted to the development of novel cancer therapeutics, do not require convincing of the difficulty of combating cancer and this issue will not be labored here. Instead, this chapter provides a broad overview of dendrimer-based nanotechnologies for the treatment of cancer with a consideration of their synthesis, the encapsulation and covalent attachment of drugs, and various strategies used for tumor specific targeting, imaging, and therapy. The discussion of specific topics begins with a description of the basic properties of dendrimers in Section 1.3 to highlight how these molecules lie at the interface between conventional synthetic polymers and the archetypical nanosized biological polymers, proteins. Section 1.4 briefly outlines the synthesis of dendrimers; exhaustive review articles (referenced therein) provide a wealth of synthetic detail beyond the scope of this discussion. This chapter aims to provide the reader with the knowledge that, by control of design parameters, the attributes of dendrimers can be tuned to incorporate the most desired features of synthetic polymers and proteins and, thereby, gain exquisite control of biological activity. Upon having established that dendrimers are synthetically-tractable, biologicallycompatible nano-devices, their unique suitability for drug delivery will be delineated in some detail in Section 1.5. Specific topics covered include the alternative drug-carrying strategies of encapsulation (Section 1.5.2) and covalent conjugation (Section 1.5.3), as well as design features needed to ensure bioactivity of the drug (Section 1.5.4) and the biocompatibility of the dendrimer (Section 1.5.5). Finally, with the multi-disciplinary set of tools required for dendrimer-based drug delivery now reaching maturity, this area of investigation is undergoing transformation from the developmental stage to ‘‘real-world’’ applications. Accordingly, Section 1.6 discusses the prospects for using dendrimer-based nanotechnologies to overcome arguably the most difficult biomedical problem now faced, the diagnosis and treatment of cancer. In particular, the general properties of dendrimers that make them attractive for cancer treatment will be outlined in Section 1.6.1, with a specific benefit – exploitation of the enhanced permeability and retention effect that allows passive accumulation at the sites of tumors – discussed in Section 1.6.2. The ability of dendrimers to serve as a technological platform for multifunctional nano-devices that include targeting, imaging, and/or cytotoxic modalities is discussed in Section 1.6.3 and their prospects for diagnosis and therapeutic applications are given in Sections 1.6.4 and 1.6.5, respectively. Finally, Section 1.6.6 gives a brief synopsis of innovations that promise to speed progress in the near future.

1.3 Basic Properties and Applications of Dendrimers

Together – while broader in scope than the typical discussion of dendrimers, drug delivery, or cancer therapy – this chapter provides an integrated look at the many considerations required for successful application of dendrimers for cancer therapy. For a more-in-depth consideration of any particular sub-topic, the interested reader is urged to consult the many original research reports and review articles cited throughout.

1.3

Basic Properties and Applications of Dendrimers 1.3.1

Structural Features and Chemical and Biological Properties Basic Features of Dendritic Macromolecules are Inspired by Nature Dendritic structures, characterized by hyperbranched subunits, are widely found in nature. Indeed, the word dendrimer is based on the Greek words ‘‘dendron’’ meaning ‘‘tree’’ or ‘‘branch’’ and ‘‘meros’’ meaning ‘‘part’’ [1, 2]. Taken literally, similarities with dendrimer macromolecules are illustrated by a tree, where the leaves of a tree are maximally displayed on a highly-branched scaffold to maximize their accessibility to the outside world to optimize functions such as light harvesting. The branches of a tree can modify the environment within them, similarly the core/ interior encapsulated within a dendrimer can provide a sheltered microenvironment with tailored chemical properties and reactivities [2]. In addition to actual trees, Nature has scaled dendrimeric structures down to the multi-centimeter level (the intricate neural pathways found in the brain), the millimeter level (ice crystals and snowflakes), and yet further to the micron level (the dendritic outgrowths of neurons). At a molecular, ‘‘nano-size’’ level, dendrimer-like molecules, such as branching polysaccharides, provide an elegant solution to a cell’s need to stably store high energy molecules like monosaccharides; the presence of many chain ends allows the rapid release of large numbers of glucose monomers when needed [3]. Unlike Nature, which provides dendritic structures in a range of sizes from real trees to the namesake molecular nano-sized structures, this chapter focuses exclusively on dendritic macromolecules that are of a synthetically tractable scale and appropriate for cancer therapy. Starburst1 clusters [4], made of poly(amidoamine) (PAMAM) units, are arguably the most-thoroughly characterized and extensivelyutilized dendrimers [5]. A basic characteristic of these molecules is their layered composition – known as ‘‘cascades’’ or ‘‘generations’’ [1] (Fig. 1.1). The overall shapes of dendrimers range from spheres to flattened spheroids (disks) to amoeba-like structures, especially in cases where surface charges exist and give the macromolecule a ‘‘starfish’’-like shape [6]. The exact morphology of a dendrimer depends both on its chemical composition (the chemical composition of PAMAM dendrimers is shown in Fig. 1.1) as well as on the generation number, as exemplified by PAMAM where the lowest generation 1.3.1.1

3

4

1 Dendrimers in Cancer Treatment and Diagnosis

Schematic representation of a generation G4 dendrimer with 64 amino groups at the periphery. This dendrimer starts from an ethylene diamine core; the branches or arms were attached by exhaustive Michael addition to methyl acrylate followed by exhaustive aminolysis of the resulting methyl ester using ethylene diamine [36]. This sequence of reactions was applied in an iterative fashion to increase the level of Fig. 1.1.

generations. The periphery of successive generations is marked by grey circles, starting from G0, G1, G2, G3 and G4. Of note, distinctive features of dendrimers, including the densely-packed membrane-like arrangement of surface functional groups, the formation of internal cavities, and the condensation into globular structures (not shown), are typically manifest at the G4 stage (and amplified in successive generations, Table 1.1).

1.3 Basic Properties and Applications of Dendrimers

structure (e.g., G0 and G1) have highly asymmetric shapes and posses open structures compared with higher-generation structures that first appear to be disk-like and then progress to increasingly spherical geometries [5] as they assume globular structures with a significant reduction in hydrodynamic volume [7]. In addition to sphere-like dendrimers – based on a ‘‘dot-like’’ core (Fig. 1.1) – increasing interest is developing in cylindrical dendrimers that are based on ‘‘rod-like’’ cores. These interesting spin-off macromolecules have been compared with spaghetti because they can be rigid like the uncooked form of this pasta or highly flexible like the cooked form; these properties can be tuned based on the chemical composition and density of packing of the dendritic branches [6]. Additional features of dendrimers are discussed below, by comparison with the two classes of molecules they are most often likened to, i.e., ‘‘conventional’’ synthetic polymers and, the most extensively studied biological polymer, proteins. Comparison of the Properties of Dendrimers and Conventional Synthetic Polymers Dendrimers have both similarities and differences when compared with traditional polymers. One similarity is the vast diversity in the basic monomeric building blocks used to create both classes of molecules and to provide the final macromolecular products with a wide range of chemical, mechanical, and biological properties. Until recently, polymer chemistry has been focused on the production of linear polymers that often have a degree of branching or crosslinking; this property, however, is dramatically limited by comparison to dendrimers whose entire identity is wrapped up in their hyperbranched character. Interestingly, highly-branched polymers of the same material can be vastly different from conventional polymers of a similar molecular weight and composition; in particular, as dendritic macromolecules progress in size, usually when becoming larger than the third generation (G3), they assume globular structures and occupy considerably smaller hydrodynamic volumes than linear polymers [1]. When dendrimers condense into globular structures, a feat rarely achieved with linear synthetic polymers, their many termini become fixed into an outwards orientation and also form a densely packed, membrane-like surface (Fig. 1.1). This structural arrangement provides numerous attachment points for covalent conjugation of bioactive molecules on the surface as well as enclosed cavities for occlusion of guest molecules within the dendrimer. This tight packing ultimately results in the reaching of a critical branched state – known as the ‘‘starburst effect’’ [4] – where growth of the dendritic macromolecule is dramatically hindered by steric constraints [8] (this state is reached at G10 or G11 for PAMAM, Table 1.1). Dendrimers also have dramatically different rheological properties than conventional polymers; viscosity tends to increase continuously with molecular mass for linear macromolecules whereas the intrinsic viscosity of dendrimers goes through a maximum at approximately the fourth generation and then declines [8, 9]. Finally, dendrimers have a negligible degree of polydispersity because, unlike classical polymerization that is random in nature and produces molecules of various sizes, the size of dendrimers can be carefully controlled during synthesis. Under ideal condi1.3.1.2

5

6

1 Dendrimers in Cancer Treatment and Diagnosis Tab. 1.1. Generation by generation specifications for PAMAM Starburst4 dendrimers. (Adapted from Ref. [5].)

Generation

G0 G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11

Physical or structural parameter Molecular weight (Daltons)

Diameter (A˚)

Surface groups (xNH2 )

Radius of gyration (A˚)

517 1430 3256 6909 14 215 28 826 58 048 116 493 233 383 467 162 934 720 1 869 780

15 22 29 36 45 54 67 81 97 114 135 167

4 8 16 32 64 128 256 512 1024 2048 4096 8192

4.93 7.46 9.17 11.2 14.5 18.3 22.4 29.1 36.4 46.0 55.2 68.3

tions, preparations of dendrimers are monodisperse, which is to say they have one molecular weight instead of the range, over tens or even hundreds of kDa, often seen for traditional synthetic polymers. Indeed, the homogeneity and uniformity of dendrimers of successive generations becomes strikingly obvious as shown by the tunneling electron microscopy (TEM) images for G5 to G10 PAMAM (Fig. 1.2) [10]. Comparison of the Properties of Dendrimers and Proteins (a Biological Polymer) As discussed above, dendrimers have unusual, often dramatically different, characteristics compared with conventional synthetic polymers. In fact dendritic molecules have often been compared with proteins, which are the workhorse biological polymers. Both classes of macromolecules are globular, are composed of precisely controlled monomeric units, have defined architectures, are of comparable size (Table 1.1), and have surfaces with chemically-reactive sites that can be endowed with biologically-compatible ligands found on proteins (such as glycosylation, Section 1.4.3.2). Moreover, the interior of a dendritic molecule, reminiscent of a protein, can harbor unique microenvironments, providing behaviors like redox chemistry, molecular recognition, ligand and substrate binding, and catalysis [11, 12]. The ability to create and exploit isolated nanoenvironments within a dendritic shell is derived from two main properties of a dendrimer. First, dendritic macromolecules adopt a semi-globular or fully globular character containing internal void 1.3.1.3

1.3 Basic Properties and Applications of Dendrimers

Transmission electron microscopy (TEM) of PAMAM dendrimers. Dendrimers were positively stained with aqueous sodium phosphotungstate and imaged by conventional TEM: (a) G10, (b) G9, (c) G8, (d) G7, (e) G6, (f ) G5. The scale bars indicate 50 nm, and a

Fig. 1.2.

small amount of G10 has been added as a focusing aid for G6 and G5. (Reprinted with permission from Jackson and coauthors [10]. Copyright 1998 by the American Chemical Society.)

spaces once they reach the fourth generation in size (Fig. 1.1) [8], enabling the encapsulation of protein-like functions, including catalysis [13, 14]. Second, these molecules have molecular flexibility and can undergo deformations, leading to rudimentary ‘‘lock and key’’ molecular recognition of the type vitally important to protein functions [15, 16]. Molecular recognition between molecules is a fundamental process in biology and chemistry without which life could not exist. The concept of molecular recognition, based on complementarity between the receptors and substrates, is very similar to the ‘‘lock and key’’ function first described by Emil Fischer over 100 years ago. In biology, the ‘‘lock’’ is the molecular receptor such as a protein or enzyme and the ‘‘key’’ can be regarded as a substrate such as a drug or ligand that is recognized to give a defined receptor–substrate complex [15]. In proteins, molecular recognition is largely driven by non-covalent forces such as hydrogen bonding, electrostatics, van der Waals forces, p–p interactions, solvent-dependent interactions including hydration forces, and conformational energy [17]; notably, all of these parameters can be controlled in dendrimers through synthetic design. The inherent ability of dendrimers to achieve molecular recognition of biological features, if it can be successfully developed to a level of sophistication where it can be exploited for the recognition of the surface biomarkers that distinguish cancer

7

8

1 Dendrimers in Cancer Treatment and Diagnosis

cells from healthy cells (Section 1.6.3), has important – and extremely beneficial – implications for drug delivery (Section 1.5). Although sharing many superficial features, a close inspection reveals important differences between dendrimers and proteins. For example, remaining on the topic of deformability and flexibility, the linear, folded chain of a protein is more tightly packed but also has a greater potential for flexibility (when a comparison is made between the fully folded and unfolded states of a peptide chain) than is possible for the branches of a dendrimer. Only a small proportion of the potential flexibility of a protein, however, is usually available for ‘‘induced fit’’ interactions because the extensive unfolding of a protein is highly thermodynamically unfavorable. By comparison, although the extensive covalent bond networks within a dendrimer prevents complete unfolding under any condition, this arrangement does provide sufficient flexibility to allow dramatic – albeit somewhat thermodynamically unfavorable – deformations fairly readily [18]. Next, to consider dendrimer surfaces in comparison with proteins, synthetic dendritic macromolecules can be given a significant repertoire of tunable characteristics not found on natural proteins; this feature has greatly propelled the development of practical applications for these molecules. In particular, the surface of a protein contains a relative sparse complement of chemically reactive and accessible functional groups because most amino acid side chains are buried with the globular structure of the protein. By contrast, virtually all of the termini of dendritic branches, which can be customized with a wide range of chemical functionalities (Fig. 1.3), are oriented outward and are highly accessible on the surface of the dendrimer (Fig. 1.1). The consequent ability of a dendrimer to be functionalized with far more surface groups than a protein of comparable size [1, 19] has provided impetus to their widespread use as drug delivery vehicles. 1.3.2

Dendritic Macromolecules Possess a Wealth of Possible Applications

Within the past decade, the success of chemists in synthesizing mimics of natural dendrimers with a plethora of interesting physicochemical properties at the nanoscale has spurred efforts to find practical uses for these versatile nanodevices. Now that efforts to synthesize these molecules have reached fruition, there is a pleasing circularity that certain applications mirror natural processes considering that dendritic molecules were initially inspired by nature. In a dramatic example, a primary function of the leaves of real trees is for light harvesting; now, synthetic dendrimers have been created with highly-efficient light-harvesting antennae as well [8, 20]. Similarly, the dendritic network of hairs found on the Gecko foot that allows amazingly strong attachment to many types of surfaces through van der Walls forces [21] has led to efforts to create new forms of adhesives that are unaffected by the roughness, smoothness, wetness, or other macroscopic properties of the surface while providing strong but reversible adhesion. In addition to these two examples, many novel applications such as the exploitation of organometallic dendrimers as quantum dots for imaging, the solubilization of hydrophilic dyes in

1.3 Basic Properties and Applications of Dendrimers

Structural options for dendrimerbased drug delivery. Dendrimers can be synthesized with neutral surfaces (1) and positive (2) or negative (3) charges at the periphery; moreover, dendritic macromolecules, generally when larger than G3, can harbor non-covalently encapsulated guest/drug molecules [4 and discussion in Section 1.5.2]. An alternative strategy for drug delivery is through covalent conjugation of ligands (‘‘A’’ in 5) to the surface of the dendrimer (Section 1.5.3). The versatility of dendrimers for drug delivery is illustrated by considering that ‘‘A’’ could be a targeting Fig. 1.3.

ligand (Section 1.6.6.3) and the active drug could be encapsulated within the same macromolecule (6). Synthetic strategies are now available for providing dendritic clusters with extremely high densities of surface ligands (7) and for providing more than one type of surface ligand, either in a random orientation (8), or in blocks (9). The latter dendrimers are now being exploited in sophisticated cancer cell targeting (Fig. 1.4) and drug release (Section 1.5.3.3) strategies where A, B, and C can be any combination of targeting agents, drugs, contrast agents, or functional groups that improve pharmacological properties.

9

10

1 Dendrimers in Cancer Treatment and Diagnosis

apolar dendritic ‘‘solvents’’ [22], use as chemical catalysts, and in electronics as insulated molecular wires, light-emitting diodes, or fiber optics [12, 23, 24] have been reported. Besides their use for drug delivery and cancer therapy, the many emerging chemical, synthetic, research, and industrial uses for dendrimers are outside the scope of this article and will not be discussed further; the interested reader can consult chapter articles [1, 2, 11].

1.4

Methods for Dendrimer Synthesis 1.4.1

History and Basic Strategies

The ability to create homogeneous molecules with defined dendritic architecture and novel physicochemical properties at the sub-nano to nano-size scales occurred in chronological synchrony with the wide-spread application of nanobiotechnology to biology and medicine. Consequently, the parallel development of synthetic chemical methodology and the ever-increasing application of nano-tools in biomedicine triggered an explosive growth in the new field of dendrimer synthesis. This growth is evidenced by a cursory search for ‘‘dendrimers and synthesis’’ in the Web of Science database, which reveals that@2000 articles have been published on this topic since 1986. Clearly, a full discussion is beyond the scope of this report; excellent accounts and review articles on the synthesis of dendrimers by pioneers of the field have appeared at regular intervals [25–32] and are cited throughout this chapter. Nonetheless, a working knowledge of the chemical properties of dendrimers is critical to successfully devise efficacious therapeutic strategies with these versatile, but temperamental, macromolecules (as described in detail in Sections 1.5 and 1.6). Accordingly, we next provide an outline of the basic strategies and building blocks employed in dendrimer synthesis, with an emphasis on families of dendritic molecules that possess special properties – such as possessing cavities in their interiors suitable for host–guest complexation similar to enzyme– substrate complexes or displaying several functional groups on their surface appropriate for sophisticated drug delivery strategies – relevant to the field of biology and medicine. Cascade Reactions are the Foundation of Dendrimer Synthesis Although the term ‘‘dendrimer’’ was coined by Tomalia and coworkers less than two decades ago, the basic cascade or iterative methods that are currently employed for synthesis were known to chemists much earlier. For example, similar schemes form the basis of solid phase peptide synthesis. In turn, biology has long exploited similar iterative strategies in biochemical synthetic pathways; one example is provided by fatty acid biosynthesis [33]. Focusing on dendrimers, these macromolecules are constructed by performing simple chemical reactions in a repetitive or iterative manner by using small building blocks. In 1978 Vo¨gtle and coworkers re1.4.1.1

1.4 Methods for Dendrimer Synthesis

Synthetic approaches to dendrimers. (A) Cascade reaction sequences developed for the synthesis of ‘‘non-skid-chain like’’ polyaza macrocyclic compounds [34]. (B) Divergent approach – synthesis of radially symmetric PAMAM dendrimers using ammonia as the trivalent core; the generations are added at each synthetic cycle (two steps), leading

Fig. 1.4.

to an exponential increase in the number of surface functional groups [36]. (C) Convergent approach – synthesis of dendrons or wedges or branches that will become the periphery of the dendrimer when coupled to a multivalent core in the last step of the synthesis [40].

ported a similar approach, termed as cascade reactions, for the construction of nonskid-chain-like poly-aza macrocyclic molecules with well-defined architectures. Cascade synthesis is defined as ‘‘reactions where a functional group (e.g., amine) is made to react in such a way as to appear twice in the subsequent molecule or product’’ [34] (Fig. 1.4A). In the first step of the synthesis a primary or secondary amine was reacted with excess acrylonitrile in a Michael reaction to obtain a product with two arms [bis(2-cyanoethyl)amines]. In the second step the nitrile groups were reduced using cobalt(ii)/sodium borohydride to generate a new set of primary amine groups on both arms. The newly generated amino groups were then subject to identical reaction sequences iteratively to obtain the desired oligo-amine compounds.

11

12

1 Dendrimers in Cancer Treatment and Diagnosis

In 1985 Newkome and coworkers reported the synthesis of cascade molecules consisting of hydrocarbon core and shell with alcohol groups on the surface. These synthetic efforts were inspired by the Leuwenberg model of arboreal design; hence they named their synthetic macromolecular tree-like molecules ‘‘arborols’’ (Latin: arbor ¼ tree). Interestingly, characterization of these molecules showed they could be considered to be unimolecular micelles possessing cavities for encapsulation [35], a property that foreshadowed today’s efforts to use dendrimers for the delivery of encapsulated small molecule drug candidates (Section 1.5.2). Dendrimer Synthesis has Expanded Dramatically in the Past Two Decades In 1986, Tomalia and coworkers coined the now popular name ‘‘dendrimers’’ (Greek: dendron ¼ branch or tree-like) for radially symmetric branched molecules and reported the application of cascade synthesis for the synthesis of starburst dendrimers [36]. These researchers obtained homogeneous dendrimers by using a synthetic sequence of two simple reactions: (a) exhaustive Michael addition of ammonia to methyl acrylate and (b) exhaustive aminolysis of the resulting tri-ester derivative by ethylene diamine. The acrylate addition and aminolysis were repeated in an iterative manner, with excellent yields in each step, to prepare various molecules with increasing molecular weight and generations (Fig. 1.4B). The products with ester groups at the exterior were defined as G(m þ 0.5) generations and those with amine groups at the exterior were defined as G(m) (Fig. 1.1). This simple methodology is both powerful and versatile as it provided the ability to synthesize dendrimers with various surface properties. For instance, the ester groups could be hydrolyzed to present negatively charged carboxylate functional groups at the periphery or the amine groups could be protonated to present positive charges at the periphery. Electron micrographic studies showed the dendrimer with carboxylate groups of generation, G ¼ 4.5, to be highly monodispersed with a diameter of 88 G 10 A˚, compared with the theoretical value of @78 A˚. These dendrimers, when covalently attached to a polymeric backbone, were called ‘‘starburst polymers’’ or, less commonly, ‘‘cauliflower polymers’’ [7, 28]. 1.4.1.2

1.4.2

Strategies, Cores, and Building Blocks for Dendritic Macromolecules Dendrimers are Constructed from Simple ‘‘Building Blocks’’ In terms of synthesis, dendrimers can be constructed by using simple chemical reactions and building blocks reminiscent to the modular assembly of ‘‘LEGO’’ toys. Due to the ease, simplicity and repetitive nature of the synthetic methods, dendrimers based on organic, inorganic and organometallic molecular building blocks with greater than hundred different compositions are currently known, and new designs continue to be reported at a fast pace. In general, dendrimers consists of three major regions – (a) an initiator core, (b) a shell with extending arms or branches made of building blocks and (c) the exterior or outer-most surface groups on the termini of the branches. 1.4.2.1

1.4 Methods for Dendrimer Synthesis

There are innumerable ways of designing dendrimers [37–39]. For instance, the symmetry of the initiator core (Fig. 1.1) can be varied by using a wide range of molecules, which have included ammonia, a,o-diaminoalkanes, tri-substituted benzene, oligo- or polyalcohols, nucleic acids, amino acids, lipids, carbohydrates, or heteroatoms; many additional permutations are possible, e.g., the number of branching units in the initiator can be increased (tri- or tetra- or higher valency cores have been reported). Once the core moiety has been selected, options for the synthesis of the dendritic branches are equally numerous as various types of building blocks can be used, either singly or in combination with each other in the same dendritic macromolecule. The lengths of the dendritic arms, the nature of the surface, and the display of terminal functional groups can all be customized. The Synthesis of Dendrimers Follows Either a Divergent or Convergent Approach Dendrimers can be synthesized by two major approaches. In the divergent approach, used in early periods, the synthesis starts from the core of the dendrimer to which the arms are attached by adding building blocks in an exhaustive and step-wise manner. This process provides dendrimers with incrementally increasing generation numbers. However, only one type of reaction can be performed at each step, giving a uniform display of only one functional group on the exterior surface; moreover, defects in successive generations can arise due to incomplete reactions or steric hindrance (Fig. 1.4B). In the convergent approach, pioneered by Fre´chet and coworkers [40], synthesis starts from the exterior, beginning with the molecular structure that ultimately becomes the outer-most arm of the final dendrimer (Fig. 1.4C). In this strategy, the final generation number is pre-determined, necessitating the synthesis of branches of the various requisite sizes beforehand for each generation [31]. Small branches or dendrons are synthesized starting from the building blocks containing surface groups; these assemblies are then condensed with a multivalent core. This approach is versatile in the sense that branches of different molecular composition can be linked to a single core molecule, introducing regional variations on the final dendrimer (Fig. 1.3); this strategy also minimizes the introduction of defects at various stages of synthesis. 1.4.2.2

1.4.3

Heterogeneously-functionalized Dendrimers Basic Description and Synthetic Considerations By simultaneously conjugating appropriate targeting moieties, drugs, and imaging agents to dendritic polymers, ‘‘smart’’ drug-delivery nanodevices can be developed to target, deliver, and monitor the progression of therapy. For example, as will described in greater detail below, a dendrimer intended for cancer therapy needs to be functionalized with the drug itself, display a moiety for targeting to the tumor cells, as well as include surface groups designed to improve the pharmacological 1.4.3.1

13

14

1 Dendrimers in Cancer Treatment and Diagnosis

properties (e.g., to ensure water solubility, avoid non-specific uptake or immunogenicity). Several synthetic strategies – primarily the convergent method discussed above (Section 1.4.2.2) – have been developed that enable multiple species to be added to a dendritic surface in an ordered manner [41] and thereby achieve multiple functionalities within the same dendritic nanodevice (Fig. 1.3). The ability to create multi-functional nanodevices based on dendritic scaffolds, however, remains a challenging endeavor because conjugating several types of different molecules to a dendrimer is likely to change its physicochemical properties and resulting biological activity. Practically, additional synthetic steps required to fine-tune bioactivity and remedy bioincompatibility if it arises may render the whole process costinefficient at best and, more troublesomely, lead to loss of product uniformity, thereby negating a key benefit of dendrimers, i.e., their monodisperse, fully defined nature [42]. Glycosylation is an Example of Surface Modification with Multiple Bioactivities An outstanding demonstration of the synthetic power of decorating the surface of dendrimers with ‘‘interesting’’ molecules comes, once again, by way of comparison of these nanodevices with proteins. Proteins, which have had the opportunity to evolve biocompatibility and systemic functions in multicellular organisms over hundreds of millions of years, have found it advantageous to decorate their surfaces with complex carbohydrates when they are displayed on the cell surface or secreted into the extracellular milieu. In the past few years, it has become clear that these sugars play many key roles in molecular recognition over short distances, such as interactions with the extracellular matrix and with neighboring cells, as well providing system-wide communication (e.g., almost all protein hormones are glycosylated). When developing dendritic nanotools requiring bioactivity similar to that found in proteins, including the ability needed by a drug candidate to seek out and evoke responses at a specific but far-removed cell type in the body, it is wise to learn from nature and consider the inclusion of sugars to be an important design parameter. The ability to provide dendrimers with oligosaccharide coatings has been facilitated by the many functional groups that can be displayed on the surface and function as chemical handles for covalent attachment of a second group. A pioneering example of sugar display on a dendritic scaffold was provided by the unusual ninecarbon sugar sialic acid [43, 44]. This sugar, when displayed on human cells, serves as a critical binding epitope for the influenza virus. The virus, however, does not bind to soluble sialic acid, or sialic acid appended to a conventional polymer. Because these forms of sialic acid do not serve as effective binding elements, they are unable to act as a molecular decoys [45] and prevent the virus from binding to its real target, sialic acid on the human cell. By contrast, when sialic acid was conjugated to the surface of a dendritic polymer, it functioned as an effective and efficient binding decoy [46, 47], opening the door to the development of new diagnostic devices and novel anti-viral therapies [48, 49]. The molecular basis of the preferential recognition of sialic acid by the influenza virus when this sugar was 1.4.3.2

1.5 Dendrimers in Drug Delivery

displayed on a highly structured dendritic scaffold was traced to the ‘‘cluster glycoside effect’’ [50]. Over the past decade it has become firmly established that carbohydrate-based recognition depends on multiple simultaneous interactions to increase specificity and affinity [45, 51]. The demonstration that dendrimers provide an ideal synthetic platform for the appropriate display of carbohydrates to achieve the cluster glycoside response [52–54], along with improved methodology to synthesize glycoconjugated-dendrimers [43, 55], has driven the expansion of this approach from a single monosaccharide to a sugar-amino acid couple (the Tn antigen, which is N-acetylgalactosamine linked to serine [56]) to disaccharides (lactose [57] and the T-antigen [58]), and, finally, to tetrasaccharides (the sialyl Lewis X epitope [59]).

1.5

Dendrimers in Drug Delivery 1.5.1

Dendrimers are Versatile Nano-devices for the Delivery of Diverse Classes of Drugs

A successful drug must perform the demanding tasks of selectively recognizing and binding to a molecular target, then triggering an appropriate biological response, all the while possessing pharmacological properties that render it ‘‘drug-like’’. In some cases, nature has supplied compounds – such as aspirin or penicillin – that can be used directly as drugs but the more common situation is that many otherwise promising therapeutic agents are not successful in the clinic because of their poor pharmacological properties. The properties of dendrimers, in particular the synthetic ability to provide them with many different biological properties, along with their capacity to carry conjugated surface molecules or encapsulated guest molecules, make them immediately attractive as potential vehicles for drug delivery. Drug delivery efforts are complicated by the diversity of molecules that hold potential therapeutic or diagnostic value; briefly reviewing three classes of drug candidates based on size demonstrates the wide applicability of dendrimers to drug delivery. First, regarding ‘‘small molecules’’, many low molecular weight drug candidates are limited by poor solubility in aqueous environments or, if they are soluble, face rapid elimination from the bloodstream through filtration in the kidney. In the past, efforts have been made to modify the molecule itself, often following the ‘‘rule-of-five’’ guidelines developed by Lipinski to raise awareness of the properties and structural features that render molecules more or less ‘‘drug-like’’ [60]. Dendrimers present an attractive alternative strategy to the redesign of the drug because they allow unfavorable properties of a small molecule, such as insolubility, to be overcome by the larger characteristics of the macromolecule. An approach for improving the pharmacological properties of higher molecular weight drug candidates, analogous to Lipinski’s guidelines for the modification of small molecule drugs, has been applied for protein therapeutics such as recombinant antibodies

15

16

1 Dendrimers in Cancer Treatment and Diagnosis

and protein toxins used in cancer treatment. In these cases, the amino acid sequences of recombinant proteins have been ‘‘humanized’’ by genetic engineering to avoid immunogenicity [61, 62] and their glycosylation patterns have been modified to increase serum half-life [63, 64]. These efforts, undertaken with actual proteins, illuminate design features that can benefit the development of protein mimics, dendrimers. In particular, the ‘‘humanizing’’ experiments show that small changes, such as the substitution of a single amino acid for another, can avoid significant problems like undesired systemic immune responses. In the same manner, small changes in the surface properties of dendrimers, such as the addition of poly(ethylene glycol) (PEG), can avoid unwanted immunogenicity. Finally, even extremely large therapeutic candidates, notably plasmids or naked viral DNAs used for non-viral gene delivery that are well beyond the size of traditional drugs, are also benefiting from dendrimer-assisted delivery. The next section outlines specific approaches for the delivery of both small and large drug candidates by dendrimers. 1.5.2

Dendritic Drug Delivery: Encapsulation of Guest Molecules Dendrimers have Internal Cavities that can Host Encapsulated Guest Molecules The flexible branches of a dendrimer, when constructed appropriately, can provide a tailored sanctuary containing voids that provide a refuge from the outside environment [2] wherein drug molecules can be physically trapped [65] (Figs. 1.1 and 1.3). Encapsulation of hydrophilic, hydrophobic, or even amphiphilic compounds as guest molecules within a dendrimer [66] can be enhanced by providing various degrees of multiple hydrogen bonding sites or ionic interactions [65, 67] or highly hydrophobic interior void spaces [68, 69]. A wide variety of molecules have been successfully encapsulated inside dendrimers. In early experiments, compounds used to demonstrate the ‘‘guest molecule’’ concept included easy-to-visualize dye molecules such as rose bengal [66] and Reichardt’s dye [69] as well as pyridine [65] and peptides [67]. More recently, actual drugs, including 5-fluorouracil [70], 5-amino salicylic acid, pyridine, mefanminic acid and diclofenac [65], paclitaxel [71, 72], docetaxel [73], as well as the anticancer agent 10-hydroxycamptothecin [69], have been successfully encapsulated. Together, these results demonstrate that encapsulation is a general strategy for the delivery of low molecular weight compounds by dendrimers. This method is anticipated to be of particular value when display of the bioactive molecule on the surface of the dendrimer induces unwanted immunogenicity or reduces biocompatibility (Section 1.5.5). 1.5.2.1

Using Dendrimers for Gene Delivery The delivery of small molecules complexed as guest molecules in internal void spaces of dendrimers is, at least in retrospect, intuitively obvious. By contrast, the delivery of extremely large macromolecules, such as MDa-sized plasmid DNA for non-viral gene therapy, is counter-intuitive because the encapsulation of a ‘‘guest’’ molecule many times the molecular weight of the dendrimer itself appears impos1.5.2.2

1.5 Dendrimers in Drug Delivery

sible. Nonetheless, experimental evidence had demonstrated that gene delivery strategies also benefit from the participation of dendrimers [74]. For example, from its original discovery of efficacy for gene delivery [75], the fractured form of PAMAM, known as Superfect TM , is now a commercially-available transfection agent for in vitro applications [76]. Typical approaches to optimize dendritic gene delivery for in vivo use have involved the surface modification of a PAMAM backbone, either with arginine [77] or hydroxyl groups [78]. Alternatively, the results reported by Kim and coworkers, who demonstrated improved gene delivery with a novel PAMAM-PEG-PAMAM triblock copolymer, show that construction of dendrimers composed of new building blocks is warranted [76]. Although still in their infancy, there are efforts afoot to exploit dendrimers for the delivery of smaller nucleic acids such as antisense oligonucleotides and short interfering RNAs (siRNA); the success of these applications is likely to depend on the continuing development of novel materials for dendrimer synthesis [79]. Release of Encapsulated ‘‘Pro-drugs’’ Once a dendrimer carrying an encapsulated drug reaches the intended site of action, the guest molecule generally must be released to gain bioactivity. Indeed, a concern is that the active drug would ‘‘leak’’ out prematurely, thereby reducing the amount available for the intended therapeutic intervention, or more ominously, result in systemic toxicity. Reassuringly, early experiments showed that the close packing of dendritic branches on the surface of the macromolecule (Fig. 1.1) effectively formed a ‘‘membrane’’ that reduced diffusion to immeasurably slow rates [66]. In other cases, the release of encapsulated guest molecules was relatively faster, occurring over a few hours, apparently through hydrolytic degradation of the dendrimer in aqueous conditions [65]. The observation that guest molecules could be liberated at different rates demonstrated that viable opportunities exist to tailor the release for either slow or rapid delivery (Fig. 1.5). At present, additional control of delivery rates is being sought; for instance, the ability of a dendrimer to instantaneously release its entire drug payload upon reaching its cellular target would be valuable. Promising steps in this direction are being taken by the development of pH-sensitive materials [65], the fine tuning of hydrolytic release conditions, and the selective liberation of guest molecules on the basis of their size or shape [80]. 1.5.2.3

1.5.3

Covalent Conjugation Strategies Dendrimers Overcome many Limitations Inherent in Polymeric Conjugation Strategies The strategy of coupling small molecules to polymeric scaffolds by covalent linkages to improve their pharmacological properties has been under experimental test for over three decades [81–84]. Unfortunately, conventional linear polymers typically used in these efforts are plagued by inherent properties that render them distinctly ‘‘un-drug-like’’, including high polydispersity and size distributions, a 1.5.3.1

17

18

1 Dendrimers in Cancer Treatment and Diagnosis

Requirements for dendrimer-based, cancer-targeted drug delivery. (a) Dendrimers with multiple surface functional groups (Section 1.4.3) can be directed to cancer cells by tumor-targeting entities that include folate or antibodies specific for tumor-associated antigens (TAAs). (b) The next step is intake into the cell, which in the case of folate Fig. 1.5.

targeting occurs by membrane receptormediated endocytosis (Section 1.6.3.2). (c) Once inside the cell, the drug generally must be released from the dendrimer, which, for the self-immolative method (Section 1.5.3.3), results in the simultaneous disintegration of the dendritic scaffold (d).

lack of defined structure, and a low density of drug payload per unit volume or mass. Properties of dendrimers that overcome these problems include monodispersity that results in the ability to select the precise sizes of nanoparticle required to a specific application (Table 1.1), a fully defined structure that allows the presentation of attached conjugates in a defined architecture, a high ratio of drug payload to volume, and enhanced control over drug release rates. Unsurprisingly, based on these many beneficial features, a wide range of biologically active molecules have

1.5 Dendrimers in Drug Delivery

already been covalently attached to dendrimers. These conjugates range from small molecule drugs, such as ibuprofen [85], fluorescent and radioactive imaging agents (Section 1.6.4), oligonucleotides, oligosaccharides and peptides, as well as much larger molecules such as monoclonal antibodies (Section 1.6.3). Biologically active molecules attached to dendrimers can have two fundamentally different relationships to the host molecule. In some cases, exemplified by vaccine applications, there is no need to liberate active drug from the dendrimer (indeed, the success of antibody production usually depends on the unique display characteristics achieved by conjugation to the dendrimer). In most cases, however, the conjugated dendritic assembly functions as ‘‘pro-drug’’ where, upon internalization into the target cell, the conjugate must be liberated to activate the drug. Dendrimer Conjugates can be Used as Vaccines Most low molecular weight substances are not immunogenic; consequently, when it is desired to raise antibodies against small molecules, they must be conjugated to a macromolecule. In the past, natural proteins have commonly been used as carriers to generate antibodies to small molecules; now an alternative strategy using dendrimers has been demonstrated. In particular, unmodified PAMAM dendrimers that fail to elicit an antibody response on their own become haptenized upon protein conjugation and generate a dendrimer-dependent antigenic response [86, 87]. A specific example of this technique is provided by the dendrimeric presentation of antigenic HIV peptides, which proved superior to other multimeric presentation strategies, such as conjugation to dextran [88]. Notably, the immunogenicity of dendrimer conjugates is not limited to peptides antigens; in one study antibodies were produced against densely penicilloylated dendrimers that were subsequently used for the diagnostic testing of patients with potential allergy to b-lactam antibiotics [89]. Finally, although carbohydrate-conjugated dendrimers (Section 1.4.3.2) are typically non-immunogenic [1], antibodies can be successfully elicited against cancer-specific oligosaccharides displayed on a dendritic scaffold, offering a method for generation of a new class of cancer vaccines (Section 1.6.6.2). 1.5.3.2

Release of Covalently-delivered ‘‘Pro-drugs’’ Similar to encapsulated guest molecules that generally require release from the void spaces of a dendrimer to gain bioactivity (Section 1.5.2.3), a covalently delivered dendritic conjugate must also be cleaved within the target cell to regenerate the active cytotoxic agent (Fig. 1.5). At the same time, to ensure systemic nontoxicity, the covalent linker must be stable in circulation [90]. Several strategies are being pursued to ensure the successful cleavage and activation of the pro-drug in the target cell or tissue while avoiding systemic release. These include activation by low pH found in endosomal vesicles, installation of enzyme-cleavable ester linkages into the linkers that attach the pro-drug to the dendritic macromolecule, or disulfide bonds that are liberated in the reducing environment of the endoplasmic reticulum, photoactivation, or sensitivity to ultrasound [1]. Briefly returning to the benefits of dendritic clusters over conventional polymers for drug delivery, problems with the delivery of covalent conjugates when conven1.5.3.3

19

20

1 Dendrimers in Cancer Treatment and Diagnosis

tional polymers, such as poly(lactic acid) (PLA) or its copolymer with glycolide (PLGA), are used include a lack of sustained drug release [91]. Generally, these and other linear, randomly oriented polymers have an initial burst where as much as 50% of drug is released followed by a dramatic drop-off. An advantage of dendrimers is that their release rates are more consistent, which has been demonstrated by polylactide-PAMAM dendrimers [91] and dendrimer-platinate [92]. Consistent release from dendrimers is likely an inherent feature of their defined three-dimensional structure as their sites of drug attachment are continuously exposed to solvent, compared with random polymers where conjugated pro-drug moieties can be internalized randomly. The unique architectural features of dendrimers offer additional elegant strategies to gain exquisite control over release of active drug. In particular, the production of dendrimers functionalized with catalytic antibodies [68] has spurred the development of dendrimers capable of ‘‘selfimmolation’’ [93–95]. Self-immolative dendrimers provide an attractive potential platform for multidrug delivery. To briefly explain, these unique assemblies have the ability to release all of their tail units (i.e., the active drug) through a self-immolative chain fragmentation, which is initiated by a single cleavage at the dendrimer’s core [96]. The first generation of dendritic prodrugs was demonstrated by Shamis and coworkers who synthesized doxorubicin and camptothecin as tail units and designed a retro-aldol retro-Michael focal trigger provided by action of the catalytic antibody 38C2 [94]. This method showed a dramatic increase in toxicity to tumor cells upon bioactivation of the pro-drug compared with tests done in the absence of the activating antibody. This technology, when fully developed into a complete chemical adaptor system that combines a tumor-targeting device (Section 1.6.3), a pro-drug, and pro-drug activation trigger, provides a sophisticated platform for future research efforts and the development of drugs for in vivo use [93]. 1.5.4

Fine-tuning Dendrimer Properties to Facilitate Delivery and Ensure Bioactivity Delivery Requires Avoiding Non-specific Uptake From the initial entry into the body, a drug candidate confronts many barriers and diversions on its route to the site of intended bioactivity. Uptake by oral ingestion is ideal for patient comfort and, while still largely speculative for dendrimers [97], there is now evidence that uptake occurs in the rat gut [98]; this route is enticing based on an increasing recognition that nanoparticle uptake across the gut is largely governed by the physicochemical properties and surface chemistries of oral drug delivery vehicles [99]. Typically, to get to the target site in the body, the drug candidate must avoid becoming trapped with the extracellular matrix, which has been shown to hinder cellular uptake and reduce the efficiency of other nanosized delivery vehicles [100]; instead entry into the bloodstream is generally required for transit to the intended site of action. Once in the bloodstream, either by successful navigation of an oral route or through direct injection, dendrimers below a certain size are at risk of filtration 1.5.4.1

1.5 Dendrimers in Drug Delivery

and removal by the kidney. This pitfall, however, can be avoided by ensuring that sufficiently large dendrimers are used. Indeed an important design feature and overriding impetus to use dendrimer delivery vehicles is to prevent the filtration of these drug candidates by the kidney. A second, off-target ‘‘trap’’ for dendrimers has been identified in a study that showed sequestration of dendrimers in the liver and spleen, in part due to their surface properties and in part due to their size [101]. As discussed elsewhere, both of these parameters can be controlled with exquisite sensitivity for dendritic macromolecules, allowing longer residence times in the blood (the longer the serum half-life, the greater the opportunity to reach the intended site of action). ‘‘Local’’ Considerations: Contact with, and Uptake by, the Target Cell Once a dendrimer has successfully entered the bloodstream and has been designed to minimize undue accumulation in non-target organs or tissues, it still faces the challenge of seeking out and interacting with its targeted site of action. The diversity of cell surface targets available for a nanodevice to bind to is vast; here we limit ourselves to specific examples related to cancer (Section 1.6.3). We will jump ahead to the point when a dendrimer has made ‘‘first contact’’ with a cell and reflect on how it interacts with the membrane. In this regard, there are provocative studies with PAMAM polymers that suggest that binding to the cell surface is facilitated by the deformable properties of dendrimers [15, 16, 18] (Section 1.3.1.3). Cellbinding induced deformations, if they prove real, have important implications for drug delivery. For example, the flattened forms of dendrimers lose their internal voids where guest molecules – such as drug payloads – are sequestered [6]. If this step occurs too soon, i.e., outside of the target cell, the drug might be ineffective, whereas if it occurs at the right moment, i.e., in the cytosol for cytosolic-acting drugs, it would provide an additional design parameter to exploit in the drug release process (Section 1.5.2.3). Notably, the deformations proposed to occur upon the interaction of a dendrimer with a cell, where the dendrimer shifts from a canonical ‘‘spherical’’ shape to a flattened disk with a significant loss in volume, have been most-extensively investigated at the dendrimer–mica interface. Clearly, the plasma membrane of a cell shares few biophysical characteristics with an extremely flat and rigid surface of mica, therefore, combined with the thermodynamically unfavorable aspects of the putative shape change, the extrapolation to drug delivery in biological systems should not be overstated. Encouragingly, shape changes also have been observed – but not thoroughly characterized – for dendrimers encountering the air–water interface, which is a better model for biological systems. Regardless of the current lack of concrete information, the intriguing nature of this potential mechanism for cell targeting and drug release merits its discussion here and also warrants further experimental investigation. Once a dendrimer is in contact with a cell, there is strong experimental evidence that the exact surface properties of the dendrimer influence cellular uptake [102]. Therefore, the ability to modulate the chemical properties of a dendrimer provides additional options for controlling the uptake of a dendritic drug delivery device into a cell and even partitioning pro-drug release into specific organelles. To elaborate 1.5.4.2

21

22

1 Dendrimers in Cancer Treatment and Diagnosis

by briefly recapitulating a series of elegant experiment from the Banaszak Holl group, these researchers used a battery of assays, ranging from dye leakage to atomic force microscopy, to demonstrate that G5–G7 PAMAM dendrimers disrupt lipid bilayers and form holes large enough (5–40 nm) to account for dendrimer internalization. Moreover, the hole formation could be tuned by the exact size of the dendrimer, as well as surface chemical properties. To be specific, G7 amineterminated PAMAM initiated hole formation while its G5 counterpart did not. The smaller G5 dendrimer, however, did expand holes at existing defects; by contrast, acetamide terminated G5 PAMAM neither initiated hole formation nor expanded existing defects [102, 103]. The mechanism of hole formation in membranes by PAMAM was proposed to involve the removal of lipid molecules from the membrane to form aggregates consisting of a dendrimer surrounded by lipid molecules [103]. Once inside a cell, there are early indications that the precise properties of a dendrimer can influence subcellular trafficking. Eventually, if these processes can be better understood and controlled, their exploitation for drug delivery will be very attractive considering that some entities, such as dendrimerdelivered ibuprofen, need to only gain access to the cytosol [85], whereas other class of drugs, such as dendrimer-delivered plasmid DNAs, have the moredemanding task of reaching the nucleus [104]. 1.5.5

Drug Delivery: Ensuring the Biocompatibility of Dendritic Delivery Vehicles 1.5.5.1 Biocompatibility Entails Avoiding ‘‘Side Effects’’ such as Toxicity and Immunogenicity To briefly reiterate, properties of dendritic polymers important for drug delivery include negligible polydispersity, a high-density payload of pro-drug, and the ability to selectively release the active form of drug precisely at its intended site of action. Although dendrimers are capable of each of these tasks, their advantages are for naught if the final dendritic complex is not ‘‘biocompatible.’’ Biocompatibility, a broad term with numerous meanings, will be considered here from three perspectives, water solubility, lack of immunogenicity, and toxicity.

Water Solubility and Immunogenicity The first two biocompatibility issues mentioned above, namely water solubility and immunogenicity, are closely related insofar as highly-hydrated macromolecules tend to be less immunogenic. With dendrimers, there are many options available to overcome difficulties that arise in these areas. For example, solubility can be readily adjusted by surface modifications to surface chemistry or by the addition of conjugated ligands (Section 1.5.3, Fig. 1.3). Moreover, dendrimers such as the commonly used G3, G5, and G7 PAMAM clusters are not inherently immunogenic [105]. Derivatized PAMAM such as the G4D-(1B4M-Gd)62 magnetic resonance imaging (MRI) contrast dendrimer, however, can become immunogenic (which is not surprising considering the deliberate efforts to render small molecules immunogenic through presentation on a dendritic scaffold). This problem – 1.5.5.2

1.5 Dendrimers in Drug Delivery

once again tying together the concepts of solubility and immunity – was overcome in one study by conjugation of poly(ethylene glycol) (PEG) to the surface of the dendrimer. Notably, PEG also had the positive effect of decreasing non-specific clearance from the blood, likely due to the increased hydration and resulting solubility of the particle [106]. Inherent and Induced Toxicity A basic issue in drug delivery is the avoidance of non-specific, systemic, or offtarget toxicity. At its simplest this issue, when applied to dendrimers, involves the biological effects of the material used to construct the polymer. Ideally, the building blocks themselves, as well as their degradation products upon delivery and release of the drug payload, are non-toxic. One strategy is to directly use natural biological molecules, such as carbohydrates [59, 107], amino acids and peptides [108], nucleic acids [109–113], or lipids [114, 115] as the building blocks. To provide additional synthetic flexibility, while maintaining biocompatibility, an increasing number of biologically compatible and generally-regarded as safe (GRAS) materials are being used in dendrimer construction. Examples include dendritic polyglycerol [116], melamine [117]; phosphate [118], polyglycerols [39], a polyester dendrimer based on poly(ethylene oxide) that has tunable molecular weights and architectures [84], and dendrimers composed of citric acid and poly(ethylene glycol) [65]. The pioneering PAMAM-based dendrimers illustrate a second issue beyond inherent toxicity of the material or breakdown products, namely ‘‘induced’’ toxicity. The PAMAM family (Table 1.1), although not explicitly designed for biocompatibility, was found to be non-toxic when generations 1 through 5 were tested [105]. Evaluation of G7 dendrimers, however, showed potential biological complications, including dose-dependent toxicity [105], thereby illustrating that, while the basic material of PAMAM is inherently non-toxic, deleterious outcomes could be ‘‘induced’’ by factors such as the size or structure of the nanodevice. Smaller generation, non-toxic, dendrimers are sufficient for some applications but larger clusters are needed to fully exploit the enhanced permeation and retention (EPR) effect important in the treatment of cancer with macromolecular therapeutics (Section 1.6.2); consequently, toxicity cannot simply be avoided by restricting use to small, safe-sized particles. Instead, one strategy devised to avoid toxicity was the re-design of the building blocks of PAMAM-based material [76, 119] while another strategy involved the development of completely new polymeric backbones [120]. The selection of ‘‘safe’’ building blocks to avoid deleterious effects in dendrimer construction is unlikely to prevent all problems. To illustrate, even very safe building blocks, such as amino acids, can be highly toxic or immunogenic when assembled into large macromolecules – in this case proteins – in the ‘‘wrong’’ way. Indeed, the toxicity of dendrimers could be the result of several factors beyond the simple properties of the unloaded scaffold. For instance, with cancer drugs intended to kill cells, systemic toxicity could result if the drug is taken up by the wrong cellular target (i.e., a healthy cell or tissue, rather than a cancer cell or tumor) or if the nanodevice was ‘‘leaky’’ (i.e., if the pro-drug was released systemically before reaching the target cell). Fortunately, many strategies exist for prevent1.5.5.3

23

24

1 Dendrimers in Cancer Treatment and Diagnosis

ing toxicity, including directing a drug to its intended site of action by targeting moieties (Section 1.6.3) and developing sophisticated release strategies (Section 1.5.3.3). Problems that arise from the surface properties of the conjugated dendrimer can be ameliorated by masking the surface with something as simple as PEG or, in more advanced schemes, by coating with sugars or peptides to make glycodendrimers or peptide dendrimers, respectively (discussed in Ref. [121]) to mimic proteins naturally found in circulation (Section 1.4.3.2).

1.6

Dendrimers in Cancer Diagnosis and Treatment 1.6.1

Dendrimers have Attractive Properties for Cancer Treatment

Cancer epitomizes the challenges faced during drug delivery: an anticancer drug must be able to seek out subtle changes that distinguish a transformed cell from the other 200 or so types of healthy cells found in the body and then provide a sufficiently high dose of a toxic agent to selectively kill the cell while not harming its healthy neighbors. Therefore, even though dendrimers can be endowed with many favorable properties for drug delivery (Section 1.5), an ultimate challenge – ergo, a ‘‘real-world’’ test – of these versatile nano-devices will be whether they can successfully meet the formidable tasks of diagnosing and treating of malignant disease. As described in Section 1.7, although significant work remains in several areas, prospects now appear bright for dendrimer-based approaches to cancer treatment. 1.6.2

Dendrimer-sized Particles Passively Accumulate at the Sites of Tumors

To begin the discussion of properties that make dendrimers attractive vehicles for cancer treatment, we revisit the concept that encapsulation (Section 1.5.2) or covalent linkage (Section 1.5.3) of small molecule drug candidates to a dendrimer enhances the pharmacological properties of the drug. In cancer chemotherapy, these desirable size-based features are reinforced by the enhanced permeability and retention (EPR) effect that improves the delivery of macromolecules to tumors. The EPR effect is based on unique pathophysiological features of a solid tumor, such as extensive angiogenesis resulting in hyper-vascularization, limited lymphatic drainage, and increased permeability to lipids and macromolecules. These features, which help ensure adequate nutrient supply to meet the metabolic requirements of rapidly growing tumors [122, 123], can be turned to the tumor’s disadvantage by the use of nano-sized therapeutic agents. The EPR effect was discovered when selective accumulation of the SMANCS conjugate (styrene-maleic anhydride-neocarzinostatin) was observed at the site of tumors while similar accumulation was not seen with neocarzinostatin alone

1.6 Dendrimers in Cancer Diagnosis and Treatment

[124, 125]. The EPR response was subsequently demonstrated for similarly-sized liposomes, thereby establishing that this effect was largely a function of particle size and did not solely depend on the chemical or biophysical properties of the macromolecule. Specifically, in one study optimal tumor delivery occurred for liposomes having a size distribution between 70 and 200 nm in diameter [126]. An independent study showed efficacy for liposomes loaded with daunorubicin in the same size range; specifically, those @142 nm in diameter exhibited an inhibitory effect against Yoshida sarcoma whereas smaller (@57–58 nm) and larger (@272 nm) liposomes had weaker or no effect [127]. Over time, cautionary notes were raised that tempered initial enthusiasm for exploiting the EPR effect for cancer treatment. For example, the porosity of the vasculature in tumors can be highly variable even with a single vessel that can be leaky to one size of particle in one region but not in another [128]. Experimentally addressing this issue was complicated by the size polydispersity of traditional nanoparticles used to exploit the EPR effect, which were typically either lipids or conventional polymers that rendered a significant proportion of intended drug inactive. Fortunately this issue – the ability to match exact and uniform sizes needed to target an individual tumor – is highly tractable with dendrimers because selection of an exactly-sized entity is possible (Table 1.1) compared with the large size distributions that plague liposome and most polymeric materials [42]. The ability to construct monodisperse populations of dendrimers in the size range needed to exploit the EPR effect is an encouraging step towards the passive exploitation of tumor properties. Once the basic issue of size was resolved, however, secondary challenges (and opportunities) arose from observations that the chemical properties of the nano-sized particle can play significant roles in modulating the EPR effect. By way of a specific example, ‘‘conventional’’ polymeric materials showed efficacy at a smaller size range, occurring at ~60 nm for both watersoluble and hydrogel forms of poly(vinyl alcohol) (PVA) [129], whereas almost identically-sized 57 nm egg phosphatidylcholine (EPC)-liposomes were ineffective [127]. As reported above, liposomes about twice this size showed maximal efficacy, so it was not unexpected that the EPC-liposomes were ineffective. Interestingly, however, hydrogenated egg phosphatidylcholine (HEPC)-liposomes in this size range (specifically, 58 nm) were active [127], illustrating that the exact chemical properties of the material is a critical design parameter. In this respect, the many options for dendrimer ‘‘building blocks’’, as well as the ability to further tune surface properties provide many opportunities to endow dendrimers with favorable ‘‘passive’’ properties for tumor targeting. 1.6.3

Multifunctional Dendrimers can Selectively Target Biomarkers found on Cancer Cells 1.6.3.1 Methods for Targeting Specific Biomarkers of Cancer As discussed above, dendrimers can achieve passive EPR-mediated targeting to a tumor simply by control of their size and physicochemical properties. Passive tar-

25

26

1 Dendrimers in Cancer Treatment and Diagnosis

geting, which localizes the nano-particle in the close vicinity of a cancer cell, can be immediately useful for diagnostic purposes (Section 1.6.4) or for the delivery of radioisotopes capable of killing any cell within a defined radius. In general, however, most delivery strategies require that the anticancer agent directly attached to, or be taken up by, the target cell. The ability to append more than one type of functionality to a dendrimer (Fig. 1.3) allows the inclusion of ligands intended to bind specifically to cancer cells in the design of a multi-functional drug-delivery nanodevice (Fig. 1.5). Although a wide range of targeting ligands have been considered, including natural biopolymers such as oligopeptides, oligosaccharides, and polysaccharides such as hyaluronic acid, or polyunsaturated fatty acids [90, 130], discussion here is limited to folate, which is an exemplary small molecule tumor-targeting agent [42], as well as monoclonal antibodies directed against tumor associated antigens (TAAs). Targeting by Folate, a Small Molecule Ligand Folate is an attractive small molecule for use as a tumor targeting ligand because the membrane-bound folate receptor (FR) is overexpressed on a wide range of human cancers, including those originating in ovary, lung, breast, endometrium, kidney and brain [131]. As a small molecule, it is presumed to be non-immunogenic, it has good solubility, binds to its receptor with high affinity when conjugated to a wide array of conjugates, including protein toxins, radioactive imaging agents, MRI contrast agents, liposomes, gene transfer vectors, antisense oligonucleotides, ribozymes, antibodies [131, 132] and even activated T-cells [133]. Upon binding to the folate receptor, folate-conjugated drug conjugates are shuttled into the cell via an endocytic mechanism, resulting in major enhancements in cancer cell specificity and selectivity over their non-targeted formulation counterparts [131, 132]. Recently, folate has been enlisted in an innovative dendrimer-based targeting schemes ([42, 134], Section 1.6.6.1). 1.6.3.2

Targeting by Monoclonal Antibodies Of the many strategies devised to selectively direct drugs to cancer cells, perhaps the most elegant (and demanding!) is the use of monoclonal antibodies that recognize and selectively bind to tumor associated antigens (TAAs) [135–138]. TAA-targeting monoclonal antibodies have been exploited as delivery agents for conjugated ‘‘payloads’’ such as small molecule drugs and prodrugs, radioisotopes, and cytokines [139, 140]. The field of ‘‘immunotherapy’’ envisioned almost a hundred years ago, and given renewed impetus a quarter century ago by the development of monoclonal antibody technologies, has nonetheless progressed erratically over the past two decades as many pitfalls have been encountered [139]. Current prospects remain mixed but hopeful; optimistically, progress marked by commercial interest with companies providing their immunotherapeutic drug candidates with flashy trademarked names, such as ‘‘Armed Antibodies TM’’ [141]. Similarly, the rosy opinion that this field is ‘‘on the verge of clinical fruition’’ has been published recently [142]. Perhaps, more realistically, one recent synopsis 1.6.3.3

1.6 Dendrimers in Cancer Diagnosis and Treatment

holds out ‘‘hope’’ for a major clinical impact for this strategy within the next 10 years [136]. Although a detailed discussion of the many pitfalls encountered in immunotherapy efforts is beyond the scope of this chapter, one key issue – readily addressed by dendrimers – is the requirement that an extremely potent cytotoxic drug be used in targeted antibody therapy. This point is illustrated by the fact that the greatest progress in this field has occurred for immunotoxins, which are antibody–toxin chimeric molecules that kill cancer cells via binding to a surface antigen, internalization and delivery of the toxin moiety to the cell cytosol. In the cytosol, protein toxins, such as those from diphtheria or pseudomonas, catalytically inhibit a critical cell function and cause cell death [143]. The high potency of immunotoxins for killing cancer cells is dramatically illustrated by ricin, where the catalytic activity of this ribosome-inactivating enzyme allows a single immunotoxin conjugate to kill a cell upon successful uptake and trafficking to the site of action [144, 145]. A drawback of immunotoxins is their significant immunogenicity, which limits repeated use [136]; from a broader perspective, their repeated use is made necessary by difficulties in providing a sufficiently high drug load to eradicate all cancer cells despite the high potency of conjugated toxin. An alternative approach of radioimmunotherapy, where high energy radionuclides are conjugated to TAA-targeting antibodies, also shows promise [146] but suffers from indiscriminate toxicity (the surrounding healthy tissues, as well as off-target tissues, become irradiated in addition to the target cancer cells). A third possible approach for immunotherapy, the conjugation of commonly-used small molecule drugs to TAAs, is hindered by the relatively low potency of most low molecular weight therapeutics. To illustrate this point, @10 000 TAAs occur on a typical cancer cell [101], making this number the upper limit for the number of targeting antibodies that can bind to the cell. The widely used anticancer drug cisplatin, to give one example, requires internalization of at least 50
this level of drug molecules for therapeutic efficacy. A numerical analysis of the cisplatin example presented above indicates that each tumor-targeting antibody would have to be modified with a large number of small molecules to be effective as an anticancer drug (in this case, roughly 50 cisplatin molecules upon superficial analysis). Modification of an antibody with multiple radioisotopes, toxins, or even small molecules to increase the efficacy of cell killing, however, diminishes or eliminates the inherent specific antigen-binding affinity of an antibody. Therefore, to maximize drug loading while minimizing the deleterious effects on the biological integrity of the host antibody, an attractive approach is to use a linker molecule, such as a dendrimer, that can be highly conjugated (or internally loaded) with drug while modifying only a single site on the surface of the antibody [147]. Methodology to covalently attach antibodies to dendrimers that preserve the activity of the antigen–antibody binding site [148, 149], e.g., by chemical modification of their carbohydrates and subsequent linkage to PAMAM [150], has opened the door for the inclusion of dendrimers in immunotherapy [151, 152], thereby enhancing the future prospects of this chronically ‘‘almost-there’’ strategy.

27

28

1 Dendrimers in Cancer Treatment and Diagnosis

1.6.4

Dendrimers in Cancer Diagnosis and Imaging Labeled Dendrimers are Important Research Tools for Biodistribution Studies The synthetic ability to attach both a tumor-targeting antibody and a potent payload of anticancer drugs to the same dendritic molecule provides a platform for multifunctional nano-scale drug delivery devices (Fig. 1.5). Before this technology can be applied in the clinic, however, its safety and efficacy must be demonstrated; towards this end, fluorescently-modified dendritic conjugates have been used extensively to characterize cell targeting, surface binding, uptake and internalization, and even sub-cellular localization [85, 151, 152]. The radiolabeled counterparts appropriate for animal studies have allowed detailed examination of the biodistribution of dendrimers. Several radio-isotopes have been conjugated to dendrimers, including 3 H [153], 14 C [105], 88 Y [154], 111 In [154, 155], and 125 I [98, 149, 156–158]. These studies have established that the chemical and physical properties of dendrimers can be tuned to favor distribution to or away from specific organs and, ultimately, to achieve favorable biodistribution to tumors. The methods used in these experiments, however, typically requiring post-administration dissection of the host animal to allow the analysis of organ sequestration and tissue distribution of the radioisotope, are clearly not applicable to clinical practice. Instead, they have served as an important stepping stone along the path towards non- or minimally-invasive diagnostic procedures, which are proceeding mainly by the development of MRI contrast agents. 1.6.4.1

Towards Clinical Use: MRI Imaging Agents Upon successful demonstration of the selective accumulation of dendrimers at the sites of tumors in animal models, a natural extension of this approach was to substitute gadolinium for the previously-tested isotopes or fluorophores. Gadolinium ( 153 Gd) is the best known and most extensively utilized magnetic resonance (MR) contrast agent [159, 160] and has previously been shown to be valuable for the improved diagnosis of cancer [161, 162]. Importantly, the in vivo efficacy of gadolinium is greatly enhanced when used as part of a macromolecular system [159]; in the past, attempts to create macromolecular gadolinium platforms have included the conjugation of chelators for this metal to both proteins [163] and conventional polymers [164]. These efforts have met with mixed (but generally limited) success. By contrast, Kobayashi and Brechbiel report that, by conjugating gadolinium to dendrimers, the unique properties of these polymers, such as exquisite size control, allowed selective targeting and imaging of the kidney, vascular, liver, or tumors [159]. Of note, tumor specific targeting and accumulation of gadolinium contrast agents is possible by use of either the folate receptor [165] or TAAs [159]. A drawback of the initial PAMAM-based MR contrast agents was their long residence time in the body; this problem, however, can be met by modifying both the surface properties [106] and basic chemical composition of the dendrimer. Specifically, diaminobutane (DAB) dendrimer-based chelators were more rapidly excreted from the 1.6.4.2

1.6 Dendrimers in Cancer Diagnosis and Treatment

body, illustrating that the development of clinically-acceptable dendrimer MR platforms is realistic [166]. 1.6.5

Steps Towards the Clinical Realization of Dendrimer-based Cancer Therapies The Stage is now set for Dendrimer-based Cancer Therapy The use of dendrimers for cancer treatment is still in its infancy with few, if any, applications successfully translated to the clinic. Consequently, their use as diagnostic agents constitutes both an important goal in and of itself, and also a valuable ‘‘baby step’’ towards the ultimate goal of curing cancer. As discussed, the process of actual killing cancer cells entails the complicated process of drug uptake followed by release of the drug into the cytoplasm or nucleus and is clearly a more demanding process than cell surface labeling, or even localization to the vicinity of the tumor, sufficient for diagnostic purposes. Nonetheless, in some cases, the transition from imaging to therapy will be closely linked, as evidenced by efforts now underway to combine antibody-targeted MR imaging nanoparticles with the delivery of antiangiogenic genes intended to inhibit the vascularization to the V2 carcinoma model in rabbits [167]. Another promising strategy – boron neutron capture therapy – has undergone impressive development over the past decade and is presented next as a successful demonstration of the promise of dendrimer-based cancer therapies. 1.6.5.1

Boron Neutron Capture Therapy Cisplatin-based therapies illustrate the need for multiple conjugations of small molecules – estimated at 50 for this platinum drug – to a targeting antibody (Section 1.6.3.3). While some efforts are underway to use dendrimeric strategies for platinum drug delivery [168], an even more demanding situation, where thousands of ligands are required per targeting antibody, is provided by boron neutron capture therapy (BNCT). Accordingly, BNCT will be discussed here as an illustrative example of how dendrimers can help overcome high hurdles in the development of innovative cancer therapies. As a brief background, BNCT is based on the nuclear reaction that occurs when boron-10, a stable isotope, is irradiated with low energy (a 0.025 eV) or thermal neutrons to yield alpha particles and recoiling lithium-7 nuclei. A major requirement for the success of BNCT is the selective delivery of a sufficient number of boron atoms (@10 9 ) to individual cancer cells to sustain a lethal 10 B(n, alpha) ! 7 Li capture reaction [169, 170]. Considering that the maximal number of antigenic sites per tumor cell is in the range of 100 000, and more commonly only 1/10 th that level, an a priori calculation suggests that each targeting antibody must be linked to at least 2000, but preferably closer to 5000, boron atoms [101]. Clearly, a single TAA-targeting antibody cannot be directly conjugated at this level and conventional polymers – e.g., polylysine conjugated with @1700 boron derivatives and linked to a targeting antibody – caused the antibody to lose in vivo tumor localizing properties [171]. By contrast, when a 1.6.5.2

29

30

1 Dendrimers in Cancer Treatment and Diagnosis

PAMAM dendrimer was used for polyvalent boron conjugation, the linked antibody maintained immuno-recognition (although in vivo tumor targeting remained problematic because the conjugated dendrimer had a strong propensity to mislocalize in the spleen and liver) [101]. Over the decade since these pioneering efforts were first reported, continued progress has been made to solve problems such as off-target tissue localization, which was traced to the size of the dendrimer and presence of a large number of amine groups on the surface of PAMAM, by exploiting the versatility of dendrimer chemistry. In short, the re-design of boronated, anti-body-targeted dendrimers has culminated in the successful treatment of gliomas in the rat [158, 169, 172] and laid the foundation for translation of this technology into clinical tests in the foreseeable future. 1.6.6

Innovations Promise to Speed Progress ‘‘Mix-and-Match’’ Strategy of Bifunctional Dendritic Clusters Two lessons are immediately apparent from the dedicated efforts to bring dendrimer-based BNCT to fruition. One is that dendritic technologies, while still at an early developmental stage, hold tremendous promise and merit continued investigation. The second is that the coupling of one treatment modality (BNCT) with one targeting strategy (antibodies to a specific type of glioma) required a staggering amount of effort. The growing realization that cancer is hundreds, if not thousands, of unique diseases at the cellular and molecular level, suggests that a commensurate number of therapeutic strategies are needed. The diversity of targeting strategies (which are not limited to folate and TAAs discussed here), coupled with the many ‘‘payload’’ possibilities (beside radioisotopes, boron, and cisplatin discussed here) used to diagnose and kill cancer cells, means that there are literally tens of thousands of individually customized therapies required to fully confront the myriad clinical manifestations of cancer. The sobering reality is that, if each of these customized treatments will require a decade long effort by a large team of researchers and clinicians, the large problem of cancer treatment will not be solved for a long time. Choi and coworkers [134] have come up with an innovative mix-and-match scheme that promises to offset this gloomy prediction. These researchers have recently reported a cancer-targeting strategy that is reminiscent of the antibody– toxin/immunoconjugate strategy where distinct, but linked, entities are used to first recognize and bind and then subsequently modify a cancer cell. Their strategy, however, has great potential to improve on both the ‘‘targeting’’ and ‘‘payload’’ aspects of cancer therapy by, at first seemingly paradoxically, completely dividing these functions into separate dendritic clusters (Fig. 1.6). The key to this approach was to include a DNA ‘‘zipper’’ on each dendrimer that allows the targeting cluster, composed of folate-derivatized PAMAM in proof-of-concept experiments [173], to be readily combined with the imaging or drug-carrying dendrimer by way of the complementary DNA strand [134]. It can be envisioned that the production of libraries of dendrimers targeted to different cancer-specific biomarkers can be pro1.6.6.1

1.6 Dendrimers in Cancer Diagnosis and Treatment

DNA–dendrimer conjugates as potential cancer targeting imaging agents or therapeutics. (Adapted from Ref. [189].) Differentially functionalized dendrimers covalently conjugated to complementary deoxyoligonucleotides can readily form duplex combinatorial nanoclusters that possess

Fig. 1.6.

cancer cell-specific ligands hybridized to an imaging agent or drug. Cell-specific targeting ligands (e.g., folic acid in one study) are appended to Dendrimer A, and Dendrimer B is conjugated with an imaging agent or drug [134].

duced by a ‘‘mix-and-matched’’ strategy by combining ‘‘off-the-shelf ’’ targeting and drug clusters as needed [42]. Development of easily-customizable nanomedicine platforms that exploit the facile duplex DNA formation for the generation of hybrid nano-clusters, thus circumventing the tedious synthesis of multiply-functionalized dendrimers, offers hope that the next ten years will witness rapid expansion of dendrimer technologies that build on the painstaking advances of the past decade. Towards Therapeutic Exploitation of Glycosylation Abnormalities found in Cancer Aberrant glycosylation, where the patterns of complex carbohydrate glycoforms found on the surfaces of cancer cells are dramatically different from those on healthy cells, is a hallmark of cancer [174–178]. Efforts to exploit these changes therapeutically, however, have long been stymied by the difficulty of controlling these complex and diverse molecules in an artificial synthetic setting. Today, with new technologies such as dendrimers that provide a platform for physiologicallyrelevant display of carbohydrates, new vistas are opening up for exploiting these molecules to intervene in malignant disease. Promising – but still early-stage – efforts in this direction include the presentation of oligosaccharides found only in cancer cells [53, 56, 58, 179–181] on a dendritic scaffold (Section 1.4.3.2) for vaccine development (Section 1.5.3.2). 1.6.6.2

Towards Targeting Metabolically-engineered Carbohydrate Epitopes As discussed above, one area of rapidly-expanding investigation is the abnormal glycosylation associated with the cancer cells; in particular dendrimeric scaffolds provide a unique platform to control the multimeric carbohydrate presentation needed to enact the ‘‘cluster glycoside effect’’ [45, 50, 51], which is crucial for tar1.6.6.3

31

32

1 Dendrimers in Cancer Treatment and Diagnosis

geting diseased tissues found in malignant diseases [1, 24]. Another approach to exploiting glycosylation for the treatment of cancer is through ‘‘chemical biology’’ strategies, such as the ability to express non-natural sialic acids on the cell surface through the use of N-acetylmannosamine (ManNAc) analogs [49, 182, 183] (Fig. 1.7). By appropriate design of the ManNAc analog, sialic acids, which are interesting nine-carbon sugars often overexpressed on cancer cells [175], can be provided with a ‘‘chemical handle’’ – such as a ketone, azide, or thiol [184–186] – for tar-

Chemoselective targeting of drugloaded dendrimers to the cell surface. (A) Overview of sialic acid engineering. (a) A dendrimer can encapsulate and assist the delivery of N-acetylmannosamine (ManNAc) analogs, such as the thiol-containing sugar ‘‘ManNTGc’’ (shown as ‘‘*’’) into a cell (Section 1.5.2). (b) Once inside the cell, ManNTGc can be metabolically converted into CMP-Neu5TGc, a compound that serves as a sugar-nucleotide needed for the glycosylation process (c) where ‘‘Neu5TGc’’ a non-natural form of sialic acid, is installed into cell surface glycoconjugates. Overall, this process replaces Fig. 1.7.

natural sialic acids, such as ‘‘Neu5Ac’’, with their thiol-containing counterparts (d), which can then be targeted by dendritic assemblies such as the bifunctional ‘‘targeting’’ and ‘‘payload’’ clusters shown in Fig. 1.6. (B) Details of the ‘‘chemoselective ligation reaction’’ required for targeting the appropriately derivatized dendrimeric assembly to the cell. In this case, a maleimideconjugated targeting capsule will selectively interact with the sialic acid-display thiols to covalently bind the dendrimer to the cell surface via thio-ether bond formation.

References

geted delivery of a second agent such as the ricin A-chain used in immunotoxins [187] or small molecule anticancer drugs [188]. Dendrimers offer assistance at several steps in this process of translating early-stage anticancer strategies like ‘‘sialic acid engineering’’ from the laboratory to clinical relevance. An enticing proposition is that the starting material – ManNAc, which like all sugars has notoriously poor pharmacological properties – can be made ‘‘drug-like’’ by encapsulation (or covalent ligation). Subsequently, after display of the target epitope on the cell surface, which is a modified thiol-bearing sialic acid in the case shown in Fig. 1.7, this can benefit from the high local density of dendritic display of maleimide to increase the rate of drug binding to the cell surface, which occurs over an unacceptably long period of several hours for current covalent coupling schemes [188]. This strategy, under evaluation in our laboratory, coupled with a high drug payload on the DNA-hybridized cluster (Fig. 1.6), provides renewed impetus for the already promising application of sugar-based therapeutic approaches to cancer. A particularly attractive aspect of this approach is that @10 8 sialic acids exist on cancer cells, greatly improving prospects to deliver adequate levels of drug to achieve therapeutic efficacy compared with TAA-targeting schemes (Section 1.6.3).

1.7

Concluding Remarks

Dendrimers, chemically-defined entities with tunable biological properties, have advanced over the past two decades to the point where they stand on the cusp of major contributions to the treatment of cancer in a meaningful way. Although, as has been apparent by the many instances cited throughout this chapter where gaps in knowledge still remain and that must be plugged before dendrimers are ready for wide clinical use, their extreme versatility combined with the extensive research efforts now underway are sure to add sophistication to drugs already in use as well as spur the development of entirely new classes of anticancer therapy.

Acknowledgments

Funding was provided by the Whitaker Biomedical Engineering Institute and Department of Biomedical Engineering at The Johns Hopkins University, the Arnold and Mabel Beckman Foundation, and The National Institutes of Health.

References 1 U. Boas, P. M. H. Heegaard,

Dendrimers in drug research. Chem. Soc. Rev. 2004. 33, 43–63. 2 G. M. Dykes, Dendrimers: A review of their appeal and applications. J.

Chem. Technol. Biotechnol. 2001. 76, 903–918. 3 A. Sunder, J. Heinemann, H. Frey, Controlling the growth of polymer trees: Concepts and perspectives for

33

34

1 Dendrimers in Cancer Treatment and Diagnosis

4

5

6

7

8

9

10

11

12

13

14

hyperbranched polymers. Chem. Eur. J. 2000. 6, 2499–2506. ‘‘Starburst2’’ is an official trademark owned by Dow Chemical that covers PAMAM based dendrimers but the term ‘‘starburst’’ is often also used generically to describe the properties of certain dendritic macromolecules. P. K. Maiti, T. C ¸ agin, G. Wang, W. A. Goddard III, Structure of PAMAM dendrimers: Generations 1 through 11. Macromolecules 2004. 37, 6236–6254. ¨ ter, J. P. Rabe, A. D. Schlu Dendronized polymers: Synthesis, characterization, assembly at interfaces, and manipulation. Angew. Chem. Int. Ed. 2000. 39, 864–883. P. G. de Gennes, H. Hervet, Statistics of ‘‘starburst’’ polymers. J. Phys. Lett. 1983. 44, L351–L360. B. Klajnert, M. Bryszewska, Dendrimers: Properties and applications. Acta Biochim. Pol. 2001. 48, 199–208. J. M. J. Fre´chet, Functional polymers and dendrimers: Reactivity, molecular architecture, and interfacial energy. Science 1994. 263, 1710–1715. C. L. Jackson, H. D. Chanzy, F. P. Booy, B. J. Drake, D. A. Tomalia, B. J. Bauer, E. J. Amis, Visualization of dendrimer molecules by transmission electron microscopy (TEM): Staining methods and Cryo-TEM of vitrified solutions. Macromolecules 1998. 31, 6259–6265. D. K. Smith, F. Diederich, Functional dendrimers: Unique biological mimics. Chem. Eur. J. 1998. 4, 1353–1361. S. Hecht, J. M. J. Fre´chet, Dendritic encapsulation of function: Applying Nature’s site isolation principle from biomimetics to materials science. Angew. Chem. Int. Ed. 2001. 40, 74–91. A. Esposito, E. Delort, D. Lagnoux, F. Djojo, J.-L. Reymond, Catalytic peptide dendrimers. Angew. Chem. Int. Ed. 2003. 42, 1381–1383. A. Clouet, T. Darbre, J.-L. Reymond, A combinatorial approach to catalytic peptide dendrimers. Angew. Chem. Int. Ed. 2004. 43, 4612–4615.

15 B. Chen, S. Piletsky, A. P. Turner,

16

17

18

19

20

21

22

23

24

25

High molecular recognition: Design of ‘‘Keys’’. Comb. Chem. High Throughput Screen. 2002. 5, 409–427. C. S. Goh, D. Milburn, M. Gerstein, Conformational changes associated with protein-protein interactions. Curr. Opin. Struct. Biol. 2004. 14, 104–109. P. O. Glantz, T. Arnebrant, T. Nylander, R. E. Baier, Bioadhesion – A phenomenon with multiple dimensions. Acta Odontol. Scand. 1999. 57(5), 238–241. A. Mecke, I. Lee, J. R. Baker Jr, M. M. Holl, B. G. Orr, Deformability of poly(amidoamine) dendrimers. Eur. Phy. J. E Soft Matter 2004. 14, 7–16. D. Farin, D. Avnir, Surface fractality of dendrimers. Angew. Chem. Int. Ed. Engl. 1991. 30, 1377–1379. S. L. Gilat, A. Adronov, J. M. J. Fre´chet, Light harvesting and energy transfer in novel convergently constructed dendrimers. Angew. Chem. Int. Ed. 1999. 38, 1422–1427. K. Autumn, Y. A. Liang, S. T. Hsieh, W. Zesch, W. P. Chan, T. W. Kenny, R. Fearing, R. J. Full, Adhesive force of a single gecko foot-hair. Nature 2000. 405, 681–185. G. M. Dykes, L. J. Brierley, D. K. Smith, T. McGrail, G. J. Seeley, Supramolecular solubilisation of hydrophilic dyes by using individual dendritic branches. Chem. Eur. J. 2001. 7, 4730–4739. R. Esfand, D. A. Tomalia, Poly(amidoamine) (PAMAM) dendrimers: from biomimicry to drug delivery and biomedical applications. Drug Discovery Today 2001. 6, 427– 436. M. Ozkan, Quantum dots and other nanoparticles: What can they offer to drug discovery? Drug Discovery Today 2004. 9, 1065–1071. D. A. Tomalia, A. M. Naylor, W. A. Goddard III, Starburst dendrimers: Molecular-level control of size, shape, surface chemistry, topology, and flexibility from atoms to macroscopic matter. Angew. Chem. Int. Ed. Engl. 1990. 29, 138–175.

References 26 H.-B. Mekelburger, W. Jawarek, F.

27

28

29

30

31

32

33

34

35

36

37

Vo¨gtle, Dendrimers, arborols, and cascade molecules: Breakthrough into generations of new materials. Angew. Chem. Int. Ed. Engl. 1992. 31, 1571–1576. J. Issberner, R. Moors, F. Vo¨gtle, Dendrimers: From generations and functional groups to functions. Angew. Chem. Int. Ed. Engl. 1994. 33, 2413–2420. D. Gudat, Inorganic cauliflower: Functional main group element dendrimers constructed from phosphorus- and silicon-based building blocks. Angew. Chem. Int. Ed. Engl. 1997. 36, 1951–1955. M. Fischer, F. Vo¨gtle, Dendrimers: From design to application – A progress report. Angew. Chem. Int. Ed. 1999. 38, 884–905. G. R. Newkome, C. N. Moorefield, F. Vo¨gtle, Dendrimers and Dendrons: Concepts, Syntheses, Applications. 2001, Weinheim, Wiley-VCH. S. M. Grayson, J. M. J. Fre´chet, Convergent dendrons and dendrimers: From synthesis to applications. Chem. Rev. 2001. 101, 3819–3868. I. Gitsov, C. Lin, Dendrimers – Nanoparticles with precisely engineered surfaces. Curr. Org. Chem. 2005. 9, 1025–1051. N. Feuerbacher, F. Vo¨gtle, Iterative synthesis in organic chemistry. Top. Curr. Chem. 1998. 197, 1–18. E. Buhleier, W. Wehner, F. Vo¨gtle, ‘‘Cascade’’- and ‘‘nonskid-chain-like’’ synthesis of molecular cavity topologies. Synthesis 1978. 155–158. G. R. Newkome, Z. Yao, G. R. Baker, V. K. Gupta, Micelles. Part 1. Cascade molecules: A new approach to micelles. A [27]-arborol. J. Org. Chem. 1985. 50, 2003–2004. D. A. Tomalia, H. Baker, J. Dewald, M. Hall, G. Kallos, S. Martin, J. Roeck, J. Ryder, P. Smith, Dendritic macromolecules: Synthesis of starburst dendrimers. Macromolecules 1986. 19, 2466–2468. D. A. Tomalia, H. D. Durst, Genealogically directed synthesis – Starburst cascade dendrimers and

38

39

40

41

42

43

44

45

46

47

hyperbranched structures. Top. Curr. Chem. 1993. 165, 193–313. T. M. Miller, T. X. Neenan, Convergent synthesis of monodisperse dendrimers based upon 1,3,5trisubstituted benzenes. Chem. Mater. 1990. 2, 346–349. R. Haag, J.-F. Stumbe´, A. Sunder, H. Frey, A. Hebel, An approach to core-shell-type architectures in hyperbranched polyglycerols by selective chemical differentiation. Macromolecules 2000. 33, 8158–8166. C. J. Hawker, J. M. J. Fre´chet, Preparation of polymers with controlled molecular architecture. A new convergent approach to dendritic macromolecules. J. Am. Chem. Soc. 1990. 112, 7638–7647. J. R. Morgan, M. J. Cloninger, Heterogeneously functionalized dendrimers. Curr. Opin. Drug Discov. Devel. 2002. 5, 966–973. Y. Choi, J. R. Baker Jr, Targeting cancer cells with DNA-assembled dendrimers: A mix-and-match strategy for cancer. Cell Cycle 2005. 4, 669–671. M. Meldal, P. M. St Hilaire, Synthetic methods of glycopeptide assembly, and biological analysis of glycopeptide products. Curr. Opin. Chem. Biol. 1997. 1, 552–563. D. Zanini, R. Roy, Synthesis of new a-thiosialodendrimers and their binding properties to the sialic acid specific lectin from Limax flavus. J. Am. Chem. Soc. 1997. 119, 2088–2095. K. J. Yarema, C. R. Bertozzi, Chemical approaches to glycobiology and emerging carbohydrate-based therapeutic agents. Curr. Opin. Chem. Biol. 1998. 2, 49–61. J. J. Landers, Z. Cao, I. Lee, L. T. Piehler, P. P. Myc, A. Myc, T. Hamouda, A. T. Galecki, J. R. J. Baker Jr, Prevention of influenza pneumonitis by sialic acid-conjugated dendritic polymers. J. Infect. Dis. 2002. 186, 1222–1230. J. D. Reuter, A. Myc, M. M. Hayes, Z. Gan, R. Roy, D. Qin, R. Yin, L. T. Piehler, R. Esfand, D. A. Tomalia, J. R. Baker Jr, Inhibition of viral

35

36

1 Dendrimers in Cancer Treatment and Diagnosis

48

49

50

51

52

53

54

55

56

57

adhesion and infection by sialic-acidconjugated dendritic polymers. Bioconjugate Chem. 1999. 10, 271–278. D. H. Charych, J. O. Nagy, W. Spevak, M. D. Bednarski, Direct colorimetric detection of a receptorligand interaction by a polymerized bilayer assembly. Science 1993. 261, 585–588. O. T. Keppler, R. Horstkorte, M. Pawlita, C. Schmidt, W. Reutter, Biochemical engineering of the N-acyl side chain of sialic acid: Biological implications. Glycobiology 2001. 11, 11R–18R. J. J. Lundquist, E. J. Toone, The cluster glycoside effect. Chem. Rev. 2002. 102, 555–578. L. L. Kiessling, S. Pohl, Strength in numbers: Non-natural polyvalent carbohydrate derivatives. Chem. Biol. 1996. 3, 71–77. W. B. Turnbull, J. F. Stoddart, Design and synthesis of glycodendrimers. J. Biotechnol. 2002. 90, 231– 255. R. Roy, M. G. Baek, Glycodendrimers: Novel glycotope isosteres unmasking sugar coding. Case study with Tantigen markers from breast cancer MUC1 glycoprotein. J. Biotechnol. 2002. 90, 291–309. R. Roy, Synthesis and some applications of chemically defined multivalent glycoconjugates. Curr. Opin. Struct. Biol. 1996. 6, 692–702. P. R. Ashton, S. E. Boyd, C. L. Brown, N. Jayaraman, J. F. Stoddart, A convergent synthesis of a carbohydrate-containing dendrimer. Angew. Chem. Int. Ed. Engl. 1997. 36, 732–735. T. Toyokuni, S.-i. Hakomori, A. K. Singhal, Synthetic carbohydrate vaccines: Synthesis and immunogenicity of Tn antigen conjugates. Bioorg. Med. Chem. 1994. 2, 1119– 1132. S. Andre´, P. J. Ortega, M. A. Perez, R. Roy, H.-J. Gabius, Lactosecontaining starburst dendrimers: Influence of dendrimer generation and binding-site orientation of receptors (plant/animal lectins and

58

59

60

61

62

63

64

65

66

67

immunoglobulins) on binding properties. Glycobiology 1999. 9, 1253–1261. R. Roy, M.-G. Baek, K. RittenhouseOlson, Synthesis of N,N 0 bis(acrylamido)acetic acid-based Tantigen glycodendrimers and their mouse monoclonal IgG antibody binding properties. J. Am. Chem. Soc. 2001. 123, 1809–1816. M. M. Palcic, H. Li, D. Zanini, R. S. Bhella, R. Roy, Chemoenzymatic synthesis of dendritic sialyl Lewis(x). Carbohydr. Res. 1997. 305, 433–442. M. S. Lajiness, M. Vieth, J. Erickson, Molecular properties that influence oral drug-like behavior. Curr. Opin. Drug Discov. Devel. 2004. 7, 470–474. N. R. Gonzales, R. De Pascalis, J. Schlom, S. V. S. Kashmiri, Minimizing the immunogenicity of antibodies for clinical application. Tumour Biol. 2005. 26, 31–43. B. Matthey, A. Engert, S. Barth, Recombinant immunotoxins for the treatment of Hodgkin’s disease (Review). Int. J. Mol. Med. 2000. 6, 509–514. K. Viswanathan, S. Lawrence, S. Hinderlich, K. J. Yarema, Y. C. Lee, M. Betenbaugh, Engineering sialic acid synthetic ability into insect cells: Identifying metabolic bottlenecks and devising strategies to overcome them. Biochemistry 2003. 42, 15 215–15 225. M. J. Betenbaugh, N. Tomiya, S. Narang, J. T. A. Hsu, Y. C. Lee, Biosynthesis of human-type N-glycans in heterologous systems. Curr. Opin. Struct. Biol. 2004. 14, 601–606. H. Namazi, M. Adeli, Dendrimers of citric acid and poly (ethylene glycol) as the new drug-delivery agents. Biomaterials 2005. 26, 1175–1183. J. F. G. A. Jansen, E. M. M. de Brabander-van den Berg, E. W. Meijer, Encapsulation of guest molecules into a dendritic box. Science 1994. 266, 1226–1229. U. Boas, S. H. M. So¨ntjens, K. J. Jensen, J. B. Christensen, E. W. Meijer, New dendrimer-peptide hostguest complexes: Towards dendrimers

References

68

69

70

71

72

73

74

75

76

as peptide carriers. ChemBioChem 2002. 3, 433–439. A. Co´rdova, K. D. Janda, Synthesis and catalytic antibody functionalization of dendrimers. J. Am. Chem. Soc. 2001. 123, 8248–8259. M. T. Morgan, M. A. Carnahan, C. E. Immoos, A. A. Ribeiro, S. Finkelstein, S. J. Lee, M. W. Grinstaff, Dendritic molecular capsules for hydrophobic compounds. J. Am. Chem. Soc. 2003. 125, 15 485– 15 489. P. K. Tripathi, A. J. Khopade, S. Nagaich, S. Shrivastava, S. Jain, N. K. Jain, Dendrimer grafts for delivery of 5-fluorouracil. Pharmazie 2002. 57, 261–264. T. Ooya, J. Lee, K. Park, Effects of ethylene glycol-based graft, starshaped, and dendritic polymers on solubilization and controlled release of paclitaxel. J. Controlled Release 2003. 93, 121–127. T. Ooya, J. Lee, K. Park, Hydrotropic dendrimers of generations 4 and 5: Synthesis, characterization, and hydrotropic solubilization of paclitaxel. Bioconjugate Chem. 2004. 15, 1221–1229. J. M. Benito, M. Gomez-Garcia, C. Ortiz Mellet, I. Baussanne, J. Defaye, J. M. Garcia Fernandez, Optimizing saccharide-directed molecular delivery to biological receptors: Design, synthesis, and biological evaluation of glycodendrimer-cyclodextrin conjugates. J. Am. Chem. Soc. 2004. 126, 10 355–10 363. T. Hudde, S. A. Rayner, R. M. Comer, M. Weber, J. D. Isaacs, H. Waldmann, D. F. Larkin, A. J. George, Activated polyamidoamine dendrimers, a non-viral vector for gene transfer to the corneal endothelium. Gene Ther. 1999. 6, 939–943. J. Haensler, F. C. Szoka Jr, Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconjugate Chem. 1993. 4, 372–379. T.-i. Kim, H. J. Seo, J. S. Choi, H.-S. Jang, J.-u. Baek, K. Kim, J.-S. Park,

77

78

79

80

81

82

83 84

85

PAMAM-PEG-PAMAM: Novel triblock copolymer as a biocompatible and efficient gene delivery carrier. Biomacromolecules 2004. 5, 2487–2492. J. S. Choi, K. Nam, J.-y. Park, J.-B. Kim, J.-K. Lee, J.-s. Park, Enhanced transfection efficiency of PAMAM dendrimer by surface modification with L-arginine. J. Controlled Release 2004. 99, 445–456. J. H. Lee, Y.-b. Lim, J. S. Choi, Y. Lee, T.-i. Kim, H. J. Kim, J. K. Yoon, K. Kim, J.-s. Park, Polyplexes assembled with internally quaternized PAMAMOH dendrimer and plasmid DNA have a neutral surface and gene delivery potency. Bioconjugate Chem. 2003. 14, 1214–1221. M. Hussain, M. Shchepinov, M. Sohail, I. F. Benter, A. J. Hollins, E. M. Southern, S. Akhtar, A novel anionic dendrimer for improved cellular delivery of antisense oligonucleotides. J. Controlled Release 2004. 99, 139–155. J. F. G. A. Jansen, E. W. Meijer, E. M. M. de Brabander-van den Berg, The dendritic box: Shapeselective liberation of encapsulated guests. J. Am. Chem. Soc. 1995. 117, 4417–4418. H. Ringsdorf, Structure and properties of pharmacologically active polymers. J. Polym. Sci. Polym. Symp. 1975. 51, 135–153. H. Bader, H. Ringsdorf, B. Schmidt, Water-soluble polymers in medicine. Angew. Makromol. Chem. 1984. 123/124, 457–485. J. Kopacek, Soluble biomedical polymers. Polym. Med. 1977. 7, 191–221. E. R. Gillies, E. Dy, J. M. J. Fre´chet, F. C. Szoka, Biological evaluation of polyester dendrimer: Poly(ethylene oxide) ‘‘bow-tie’’ hybrids with tunable molecular weight and architecture. Mol. Pharm. 2005. 2, 129–138. P. Kolhe, J. Khandare, O. Pillai, S. Kannan, M. Lieh-Lai, R. M. Kannan, Preparation, cellular transport, and activity of polyamidoamine-based dendritic nanodevices with a high drug payload. Biomaterials 2006. 27, 660–669.

37

38

1 Dendrimers in Cancer Treatment and Diagnosis 86 S. C. Lee, R. Parthasarathy, K.

87

88

89

90

91

92

93

94

Botwin, D. Kunneman, E. Rowold, G. Lange, J. Klover, A. Abegg, J. Zobel, T. Beck, T. Miller, W. Hood, J. Monahan, J. P. McKearn, R. Jansson, C. F. Voliva, Biochemical and immunological properties of cytokines conjugated to dendritic polymers. Biomed. Microdevices 2004. 6, 191–202. F. Chaves, J. C. Calvo, C. Carvajal, Z. Rivera, L. Ramirez, M. Pinto, M. Trujillo, F. Guzman, M. E. Patarroyo, Synthesis, isolation and characterization of Plasmodium falciparum antigenic tetrabranched peptide dendrimers obtained by thiazolidine linkages. J. Pept. Res. 2001. 58, 307–316. L. J. Cruz, E. Iglesias, J. C. Aguilar, L. J. Gonza´lez, O. Reyes, F. Albericio, D. Andreu, A comparative study of different presentation strategies for an HIV peptide immunogen. Bioconjugate Chem. 2004. 15, 112–120. F. Sa´nchez-Sancho, E. Pe´rezInestrosa, R. Suau, C. Mayorga, M. J. Torres, M. Blanca, Dendrimers as carrier protein mimetics for IgE antibody recognition. Synthesis and characterization of densely penicilloylated dendrimers. Bioconjugate Chem. 2002. 13, 647–653. S. Jaracz, J. Chen, L. V. Kuznetsova, I. Ojima, Recent advances in tumortargeting anticancer drug conjugates. Bioorg. Med. Chem. 2005. 13, 5043–5054. Q. Cai, Y. Zhao, J. Bei, F. Xi, S. Wang, Synthesis and properties of star-shaped polylactide attached to poly(amidoamine) dendrimer. Biomacromolecules 2003. 4, 828–834. N. Malik, E. G. Evagorou, R. Duncan, Dendrimer-platinate: A novel approach to cancer chemotherapy. Anticancer Drugs 1999. 10, 767–776. D. Shabat, R. J. Amir, A. Gopin, N. Pessah, M. Shamis, Chemical adaptor systems. Chem. Eur. J. 2004. 10, 2626–2634. M. Shamis, H. N. Lode, D. Shabat, Bioactivation of self-immolative

95

96

97

98

99

100

101

102

103

dendritic prodrugs by catalytic antibody 38C2. J. Am. Chem. Soc. 2004. 126, 1726–1731. R. J. Amir, N. Pessah, M. Shamis, D. Shabat, Self-immolative dendrimers. Angew. Chem. Int. Ed. 2003. 42, 4494–4499. F. M. H. de Groot, C. Albrecht, R. Koekkoek, P. H. Beusker, H. W. Scheeren, ‘‘Cascade-release dendrimers’’ liberate all end groups upon a single triggering event in the dendritic core. Angew. Chem. Int. Ed. 2003. 42, 4940–4494. A. T. Florence, T. Sakthivel, I. Toth, Oral uptake and translocation of a polylysine dendrimer with a lipid surface. J. Controlled Release 2000. 65, 253–259. ˜ oR. Wiwattanapatapee, B. Carren Go´mez, N. Malik, R. Duncan, Anionic PAMAM dendrimers rapidly cross adult rat intestine in vitro: A potential oral delivery system? Pharm. Res. 2000. 17, 991–998. A. T. Florence, N. Hussain, Transcytosis of nanoparticle and dendrimer delivery systems: Evolving vistas. Adv. Drug Delivery Rev. 2001. 50, S69–S89. M. Ruponen, P. Honkakoski, S. Ro¨nkko¨, J. Pelkonen, M. Tammi, A. Urtti, Extracellular and intracellular barriers in non-viral gene delivery. J. Controlled Release 2003. 93, 213–217. R. F. Barth, D. M. Adams, A. H. Soloway, F. Alam, M. V. Darby, Boronated starburst dendrimermonoclonal antibody immunoconjugates: Evaluation as a potential delivery system for neutron capture therapy. Bioconjugate Chem. 1994. 5, 58–66. S. Hong, A. U. Bielinska, A. Mecke, B. Keszler, J. L. Beals, X. Shi, L. Balogh, B. Orr, J. R. Baker Jr, M. M. Banaszak Holl, Interaction of poly(amidoamine) dendrimers with supported lipid bilayers and cells: Hole formation and the relation to transport. Bioconjugate Chem. 2004. 15, 774–782. A. Mecke, S. Uppuluri, T. M. Sassanella, D.-K. Lee, A.

References

104

105

106

107

108

109

110

111

112

113

Ramamoorthy, J. R. Baker Jr, B. G. Orr, M. M. Banaszak Holl, Direct observation of lipid bilayer disruption by poly(amidoamine) dendrimers. Chem. Phys. Lipids 2004. 132, 3–14. G.-D. Zhang, A. Harada, N. Nishiyama, D.-L. Jiang, H. Koyam, T. Aida, K. Kataok, Polyion complex micelles entrapping cationic dendrimer porphyrin: Effective photosensitizer for photodynamic therapy of cancer. J. Controlled Release 2003. 93, 141–150. J. C. Roberts, M. K. Bhalgat, R. T. Zera, Preliminary biological evaluation of polyamidoamine (PAMAM) Starburst TM dendrimers. J. Biomed. Mater. Res. 1996. 30, 53–65. H. Kobayashi, S. Kawamoto, T. Saga, N. Sato, A. Hiraga, T. Ishimori, J. Konishi, K. Togashi, M. W. Brechbiel, Positive effects of polyethylene glycol conjugation to generation-4 polyamidoamine dendrimers as macromolecular MR contrast agents. Magn. Reson. Med. 2001. 46, 781–788. N. Ro¨ckendorf, T. K. Lindhorst, Glycodendrimers. Top. Curr. Chem. 2001. 217, 201–238. L. Crespo, G. Sanclimens, M. Pons, E. Giralt, M. Roho, F. Albericio, Peptide and amide bond-containing dendrimers. Chem. Rev. 2005. 105, 1663–1681. M. S. Shchepinov, I. A. Udalova, A. J. Brigman, E. M. Southern, Oligonucleotide dendrimers: Synthesis and use as polylabelled DNA probes. Nucleic Acids Res. 1997. 25, 4447–4454. T. W. Nilsen, J. Grayzel, W. Prensky, Dendritic nucleic acid structures. J. Theoret. Biol. 1997. 187, 273–284. R. H. E. Hudson, M. J. Damha, Nucleic acid dendrimers: Novel biopolymer structures. J. Am. Chem. Soc. 1998. 115, 2119–2124. Y. Suzuki, T. Otomo, H. Okazi, H. Sawai, Synthesis and properties of a new type DNA dendrimer. Nucleic Acids Symp. Ser. 2000. 44, 125–126. Y. Li, Y. D. Tseng, S. Y. Kwon, L. D’Espaux, J. S. Bunch, P. L.

114

115

116

117

118

119

120

121

122

123

McEuen, D. Luo, Controlled assembly of dendrimer-like DNA. Nat. Mater. 2004. 3, 38–42. K. T. Al-Jamal, T. Sakthivel, A. T. Florence, Dendrisomes: Vesicular structures derived from a cationic lipidic dendron. J. Pharm. Sci. 2005. 94, 102–113. S. Takeoka, K. Mori, H. Ohkawa, K. Sou, E. Tsuchida, Synthesis and assembly of poly(ethylene glycol)lipids with mono-, di-, and tetraacyl chains and a poly(ethylene glycol) chain of various molecular weights. J. Am. Chem. Soc. 2000. 122, 7927–7935. H. Frey, R. Haag, Dendritic polyglycerol: A new versatile biocompatible-material. J. Biotechnol. 2002. 90, 257–267. M. F. Neerman, W. Zhang, A. R. Parrish, E. E. Simanek, In vitro and in vivo evaluation of a melamine dendrimer as a vehicle for drug delivery. Int. J. Pharm. 2004. 281, 129–132. A.-M. Caminade, J.-P. Majoral, Nanomaterials based on phosphorus dendrimers. Acc. Chem. Res. 2004. 37, 341–348. X.-Y. Wu, S.-W. Huang, J.-T. Zhang, R.-X. Zhuo, Preparation and characterization of novel physically cross-linked hydrogels composed of poly(vinyl alcohol) and amineterminated polyamidoamine dendrimer. Macromol. Biosci. 2004. 4, 71–75. H. R. Ihre, O. L. Padilla De Jesu´s, F. C. Szoka Jr, J. M. J. Fre´chet, Polyester dendritic systems for drug delivery applications: Design, synthesis, and characterization. Bioconjugate Chem. 2002. 13, 443–452. M. J. Cloninger, Biological applications of dendrimers. Curr. Opin. Chem. Biol. 2002. 6, 742–748. L. S. Heuser, F. N. Miller, Differential macromolecular leakage from the vasculature of tumors. Cancer 1986. 57, 461–464. H. Maeda, J. Wu, T. Sawa, Y. Matsumura, K. Hori, Tumor vascular permeability and the EPR effect in

39

40

1 Dendrimers in Cancer Treatment and Diagnosis

124

125

126

127

128

129

130

131

132

133

macromolecular therapeutics: A review. J. Controlled Release 2000. 65, 271–284. Y. Matsumura, H. Maeda, A new concept for macromolecular therapeutics in cancer chemotherapy: Mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res. 1986. 46, 6387–6392. L. W. Seymour, Passive tumor targeting of soluble macromolecules and drug conjugates. Crit. Rev. Ther. Drug Carrier Syst. 1992. 9, 135–187. D. Liu, A. Mori, L. Huang, Role of liposome size and RES blockade in controlling biodistribution and tumor uptake of GM1-containing liposomes. Biochim. Biophys. Acta 1992. 1104, 95–101. A. Nagayasu, T. Shimooka, Y. Kinouchi, K. Uchiyama, Y. Takeichi, H. Kiwada, Effects of fluidity and vesicle size on antitumor activity and myelosuppressive activity of liposomes loaded with daunorubicin. Biol. Pharm. Bull. 1994. 17, 935–939. R. K. Jain, Barriers to drug-delivery in solid tumors. Sci. Am. 1994. 271, 58–65. Y. Tabata, Y. Murakami, Y. Ikada, Tumor accumulation of poly(vinyl alcohol) of different sizes after intravenous injection. J. Controlled Release 1998. 50, 123–133. S. Zalipsky, N. Mullah, J. A. Harding, J. Gittelman, L. Guo, S. A. DeFrees, Poly(ethylene glycol)-grafted liposomes with oligopeptide or oligosaccharide ligands appended to the termini of the polymer chains. Bioconjugate Chem. 1997. 8, 111–118. J. A. Reddy, V. M. Allagadda, C. P. Leamon, Targeting therapeutic and imaging agents to folate receptor positive tumors. Curr. Pharm. Biotechnol. 2005. 6, 131–150. C. P. Leamon, J. A. Reddy, Folatetargeted chemotherapy. Adv. Drug Delivery Rev. 2004. 56, 1127–1141. E. J. Roy, U. Gawlick, B. A. Orr, D. M. Kranz, Folate-mediated targeting of T cells to tumors.

134

135

136

137

138

139

140

141

142

143

144

Adv. Drug Delivery Rev. 2004. 56, 1219–1231. Y. Choi, T. Thomas, A. Kotlyar, M. T. Islam, J. R. Baker, Synthesis and functional evaluation of DNAassembled polyamidoamine (PAMAM) dendrimer clusters with cancer cellspecific targeting. Chem. Biol. 2005. 12, 35–43. D. Laheru, E. M. Jaffee, Immunotherapy for pancreatic cancer – science driving clinical progress. Nat. Rev. Cancer 2005. 5, 549–467. M. Harris, Monoclonal antibodies as therapeutic agents for cancer. Lancet Oncol. 2004. 5, 292–302. M. Z. Lin, M. A. Teitell, G. J. Schiller, The evolution of antibodies into versatile tumor-targeting agents. Clin. Cancer Res. 2005. 11, 129–138. J.-Y. Zhang, Tumor-associated antigen arrays to enhance antibody detection for cancer diagnosis. Cancer Detect. Prev. 2004. 28, 114–118. R. E. Kontermann, Recombinant bispecific antibodies for cancer therapy. Acta Pharmacol. Sin. 2005. 26, 1–9. P. A. Trail, D. H. King, G. M. Dubowchik, Monoclonal antibody drug immunoconjugates for targeted treatment of cancer. Cancer Immunol. Immunother. 2003. 52, 328–337. G. C. McDonald, N. Glover, Effective tumor targeting: Strategies for the delivery of armed antibodies. Curr. Opin. Drug Discov. Devel. 2005. 8, 177–183. S. V. Govindan, G. L. Griffiths, H. J. Hansen, I. D. Horak, D. M. Goldenberg, Cancer therapy with radiolabeled and drug/toxinconjugated antibodies. Technol. Cancer Res. Treat. 2005. 4, 375–392. D. J. FitzGerald, R. Kreitman, W. Wilson, D. Squires, I. Pastan, Recombinant immunotoxins for treating cancer. Int. J. Med. Microbiol. 2004. 293, 577–582. K. Sandvig, S. Grimmer, T. G. Iversen, K. Rodal, M. L. Torgersen, P. Nicoziani, B. van Deurs, Ricin transport into cells: Studies of endocytosis and intracellular

References

145

146

147

148

149

150

151

152

153

transport. Int. J. Med. Microbiol. 2000. 290, 415–420. S. Olsnes, The history of ricin, abrin and related toxins. Toxicon 2004. 44, 361–370. A. Gruaz-Guyon, O. Raguin, J. Barbet, Recent advances in pretargeted radioimmunotherapy. Curr. Med. Chem. 2005. 12, 319–338. J. C. Roberts, Y. E. Adams, D. A. Tomalia, J. A. Mercer-Smith, D. K. Lavallee, Using starburst dendrimers as linker molecules to radiolabel antibodies. Bioconjugate Chem. 1990. 1, 305–308. P. Singh, Terminal groups in Starburst dendrimers: Activation and reactions with proteins. Bioconjugate Chem. 1998. 9, 54–63. H. Kobayashi, N. Sato, T. Saga, Y. Nakamoto, T. Ishimori, S. Toyama, K. Togashi, J. Konishi, M. W. Brechbiel, Monoclonal antibodydendrimer conjugates enable radiolabeling of antibody with markedly high specific activity with minimal loss of immunoreactivity. Eur. J. Nucl. Med. Mol. Imaging 2000. 27, 1334–1339. N. Fischer-Durand, M. Salmain, B. Rudolf, A. Vessie`res, J. Zakrzewski, G. Jaouen, Synthesis of metalcarbonyl-dendrimer-antibody immunoconjugates: Towards a new format for carbonyl metallo immunoassay. ChemBioChem 2004. 5, 519– 525. T. P. Thomas, A. K. Patri, A. Myc, M. T. Myaing, J. Y. Ye, T. B. Norris, J. R. Baker Jr, In vitro targeting of synthesized antibody-conjugated dendrimer nanoparticles. Biomacromolecules 2004. 5, 2269–2274. A. K. Patri, A. Myc, J. Beals, T. P. Thomas, N. H. Bander, J. R. Baker Jr, Synthesis and in vitro testing of J591 antibody-dendrimer conjugates for targeted prostate cancer therapy. Bioconjugate Chem. 2004. 15, 1174–1181. S. S. Nigavekar, L. Y. Sung, M. Llanes, A. El-Jawahri, T. S. Lawrence, C. W. Becker, L. Balogh, M. K. Khan, 3 H Dendrimer nano-

154

155

156

157

158

159

160

particle organ/tumor distribution. Pharm. Res. 2004. 21, 476–483. H. Kobayashi, C. Wu, M.-K. Kim, C. H. Paik, J. A. Carrasquillo, M. W. Brechbiel, Evaluation of the in vivo biodistribution of indium-111 and yttrium-88 labeled dendrimer-1B4MDTPA and its conjugation with antiTac monoclonal antibody. Bioconjugate Chem. 1999. 10, 103–111. M. Mamede, T. Saga, H. Kobayashi, T. Ishimori, T. Higashi, N. Sato, M. W. Brechbiel, J. Konishi, Radiolabeling of avidin with very high specific activity for internal radiation therapy of intraperitoneally disseminated tumors. Clin. Cancer Res. 2003. 9, 3756–3762. D. S. Wilbur, P. M. Pathare, D. K. Hamlin, K. R. Buhler, R. L. Vessella, Biotin reagents for antibody pretargeting. 3. Synthesis, radioiodination, and evaluation of biotinylated starburst dendrimers. Bioconjugate Chem. 1998. 9, 813–825. N. Malik, R. Wiwattanapatapee, R. Klopsch, K. Lorenz, H. Frey, J. W. Weener, E. W. Meijer, W. Paulus, R. Duncan, Dendrimers: Relationship between structure and biocompatibility in vitro, and preliminary studies on the biodistribution of 125 I-labelled polyamidoamine dendrimers in vivo. J. Controlled Release 2000. 65, 133–148. W. Yang, R. F. Barth, G. Wu, A. K. Bandyopadhyaya, B. T. S. Thirumamagal, W. Tjarks, P. J. Binns, K. J. Riley, H. Patel, J. A. Coderre, M. J. Ciesielski, R. A. Fenstermaker, Boronated epidermal growth factor as a delivery agent for neutron capture therapy of EGF receptor positive gliomas. Appl. Radiat. Isot. 2004. 61, 981–985. H. Kobayashi, M. W. Brechbiel, Dendrimer-based macromolecular MRI contrast agents: Characteristics and application. Mol. Imaging 2003. 2, 1–10. S.-E. Stiriba, H. Frey, R. Haag, Dendritic polymers in biomedical applications: From potential to clinical use in diagnostics and therapy. Angew. Chem. Int. Ed. 2002. 41, 1329–1334.

41

42

1 Dendrimers in Cancer Treatment and Diagnosis 161 T. Kaminaga, T. Takeshita, I.

162

163

164

165

166

167

168

Kimura, Role of magnetic resonance imaging for evaluation of tumors in the cardiac region. Eur. Radiol. 2003. 13(Suppl 4), L1–L10. P. de Graaf, F. Barkhof, A. C. Moll, S. M. Imhof, D. L. Knol, P. van der Valk, J. A. Castelijns, Retinoblastoma: MR imaging parameters in detection of tumor extent. Radiology 2005. 235, 197–207. M. D. Ogan, U. Schmiedl, M. E. Mosely, W. Grodd, H. Paajanen, R. C. Brasch, Albumin labeled with Gd-DTPA. An intravascular contrastenhancing agent for magnetic resonance blood pool imaging: Preparation and characterization. Invest. Radiol. 1987. 22, 665–671. L. D. Margerum, B. K. Campion, M. Koo, N. Shargill, J. Lai, A. Marumoto, P. C. Sontum, Gadolinium(III) DO31 macrocycles and polyethylene glycol coupled to dendrimers. Effect of molecular wieght on physical and biological properties of macromolecular magnetic resonance imaging agents. J. Alloys Compd. 1997. 249, 185–190. S. D. Konda, S. Wang, M. Brechbiel, E. C. Wiener, Biodistribution of a 153 Gd-folate dendrimer, generation ¼ 4, in mice with folate-receptor positive and negative ovarian tumor xenografts. Invest. Radiol. 2002. 37, 199–204. H. Kobayashi, S. Kawamoto, S.-K. Jo, H. L. Bryant Jr, M. W. Brechbiel, R. A. Star, Macromolecular MRI contrast agents with small dendrimers: Pharmacokinetic differences between sizes and cores. Bioconjugate Chem. 2003. 14, 388–394. S. Guccione, K. C. Li, M. D. Bednarski, Molecular imaging and therapy directed at the neovasculature in pathologies. How imaging can be incorporated into vascular-targeted delivery systems to generate active therapeutic agents. IEEE Eng. Med. Biol. Mag. 2004. 23, 50–60. K. Onitsuka, M. Fujimoto, H. Kitajima, N. Ohshiro, F. Takei, S. Takahashi, Convergent synthesis of

169

170

171

172

173

174

175

176

platinum-acetylide dendrimers. Chem. Eur. J. 2004. 10, 6433–6446. R. F. Barth, A. H. Soloway, Boron neutron capture therapy of primary and metastatic brain tumors. Mol. Chem. Neuropathol. 1994. 21, 139–154. L. Liu, R. F. Barth, D. M. Adams, A. H. Soloway, R. A. Reisfeld, Bispecific antibodies as targeting agents for boron neutron capture therapy of brain tumors. J. Hematother. 1995. 4, 477–483. F. Alam, A. H. Soloway, R. F. Barth, N. Mafune, D. M. Adams, W. H. Knoth, Boron neutron capture therapy: Linkage of a boronated macromolecule to monoclonal antibodies directed against tumorassociated antigens. J. Med. Chem. 1989. 32, 2326–2330. R. F. Barth, G. Wu, W. Yang, P. J. Binns, K. J. Riley, H. Patel, J. A. Coderre, W. Tjarks, A. K. Bandyopadhyaya, B. T. S. Thirumamagal, M. J. Ciesielski, R. A. Fenstermaker, Neutron capture therapy of epidermal growth factor (þ) gliomas using boronated cetuximab (IMC-C225) as a delivery agent. Appl. Radiat. Isot. 2004. 61, 899–903. A. Quintana, E. Raczka, L. Piehler, I. Lee, A. Myc, I. Majoros, A. K. Patri, T. Thomas, J. Mule´, J. R. Baker Jr, Design and function of a dendrimer-based therapeutic nanodevice targeted to tumor cells through the folate receptor. Pharm. Res. 2002. 19, 1306–1310. J. W. Dennis, S. Laferte, C. Waghorne, M. L. Breitman, R. S. Kerbel, ß1-6 branching of Asn-linked oligosaccharides is directly associated with metastasis. Science 1987. 236, 236–239. S. Sell, Cancer-associated carbohydrates identified by monoclonal antibodies. Hum. Pathol. 1990. 21, 1003–1019. A. Kobata, J. Amano, Altered glycosylation of proteins produced by malignant cells, and application for the diagnosis and immunotherapy of tumours. Immunol. Cell Biol. 2005. 83, 429–439.

References 177 J. W. Dennis, M. Granovsky, C. E.

178

179

180

181

182

Warren, Protein glycosylation in development and disease. BioEssays 1999. 21, 412–421. L. Tong, G. Baskaran, M. B. Jones, J. K. Rhee, K. J. Yarema, Glycosylation changes as markers for the diagnosis and treatment of human disease, in Biochemical and Genetic Engineering Reviews, S. Harding (Ed.), 2003, Intercept Limited, Andover, Hampshire, UK. pp. 199–244. J. R. Allen, C. R. Harris, S. J. Danishefsky, Pursuit of optimal carbohydrate-based anticancer vaccines: Preparation of a multiantigenic unimolecular glycopeptide containing the Tn, MBr1, and LewisY antigens. J. Am. Chem. Soc. 2001. 123, 1890–1897. J. R. Allen, J. G. Allen, X. F. Zhang, L. J. Williams, A. Zatorski, G. Ragupathi, P. O. Livingston, S. J. Danishefsky, A second generation synthesis of the MBr1 (Globo-H) breast tumor antigen: New application of the N-pentenyl glycoside method for achieving complex carbohydrate protein linkages. Chemistry 2000. 6, 1366–1375. V. Kudryashov, P. W. Glunz, L. J. Williams, S. Hintermann, S. J. Danishefsky, K. O. Lloyd, Toward optimized carbohydrate-based anticancer vaccines: Epitope clustering, carrier structure, and adjuvant all influence antibody responses to LewisY conjugates in mice. Proc. Natl. Acad. Sci. U.S.A. 2001. 98, 3264–3269. H. Kayser, R. Zeitler, C. Kannicht, D. Grunow, R. Nuck, W. Reutter, Biosynthesis of a nonphysiological

183

184

185

186

187

188

189

sialic acid in different rat organs, using N-propanoyl-D-hexosamines as precursors. J. Biol. Chem. 1992. 267, 16 934–16 938. K. J. Yarema, L. K. Mahal, R. E. Bruehl, E. C. Rodriguez, C. R. Bertozzi, Metabolic delivery of ketone groups to sialic acid residues. Application to cell surface glycoform engineering. J. Biol. Chem. 1998. 273, 31 168–31 179. L. K. Mahal, K. J. Yarema, C. R. Bertozzi, Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis. Science 1997. 276, 1125–1128. E. Saxon, C. R. Bertozzi, Cell surface engineering by a modified Staudinger reaction. Science 2000. 287, 2007–2010. S.-G. Sampathkumar, A. V. Li, M. B. Jones, Z. Sun, K. J. Yarema, Metabolic installation of thiols into sialic acid modulates adhesion and stem cell biology. Nat. Chem. Biol. 2006. 2, 149–152. L. K. Mahal, K. J. Yarema, G. A. Lemieux, C. R. Bertozzi, Chemical approaches to glycobiology: Engineering cell surface sialic acids for tumor targeting, in Sialobiology and Other Novel Forms of Glycosylation, Y. Inoue, Y. C. Lee, F. A. Troy II (Eds.), 1999, Gakushin Publishing Company, Osaka, Japan, pp. 273–280. D. A. Nauman, C. R. Bertozzi, Kinetic parameters for small-molecule drug delivery by covalent cell surface targeting. Biochim. Biophys. Acta 2001. 1568, 147–154. S.-G. Sampathkumar, K. J. Yarema, Targeting cancer cells with dendrimers. Chem. Biol. 2005. 12, 5–6.

43

44

2

Nanoparticles for Optical Imaging of Cancer Swadeshmukul Santra and Debamitra Dutta 2.1

Introduction

The word ‘‘cancer’’ comes from the Latin word for crab. Historically, an ancient physician from Greece noticed the resemblance of the swollen mass of blood vessels around a malignant tumor to the shape of a crab and so named the disease. The malignant tumor is also seen to adhere to surrounding tissues that it can seize upon in a stubborn manner, similar to a crab. Human cancer consists of more than 200 different diseases [1] in which cells multiply at an exponential growth rate in an uncontrolled fashion. This abnormal growth rate leads to the formation of a lump, called malignant tumor. Gradually the tumor tissue grows and invades the adjacent tissues and organs, obstructing normal physiological functions. In some cases, the cancerous cells can detach from its origin and migrate through circulation to different parts of the body, forming a new tumor site. This is termed as cancer metastasis. Over a period of time, malignant tumors cause malfunctioning of various organs, which turns fatal. According to the American Cancer Society’s annual report [2], about 570 280 people are expected to die of various cancers in the year 2005 in the United States of America (USA). During the last century, cancer has slowly advanced to become the leading cause of death for patients below the age of 85 in USA, despite rapid advances in global cancer research towards the understanding of cancer biology in the past several decades. The formation of malignant tumors is associated with six different cellular characteristics [3, 4], each of which is unique for cancer development. These characteristics are self-sufficiency in growth signals, evading apoptosis (a process by which a cell is ‘‘commanded’’ by the environment to die), insensitivity to anti-growth signals (an inherent mechanism for preventing undesirable cell growth), sustained angiogenesis (a process of growth of new blood vessels), tissue invasion and metastasis and limitless replicative potential. Cancer can develop in any living organ or tissue in the body. The part of the body in which the cancer first develops is referred to as the primary site. The most common cancer developing sites include the skin, lungs, female breasts, prostate, coNanotechnologies for the Life Sciences Vol. 7 Nanomaterials for Cancer Diagnosis. Edited by Challa S. S. R. Kumar Copyright 8 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31387-7

2.1 Introduction

lon and rectum, and corpus uteri. The secondary site refers to the body part where metastasized cancer cells grow and form secondary tumors. Even if a cancer has spread to another part of the body, it is always described with respect to the primary site. For example, advanced breast cancer that has spread to the lymph nodes under the arm and to the lungs is always considered breast cancer. The diagnosis of cancer means an attempt to accurately identify the anatomical site of origin of the malignancy and the type of cells involved. The presence of cancer may be preliminarily suspected by some other disease-like symptoms. For example, weight loss and abdominal pain can be caused by stomach cancer or an ulcer. However, to confirm the diagnosis of cancer, a biopsy (removal of tissue for microscopic evaluation) is usually done. Biopsies can provide information about histological type, classification, grade, potential aggressiveness and other information that may help determine the best treatment. A biopsy together with advanced imaging technologies, can not only confirm the presence of cancer, but also can pinpoint the primary and secondary cancer sites. Early cancer diagnosis, in combination with the precise cancer therapies, could eventually save millions of lives. The diagnosis of cancer at the early stage is extremely challenging and has been an active research area of great interest in current times. If the tumor is located near the body’s surface, a tissue sample can be easily retrieved for a biopsy (removal of tissue for microscopic evaluation) and the tissue abnormality can be confirmed at the cellular level. However, if the tumor mass is inaccessible for a biopsy, one has to then rely upon the existing imaging techniques for the detection of the tumor location. Existing diagnostic non-invasive imaging techniques such as Computed Tomography (CT), Magnetic Resonance (MR), Positron Emission Tomography (PET), Single Photon emission CT (SPECT), Ultrasound (US) and optical imaging are effective for macroscopic visualization of tumors. However, none of these techniques are sensitive enough for the diagnosis of abnormalities in the microscopic level. Substantial research efforts are being made for the development of better cancer imaging techniques. Of them, optical imaging has shown a great promise with respect to the image resolution [5]. The feasibility of developing optical imaging technique for the sensitive detection of cancer has been recently demonstrated [6, 7] using nanoparticle-based highly sensitive optical contrast agents. This chapter provides a knowledge base platform to readers interested in learning about nanoparticle technology and its implications in diagnostic cancer imaging. An overview of existing cancer imaging techniques with special emphasis on optical-based imaging techniques has been incorporated. Optical imaging has strong potential in becoming an attractive alternative to existing cancer imaging techniques. Optical imaging is a highly sensitive, non-invasive, non-ionizing, relatively inexpensive and simple technique. With the aid of better contrast agents, this imaging technology could be transferred to a clinical setup for human applications for early cancer diagnosis in the near future. Recent developments which are directly associated with the improvement of optical image contrast, such as the use of sophisticated laser technology, highly sensitive charged-coupled device (CCD) technology and powerful mathematical modeling of light propagation through the

45

46

2 Nanoparticles for Optical Imaging of Cancer

biological systems, have been integrated into the imaging components. However, presently, significantly limited numbers of appropriate in vivo optical contrast agents are available. Therefore, there is a great demand of developing highly sensitive, stable and clinically safe in vivo optical contrast agents. Nanoscience and nanotechnology is an interdisciplinary research area that brings various traditional disciplines such as chemistry, physics, materials science, biomedical, molecular biology, and many others together under one umbrella. Several edited books [8–12] on nanoscience and nanotechnology include the development of various nanoparticles such as metals, semiconductors, etc. for biological applications. Several review articles [5, 7, 9, 13–22] have recently reported advance research highlights on optical-based contrast agents [15–17, 21], some of which emphasize nanoparticle-based optical contrast agents [7, 18–20, 22] for bioimaging. However, none of these articles captured thoroughly recent developments on nanoparticle-based optical contrast agents suitable for cancer imaging. In this chapter, we made every effort to provide extensive details of nanoparticle-based contrast agents developments, including separate sections on nanoparticle design, synthesis strategies, nanoparticle dispersion, surface modification, bioconjugations and cancer imaging applications. We hope that this chapter will be useful for students, teachers, research scientists, general audiences who are interested in learning more about early cancer diagnosis and others. We have attempted to explain each section and sub-section of this chapter in a simple manner so that readers could easily grasp the general strategy of various nanoparticle-based optical contrast agent development. Since the field of cancer imaging is expanding rapidly, we start with an introduction to highlight the contents of this chapter (Section 2.1). Section 2.2 briefly overviews various existing cancer-imaging techniques, allowing readers to understand merits and demerits of these techniques. Section 2.3 describes the basics of optical imaging, optical imaging techniques and optical contrast agents. This chapter provides a clear understanding of the merits and challenges of developing opticalbased imaging techniques and contrast agents. Section 2.4 details recent advances in nanoparticle-based optical contrast agents. While Section 2.4.1 provides the reasons for developing such contrast agents, Section 2.4.2 provides some literature review on the development of nanoparticle-based various contrast agents such as quantum dots, dye-doped nanoparticles and gold nanoparticles. Various cancer imaging applications using nanoparticles are reviewed in Section 2.5. Section 2.6 covers some miscellaneous nanoparticles such as up-converting phosphors, fluorescent polymer particles, etc. that are potential contrast agents. Section 2.7 provides concluding remarks and the perspectives of nanoparticle-based optical imaging of cancers.

2.2

Cancer Imaging Techniques

Some of the major imaging techniques routinely used in hospital setup for cancer imaging in humans are briefly described below.

2.2 Cancer Imaging Techniques

2.2.1

Computed Tomography (CT) Scanning

The detection components of a typical CT scanner include the X-ray tube, detectors, image reconstruction computer and visual display monitor. The X-ray tube generates a beam of X-rays that are made to pass through the body of the patient. The detectors are positioned to absorb the X-rays coming out through the body. While processing the information, the reconstruction computer takes into consideration that X-rays passing through denser tissues like bones are attenuated to a higher extent than those passing through softer tissue such as lungs. Thus the X-ray beams of varying strengths that come out from the body create a differential profile. This profile is measured by the detectors and finally imaged by the display monitor. The complete setup rotates around the patient and acquires about 1000 snapshots for every 360 rotation, when a slice is completed. After each rotation, the information from the detectors is collected together and processed by the computer to construct a two-dimensional image (slice) on the display monitor. Image resolution is on the order of 50–100 mm with data acquisition time varying from 5 to 30 min. Recently, there have been continuing efforts to merge the CT modality with other modules like nuclear imaging modalities, which have limited spatial resolution, to generate better images. 2.2.2

Magnetic Resonance (MR)

In this technique, patients are placed in a strong electromagnetic field that causes the hydrogen atoms of water molecules present in the body fluid to align with the field. A short, powerful radio signal is then sent through the body at a desired level (slice) perpendicular to the original field. Hydrogen atoms with similar frequencies resonate with the radio signal and get excited. When the radio signal is switched off, the excited atoms will release their excitation energy in the form of radio waves and return to their normal state. The time taken for the hydrogen atoms to release their energy is characteristic of the physical properties of the tissue. These radio waves are detected and the time taken is measured and analyzed by a computer to construct an image of the tissues. Usually, it is difficult to distinguish tumors from normal tissues in the body using an MR image. Therefore, patients are injected with contrast agents that selectively highlight the tumors. Standard MR images (1.5 Tesla, the magnetic field strength) provide a spatial resolution of 1 mm, which could be increased to about 10 mm with certain modifications. 2.2.3

Positron Emission Tomography (PET)

In this technique, a positron-emitting isotope ( 11 C or 18 F) is attached to a biological molecule that has an affinity to tumor cells and introduced into the patients. The decaying isotopes emit positrons, which collide with a nearby electron and annihilate to release g-rays. These rays are detected and analyzed by a computer. The po-

47

48

2 Nanoparticles for Optical Imaging of Cancer

sition of the tumor in the body can be located by tracking the density of the positrons in a particular region. 2.2.4

Single-photon Emission CT (SPECT)

This imaging technique is similar to PET, except that the SPECT isotope itself emits a single g-ray instead of a positron. This technique is very inexpensive when compared with PET. However the spatial resolution is not as good as PET. 2.2.5

Ultrasonography (US)

In this form of imaging, ultrasonic sound waves are sent through the body. The waves are partially reflected from the interfaces of different tissues. The intensity of reflected waves depends on the density of the tissues. The time taken for the reflected wave to reach the detector gives a measure of the depth of the tissue location. The advantage of ultrasonography is that the images are generated in real time with very high temporal resolution. However, ultrasonic waves cannot travel through bone and therefore cannot detect tissues behind bony structures such as the brain behind the skull.

2.3

Optical Imaging 2.3.1

Basics of Optical Imaging

Optical imaging is a sensitive, non-invasive, non-ionizing (clinically safe) and relatively inexpensive technique that has strong potential for diagnostic cancer imaging. Two major components are associated with an optical imaging system: an imaging component and an optical contrast-enhancing component (i.e., contrast agent). Recent advances in optical imaging have utilized sophisticated laser technology, highly sensitive charged-coupled device (CCD) technology and powerful mathematical modeling of light propagation through the biological systems; all these developments have formed a solid basis for the imaging component. Molecular fluorescent probes have been successfully used as optical contrast agents for imaging various cancer tissues in the past [5, 15–17, 23–27]. However, the sensitivity of the contrast agent has become the major obstacle in obtaining a highresolution image. Again, in vivo deep tissue optical imaging has been limited because of the low penetration depth of the light in the ultraviolet (UV) and visible spectral range (the approximate tissue penetration depth is about 1–2 mm). Nearinfrared (NIR) light in the spectral range 650–900 nm can, however, penetrate much deeper (up to several centimeters) into the tissue and skull [5, 15]. This is due to the relatively low absorption of tissue components (water and hemoglobin)

2.3 Optical Imaging

in the NIR spectral range. Therefore, the development of an NIR-based optical imaging system has attracted tremendous attention in the cancer imaging community in recent years. For developing optical-based imaging system, it is important to understand how light interacts with biological tissues. Simply, a tissue can interact with light photons by absorption, scattering and reflection. Since biological tissue represents a complex system in terms of light propagation, it is expected that the optical image would be somewhat distorted. A robust mathematical modeling is thus necessary to improve image quality. Again, all biological tissues somewhat autofluoresce upon interaction with the light in the UV and visible spectrum. The tissue autofluorescence originates from the natural tissue fluorescent molecules such as nicotinamide, flavins, collagen, and elastin [25]. To develop a robust optical imaging system it is thus important to address all sorts of light interaction with tissue as well as tissue autofluorescence. 2.3.2

Optical Imaging Techniques

Optical-based imaging methods such as confocal imaging, multiphoton imaging, microscopic imaging by intravital microscopy or total internal reflection fluorescence microscopy have been used traditionally to image fluorescence events that originates in vivo from surface and subsurface region. Recently, advanced imaging technologies that use photographic systems with continuous or intensitymodulated light and tomographic systems have shown great potential for deep tissue imaging. With the aid of highly sensitive contrast agents such as nanoparticles, it may be possible to transfer optical imaging technology to human application. There are several potential optical imaging techniques, such as reflectance fluorescence imaging and fluorescence-mediated molecular tomography (FMT), that use the diffuse component of light for probing molecular events deep in tissue samples. These techniques are briefly described below. In a typical reflectance imaging technique, a simple ‘‘photographic method’’ is used where the light source and the detector reside on the same side of the imaging object (e.g., an animal). This technique is currently used for in vivo assessment of fluorescent dyes [such as green fluorescent proteins (GFP), bioluminescent molecules, etc.]. In a reflectance imaging system, the light source can be either an appropriate laser for the target fluorescent molecules or a white light with the appropriate low-pass filter. The laser excitation source is preferable because it provides a narrow and well-defined spectral window (G3 nm) when compared with white light (G10 nm). A high-sensitivity CCD camera is usually used as detector. Reflectance imaging has been successfully used to image cathepsin B [28], cathepsin D [29], matrix metalloproteinase 2 (MMP-2) [30], using activatable probes that are dark in the native (quenched) state and fluoresce upon interaction with a specific enzyme. This technique has been used for the elucidation of MMP-2 (a biomarker) expression levels in two different breast cancers, MMP-2 positive human HT1080 fibrosarcoma and MMP-2 negative BT20 mammary adenocarcinoma [31]. A NIR probe was activated upon interaction with both the tumors. The level of MMP-2

49

50

2 Nanoparticles for Optical Imaging of Cancer

expression was directly related to the number of probe molecule activation. As expected, great probe activation was found in human HT1080 fibrosarcoma. Reflectance imaging technique has also been used for targeting cell-surface receptors in vivo using peptide-NIR dye conjugate [26, 32] and for studying gene expression [33]. Reflectance imaging is a simple, fast and highly sensitive imaging and screening technique for capturing surface fluorescent events in vivo or in excised tissues. This imaging system can be, inexpensively, made portable for the laboratory bench. However, this technique has several limitations. Firstly, the technique will allow imaging of a few millimeters thick tissue. As a result, the appearance of deeper lesions is significantly blurred. Secondly, quantitative information cannot be extracted from the reflectance imaging technique. For example, the surface appearance of a small structure with high dye loading that is located in a deeper tissue could be similar with a larger structure of low dye loading that is closer to the surface. Fluorescence-mediated molecular tomography is a powerful technique to resolve and quantify deep tissue fluorescence signal. This technique, usually termed as diffuse optical tomography (DOT) utilizes advanced photon sources, a detection system and rigorous mathematical modeling of light propagation in tissue. The DOT technique uses multiple projections and measures light around the boundary of the illuminated body followed by a complex mathematical modeling to construct the three-dimensional tomographic image. This technique has recently been applied clinically for imaging tissue oxy- and deoxy-hemoglobin concentration and blood saturation [34–37]. Based on the same principle, fluorescence molecular tomography (FMT) has been developed where measurements of fluorescent molecular probes at both the emission and excitation wavelengths were considered. The FMT technique has been recently used for imaging cathepsin B activity in deep tissue structure of 9L gliosarcomas [38–40]. An optical imaging system that can image both reflected light and fluorescence light to generate multi-spectral digital imaging of tissue morphology from a large field of view with mm resolution has also been developed [21]. Readers are encouraged to read a few recent review articles [5, 21, 41] that describe the topic on optical imaging techniques. 2.3.3

Optical Contrast Agents

Optical tissue contrast agents are used in biological systems (e.g., cells, tissues, etc.) to enhance the optical contrast by virtue of their contrast enhancing properties (e.g., fluorescence, scattering, etc.). Tissue contrast agents, for example, are capable of reducing the background signal and improving the image resolution. Fluorescent molecular contrast agents, mostly organic fluorescent compounds, possess high extinction coefficient and quantum yield and have the potential to drastically suppress tissue autofluorescence and hence background signal. Effective delivery (loading) of these contrast agents to the target tissue has also been realized to be one of the most important factors for achieving better image contrast, other than its intrinsic fluorescent characteristics (extinction coefficients, quantum yield, etc.). The concentration of contrast agent per unit volume of target tissue would

2.4 Nanoparticles for Optical Imaging

determine the signal strength. Therefore, a higher loading of contrast agent is always desirable for better image resolution and hence in obtaining a sharp marginal contrast between the normal and the pathological (e.g., a tumor) tissues. A few important features of contrast agents have to be kept in mind prior to using or developing new contrast agents for diagnostic cancer imaging. Firstly, contrast agents with the excitation and emission band maxima in the NIR range (650 to 900 nm) are highly preferable for deep tissue imaging. Secondly, contrast agents should have a high extinction coefficient for effective absorption and a high quantum yield for obtaining strong fluorescence signal. Thirdly, they should be photostable and should not have any photo-sensitizing effects (i.e., photodynamic effect causing the damage of cellular DNA and hence cell death; also termed as photosensitized cell death). Fourthly, contrast agents should be hydrophilic so that an aqueous-based formulation can be easily made. Fifthly, contrast agents should have low toxicity so that they can be administered safely. Lastly, for cancer imaging, contrast agents should be attached to appropriate cancer specific delivery systems (e.g., antibodies, peptides, folates, etc.) for targeting. Organic fluorescent contrast agents, although studied extensively for various bioimaging applications [42], starting from cellular to tissues to whole animal fluorescence imaging have, however, several limitations for them to be considered as robust contrast agents. Firstly, organic fluorescent contrast agents (dyes) rapidly undergo photobleaching. As a result, the fluorescence signal fades away when exposed to the excitation light source (particularly when a laser is used for the excitation), limiting sensitive detection of the target. Secondly, fluorescent dyes are usually hydrophobic. To make aqueous-based formulation, chemical modifications (e.g., sodium salt) are often required that sometimes compromises their spectral characteristics. Thirdly, a handful of fluorescent compounds have been shown to possess low toxicity. Lastly, a few dyes have excitation and emission bands in the NIR spectral range (e.g., cyanine dyes). Another class of fluorescent contrast agents is fluorescent proteins. Fluorescent proteins such as green fluorescent protein (GFP) represent one class of imaging marker genes (IMG, artificial genes) with an optical signature where GFP is the transcriptional product of IMG. The GFPbased optical imaging has been successfully used to study various gene expressions in vivo. For example, human and rodent tumor cell lines, transected with GFP, could be visualized in vivo for monitoring tumor growth and metastasis [43–45]. The major drawback of GFP is limited penetration depth since the tissue can highly absorb the green emission of GFP.

2.4

Nanoparticles for Optical Imaging 2.4.1

Why Nanoparticles for Optical Imaging?

Nanoparticle (NP)-based contrast agents present a whole new class of robust nanometer size (between 1 and 100 nm) particulate materials that has strong potential

51

52

2 Nanoparticles for Optical Imaging of Cancer

for optical imaging of cancer. The use of NPs for bioimaging applications has several advantages. Firstly, the sensitivity of the optical imaging could be greatly improved using nanoparticle-based contrast agents. A classic example is the fluorescent quantum dots (Qdots) and their applications in cancer imaging [6, 13, 21, 46–65]. Qdots are usually crystalline cadmium sulfide (CdS) and cadmium selenide (CdSe)-based semiconductor particulate materials. They are small (10 ns), leading to the emission of a photon in a narrow and symmetric energy band. These spectral characteristics of Qdot materials are different from a typical organic fluorescent molecule with red-tailed broad emission band and short fluorescence lifetimes. In comparison to traditional fluorescent molecules (fluorophores) or fluorescent proteins (e.g., GFP), Qdots have several attractive optical features that are desirable for long-term, multi-target and highly sensitive bioimaging applications. Some of the major optical features of Qdots are described below. (a) Large molar extinction coefficient: Qdots are highly sensitive fluorescent agents (or fluorescent tags) for labeling cells and tissues. Unlike organic fluorescent compounds, Qdots have very large molar extinction coefficients [99], typically of the order of 0.5–5  10 6 m1 cm1 which means that Qdots are capable of absorbing excitation photons very efficiently (the absorption rate is approximately 10– 50 faster than organic dyes). The higher rate of absorption is directly correlated to the Qdot brightness and it has been found that Qdots are approximately 10–20 brighter than organic dyes [100–102], allowing highly sensitive fluorescence imaging.

2.4 Nanoparticles for Optical Imaging

(b) Excellent photostability: Qdots are several thousand times more photostable than organic dyes. This feature allows real-time monitoring of biological processes over a long period. (c) Much longer lifetime: Qdots are highly suitable for time-correlated lifetime imaging spectroscopy. This is possible due to the longer excited state lifetime of Qdots (about one order of magnitude longer than that of organic dyes), allowing effective separation of Qdot fluorescence from the background fluorescence. This will improve the image contrast by reducing the signal-to-noise ratio dramatically [103, 104] in the time-delayed data acquisition mode. (d) Large Stokes shift: Unlike in organic dyes, the excitation and emission spectra of Qdots are well separated (i.e., large Stokes shift value; up to 400 nm, depending on the wavelength of the excitation light). This allows further improvement of sensitivity of the detection by reducing the high autofluorescence background often seen in biological specimens [6]. (e) Multiple targeting capability. The wavelength of Qdot emission is size dependent. This is a unique feature of Qdot materials in comparison to organic fluorescent dyes. The size dependent emission of Qdots allows imaging and tracking of multiple targets simultaneously using a single excitation source. This feature is particularly important in tracking a panel of disease-specific molecular biomarkers simultaneously, allowing classification and differentiation of various complex human diseases [105]. The development of Qdot-based fluorescent probes involves a multi-step process: synthesis, surface capping and bioconjugation. Each step is described below in detail. Qdot Synthesis Qdot nanocrystals are made out of hundreds to thousands of atoms that typically belong to group II and VI elements or group III and V elements in the periodic table. For example, CdSe, CdTe, and ZnSe are group II–VI semiconductor Qdots, whereas InP and InAs Qdots are group III–V semiconductors. The Qdot emission can be continuously tuned from 400 to 2000 nm by changing both the particle size and chemical composition. Herein, we briefly describe two robust synthesis techniques that produce high quality Qdots. Hot Solution-phase Mediated Qdot Synthesis This is most popular technique of synthesizing high quality Qdots. Typically, Qdots are synthesized at elevated temperature in high boiling point non-polar organic solvents. Bawendi’s group have reported [106] the synthesis of highly crystalline and monodisperse (size distribution 8–11%) CdSe Qdots using high-temperature growth solvents/ligands (mixture of trioctylphosphine/trioctylphosphine oxide, TOP/TOPO). A combination of TOPO and hexadecylamine can also be used [96]. The purpose of using hydrophobic organic molecules as mixed solvents or as a solvent/ligand mixture is two-fold. The mixture serves as a robust reaction medium and also coordinates with unsaturated metal atoms on the Qdot surface to prevent the formation of bulk semiconductors. Following a similar synthesis strategy, Qu et al. have reported the formation high

55

56

2 Nanoparticles for Optical Imaging of Cancer

quality CdSe nanocrystals having fluorescence quantum yields as high as 85% at room temperature [107]. Reverse-micelle Mediated Qdot Synthesis The reverse micelle synthesis of high quality CdS:Mn/ZnS core–shell Qdots has been reported [86, 89, 90, 108–110]. Reverse micelles [also called water-in-oil (W/O) microemulsion system] are an isotopic, thermodynamically stable homogeneous mixture of oil, water and surfactant molecules where the surfactant capped (stabilized) water droplets remain uniformly dispersed in the bulk oil phase. The water droplets serve as a tiny reactor (nano-reactor) for the synthesis of Qdots. This is a simple procedure that does not require extreme reaction conditions such as high temperature or high pressure. This is a robust method that allows room temperature synthesis of monodisperse Qdots at normal atmospheric pressure. Yang et al. have reported the synthesis of manganese-doped cadmium sulfide core and zinc sulfide shell (CdS:Mn/ZnS) Qdots using AOT (dioctylsulfosuccinate sodium salt, a surfactant)/heptane (an oil)/water reverse micelle system [90, 108, 109]. The bright yellow emitting CdS:Mn/ZnS Qdots are small (average Qdot size was 3.2 nm) and highly photostable. Qdot Surface Passivation and Aqueous Stabilization Effective surface passivation of the Qdot nanocrystal core with wide bandgap semiconductor materials (shell) is extremely important [100, 111] for the following reasons. For example, with cadmium selenide/zinc sulfide (CdSe/ZnS) core–shell Qdots, the epitaxially matched ZnS layer effectively passivates the surface defects of the CdSe core [100, 108, 112], protects the core from oxidation, prevents leaching of highly toxic Cd 2þ ions, and also drastically improves the quantum yield by reducing surface defects (that act as exciton traps, leading to non-radiative recombination processes). Surface passivation with silica is effective for the CdSe core [90, 113, 114] nanocrystals. TOP/TOPO-capped Qdots prepared using hot solution phase mediated synthesis route are hydrophobic. For biological applications, however, it is necessary to obtain aqueous dispersible Qdots. Therefore, phase transfer from the organic (e.g., toluene, hexanes, chloroform) to aqueous solution is usually performed by surface functionalization with hydrophilic ligands. There are three major routes to surface functionalization. Firstly, the ‘‘cap exchange’’ route that involves replacement of TOP/TOPO capping with bifunctional ligands. The bifunctional ligands [102, 115–118] have two functional moieties, Qdot surface anchoring (e.g., thiol) and hydrophilic moieties (e.g., hydroxyl, carboxyl). Secondly, the formation of a hydrophilic silica shell [86, 89, 90, 101, 108, 119] that encapsulates the Qdot. Lastly, the over-coating of TOP/TOPO-capped Qdots with amphiphilic ‘‘diblock’’ and ‘‘triblock’’ copolymers and phospholipids [6, 64, 120–124]. Notably, Qdots capped with mono-mercapto ligands have short shelf-lives, about a week, due the weak (dynamic) thiol–ZnS interaction [125], although polydentate thiolated ligands (containing more than one thiol groups) afford better stability (from a week to a couple of years) [116, 118, 125]. Applying a silica shell over the Qdots has several advantages with respect to long-term stability (shelf-life) and bio-

2.4 Nanoparticles for Optical Imaging

compatibility. Also, a silica coating remains stable upon pH fluctuation (below pH 8) and a further coating with a multifunctional hybrid silica is possible using appropriate silane reagents [86, 89]. The polymer/phospholipid encapsulation is also robust in terms of long-term stability. Both the silica and the polymer/ phospholipid coating increase the particle size (@20–30 nm). However, Yang et al. reported the W/O microemulsion-mediated synthesis of silica-overcoated CdS:Mn/ ZnS Qdots [90] where the silica shell thickness was approximately 2–3 nm. Qdot Bioconjugation Qdot bioconjugation represents the attachment of biomolecules (e.g., proteins, antibodies, peptides, DNA, etc.) to the Qdot surface, forming a hybrid structure, interfacing both the inorganic and the biological materials, for targeting to biological systems such as cells, tissues, etc. either specifically or nonspecifically. Qdots are comparable or slightly larger than many proteins. For example, a 510 nm green-emitting Qdot size is comparable to GFP and a 650 nm redemitting Qdot size is comparable to DyRed (a red-emitting fluorescent protein) [126]. Medintz et al. have shown that about 15–20 maltose binding proteins (Mr @ 44 kDa) can be conjugated to a single 6-nm Qdot [127]. There are three major ways to attach proteins to Qdot surface. Firstly, using carbodiimide [e.g., EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide] coupling chemistry, carboxylated Qdots are covalently conjugated to the protein molecules through the formation of stable amide bond. Secondly, disulfide bonds can be formed between Qdot surface sulfur atoms (from the ZnS surface) and peptides containing cysteine residues [128, 129]. Histidine-expressing proteins [130] or peptides containing polyhistidine residues [131–133] can also be directly attached to the Zn atom on the Qdot surface. Lastly, engineered proteins containing positively charged domains can be non-covalently adsorbed onto the negatively charged Qdot surface via electrostatic interaction [116, 134, 135]. Although various bioconjugation strategies have been tested, none of them can control the ratios of proteins per Qdots. There is certainly a lack of experimental tools with which to discern the orientation of a protein immobilized on a Qdot surface. For specific targeting, it is highly desirable that the delivery proteins (e.g., antibody) are properly oriented and fully functional. The Qdot bioconjugation step is, therefore, extremely important in obtaining success in bioimaging. Gold Nanoparticles For over 30 years, nanometer-sized gold particles have been used to stain cells and tissue samples for electron microscopy. The basic principle of interactions between gold particles and biomolecules, like proteins, has been well studied for immunocytochemical staining applications. Although nanosize metals like gold and silver do not fluoresce they can effectively scatter light due to the collective oscillation of the conduction electrons induced by the incident electric field (light). This is known as ‘‘surface plasmon resonance’’ [20]. Thus, colloidal gold particles exhibit a range of intense colors in the visible and NIR spectral regions. Gold nanoparticles, because of their strong SPR properties, have attracted considerable attention in bioimaging in recent years. The SPR signal originates from 2.4.2.2

57

58

2 Nanoparticles for Optical Imaging of Cancer

the collective oscillation of conduction electrons upon interaction with absorption photons [136]. The SPR frequency depends on various factors, e.g., particle size [137] shape [138–140], dielectric properties [141, 142], aggregate morphology [143–146], surface functionalization [147, 148] and the refractive index of the surrounding medium [149–152]. Gold nanoparticles have high absorption [146, 151, 153] and scattering cross section [154, 155]. For example, the absorption cross section of a 5 nm diameter gold particle is about 3 nm [156] at a wavelength of 514 nm [136], which is about two orders of magnitude higher than that of organic fluorescent molecules at room temperature. The scattering cross section of gold nanoparticles is much larger than polymeric spherical particles of similar size, especially in the red region of the spectrum, i.e., red to NIR range, having potential in deep tissue imaging. For example, using composite core–shell gold (dielectric silica core and gold shell) particles, it is possible to tune the scattering from 600 to 1200 nm [141]. Due to excellent biocompatibility [157–159], gold nanoparticles have been widely used in immunohistochemistry (gold-based staining) and in ultra-sensitive DNA detection assays [146, 160, 161]. However, a few literature reports are available on gold nanoparticle-based cancers imaging. Gold Nanoparticle Synthesis Various methods have been reported for the synthesis of gold nanoparticles. There are two general approaches (so-called ‘‘top-down’’ and ‘‘bottom-up’’) that primarily categorize most reported synthesis strategies. Synthesis of gold nanoparticles by employing laser ablation technique is an example of a ‘‘top-down’’ approach, where the embryonic (nascent) particles are formed from the ionized gold atoms via nucleation and growth processes. The challenge still remains how to stabilize particles in the solution phase. Using a surfactant-based capping agent (sodium dodecylsulfate, an ionic surfactant), Kondow et al. have successfully stabilized ultrafine (@5 nm) particles [162]. The capping agents, in general, control the particle size and size distribution, prevents particle aggregation, and stabilize particle solution (such as in aqueous-based medium). In the ‘‘bottom up’’ approach, gold nanoparticles and gold nanocomposites (e.g., composite of gold and silica) have been chemically synthesized by reducing gold precursors. Various reduction methods have been reported. The major synthesis routes are as follows. (a) Reduction of gold precursors (e.g., hydrochloroauric acid, HAuCl4 ) using appropriate reducing agents, such as citrate [163–166], sodium borohydride [167], ascorbic acid [168], etc. The citrate reduction of the gold(iii) ions has been widely used. While sodium citrate reduces [AuCl 4 ] ions in hot aqueous solution, it forms a colloid. The reported average particle size is about 20 nm. Both the citrate ions and the oxidation products (e.g., acetone dicarboxylate) act as capping agents [163–165]. In conjunction with citrate ions, amphiphile surfactants have also been used that allowed particle size tuning upon varying the gold/stabilizer ratio [166]. A two-phase synthesis of gold nanoparticles (the Brust–Schiffrin method) has been reported in which a phase-transfer agent (tetraoctylammonium bromide) is used to transfer [AuCl 4 ] ions from an aqueous phase to an organic phase (tol-

2.4 Nanoparticles for Optical Imaging

uene) containing alkanethiol stabilizer. The Au(iii) in organic phase is reduced by the addition of aqueous sodium borohydride. The resulting Au clusters are then capped immediately by alkanethiols. The Brust–Schiffrin method produces monodisperse particles (approx 1.4 G 0.4 nm) in the diameter range 1.5–5.2 nm [167, 169]. (b) Microemulsions [170–172], copolymer micelles [173], reversed micelles [172], surfactant, membranes, and other amphiphiles have been widely used for the synthesis of stabilized gold nanoparticles. Wilcoxon and coworkers have studied the synthesis of gold nanoparticles formed in aqueous media and in reverse micelles, using chemical and photolytic reduction [174]. The chemical reduction method was achieved using reduction agents such as hydrazine, sodium borohydride, and metallic sodium. The advantages of using reverse micelles for the synthesis of gold nanoparticles are (i) it produces monodisperse particles, (ii) particles remain coated by surfactant molecules that prevent particle aggregation surrounding the molecule and (iii) the size of the nanoparticles can be easily varied by changing reaction parameters such as concentrations of the reagents, water-tosurfactant molar ratio, temperature, time allowed for ripening of particles, etc. (c) Another popular, long standing, method is the seed mediated route [175]. Indeed, the use of preformed metallic seeds as nucleation centers in nanoparticle synthesis has a long history [175–182]. Various references are available on the seed mediated growth of gold nanoparticles and also of gold nanorods [183–189]. Further nucleation during the ‘‘growth’’ part of the reaction often leads to non-uniform size distribution [190, 191]. The presence of seeds appears to cause additional nucleation (sometimes referred to as secondary nucleation) [184]. The step-bystep particle enlargement is considered more effective than the one-step seeding method to avoid additional nucleation [192]. More recently a modification of seed mediated method was used by Loo et al. [193] to achieve gold coating on the silica nanoparticles. Gold shells were grown using the method of Duff et al. [194]. Briefly, small gold colloid (1–3 nm) was adsorbed onto the aminated (amine functionalized) silica nanoparticle surface. More gold was then reduced onto these colloid nucleation sites using potassium carbonate and HAuCl4 in the presence of formaldehyde. Gold shell particles have been used for whole blood immunoassay [195], photothermal tumor ablation [196] molecular imaging in live cells [197] and for cancer imaging and therapy [198]. (d) Reduction of gold precursors using a combination of appropriate reducing agents and radiation such as UV [188, 199–201], ultrasound [202–207], heat [208–210]. The UV irradiation method has been used to prepare gold nanoparticles [188, 199, 200, 211, 212], including when in synergy with micelles [211] or seeds [188]. The ‘‘gold seed particles’’ are prepared photochemically by UV irradiation, preferably in the presence of a neutral micelle of a non-ionic surfactant, Triton X-100 [poly(oxyethylene) iso-octyl phenyl ether] [201]. The seed particles subsequently grow by successive addition of metal ions and, again, exposure to UV irradiation under the same experimental conditions. Several reports are available on the sonochemical synthesis (chemical synthesis using ultrasound radiation) of gold nanoparticles [202–207]. The general mecha-

59

60

2 Nanoparticles for Optical Imaging of Cancer

nism of reduction is briefly described here. When a solution is exposed to ultrasound radiation of sufficient intensity it produces a cavitation field made up of a large distribution of vapor and gas-filled bubbles, which pulsate continuously. When the pressure inside the bubble falls below the vapor pressure of the liquid the bubble fills with vapor and grows. At a certain point when the pressure turns positive the bubble collapses, resulting in extreme temperatures and pressures in the interior. The localized hot point causes ionization of molecular species present within the interior of the collapsing bubble. For water vapor, this dissociation results in the production of H and OH radicals [213]. The radicals produced in the interior of the bubble can then diffuse into the bulk solution and reduce metal ions, yielding nanosized metallic particles such as gold nanoparticles. Gold has also been fabricated by employing thermolysis [208–210] of organic derivatives of gold. This novel strategy involves the reductive elimination of thiolate ligands with simultaneous attachment of an organic moiety on the growing nuclei [210]. Surface Functionalization and Bioconjugation As mentioned in the previous section, gold nanoparticles have been widely used as immunostaining agents for labeling cell, tissue section, blots, etc. In general, protein conjugated gold nanoparticles are mostly used as labeling probes. Although the actual mechanism of macromolecule (e.g., proteins) binding to gold particles is poorly understood, some of the accepted mechanisms are [214]:

(1) Protein binding via electrostatic (ionic) interaction. Negatively charged gold nanoparticles can bind to positively charged protein domains via electrostatic interactions. (2) Protein binding via hydrophobic interaction. Hydrophobic domains present in the protein structure can interact with the metal surface of the particle. (3) Protein binding via chemical interaction. Protein molecules containing sulfohydryl (aSaH) groups can chemically interact with the gold atoms. This is also called dative binding. Other biomolecules, such as protein A, antibodies, lectins, avidins (or streptavidins), etc., have also been conjugated to gold nanoparticles to be used as sensitive probes. Some of the binding strategies are described below. Protein A–Gold Conjugate Protein A–gold conjugates are generally prepared by adsorbing protein A onto the gold surface. Following a similar method, many other immunoglobulin binding proteins can also be attached to gold nanoparticles. These probes have been used as ‘‘universal’’ probes for labeling cells, tissue sections and various blots. In a typical tissue labeling experiment, primary antibodies are specifically targeted to the tissue antigens. In the following step, protein A– gold conjugates bind to the antibodies. The advantage of this labeling technique is that the same protein A–gold conjugate can be used for various immunochemical procedures.

2.4 Nanoparticles for Optical Imaging

Antibody–Gold Conjugate Antibody–gold conjugated probes are prepared by coating antibodies directly on to the gold nanoparticle surface. These probes have been successfully used for the detection, localization and quantification of antigens on the target specimens. This is a powerful technique for detection of pathogens, intracellular foreign substances, monitoring cellular metabolic processes, etc. Lectin–Gold Conjugate Lectin-coated gold nanoparticle probes have been used for the detection of sugar-binding receptors that are expressed on the cell membranes. Lectin molecules have specific carbohydrate binding sites. In this assay, a specific carbohydrate molecule is sandwiched between the lectin molecule and the cellular receptor. The objective of this assay is to localize glycoproteins, glycolipids, etc. on cell surfaces. Avidin (or Streptavidin)–Gold Conjugate Avidin–gold conjugated probes have been used to localize, detect and quantify biotin molecules. This assay is similar to protein A–gold complex-based assays except that the primary antibodies are attached to biotin molecules. 2.4.2.3 Dye-doped Silica Nanoparticles Amorphous silica (silicon dioxide) nanoparticles that are produced via Stober’s sol–gel [67, 215, 216] or microemulsion technique [66, 68, 91, 93, 217–225] have recently found applications in the area of bioimaging. Unlike Qdots or gold nanoparticles, silica does not have inherent strong fluorescence that can be exploited for sensitive imaging applications. However, silica nanoparticles can be made fluorescent by incorporating fluorescent dye molecules inside the silica matrix (dyedoping). Another approach could be attaching fluorescent dye molecules (via covalent binding) on the silica surface. For bioimaging applications, it is preferable that dye molecules remain encapsulated by the silica matrix for the following reasons. Silica-based nanoparticles exhibit several attractive features, e.g., silica is water dispersible and is resistant to microbial attack. The size of silica particles remains unchanged by changing solvent polarity (i.e., resistant to swelling) and, therefore, silica porosity remains unaltered in a wide selection of solvents, including aqueous-based neutral and acidic solutions. A silica matrix is optically transparent, allowing excitation and emission light to pass through efficiently. Moreover, fluorescent dyes can be effectively entrapped inside the silica particles. The spectral characteristics of the dye molecules remain almost intact. Silica encapsulation provides a protective layer around dye molecules, reducing oxygen molecule penetration (which causes photodegradation of dye molecules) both in air and in aqueous medium (in the latter case dissolved oxygen). As a result, photostability of dye molecules increases substantially compared with bare dyes in solution. Amorphous silica appears to be a biocompatible [22] and non-toxic [23] material, and has potential biological applications. The surface of a silica particle can be easily modified to attach biomolecules such as proteins, peptides, antibodies, oligonucleotides, etc., using conventional silanebased chemistry. For example, carboxylated silica nanoparticles can be covalently

61

62

2 Nanoparticles for Optical Imaging of Cancer

attached to the amine groups of proteins, antibodies etc. through the formation of stable amide bond [216]. Peptides containing a cysteine residue (through a aSaH group) can be attached to the aminated silica nanoparticles [89] through (SPDP) coupling chemistry. A general synthesis strategy of fluorescent silica nanoparticles is the incorporation of organic or metalloorganic dye molecules inside the silica matrix [66, 93, 226–231]. For example, a metalloorganic dye, tris(2,2 0 -bipyridyl)dichlororuthenium(ii) (Rubpy), has been entrapped inside silica nanoparticles using a reverse microemulsion-based synthesis approach [66] where the positively charged Rubpy molecules were electrostatically bound to the negatively charged silica matrix. Dyedoped silica-based imaging probes are non-isotopic, sensitive and relatively photostable in the physiological environment. Additionally, the interaction potential of the silica surface can be easily manipulated to facilitate the interaction with cells [232–234]. Due to these novel features, functionalized silica nanoparticles (FSNPs) have found widespread applications in bioanalysis and bioimaging applications. Synthesis There are two reported synthesis routes to dye-doped silica nanoparticles: Stober’s sol–gel method and the reverse microemulsion method. Stober’s Method In a typical Stober’s method, alkoxysilane compounds [e.g., tetraethylorthosilicate (TEOS), tetramethylorthosilicate (TMOS), various TEOS or TMOS derivatives, etc.] undergo base-catalyzed hydrolysis and condensation in an ammonia–ethanol–water mixture, forming a stable alcohol. This method has been widely used for synthesizing both pure and hybrid (when more than one silane compound are used, such as dye-doped silica particles) silica nanoparticles with particle diameters ranging from a few tens to several hundreds of nanometers (sub-micron size). Following Stober’s protocol with a slight modification, fairly monodisperse organic dye doped fluorescent silica nanoparticles have been synthesized. Since organic dyes are normally hydrophobic, doping them inside the hydrophilic silica matrix is not straightforward. Typically, a reactive derivative of organic dye (e.g., amine-reactive fluorescein isothiocyanate, FITC) is first reacted with an amine-containing silane compound (e.g., APTS), forming a stable thiourea linkage. Then FITC conjugated APTS and TEOS are allowed to hydrolyze and condense to form FITC conjugated silica particles. Note that particles so formed will have some amount of bare dye molecules on the particle surface that is covalently attached. These bare dyes, due to their hydrophobic nature, will somewhat compromise the overall particle aqueous dispersibility and, also, they will be prone to photobleaching. Therefore, an additional coating with pure silica is usually applied around the dye-conjugated silica nanoparticles. Using Stober’s method, bulk amounts (kilograms) of silica particles can be easily produced in a typical laboratory setup. Reverse Microemulsion (W/O) Method This method is used for the synthesis of pure silica, as well as inorganic and organic dye-doped silica nanoparticles. Figure 2.3 shows a schematic representation of dye-doped silica nanoparticle synthesis steps. The W/O microemulsion is a robust technique for producing monodisperse

2.4 Nanoparticles for Optical Imaging

Scheme of a water-in-oil (W/O) microemulsion mediated synthesis of dyedoped silica nanoparticles. (A) An immiscible mixture of water and oil (bulk phase). Upon addition of an appropriate surfactant, a W/O microemulsion is formed (B), where each tiny water droplet (nanosize water pool) is stabilized in the bulk oil phase with a surfactant coating (C). In reality each nanosize

Fig. 2.3.

water droplet serves as a nanoreactor for the synthesis of nanoparticles (D). The nanoparticle core (shown as filled circles), intermediate shell (inner ring) and outermost shell (outer ring) of the dye-doped silica nanoparticles are constructed in modular fashion by adding appropriate silane-based reagents at various stages of the synthesis process.

particles in the nanometer size range (tens to a few hundred nanometers). Figure 2.4 shows a typical transmission electron microscopic image of dye-doped silica nanoparticles synthesized using the W/O microemulsion technique. The W/O microemulsion is an isotropic, single-phase system that consists of surfactant, oil (as the bulk phase) and water (as nanosize droplets). Each surfactant-coated water droplet that is stabilized in the oil phase serves as an individual nanoreactor for the synthesis of silica nanoparticles. The water droplets undergo rapid and spontaneous collision and coalescence (fusion followed by separation) processes. As a result droplet contents (e.g., water-soluble reagents) are mixed together and chemical reactions (e.g., precipitation, hydrolysis and condensation reactions, etc.) take place. The surfactant present at the interface of oil and water nanodroplet is responsible for the thermodynamic stability of the W/O microemulsion system. Nucleation and growth processes are carried out inside the confined spherical volume of the nanoreactor. Varying the water-to-surfactant molar ratio and the dynamic properties of the microemulsion system helps to control the size of the nanoparticles.

63

64

2 Nanoparticles for Optical Imaging of Cancer

Typical transmission electron microscopic image of dye-doped silica nanoparticles about 100 nm in size. Particles were synthesized using the water-in-oil (W/O) microemulsion technique. As expected, nanoparticles were highly monodisperse, Fig. 2.4.

confirming that the W/O microemulsion is a robust technique for the synthesis of dyedoped silica nanoparticles. This technique can be easily adopted to produce other types of nanoparticles such as quantum dots and multimodal nanoparticles.

The fluorescence brightness of dye-doped silica nanoparticles can be improved by incorporating high-quantum-yield organic dyes having large absorption coefficients. In other words, brighter probes will improve the image resolution if encapsulated fluorescent dyes do not experience substantial photobleaching during imaging. Surface Functionalization and Bioconjugation For bioimaging (e.g., cancer imaging), it is highly desirable that dye-doped silica nanoparticles are appropriately surface modified with cancer targeting molecules such as cancer specific antibodies, folates. This surface modification involves a few steps. Firstly, the particle surface should be modified to obtain appropriate functional groups such as, amines, carboxyls, thiols, etc. Secondly, using suitable coupling reagents, nanoparticles are attached to the bio-recognition molecules (such as antibodies, folates, etc.). Lastly, bioconjugated particles are targeted to cancers. Note that all these steps are usually carried out in aqueous-based solutions. A few bioconjugation methods are briefly mentioned below.

(1) Bioconjugation with carboxylated particles. This is one of the most common bioconjugation techniques to immobilize protein molecules on silica nanoparticles. The surface of the nanoparticle is modified to obtain carboxyl groups (aCOOH) by using a carboxylated silane reagent. Biomolecules such as proteins, antibodies, etc. containing free amine functional groups are then covalently attached to the carboxyl functionalized nanoparticle, using carbodiimide-coupling chemistry [235]. (2) Bioconjugation with aminated particles: Many cancer cells overexpress folate receptors. Cancer targeting with folate-conjugated nanoparticles has been

2.5 Optical Imaging of Cancer with Nanoparticles

recently reported [67]. Folates are chemically attached to aminated silica nanoparticles using carbodiimide chemistry. (3) Bioconjugation with avidin–biotin binding: Avidin is a protein molecule that contains four specific binding pockets for biotin molecules. A strong binding affinity exists between avidin and biotin molecules, which is comparable to covalent binding. Avidin-coated nanoparticles are typically attached to biotinylated molecules such as antibodies, proteins, etc. [236]. (4) Bioconjugation through disulfide bonding: Sulfohydryl-modified nanoparticles are conjugated to disulfide-linked oligonucleotides (e.g., DNAs). In this method, oligonucleotides are attached to nanoparticles through di-sulfide bond formation [237]. (5) Bioconjugation using cyanogen bromide chemistry: Nanoparticles with hydroxyl groups (such as silica) can be activated with cyanogen bromide to form a reactive aOCN derivative of the nanoparticles. The OCN derivative then readily reacts with proteins (via amine groups), forming a ‘‘zero-length’’ bioconjugate as there is no spacer between the particle surface and the protein molecule [66].

2.5

Optical Imaging of Cancer with Nanoparticles

Here we discuss the use of nanoparticle-based optical contrast agents in in vitro and in vivo experiments to image cancerous tissues. These nanoparticle-based contrast agents should provide a new gateway to characterize cancer at the molecular level. As we have already realized, these ultra-sensitive and specific probes provide a viable alternative to rapidly and non-invasively image the uptake, distribution and binding of nanoparticles to tumors. To establish the widespread use, it is important to understand the delivery, interaction and recognition mechanism of these contrast agents with cancer cells. Various delivery vehicles with varying specificity have been used to target cancer tissues, mainly for drug delivery applications, some of which are folates, antibodies, lectins, growth factors, cytokines, hormones and low-density lipoproteins. Obviously, most of these carriers can be similarly used for molecular imaging applications. These can be broadly classified [6, 238] as active and passive targeting. 2.5.1

Active Targeting

This refers to the conjugation of targeting ligands to nanoparticles to provide preferential accumulation into the tumor antigens and blood vessels with high affinity and specificity. This relies on specific interactive forces between lectins– carbohydrate, ligand–receptors and antibody–antigens [214]. Lectins can recognize and bind to glycoproteins that occur on the surface of cells. These proteins can bind to certain carbohydrates in a specific manner. Direct

65

66

2 Nanoparticles for Optical Imaging of Cancer

and reverse lectin targeting have made used of this specific interactions to receptors or antigens expressed by the plasma membrane. Folate receptor-based interactions are an excellent example of ligand–receptor based active targeting. Folate receptors are overexpressed on the surface of various cancers like those of the brain, ovary, kidney, breast and lungs. Confocal microscopic studies have demonstrated the selective intake and receptor-mediated endocytosis of folate-conjugated nanoparticles by tumor cells. Antibody-mediated tumor targeting has been performed for detecting the presence of antigenic moieties on the surface of cancer cells. Tumor targeting ligands like monoclonal antibodies are attached to nanoparticles to target the specific receptors. These moieties are minimally present on the surface of normal tissues. Only certain antigens are actually tumor-specific and are referred to as tumorspecific antigens. 2.5.2

Passive Targeting

This mode of targeting particles to tumors includes strategies like using the enhanced permeation and retention (EPR) effect, use of a unique tumor environment and a direct local delivery of imaging agents to tumors. In the EPR strategy, nanoparticles with a hydrophilic surface and diameter < 100 nm are made to accumulate at the tumors. The nanoparticles are engineered to prevent their uptake by the reticuloendothelial system, resulting in faster circulation and enhanced targeting ability in the physiological environment. Various researchers have demonstrated the strategy of exploiting the unique tumor environment to trigger the release of therapeutic drugs. The drug is conjugated to tumor specific ligands and remains inactive till it reaches its target site. On reaching the tumor, the linkages are hydrolyzed either by the enzymes present, or by a change in pH and the drug is released by the nanoparticles. Sometimes the imaging agents can be locally delivered to avoid its systemic circulation. But this is a challenging procedure as it involves the precise delivery using injections or surgical procedures that can be frequently cumbersome. 2.5.3

Cancer Imaging with Quantum Dots

Surface-functionalized quantum dots have been used to image various tumor cells and tissues in in vitro and in vivo experiments. Some of the different cell lines that have been used are human mammary epithelial tumor (MDA-MB-231) [239], human breast cancer (MDA-MB-435S [240], MDA-MD-435 [129], MCF 7 [240], and SK-BR-3 [64]), human prostate cancer [6], squamous carcinoma [19, 241] B16 melanoma (skin cancer) [56], human neuroblastoma (SK-N-SH) [242], colon tumor (SW480) [240], lung tumor (NCI H1299) [240], and bone tumor (Saos-2) [240] cells.

2.5 Optical Imaging of Cancer with Nanoparticles

In 2002, Parak and coworkers used water-soluble [119] siloxane-coated quantum dots of two different sizes (2.8 and 4.1 nm cores), functionalized with thiol and/or amine groups, to label human mammary epithelial tumor cells (MDA-MB-231) [239]. These Qdots emitted at 554 and 626 nm, respectively, and were more photo-stable than ordinary organic dyes. Confocal microscopic images verified the presence of nanocrystals ingested rapidly inside the cells, and not on the surface. The quantum dot crystals were found in the perinuclear region of the cells even after a week. Almost at the same time, Ackerman and coworkers [129] incubated human breast carcinoma MDA-MD-435 cells with peptide coated quantum dots. These cells were then injected into mice to create tumor grafts. The mice were imaged 8–12 weeks after tumor inoculation. Although quantum dot probes were not detected inside the mice, in vitro tests showed that peptide-coated Qdots could specifically target tumor cells. Wu et al. have used streptavidin-conjugated commercial Qdots, QD 560 (emission maximum 560 nm) and QD 608 (emission max. 608 nm), to detect Her2 cancer markers on the surface of human breast cancer cells (SK-BR3) [64]. The nanocrystals effectively labeled the cancer cells with negligible affinity to normal cells. Recently, Nie and coworkers [6] have developed multifunctional nanoparticle probes (2.5 nm radius core protected by a 1-nm TOPO cap with 2-nm polymer coating and 5 nm PEG/affinity ligand shell) for imaging. The quantum dots were used for in vivo imaging to target to tumor sites either through a slow passive targeting process or a more efficient active targeting process. Squamous carcinoma cells have been labeled with quantum dots conjugated to epidermal growth factors (EGF) [19, 241]. These probes with a broad fluorescence peak in the NIR at 770 nm can specifically bind and activate the EGF receptors [241] of cancer cells in C3H mice [19]. Similarly dihydroxylipoic acid (DHLA)-capped quantum dots can be efficiently delivered into B16 melanoma (skin cancer) cells [56]. The melanoma cells were labeled with the Qdots and injected into live mice to track tumor cell extravasation. The Qdots did not pose a detectable threat [56] to the labeled cells or the host animal and behaved as ‘‘inert fluorescent tags.’’ Quantum dots have also been used to detect integrin av subunits in human neuroblastoma cells (SK-NSH) [242] and label mouse lymphocytes (EL-4 cells derived from murine T-cell lymphoma) [243]. In vivo imaging to map sentinel lymph nodes (SLN) in rats and pigs has also been achieved using Qdots [238, 244, 245]. The presence of lymph node metastases is an early warning signal for breast and lung cancer. NIR nanocrystals with oligomeric phosphine coating (for solubility in aqueous buffers) was used to guide a surgeon during cancer surgery. A new quantum dot based tool, called ‘‘Quantum Dot Phagokinetic Track assay’’ has been developed [240] to quantify the invasive potential of different tumor cells. When cancer cells move through a bed of Qdots, they engulf the nanocrystals and leave behind a phagokinetic trail depleted in Qdots. Cells with higher metastatic potential engulf more Qdots and leave a clearer trail than those with weak metastatic potential. Seven different cancerous and non-cancerous cell lines were used

67

68

2 Nanoparticles for Optical Imaging of Cancer

to compare the different invasive potentials, including breast epithelial MCF 10A, breast tumor MDA-MB-231, MDA-MB-435S, MCF 7, colon tumor SW480, lung tumor NCI H1299, and bone tumor Saos-2. This assay rapidly discriminates between invasive and non-invasive cancer cell lines with greater sensitivity than the conventional Boyden chamber invasion assay. 2.5.4

Cancer Imaging with Gold Nanoparticles

Gold bioconjugates have been used for vital imaging of precancerous and cancerous cells by researchers for in vitro and in vivo experiments. The unique optical property of the metal in the nanosized range has been used for detecting breast carcinoma cells (SK-BR-3) [246] breast cancer markers like HER2, oral epithelial live cancer cells (HOC 313 clone 8 and HSC 3) [73] and neoplastic cervical biopsies [75]. Gold nanocages