Design and Applications of Theranostic Nanomedicines 0323899536, 9780323899536

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Woodhead Publishing Series in Biomaterials

Design and Applications of Theranostic Nanomedicines Edited by

Somasree Ray Professor, Department of Pharmaceutics, Gupta College of Technological Sciences, Asansol, West Bengal, India

Amit Kumar Nayak Professor, Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Jharpokharia, Mayurbhanj, Odisha, India

Woodhead Publishing is an imprint of Elsevier 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, OX5 1GB, United Kingdom Copyright © 2023 Elsevier Ltd. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-323-89953-6 For information on all Woodhead Publishing publications visit our website at https://www.elsevier.com/books-and-journals Publisher: Matthew Deans Acquisitions Editor: Sabrina Webber Editorial Project Manager: Andrea Gallego Ortiz Production Project Manager: Prem Kumar Kaliamoorthi Cover Designer: Matthew Limbert Typeset by TNQ Technologies

Dedicated to our beloved teacher Prof. (Dr.) Biswanath Sa.

List of contributors

Bandar E. Al-Dhubiab Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa, Saudi Arabia Ashique Al Hoque Department of Pharmaceutical Technology, Jadavpur University, Kolkata, West Bengal, India Abul Kalam Azad Faculty of Pharmacy, Pharmaceutical Technology Unit, AIMST University, Kedah, Malaysia Jaya Bajpai Bose Memorial Research Lab, Department of Chemistry, Government Autonomous Science College Jabalpur, Madhya Pradesh, India A.K. Bajpai Bose Memorial Research Lab, Department of Chemistry, Government Autonomous Science College Jabalpur, Madhya Pradesh, India Saad Bakrim Laboratory of Molecular Engineering, Valorization and Environment, Department of Sciences and Techniques, Polydisciplinary Faculty of Taroudant, Ibn Zohr University, Taroudant, Souss-Massa, Morocco Abdelaali Balahbib Laboratory of Biodiversity, Ecology, and Genome, Faculty of Sciences, Mohammed V University in Rabat, Rabat, Rabat-Salé-Kénitra, Morocco Souvik Basak Dr. B.C. Roy College of Pharmacy & Allied Health Sciences, Dr. Meghnad Saha Sarani, Bidhan Nagar, Durgapur, West Bengal, India Anindita Behera School of Pharmaceutical Sciences, Siksha ‘O’ Anusandhan Deemed to be University, Bhubaneswar, Odisha, India Uttam Kumar Bhattacharyya West Bengal, India

Gupta College of Technological Sciences, Asansol,

Abdelhakim Bouyahya Laboratory of Human Pathologies Biology, Department of Biology, Faculty of Sciences, and Genomic Center of Human Pathologies, Faculty of Medicine and Pharmacy, Mohammed V University in Rabat, Rabat, Morocco Elizabeth Carvajal-Millan Biopolymers, Research Center for Food and Development, CIAD A.C., Carretera Gustavo E. Astiazaran Rosas No. 46, Hermosillo, Sonora, Mexico

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List of contributors

Samrat Chakraborty Department of Pharmaceutical Technology, Jadavpur University, Kolkata, West Bengal, India; Gupta College of Technological Sciences, Asansol, West Bengal, India Apala Chakraborty Department of Pharmaceutical Technology, Jadavpur University, Kolkata, West Bengal, India Imane Chamkhi Centre GEOPAC, Laboratoire de Geobiodiversite et Patrimoine Naturel Université Mohammed V de, Institut Scientifique Rabat, Rabat, Rabat-SaléKénitra, Morocco Pronobesh Chattopadhyay Division of Pharmaceutical Technology, Defence Research Laboratory, Tezpur, Assam, India Rashmi Choubey Bose Memorial Research Lab, Department of Chemistry, Government Autonomous Science College Jabalpur, Madhya Pradesh, India Hira Choudhury Department of Pharmaceutical Technology, School of Pharmacy, International Medical University, Kuala Lumpur, Malaysia Avik Das

Gupta College of Technological Sciences, Asansol, West Bengal, India

Monodip De Dr. B.C. Roy College of Pharmacy & Allied Health Sciences, Dr. Meghnad Saha Sarani, Bidhan Nagar, Durgapur, West Bengal, India Piyali Dey Faculty of Pharmaceutical Science, Assam down town University, Guwahati, Assam, India; Piyali Dey, Assistant Professor, Assam down town University, Guwahati, Assam, India Ibrahim M. El-Sherbiny Nanomedicine Research Laboratories, Center for Materials Science, Zewail City of Science and Technology, Giza, Egypt Naoual Elmenyiy Laboratory of Physiology, Pharmacology and Environmental Health, Faculty of Science, University Sidi Mohamed Ben Abdellah, Fez, FezMeknes, Morocco Nasreddine El Omari Laboratory of Histology, Embryology, and Cytogenetic, Faculty of Medicine and Pharmacy, Mohammed V University in Rabat, Rabat, RabatSalé-Kénitra, Morocco Ouadie Mohamed El Yaagoubi Laboratory of Biochemistry, Environment and Agri-Food (URAC 36)dFaculty of Sciences and TechniquesdMohammedia, Hassan II University Casablanca, Casablanca, Casablanca-Settat, Morocco Muhammad Asim Farooq Drug Delivery, Disposition, and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, VIC, Australia Bapi Gorain School of Pharmacy, Faculty of Health and Medical Science, Taylor’s University, Subang Jaya, Selangor, Malaysia; Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra, Ranchi, India

List of contributors

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Maryam Hakkour Laboratory of Biodiversity, Ecology, and Genome, Faculty of Sciences, Mohammed V University in Rabat, Rabat, Rabat-Salé-Kénitra, Morocco Md Saquib Hasnain Department of Pharmacy, Palamau Institute of Pharmacy, Chianki, Daltonganj, Jharkhand, India Amna Jabeen Faculty of Pharmacy, Lahore College of Pharmaceutical Sciences, Lahore, Punjab, Pakistan Suman Mallik

Narayana Super Speciality Hospital, Kolkata, West Bengal, India

Amira Mansour Nanomedicine Research Laboratories, Center for Materials Science, Zewail City of Science and Technology, Giza, Egypt Mayra A. Mendez-Encinas Department of Chemical Biological and Agropecuary Sciences, University of Sonora, Avenida Universidad e Irigoyen, Caborca, Sonora, Mexico Biswajit Mukherjee Department of Pharmaceutical Technology, Jadavpur University, Kolkata, West Bengal, India Anroop B. Nair Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa, Saudi Arabia Amit Kumar Nayak Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Jharpokharia, Mayurbhanj, Odisha, India Santwana Padhi KIIT Technology Business Incubator, KIIT Deemed to be University, Bhubaneswar, Odisha, India Anjali Pal Bose Memorial Research Lab, Department of Chemistry, Government Autonomous Science College Jabalpur, Madhya Pradesh, India Parthasarathi Panda Dr. B.C. Roy College of Pharmacy & Allied Health Sciences, Dr. Meghnad Saha Sarani, Bidhan Nagar, Durgapur, West Bengal, India Brahamacharry Paul Department of Pharmaceutical Technology, Jadavpur University, Kolkata, West Bengal, India Ng Yen Ping Faculty of Pharmacy, Clinical Pharmacy Unit, AIMST University, Kedah, Malaysia Shilpi Rawat Bose Memorial Research Lab, Department of Chemistry, Government Autonomous Science College Jabalpur, Madhya Pradesh, India Somasree Ray India Malini Sen

Gupta College of Technological Sciences, Asansol, West Bengal,

Gupta College of Technological Sciences, Asansol, West Bengal, India

Ramkrishna Sen Department of Pharmaceutical Technology, Jadavpur University, Kolkata, West Bengal, India

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Shalmoli Seth India

List of contributors

Gupta College of Technological Sciences, Asansol, West Bengal,

Natalie Trevaskis Drug Delivery, Disposition, and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, VIC, Australia Dickson Pius Wande Department of Pharmaceutics and Pharmacy Practice, School of Pharmacy, Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania

Preface

In the recent scenario, theranostic nanomedicines facilitate multifunctional activities including diagnosis and therapy of various diseases. Over the past few years, numerous therapeutic and diagnostic agents are being delivered at the targeted site with minimum side effects and proper therapeutic/diagnostic action(s) for an extended period of time. An ideal theranostic nanomedicine not only genuinely diagnoses and detects any disease at its preliminary stage but also provides the most favorable treatment. This book entitled “Design and Applications of Theranostic Nanomedicines” covers the recent innovations in the designing of nanomedicines composed of natural and/or synthetic polymers and inorganic nanomaterials with their helpful theranostic applications. It also provides a concise overview of utility of the amalgamated actions of these theranostic nanostructures as nanomedicines in the pharmaceutical and healthcare industry. This book also acts as an important reference for the readers and provides detail information about targeted delivery of nanotheranostics and how they work as both diagnostic and therapeutic tools in treating complex diseases. This book is a collection of 15 chapters presenting different key topics related to nanomedicines, their designing and theranostic applications by the leading academicians, scientists, and researchers across the world. A concise sketch on each chapter contents has been presented for the readers to provide a clear overview of this book. Chapter 1 entitled “Theranostic nanostructures as nanomedicines: Benefits, costs, and future challenges” focuses on the current research on nanostructurebased therapeutics and diagnostics systems, including benefits, costs, and future challenges. Chapter 2 entitled “Theranostic nanogels: Design and applications” describes the designs and applications of theranostic nanogels with a particular emphasis in discussing the imaging modality used for the diagnostic function. Chapter 3 entitled “Exosomes: A novel tool for diagnosis and therapy” highlights various features of exosomes, their roles in diagnosis, and therapeutic applications. Chapter 4 entitled “Engineered liposomes as drug delivery and imaging agents” reviews the development, progress, and applications of various engineered liposomes for delivery of drugs and imaging agents, individually or in combinations as theranostics. Chapter 5 entitled “Polymeric micelles for theranostic uses” addresses comprehensive discussions involving description and preparation of polymeric micelles, mechanism of drug release from micelles, and their potential theranostic applications specially in different stages of cancer therapy.

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Preface

Chapter 6 entitled “Dendrimers: An effective drug delivery and therapeutic approach” highlights the usefulness of dendritic structures and their potential applications in the treatment of various diseases. Chapter 7 entitled “Nanocochleates: A novel lipid-based nanocarrier system for drug delivery” covers recent advancements in nanocochleate-based drug delivery systems with multiple aspects of nanocochleates such as their chemistry, components, mechanism of actions, methods of preparation, stability, advantages, characterization, applications, and current commercial status. Chapter 8 entitled “Theranostic applications of nanoemulsions in pulmonary diseases” deals with comprehensive discussions on the preparation and characterization of nanoemulsions, advantages and disadvantages of nanoemulsions, clearance of nanoemulsions, applications as novel theranostic tools (as nanoemulsion-based drug delivery systems and nanoemulsion-based diagnostics) in treatment/management of pulmonary diseases. Chapter 9 entitled “Polymeric nanoparticles as tumor-targeting theranostic platform” reviews different strategies developed for the application of polymeric nanoparticles for tumor-targeting diagnosis and therapy, with a closer look at the recent studies, and discusses how the strategic development has progressed throughout the years for the bench-to-market conversion of concept to commercialization. Chapter 10 entitled “Site-specific theranostic uses of stimuli responsive nanohydrogels, design, and applications of theranostic nanomedicines” summarizes the importance of nanohydrogels’ response to the internal stimuli like pH, redox potential, etc., and external stimuli like temperature, light, magnetic field, etc., used for theranostic applications like delivery of drugs at the specific target sites with controlled release kinetics to tumor tissue and other disease conditions as well as disease diagnosis. Chapter 11 entitled “Ligand appended theranostic nanocarriers for targeted bloodebrain barrier” encompasses preliminary introduction of ligand-appended nanocarriers, their synthesis, characterization, and theranostic applications in crossing bloodebrain barrier with targeting abilities. The underlying challenges and future prospects have also been highlighted for stimulating advanced research in this area. Chapter 12 entitled “Nanotheranostics in CNS malignancy” presents a brief discussion on various types of nanotheranostic agents that are used in the treatment of glioma and central nervous system (CNS) malignancy including gold nanoparticles, quantum dots, magnetic nanoparticles, mesoporous silica nanoparticles, solid lipid nanoparticles, dendrimers, liposomes, etc. Chapter 13 entitled “Application of nanotheranostics in cancer” reviews and evaluates the advances in the developments of nanomedicines for the treatments, diagnostics, and theranostics of cancer. This chapter has also discussed the limitations in the provision of effective clinical usages of cancer nanotheranostics. Chapter 14 entitled “Self-assembled protein nanoparticles for multifunctional theranostic uses” discusses self-assembled protein-based nanomaterials in depth, with a focus on nanoparticles. Their multifunctional theranostic uses in delivering therapeutic medicines have also been explored with a practical discussion on how they might be useful as prospective techniques for efficient and safe delivery in the treatment/management of different diseases.

Preface

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Chapter 15 entitled “Nanotheranostics: The toxicological implication” describes the relevance of the emergence of nanotheranostics as an effective branch of medicine with a specific focus on the toxicological concern. In addition, various in vitro and in vivo systems available for toxicity testing of nanotheranostic agents have also been outlined. We, the editors, are happy to express our special thanks to all the distinguished authors, who have contributed quality chapters in a timely manner. We especially express our gratitude to Elsevier Inc., Andre Gerhard Wolff, Sabrina Webber, Chiara Giglio, and Clodagh Holland-Borosh for their invaluable support in organization of the book-editing process. We would like to express our sincere thanks to Prem Kumar Kaliamoorthi (Senior Project Manager) for the development as well as production of finished book and Mohanraj Rajendran (Copyright Coordinator) for outstanding supports in obtaining copyright permissions. All the permissions for the reproduction of copyright contents and reprinting permission licenses from different copyright sources have duly been gratefully acknowledged. Finally, we must appreciate our family members, all respected teachers, friends, colleagues, and students, for their continuous encouragements, inspirations, and moral supports during the book-editing process of this book. Together with all the contributing authors and the publisher, we will be extremely happy if our endeavor fulfills the needs of academicians, researchers, students, pharmaceutical experts, drug delivery formulators, polymer engineers, biomedical experts, and others. Prof. Somasree Ray Gupta College of Technological Sciences, India Prof. Amit Kumar Nayak Seemanta Institute of Pharmaceutical Sciences, India

Theranostic nanostructures as nanomedicines: benefits, costs, and future challenges

1

Dickson Pius Wande 1 , Natalie Trevaskis 2 , Muhammad Asim Farooq 2 , Amna Jabeen 3 and Amit Kumar Nayak 4 1 Department of Pharmaceutics and Pharmacy Practice, School of Pharmacy, Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania; 2Drug Delivery, Disposition, and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, VIC, Australia; 3Faculty of Pharmacy, Lahore College of Pharmaceutical Sciences, Lahore, Punjab, Pakistan; 4Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Jharpokharia, Mayurbhanj, Odisha, India

1.1

Introduction

Nanomedicine is a developing field merging nanoscience, nanoengineering, and nanotechnology with life sciences, revealing the valuable results for healthcare [1]. Although there are numerous nanomedicine applications, nanotechnology-based drug delivery systems and nanoimaging agents are of the utmost interest in medicine and pharmacy. “Theranostic” refers to the simultaneous integration of diagnosis and therapy [2]. Theranostic nanomedicines (or nanotheranostics) combine the use of theranostics with nanosized constructs that give multiple properties, such as targeted drug delivery, controlled release, greater transport efficiency via endocytosis, stimuli-responsive systems, and the amalgamation of therapeutic approaches, such as multimodality diagnosis and therapy [3]. Nanotheranostics unite the three stages in a single process, supporting early-stage diagnosis and treatment to overcome some of the issues with sensitivity and specificity of current medicines. An ideal nanotheranostic system should circulate for a long time in the body, provide sufficient release behavior, show tissue target specificity and penetration, imaging capability, and high target to background ratio [4]. Recently, novel and promising nanotherapeutic applications in the diagnosis and treatment have been revealed in various diseases, such as cancer [5,6]. However, the development of novel tools with improved imaging characteristics, which can lead to the early detection of diseases, is still of high importance. Aside from the applications for diagnosis and nanotherapeutics are being increasingly designed and applied to treat a range of serious diseases [7]. At present, nanotheranostics are viewed as one of the key upcoming new strategies to tackle cancer, based on the postulation that if cancer progression can be hindered during an initial diagnostic procedure, the consequent anticancer therapy would be much easier since cancer growth will be

Design and Applications of Theranostic Nanomedicines. https://doi.org/10.1016/B978-0-323-89953-6.00008-8 Copyright © 2023 Elsevier Ltd. All rights reserved.

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Design and Applications of Theranostic Nanomedicines

retarded and the overall cancer burden will be reduced [8,9]. The main challenge is to develop a system for molecular therapy capable of circulating in the bloodstream undetected by the immune system of body and capable of recognizing the required target and signaling for effective drug delivery or gene silencing. As a result, nanotechnology plays a vital role in providing new types of nanotherapeutics for diseases that can provide effective treatments with negligible side effects and high specificity [10,11]. Generally, theranostic nanomedicines can be engineered in several ways using technologies, such as polymeric nanoparticles [12], carbon-based nanomaterials [13,14], lipid-based nanovesicles [15,16], protein-based nanostructures [17], dendrimers [18], ceramic nanostructures [19], metallic inorganic nanocarriers [20], and graphene quantum dots [21].

1.2

Nanotechnology, nanoscale, and nanostructures

Nanoscale science and technology often referred to as “nanoscience” or “nanotechnology” are science and engineering carried out on the nanometer scale, that is, 10 9 m [22]. Nanotechnology is the field of research and innovation concerned with building materials, devices, and systems on the scale of atoms and molecules [23]. The US National Nanotechnology Initiative states: “The essence of nanotechnology is the ability to work at the molecular level, atom by atom, to create large structures with the fundamentally new molecular organization” [24]. The purpose is to exploit these properties by gaining control of structures and devices at atomic, molecular, and supramolecular levels to learn how to manufacture and use these devices, efficiently. The United States National Science Foundation defines nanoscience/nanotechnology as studies dealing with materials and systems displaying three key properties: dimensiondat least one dimension from 1 to 100 nanometers (nm); processd designed with methodologies that show fundamental control over the physical and chemical attributes of molecular-scale structures; building block propertydthey can be combined to form larger structures [23e25]. In a general sense, nanoscience is quite natural in microbiological sciences considering that the sizes of many bioparticles dealt with by the body (like enzymes, viruses, etc.) fall within the nanometer range [24]. Several nanoscale technologies are now available in the market. For example, specially prepared nanosized semiconductor crystals (quantum dots) are used as a tool for the analysis of biological systems [25]. Upon irradiation, these dots fluoresce specific colors of light based on their size. Quantum dots of different sizes can be attached to different molecules in a biological reaction, allowing researchers to follow all the molecules simultaneously during biological processes with only one screening tool. These quantum dots can also be used as a screening tool for quicker, less laborious DNA and antibody screening than is possible with more traditional methods [22,26]. Nanostructures can be categorized depending on their size, shape, composition, surface characteristics, functionalization, and origin. The ability to predict the properties of nanostructures based on their classification is very useful to their applications [27].

Theranostic nanostructures as nanomedicines: benefits, costs, and future challenges

5

In terms of shape, nanostructures can be classified into spherical, conical, spiral, cylindrical, tubular, flat, hollow, or irregular in shape and can be from 1 to 100 nm in size [27,28]. Most nanostructured materials can be generally classified into four materialsbased categories (organic, inorganic, composite, and carbon-based) [29]. Among these nanostructures, the use of a combination of more than one nanostructure to form a hybrid-nanostructure system is not uncommon [30]. Hybrid nanostructures are carefully designed with a combination of different materials, which have different physiochemical properties and load different types of drugs. Hybrid nanostructures can also encompass nanoparticles that contain both structural (therapeutic) and functional (diagnostic) nanocomponents. The drug can be either encapsulated or bound to the surface of nanostructures depending on the physicochemical properties of the nanocarriers. These hybrid structures have a higher surface area. They are often engineered to increase the drug loading capacity. Sometimes, nanocarriers can be designed to respond to specific endogenous or exogenous stimuli for controlled drug release [25]. Targeted drug delivery can be monitored externally by fluorescence dyes or intrinsic optical/magnetic/electrical properties of nanostructures. The development of hybrid nanostructures signifies an important step toward an efficient delivery of a range of therapeutics and imaging agents as hybrid nanostructures may allow the delivery of a combination of multiple drugs, DNA, RNAs, and diagnostic agents [29].

1.2.1

Carbonaceous-based hybrid nanostructures

Carbon nanotubes are hydrophobic in nature and compromise the tubular nanostructures with diameters in the order of less than 50 nm [31,32]. They display remarkable mechanical and optical properties [33,34]. These features have been harnessed in biomedical applications, such as photodynamic therapy [35], diagnostic imaging [36], and drug delivery [37]. Carbon nanotubes are essentially nontoxic, have high biocompatibility in the body, and are excreted via renal or biliary pathways depending on their surface chemistry [38]. They often have a longer blood circulation time than many other nanoparticles [39]. Graphene is an allotrope of carbon composed of a hexagonal network of a honey-comb structure made up of carbon atoms consisting of sp2-hybridized bonded carbon on a 2D planar surface [40,41]. The thickness of a graphene sheet is about 1 nm. Fullerenes (C60) are carbon-based molecules and spherical in morphology [42]. These are made up of carbon atoms held together via sp2 hybridization. Generally, the other fullerenes (0D), such as C76, C80, and C240, are synthesized from larger numbers of carbon atoms [29]. Fullerenes are comprised of between 28 and 1500 carbon atoms that form spherical structures [43]. Single-layer fullerenes have diameters up to 8.2 nm, while multilayer fullerenes have diameters between 4 and 36 nm.

1.2.2

Organic-based nanostructures

Dendrimers, liposomes, and polymeric micelles are usually known as organic nanostructures [44]. These include nanostructures mostly made of organic materials. These

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Design and Applications of Theranostic Nanomedicines

nanostructures are generally nontoxic and biodegradable in nature. Sometimes, these are sensitive to electromagnetic and thermal radiation. Organic nanostructures are most employed in pharmaceuticals for drug delivery systems because of their high biocompatibility [44,45]. Dendrimers are prepared from monomers by either convergent or divergent step-growth polymerization [46]. The surface of a dendrimer encompasses several chains that can be modified to accomplish specific and specialized biochemical functions. Polymeric micelles possess amphiphilic block copolymers assembled to form nanoscopic core-shell structures [47,48]. Both the intrinsic and modifiable properties of polymeric micelles make them well suited for the systemic delivery of poorly water-soluble medicines.

1.2.3

Inorganic-based nanostructures

Inorganic nanostructures encompass structures that are not made from carbon-based or organic-based systems. Inorganic-based nanostructures have been of particular interest for bioimaging applications due to their high thermal conversion efficiency, ease of synthesis, and possible surface modifications [49]. These nanostructures include metal and metal oxide nanostructures. Metal-based and metal oxide-based nanostructures are commonly categorized as inorganic nanostructures. These nanostructures can be synthesized into metal nanostructures, such as palladium or gold, metal oxide nanostructures like titanium dioxide, and semiconductors, such as ceramics and silicon. Metal-based nanoparticles have fascinated scientists for over a century and are nowadays heavily utilized in biomedical and material sciences. Almost all metals can be synthesized into nanostructures or nanoparticles. Generally, aluminum (Al), gold (Au), silver (Ag), copper (Cu), cobalt (Co), cadmium (Cd), lead (Pb), iron (Fe), and zinc (Zn) metals are used for nanostructure synthesis [50e52]. Inorganic metal nanoparticles possess unique properties, such as large surface areas, surface charge densities, pore sizes, and stability. These kinds of nanoparticles can be cylindrical or spherical in shape, crystalline or amorphous in structure, and typically less than 100 nm in size [53]. Metal oxide-based nanostructures are synthesized mainly because of their increased efficiency and reactivity. Metal oxide-based nanostructures are prepared in order to modify the physicochemical properties of their respective metal-based nanostructures. The most commonly used metal oxides for the synthesis of nanostructures that have been well characterized include zinc oxide and iron oxide nanostructures [54e56]. Iron oxide nanostructures have generated incredible interest in nanomedicine due to their many beneficial properties [57]. Specifically, it has been found that when iron oxide nanostructures are reduced to a size of 1000 nm [39]. Again ULVs are differentiated into small unilamellar vesicles (SUVs) having dimension of 20e100 nm, large unilamellar vesicles (LUVs) having size more than 100 nm), and giant unilamellar vesicles (GUVs) having dimension more than 1000 nm. ULVs contain single bilayers of phospholipids and have more entrapment efficiency toward hydrophilic drugs. MLVs contain two or more lipid bilayers and favor the encapsulation of hydrophobic compounds. MVVs contain number of nonconcentric vesicles enclosed within single lipid bilayers and encloses large amount of hydrophilic drugs [39,40]. Number of lipid bilayers and size of vesicle determines the quantity of drugs to be enclosed in liposomes. Multicompartment liposomes (MCLs) are another type of vesicle type formulation in

Figure 4.2 Different types of liposomes depending on size: SUV representing Small Unilamellar Vesicles, LUV representing Large Unilamellar Vesicles, MLV representing Multilamellar Vesicles, and MVV representing Multivesicular Vesicles [27].

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Design and Applications of Theranostic Nanomedicines

which a tight bilayer interface connects two unlike vesicles and develops into a single vehicle delivery system for co-delivery of combined drugs [27].

4.3

Preparation of liposomes

Liposomes are generally prepared at a temperature higher than phase transition temperature of lipids (Tg). The size, distribution of size, and number of lipid bilayers are affected by methods of preparation, type and composition of lipid, type of organic solvent, and surfactants involved in the method of preparation [41]. Both conventional and novel methods are used for preparation of liposomes [27]. In both the techniques, four steps are followed for preparation of liposomes [27]: I. II. III. IV.

Solubilization of lipid in solvents (organic) Exclusion of solvent (organic) Isolation with purification of liposomes Characterization of formed liposomes

The methods used as conventional methods for preparation of liposomes include thin film hydration, reverse phase evaporation, solvent injection technique, and removal of detergent [27]. Recently, the modified methods are being adopted to solve the problems involved in the conventional methods.

4.3.1 4.3.1.1

Conventional methods Hydration method

In conventional method of hydration, MLVs can be prepared by hydrating the thin lipid layer with an excess volume of aqueous medium. Mechanical methods like sonication and extrusion can convert MLVs to SUVs and LUVs. GUVs can also be prepared easily by hydrating the lipid layer gradually for prolonged duration of about 1e48 h without shaking or maintaining the low ionic concentration in the buffer solution if buffer is replaced with water. The limitations of this method are heterogeneity in the size, poor entrapment of drugs, and difficulty in scale up [41].

4.3.1.2

Electroformation method

Angelova and Dimitrov [42] introduced electroformation method for preparation of GUVs in the presence of electric field for hydration of films of lipids deposited on the electrodes. Though the electric fields can be applied by both DC and AC current, the AC current is preferred as DC current can cause electrolysis of water and produce bubbles. Rodriguez et al. differentiated the GUVs formed by electroformation method and gentle hydration method. These two methods produced unilamellar vesicles with doubled yield in electroformation method (80%) as compared to gentle hydration method (%). Presence of small concentration of electrolyte in water can produce GUVs faster as compared to pure aqueous medium [43].

Engineered liposomes as drug delivery and imaging agents

4.3.1.3

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Bulk methods

Bulk methods produce LUVs and SUVs. Three different methods are included in bulk methods; they are reverse phase evaporation, solvent dispersion methods, and detergent depletion method. Reverse phase evaporation involves addition of aqueous phase to lipid solution prepared in solvents like diethyl ether/isopropyl ether or a mixture of both to form W/O emulsion which on evaporation produces the vesicles. In this method, low concentration of lipid produces higher yield of LUVs as compared to MLVs [44]. In solvent dispersion method, lipid solutions either in diethyl ether, diethyl-methanol mixture or ethanol are injected to a fast agitating aqueous or buffer solution producing SUVs rapidly [45]. Detergent depletion method involves preparation of LUVs by removal of detergents by controlled dialysis from the detergent-lipid micelles [46]. The major limitations of bulk methods are the presence of traces of organic solvent or surfactants, reduced entrapment efficiency, particle size heterogeneity, problem with scale-up and low storage stability [41].

4.3.2 4.3.2.1

Novel methods Recent hydration techniques

Heating method Mozafari in 2005 introduced this method for large scale production of liposomes without inclusion of any harmful chemicals in the method [47]. In this process, the aqueous phase containing 3% (v/v) glycerol is used for hydration of lipid mixture and heating at 120
C. Glycerol increases the stability of the liposomes by preventing sedimentation and coagulation of vesicles. From the final product of liposomes, glycerol is not required to be separated, and it is nontoxic, biocompatible, and watersoluble. This method was found to be successful in entrapping plasmid DNA (pCMV-GFP) with an enhanced entrapment efficiency of 81% [48].

Curvature tuning method Hauser et al. first introduced the method of rapid vesiculation with fast change of pH for preparation of liposomes [49,50]. Genc et al. reported the preparation of monodispersed SUVs by spontaneous single step method using pH jump technique employing addition of sodium hydroxide (pH ¼ 11) and immediately changing the pH to 7.4 using hydrochloric acid for a specific duration. This method avoids the steps like lipid film preparation and use of organic solvents. In this method, operating temperature, equilibration time, pH jumping time interval, and type of lipid are the major factors affecting the size and size distribution of liposomes [51].

Packed bed-assisted hydration method This method was introduced by Sundar and Tirumkudulu based on single step hydration to prepare liposomes of size less than 100 nm. This technique doesn’t require the post-processing of formulated liposomes. This method involves solubilization of lipid in organic solvent and dehydrated in a packed alumina bed containing asymmetric

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Design and Applications of Theranostic Nanomedicines

colloidal particles of uneven surfaces. Then SUVs are prepared by hydrating the packed bed with an aqueous medium [52].

Localized IR heating method Billerit et al. introduced this method for fast generation of GUVs from zwitterionic, complex, or charged lipids in presence of high or low ionic concentration [53]. In this method, a film of lipid is coated over a surface of silicon dioxide mounted on a chamber with PBS, and the target site is heated with an infrared laser by an optical tweezer. Heating process is continued until required size liposomes are produced [53].

Osmotic shock method This is a multistep process used in preparation of GUVs under physiological saline condition. In this method, the small vesicles produce dry membranes which are disrupted by osmotic shock which leads to breaking of liposomes spontaneously and synchronously. This causes formation of floating bilayers which combine and reseal as soon as possible. The SUVs get dehydrated and rehydrated with aqueous phase and facilitates the formation of GUVs due to osmotic shock. Motta et al. used this method to incorporate proteins into GUVs [54].

Spray drying method This method can be used for large scale production of liposomes. In this method, mannitol and lipids are mixed with solvents like chloroform, and then a spray dryer can dry the suspension. The amorphous dried product can be hydrated efficiently with water followed by agitation to produce liposomes. Mannitol enhances the surface area of the lipid and causes hydration of spray dried substance. Size distribution is lesser and entrapment efficiency is higher [55].

Freeze drying and lyophilization method This method is used to prepare pyrogen-free and sterile liposomes. In this method, the aqueous medium containing sucrose and the lipid are dissolved in water/tertiary butyl alcohol to form a cake of an isotropic monophasic solution. The dried material is sterilized and freeze dried followed by hydration with water medium to prepare a biphasic suspension of liposomes of dimension 100e400 nm [56]. Wang et al. modified the method to prepare unilamellar sterile liposomes with increased entrapment efficiency. In this modified method, freeze drying of a double emulsion system i.e., W1/O/W2 in which “W1” and “W2” contain the aqueous medium with sugar and “O” stands for the lipid medium [57]. Yin et al. introduced another modified method known as ultrasonic spray drying freeze drying to prepare dried powder of liposomes [58].

Gel assisted hydration This technique is an alternative to electroformation to prepare GUVs at physiological condition. This method involves drying of a polymeric film (can be transformed to a hydrogel) on a coverslip of glass and a thin film of lipid is deposited. The GUVs are produced at the interface of swollen hydrogel and water by hydration with a buffer solution [59].

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Hydration on glass beads Bayerl and Bloom used submillimeter beads of glass and added to a suspension containing heated small vesicles followed by vigorous agitation. The vesicles are broken and lipid bilayers support on glass bead surface [60]. Later, Nourian et al. prepared GUVs by hydration of lipid-coated beads [61]. Tanasescu et al. reported that simultaneous shaking produces more monodisperse GUVs as the liposomes formed are sheared between the beads [62].

4.3.2.2

Recent electroformation method

Modified electroformation method Conventionally, electroformation method produces GUVs at very low ionic concentration (10 mM NaCl) [63]. On the other hand, in physiological ionic solutions, charged lipids in lipid mixture are used for preparation of GUVs [64]. But high concentration of charged lipids interferes with formation of GUVs [65]. So Estes and Mayer reported the preparation of giant liposomes (10e100 mm) in a concentrated ionic solution to show the interaction between the membrane and binding protein at physiological conditions [66]. Bhatia et al. prepared monodispersed proteo-GUVs by mixing two SUVs suspension composed of different lipids, before dehydration followed by electroformation [67].

Electroformation in microfluidics Microfluidics utilizes electroformation successfully for preparation of liposomes. Kuribayashi et al. used the concept of electroformation in micro-sized fluidics and prepared different types of GUVs with polystyrene beads enclosed within. The method involved the sandwiching of channels between ITO electrodes coated glass slides, and GUVs are produced by use of AC voltage in the electrodes. The major merit of the method is the decrease in time required for the preparation of GUVs from >2 h (by gentle hydration method) to 10 min producing monodisperse liposomes [68].

4.3.2.3

Recent bulk methods

Membrane contractor This is a single step method, introduced by JaafareMaalej in 2011 and it’s a modified ethanol injection method used to produce liposomes. This method involves heating of a lipid solution in ethanol above Tg of the lipid and then allowed to enter into a tube to the module with membrane contactor of a fixed pore size. At the same time, water circulates on the membrane contactor tangentially which washes the formed vesicles to the module [69].

Microfluidics This method resembles ethanol injection method. The technique involves controlled incorporation of fluids in a microfluidic channel, and it’s a challenging procedure [70].

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Supercritical fluids technique

Supercritical fluids (SCFs) possess the properties of both liquid and gases. The solvent property of SCFs varies according to their critical parameters of temperature and pressure. Carbon dioxide is the most suitable SCF as its critical parameters (Tc ¼ 31. 1
C and Pc ¼ 73.8 bar) can be easily maintained along with other advantages such as nontoxic, noncorrosive, and easily available [71]. Castor et al. proposed two methods using SCFs for preparation of liposomes. The first method is an injection method in which a mixture of SCF, phospholipid, and organic cosolvent are mixed into the aqueous phase through a nozzle. The next method is a decompression method to form liposomes in which the aqueous phase, SCF, phospholipid, and organic cosolvent are mixed and decompressed [72,73].

Stationary phase interdiffusion (SPI) method Phapal et al. introduced this method which results in highly concentrated LUVs in a single step. This method of liposome formation involves addition of two miscible lipid phases by diffusion causing self-assemblage of lipidic molecules. A stationary interface is created by interaction of lipid in ethanol solution with aqueous phase in a vertical cuvette. Size of the liposomes depends on the type of lipid and the temperature. This method needs 6 h to take out the sample for measurement of size of vesicles [74].

Modified detergent depletion technique Peschka et al. introduced this method in 1998. This method produced liposomes of uniform size with high stability. This method is a combination of conventional detergent depletion method with cross-flow method. This utilizes a cross-flow filtration unit with a reservoir, a pump, a membrane filtration device, and tubing with a rotary slide valve and a manometer to control the pressure. The removal of detergent is faster due to increased pressure on the membrane. This method is very fast with high yield of liposomes [75].

4.4

Rationale for the development of engineered liposomes

Current challenges associated with liposomes with single functionality have been replaced by multifunctional liposomes prepared by surface engineering. The surface functionalization produces liposomes in the nanoscale range. Engineering of liposomes can be achieved by ligation, conjugation of PEGylation to obtain nano liposomes of different characteristics, and functionality as represented in Fig. 4.3 [27]. Engineering of liposomes can lead to target specificity using different surface functionalization and modified methods of preparations. This causes key benefits of the liposomes in terms of enhanced circulation time, increased cellular uptake, and high accumulation of entrapped drug at the tumor cells by active targeting at the tumor site [12]. Surface engineering of liposomes is achieved by an appropriate ligand like antibodies or antibody fragments, peptides, or small molecules for targeted delivery

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Figure 4.3 Surface engineering of liposomes with different functionalization for drug delivery and imaging [27].

of cancer imaging agents or anticancer drugs at the site of tumor. The engineered liposomes have minimal off-target toxicity to the healthy tissues around tumor cells [76]. Conjugation of antibody or antibody fragment generated immunoliposomes which shows extremely high specificity toward target antigens [16]. These advances in surface engineered liposomes overcome the challenges and widen the path for targeted drug delivery and diagnosis to tumor targets [77]. Due to differential vasculature and poor lymphatic drainage within the tumor microenvironment, enhanced permeability and retention (EPR) effect can be achieved by passive targeting. Active targeting involves the targeting of drug by responding to the environmental changes that occur due to growth of tumors. Engineered liposomes encourage the accumulation of anticancer drugs and diagnostics at the tumor site [21].

4.4.1 4.4.1.1

Engineered liposomes PEGylated liposomes

Conventional liposomes consists bilayers of phospholipids; they possess short circulation time as they are administered by intravenous route; and they interact with reticulo endothelial system (RES). A serum protein, opsonin, responds to the liposomes as a foreign entity and destroys it by phagocytes. So the challenge of increasing the

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circulation time can be achieved by coating the exterior of liposomes with a hydrophilic polymer like PEG. PEG increases the repulsive force between serum components and liposomes and reduces the rate of degradation of PEGylated or stealth liposomes [78]. PEG is hydrophilic, biocompatible, and flexible and attached to the lipid bilayers of liposomes shows a hydrated steric interaction between the liposomes and the serum protein reducing the liver and spleen uptake and increases the circulation time. Molecular weight and density of PEG on the surface of liposomes correlates the benefits of PEGylated liposomes. About w5% by weight of PEG can escape the protein binding and increase the blood circulation time [79]. Lee et al. reported that the liposomes having PEG less than 9.6% are lesser uptaken by liver and spleen, but accumulate in the spleen significantly when the PEGylation is achieved with 13.7% of PEG [80]. PEG 750 can’t enhance the blood circulation time and PEG 5000 has problem with binding of ligand [80,81]. Yang et al. reported the enhanced pharmacokinetic profile of paclitaxel-loaded PEGylated liposomes as compared to conventional paclitaxelloaded liposomes and free taxol [82]. Two methods are followed to PEGylation of liposomes, i.e., pre-insertion and postinsertion. Pre-insertion leads to low drug entrapment and stealth effect as PEG is inserted to the surface of liposomes and at the inner side of the lipid bilayer in a reverse orientation. Postinsertion shows increased therapeutic efficacy in in vivo and high circulation time [83]. Two major limitations with PEGylation are that less accumulation of the drug (less than 5%) at the tumor site and nontargeted distribution of the PEGylated liposomes [84,85]. These problems can be overcome by preparing PEGylated liposomes in nanometer size, and functionalizing with a ligand to target the tumor site which increases the cellular uptake and internalization. The recognition of target by the ligand and the steric interaction of PEG chains on the surface must be taken into account for selection of suitable PEG of appropriate molecular weight, chain length, and the multivalent binding potential of PEG [86,87].

4.4.1.2

Engineering of liposomes with peptides

Peptides (short chain amino acids) are bonded to liposomes by covalent and noncovalent binding to prepare engineered liposomes. Covalent bonding of peptides with liposomes can be done by variety of linkages like peptide bond, disulfide bond, sufanyl bond, and phosphatidyl etanolamine bond [88]. Non-covalent bonding is found in surface engineered liposomes with amphipathic peptides [89]. Targeted drug delivery of peptide linked liposomes is conserved as it is linked to outer surface of liposomes as represented in Fig. 4.4 [90]. Surface engineering of liposomes with peptides has become a lead for nano delivery of therapeutics. The peptides possess some structural and biological functionality which can target the overexpressed receptors on tumor cells, tissues, and vasculatures [90]. Surface functionalized liposomes with peptides used in terms of nanocarriers are classified into two types: cell-targeting peptide (CTP) and cell-penetrating peptide (CPP). CTP specifically bind to the receptors and CPP has nonspecific binding for cellular internalization, respectively [91]. Engineered liposomes of cantharidin and a

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Figure 4.4 Peptide-linked liposomes for targeted drug delivery by an active targeting targeted toward overexpressed receptors on cancer cells. Active targeting of peptide-engineered liposomes involves selective interaction with overexpressed receptors on the cancer cells internalization by receptor-mediated endocytosis. It can also penetrate into the cancer cells by cell-penetrating peptide (CPP)-mediated uptake. Therapeutic actives and diagnostic or imaging agents can be liberated through lysosomal or endosomal degradation of the lipid layers [90].

tumor specific cell-penetrating peptide BR2 showed improved uptake by hepatocellular tumor cells in comparison to nontargeted liposomes [92].

4.4.1.3

Engineering of liposomes with antibody

Immunoliposomes can be prepared by conjugating the antibody or fragment of antibody to the surface of liposomes using various techniques of surface engineering. One of the techniques used for covalent linkage between the antibody or fragment of antibody to the lipid layer of the phospholipid. Another approach involves chemical modification of antibody which increase the hydrophobic property of antibody by substituting with suitable substituent leading to increase in affinity of antibody toward lipid bilayers. During preparation of immunoliposomes, surface functionalization of liposomes with fragments of antibody reduces the risk of deactivation of antibody [93]. Thiolation of antibodies is done by Traut’s reagent (2-iminothiolane) and forms sulfhydryl group. For active targeting the functional groups of antibody like amine, carboxyl, and thiol ends can be modified. Sulfhydryl groups formed in the antibody are prone to oxidation, so to avoid the problem, oxygen can be replaced by ethylenediamine tetra acetic-acid [94]. Two carbonic anhydrase (CAs) (IX and XII) are overexpressed in many cancer cells, mostly brain and lungs cancer due to hypoxia. Lin et al. reported the higher efficacy of liposomes containing triptolide functionalized with anti-CA-IX antibody in mice bearing lung cancer [95].

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Engineering of liposomes with aptamers

Synthetic, single stranded, and short molecules of RNA or DNA with superior specificity and affinity are known as aptamers [38]. Uniqueness of aptamers are ease of synthesis, small size, increased affinity, low immunogenicity, high selectivity toward target, and stability in various physicochemical conditions makes it an ideal ligand for surface engineering of liposomes. During the synthesis of aptamers, the risk of viral or bacterial infection is reduced as it is not synthesized in biological systems [96]. Small size of aptamers favors its attachment to surface of liposomes without any steric hindrance. Aptamers of high affinity and selectivity toward the target site are evolved by systematic evolution of ligands by exponential enrichment (SELEX) technology [97]. Surface engineering of liposomes by conjugation of aptamers are done by both physical and chemical methods. Negatively charged aptamers attach to positively charged liposomes by electrostatic coupling. These electrostatically engineered liposomes are good carriers for nucleic acid based gene delivery [98]. Positively charged liposomes can also be conjugated by covalent bonding which provides a higher stability in biological condition and the damaging factors like change in temperature and pH are also minimized [98]. Covalent bonding of aptamers can link directly to the head of phospholipids of PEGylated liposomes or can attach to free terminus of lipid chains of PEG chains as represented in Fig. 4.5 [38]. The functionalization of liposomes with aptamers has high specificity, increased accumulation at the tumor site with high efficiency. Aptamer functionalized liposomes has no offtarget toxicity and very less or no adverse effect on the pharmacokinetic profile of liposomes. Dong et al. reported a gene delivery system of EGFR-LPDS (epidermal growth factor receptor aptamer-conjugated liposome-polycation-DNA complex loaded with SATB1 siRNA) targeting choriocarcinoma cells. This targeted delivery of siRNA inhibited expression of SATB1 and growth of choriocarcinoma cells by 81.5% [99].

Figure 4.5 PEGylated-liposomes with aptamers: (a) direct conjugation of aptamers to the head of phospholipid of PEGylated liposomes; (b) conjugation of aptamers to the free terminus of PEG [38].

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Jiang et al. reported the increased efficacy of miRNA-29b against ovarian cancer. Aptamer (AS1411) conjugated liposomes loaded with miRNA-29b showed significant concentration dependent cytotoxicity in A2780 ovarian cell lines [100].

4.4.1.5

Engineering of liposomes with small molecules

A number of small molecules are being identified as ligands for surface engineering of liposomes. Carbohydrate, folate, and affibody are conjugated to the surface of liposomes for targeted delivery of anticancer drugs for management of cancer. Ye et al. designed folate receptor targeted imatinib loaded liposomes against cervical cancer HeLa cell lines. Imatinib is the platelet-derived growth factor receptor (PDGFR) inhibitor, but its rapid clearance and protein binding decreases its efficacy. So the folate conjugated liposomes showed high encapsulation efficiency (>90%) and pH sensitive release (>25% at pH ¼ 5.5) with decrease in IC50 value by 6 times with enhanced HeLa cell apoptosis. The circulation time of imatinib loaded liposomes also increased with improved pharmacokinetic profile [101]. Similarly, doxorubicin loaded PEGylated liposomes were targeted toward folic acid and transferrin individually and in combination. The dual targeted (folic acid þ transferrin) doxorubicin loaded PEGylated liposomes showed sevenfold efficacy as compared to single targeted delivery systems toward cervical cancer cell line (HeLa) and ovarian cancer cell lines (A2780-ADR) [102].

4.4.1.6

Biopolymer-coated liposomes

Biopolymer conjugated liposomes are the most studied nanodelivery systems due to simple preparation methods and increased efficiency. Cationic guar gum is conjugated with anionic liposomes by electrostatic interaction. The cationic quaternary ammonium group of guar gum interacts with the anionic phosphate group of liposomes. Guar gum enhanced the packing of lipid bilayers by decreasing the membrane fluidity [103]. Doxorubicin loaded hyaluronic acid conjugated liposomes (DOX-HA-Lip) showed improved internalization into B16F10 melanoma cells. DOX-HA-Lip has enhanced anticancer activity when co-administered with iRGD (a tumor cell entering peptide) [104]. Thiolated chitosan conjugated to curcumin showed increased encapsulation efficiency, drug loading, stability, cellular uptake, and antitumor activity against MCF-7 cell lines [105]. Jeon et al. utilized layer-by-layer technology to develop multilayered liposomes with alternating sodium hyaluronate and chitosan for a transdermal delivery system. Quercetin was loaded into the anionic liposomes coated first with cationic chitosan followed by anionic sodium hyaluronate layer by layer as represented in Fig. 4.6. These polyelectrolytes provided an enhanced stability and skin permeation of the antioxidant drug, quercetin [106].

4.4.1.7

Radiolabeled liposomes

Liposomes can be loaded with radiolabeled molecules for targeted delivery of kidney, liver, spleen, inflammation, and tumor sites [107]. Incorporation of radiolabeled molecules into the liposomes can be done by two ways: attachment of radionuclide to the

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Figure 4.6 Layer-by-layer fabrication of quercetin loaded liposomes by cationic chitosan followed by anionic sodium hyaluronate [106].

Figure 4.7 Different methods for loading of radiolabeled molecules of liposomes. (a) Attachment of radionuclide to the surface of lipid bilayer via a chain of PEG or conjugated directly with the lipid bilayer. (b) The aqueous core of the liposomes encapsulate the radionuclide [108].

lipid bilayers (Fig. 4.7a); in the intra-liposomal space as represented in Fig. 4.7b [108]. Mostly, liposomes loaded with radiolabeled molecules are used for diagnosis of cancer and its stage of growth. It is also used for monitoring of response toward the treatment so that the dose adjustment and patient compatibility to the treatment can be accessed [109].

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F-radioisotope of fluorine labeled to dasatinib loaded liposomes was targeted for inhibition of a platelet-derived growth factor receptor (PDGFR). Biological activity of liposomes containing fluorine and radiolabeled fluorine were compared in brain tumor cells. The radiolabeled nano-formulations used in the study (micelles and liposomes) showed increased cellular uptake and better tumor growth inhibition as compared to nonlabeled formulations [110]. 18

4.5

Engineered liposomes in drug delivery

Liposomes is one of the most suitable targeted nanodelivery system, due to stabilizing the therapeutic active drugs, increasing the tissue and cellular uptake, thereby improving the biodistribution and bioavailability in vivo [35,111,112]. Composition of lipid bilayers, transition temperature of lipid, methods of preparation, and the target site intended for delivery are the key factors in designing or surface engineering of liposomes [109]. A liposomal formulation was designed with amphiphilic copolymer poly (b-amino esters)-PEG conjugated with an antimicrobial peptide enclosing docetaxel. The liposomal formulation acted in response to stimuli of pH change inside the tumor cells following the active targeting. Once the formulation entered the acidic endosomal environment, the nanodelivery system releases both the drug and the peptide imparting the antitumor activity [113]. Zhang et al. also reported a similar kind of study by incorporating an antimicrobial peptide along with paclitaxel into a pH sensitive liposomal formulation. The drug release was observed below pH 6.3 with improved efficiency and increased tumor cell cytotoxicity [114]. A doxorubicin loaded liposomal formulation was designed containing PEG with stearate end with cell penetrating peptides (CPPs). The accumulation of doxorubicin increased by 109-fold as compared to conventional PEGylated liposomes leading to cellular apoptosis [115]. Yang et al. designed a dual peptide modified liposomal formulation for targeted delivery of docetaxel and siRNA to the low-density lipoprotein receptor-related protein receptor (Angiopep-2) and neuropilin-1 receptor (tLyP-1) in brain tumor cell lines (U87 MG and BMVEC). The docetaxel targets the tumor cells and cause efficient apoptosis and siRNA has the anti-angiogenic activity. Both the drugs have different solubility properties, docetaxel is a small lipophilic molecule and siRNA is a large hydrophilic molecule. The lipophilic one entered into the tumor cells by passive diffusion whereas the hydrophilic one entered by receptor mediated endocytosis. The dual peptide conjugated liposomes showed strong binding to glioma cells and enhanced internalization by endocytosis and penetration into the tumor tissues. So the modified liposomes with siRNA and docetaxel showed antiproliferation activity and gene silencing as represented in Fig. 4.8 and was found to be more efficacious than single modified or non-modified liposomes [116]. Similarly, a dual-functionalized liposomal delivery system was designed by Lakkadwala and Singh for targeted delivery of 5-fluorouracil (5-FU) to glioblastoma cells by enhanced permeation to blood brain barrier (BBB). The transport of 5-FU across BBB into the tumor cells was increased by conjugating the cell penetrating peptide

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Figure 4.8 Targeted delivery of liposomes of two receptor-specific peptides Angiopep-2 and tLyP-1. Liposomes modified with dual peptides acts by receptor mediated endocytosis and EPR effect. Apoptosis-inducing chemotherapeutics docetaxel and anti-angiogenesis gene (VEGF siRNA) were used by combination therapy. Reproduced with permission from Yang Z, Li J, Wang Z, Dong D, Qi X. Tumor-targeting dual peptides-modified cationic liposomes for delivery of siRNA and docetaxel to gliomas. Biomaterials. 2014;35(19): 5226e39.

“penetratin” to transferring-liposomes (Tf-Pen-conjugated liposomes). The dual functionalized liposomes showed an increased uptake by brain endothelial (bEnd.3) monolayers and glioblastoma (U87) cells with significant apoptosis in U87 cells [117]. Shi et al. designed paclitaxel loaded pH responsive CPP (TR) conjugated liposomes (PTX-TR-Lip) and targeted to integrin aѵb3 which showed facilitated permeation across B16F10 cell lines. The (PTX-TR-Lip) showed increased cellular intake and accumulation inside the tumor cells with improvement in the survival of the in vivo B16F10 tumor-bearing mice at pH ¼ 6.5 as compared to other formulations of paclitaxel [118]. Yin et al. designed co-delivery of a chemotherapeutic drug paclitaxel (PTX) with an autophagy inhibitor hydroxychloroquine (HCQ) in a liposomal formulation conjugated with R8-dGR (PTX/HCQ-R8-dGR-Lip) and targeted toward integrin aѵb3 and neuropilin-1 receptors on B16F10 melanoma cells as represented in

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Figure 4.9 Mechanism of action of PTX/HCQ-R8-dGR-Lip as targeted delivery to cancer cells. Reproduced with permission from Yin S, Xia C, Wang Y, Wan D, Rao J, Tang X et al. Dual receptor recognizing liposomes containing paclitaxel and hydroxychloroquine for primary and metastatic melanoma treatment via autophagy-dependent and independent pathways. J Control Release 2018;288:148e60.

Fig. 4.9. PTX/HCQ-R8-dGR-Lip exhibited in vitro inhibition of migration, proliferation and resistance of B16F10 cells and exhibited increased efficacy for inhibition of growth of primary tumor and reduced metastasis in lung cancer in vivo. Antimetastasis study confirmed the co-delivery of PTX and HCQ suppressed degradation of MMP2, MMP9, and paxilin [119]. In another study, peptide-conjugated doxorubicin liposomes were designed to identify and bind selectively to the marker protein Claudin 7 (CLDN7) [120]. In this study, the initial dose of ionizing radiation in a gastric tumor xenograft mouse model was followed by a phage displayed peptide library injection. Improved therapeutic efficacy with irradiated diagnostic imaging was found in peptide conjugated liposomes loaded with doxorubicin [120]. Deshpande et al. designed a surface engineering of doxorubicin loaded PEGylated liposomes with arginine 8 (R8) and transferrin to prepare DUAL-DOX-L toward targeted delivery of doxorubicin in A2780 ovarian cancer cells. The DUAL-DOX-L showed enhanced efficacy in in vivo inhibition of tumor growth in A2780 ovarian xenograft model [121]. Integrin a(5)b(1) antagonist N-acetyl-proline-histidine-serine-cysteine-asparagines-amide (Ac-PHSCN-NH(2)) is a new targeting peptide for anticancer therapy. Dai et al. designed a ligand targeted liposomal delivery by conjugating the Ac-PHSCN-NH (2) with doxorubicin for delivery to B16F10 cells. PHSCNK-PL-DOX showed enhanced cellular uptake with increased cytotoxicity against B16F10 cells as compared to stealth liposomes of doxorubicin [122]. Ding et al. designed a pH sensitive cell-penetrating peptide conjugated doxorubicin loaded liposomes with PEG2000-Hz-stearate on the surface. The formulation showed 1.9-fold increase in accumulation, with long circulation time and improved efficacy and selectivity in breast cancer cell lines [115].

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Co-delivery of daunorubicin (DNR) and dioscin was achieved by a surface modified neutral cell penetrating peptide PFVYLI (PFV) and targeted for treatment of cancer with retardation of metastasis. Wang et al. synthesized the liposomes containing daunorubicin and dioscin modified with CPPs and reported the role of dioscin in increasing the effect of daunorubicin for inhibition of A549 cells and tumor metastasis. Inhibition of metastasis involved the downregulation of vascular endothelial cadherin, matrix metalloproteinase-2 (MMP-2), hypoxia inducible factor-1a and transforming growth factor-b1 [123]. A peptide conjugated liposomes was designed and targeted to luteinizing hormone-releasing hormone (LHRH) receptor containing mitoxantrone which showed improved accumulation and inhibition of tumor growth in breast cancer cells (MCF-7) in vivo [124]. RNA aptamer conjugated doxorubicin liposome was designed for targeted delivery to prostate cancer cells (PSMA positive). The formulation exhibited efficient binding to the target cells with decrease in size of tumor. The ligation of aptamer to the surface of liposomes by post-insertion method increased the tumor accumulation of liposomes and inhibited the growth of tumor [125]. Tian et al. used the layer by layer technology to prepare the curcumin loaded liposomes coated with carboxymethyl chitosan and quaternary ammonium chitosan as represented in Fig. 4.10. The CMCS/TMC-LPs enhanced the oral bioavailability (38%) by six- and threefold as compared to curcumin loaded liposomes and curcumin loaded quaternary ammonium chitosan liposomes respectively. The CMCS/TMC-LPs increased the circulation time of curcumin and higher accumulation was reported in spleen, lungs, and liver [126]. Seong et al. developed a pH sensitive quercetin loaded multilayered liposome with layer-by-layer technique using N-succinyl chitosan and chitooligosaccharide. The formulation showed enhanced stability against surfactant like Triton X-100, and released quercetin efficiently at pH 5.5 in the skin due to the sugar moieties present in N-succinyl chitosan and chitooligosaccharide which can absorb water in huge quantity and disturbs the stratum corneum barrier of skin by hydration [127].

4.6

Engineered liposomes in imaging

Liposomes loaded with radiolabeled nuclei are known as diagnostic nanoparticles and used for imaging agents to visualize tumors, ERS imaging and imaging of cardiovascular diseases [109,128,129]. Loading of radiolabeled nuclei in liposomes is done in the inner aqueous core or inside/on the surface of the lipid membrane. Liposomes designed for imaging are loaded with 99mTc, 111In, and 64Cu radionuclide. Imaging techniques used to envisage tumors, different types of molecules such as antibodies, enzyme inhibitors, peptides or known radiopharmaceuticals, such as 18F-FDP and 18 F-FDG, 99mTc-DISIDA, 99mTc-MIBI, 99mTc-HMPAO, 99mTc-HAS, 99mTc-DTPA, 99m Tc-streptokinase, 99mTc-BMEDA, and 99mTc-Biotin are inserted within liposomes [109]. The limitation of tumor imaging with radiolabeled liposomes is associated with slow accumulation [108].

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Figure 4.10 (a) curcumin loaded liposomes designed with layer-by-layer coating of carboxymethyl chitosan (CMCS) and quaternary ammonium chitosan (TMC); (b) Representation of mechanisms of transport of curcumin loaded CMCS/TMC-Liposomes in small intestine (AJs: adherens junctions, TJs: tight junctions); (c) Delivery of curcumin loaded CMCS/TMCliposomes to the gastrointestinal tract by oral route [126].

Lee et al. developed liposomal formulation labeled with 64Cu as an efficient positron emission tomography (PET) probe for imaging of bone marrow. They synthesized liposomes functionalized with DOTA using 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, N-succinyl 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-(succinyl DPPE), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol) 2000] (mPEG2000-DSPE) and DSPE surface modified with bifunctional chelator DOTA (DOTA-Bn-DSPE) in the ratio of (60:30:10:1:0.1) as optimized formulation of an average size of 90 nm. The formulation was then doped with DOTA-Bn-DSPE for stable incorporation of 64Cu. Fig. 4.11 represents the PET/CT images of 64Cu-DOTA-Bn-DSPE liposome (90 nm) after 24 h of administration. In spite of limitations, the spine, sacrum, and tibial and femoral heads could be clearly identified from the image. The results showed that the PET 64Cu labeled liposomes targeted efficiently in imaging of bone marrow and thereby can be used as an effective imaging agent for bone marrow [130].

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Figure 4.11 PET/CT images 64Cu-labeled liposomes (90 nm) targeting bone marrow of mice. (a) CT, PET, and PET/CT fusion images of 64Cu-liposome treated mice after 24 h of administration. The arrows designate the PET images of bone marrow. (b) Magnified CT, PET, and PET/CT fusion images of tibia of the same mice; and (c) CT, PET, and PET/CT fusion images of tibia demonstrating the specific deposition of liposomes in bone marrow [130].

The limitation of slow accumulation due to poor penetrability, the penetration of the Tc-labeled liposomes imaging agents was improved by using microwave assisted synthesized stearyl 6-(benzylidenehydrazinyl) nicotinamide lipid and the loading of radiolabeled nuclei had radiochemical purity of 94 þ 1.7%. The lipid was incorporated in two liposome formulations, i.e., 99mTc-HYNIC liposome and 99mTc-HYNIC-PEG liposome. Tumor uptake of both the liposomal formulation was significantly higher. Biodistribution study in C57BL/6 mice was done at 4 and 24 h post administration. After 4 h, the cellular uptake of 99mTc-HYNIC liposome and 99mTc-HYNIC-PEG liposome were found to be 1.1  0.6 and 2.5  0.4, respectively. Post 24 h post administration, the biodistribution were found to be 1.8  0.5 and 3.0  1.1, respectively. So this formulation can be adopted for a noninvasive targeting imaging agent for in vivo study of permeability through the tumor cells [131]. 99m

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Liposomes coated with 99mTc-radiolabeled antisense oligonucleotides (ASONs) targeting MDM2 messenger RNA (mRNA) were designed for in vivo imaging of MDM2 expression. The liposomes showed increase in cellular uptake by breast tumor cell lines (MCF-7) by threefold [132]. Kang et al. synthesized liposomes conjugated with 64Cu-labeled tetraiodothyroacetic acid (tetrac)dfor imaging of tumor angiogenesis. Tetrac arrests angiogenesis regulating factor integrin avb3. Two formulationsd DOTA-PEG-DSPE and Tetrac-PEG-DSPEdwere prepared loaded with 64Cu. Imaging study for both the liposomal formulations were conducted to study the tissue uptake and distribution in U87MG tumor cells. MicroPET imaging with tetrac/64CuDOTA-liposomes exhibited higher deposition of spleen and liver. Uptake of tetrac/64Cu-DOTA-liposomes by tumor cells was found to be 1.75 þ 0.03%ID/g as compared to 64Cu-DOTA-liposomes (0.36 þ 0.01%ID/g) respectively post administration [133]. Van der Geest et al. designed a surface labeling of 111In with DTPA-DSPE long circulating liposomes (DTPA-DSPE-LCL). In this study, the in vitro properties of LCL, DTPA-LCL, and DTPA-DSPE-LCL were compared. The stability of three formulations were conducted by DTPA challenge assay by incubation with 103 M DTPA for 24 h and the retention of activity was found to be 2%, 46%, and 93% for LCL, DTPA-LCL, and DTPA-DSPE-LCL, respectively. The in vivo targeting study of the formulations engineered with 111I was conducted in NMRI mice with an S. aureus abscess in muscles of left side thigh. The formulations were administered intravenously after 24 h of initiation of infection and the SPECT/CT imaging (after 1, 24 and 72 h) displayed the accumulation of 111In-DTPA-DSPE-LCL, and 111InDTPA-LCL in liver, spleen, and abscess as represented in Fig. 4.12 [134]. Qingshan et al. designed a dual mode imaging probe utilizing the concept of nearinfrared fluorescence (NIRF) and magnetic resonance imaging (MRI) and verified its applicability in in vivo models of liver cancer. The probes consisted of liposomes coating the superparamagnetic iron oxide (SPIO) nanoparticles. The liposomes are conjugated with a tumor targeting peptide RGD (argininedglycinedAspartate) and a NIRF dye ICG (indocyanine green). The imaging probe was also studied for preoperative diagnosis and intraoperative assistance in mouse with orthotropic liver tumor and intrahepatic liver metastasis. The probe injection of SPIO@Liposome-ICGRGD study exhibited easy and clear portrayal of tumor detection and surgery [135]. Bandekar et al. reported PSMA specified aptamers conjugated liposomes for the targeted delivery of radioactive actinium (225Ac) for antivascular radiotherapy [136]. Zhang et al. designed Gd-DTPA encapsulated thermosensitive liposome conjugated with AS1411 as an efficient imaging probe targeted toward overexpressed nucleolin receptors on tumor cells [137]. Ribeiro et al. designed cyclic RGD conjugated magnetoliposomes as MRI contrast agent and a targeted drug delivery to tumor cells. The formulation was studied for targeting the avb3 integrin overexpressed in neovascularization, glioma, and ovarian cancer. Efficacy of the formulation was studied in Swiss nude mice with human ovarian SKOV-3 cells. cRGD-magnetoliposome showed higher uptake by SKOV-3 cells and visualized by both fluorescence imaging (FLI) and MRI [138]. Similarly, a dual

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Figure 4.12 Representative SPECT/CT image for increased uptake of 111In-DTPA-DSPE-LCL by liver and spleen (top row), as compared to 111In-DTPA-LCL (bottom row). Uptake and accumulation of the formulations in the abscess area was not significantly different [134].

targeted paramagnetic liposomes surface engineered with two antiangiogenic ligands, the avb3 integrin-specific RGD (Arg-Gly-Asp), and the neuropilin-1 (NRP-1) receptor-specific ATWLPPR (Ala-Thr-Trp-Leu-Pro-Pro-Arg) (A7R) was designed by Song et al. The liposomes were loaded with MRI contrast agent gadoliniumdiethylenetriamine pentaacetic acid (Gd-DTPA) and in vitro tumor cell uptake and inhibition assay displayed enhanced binding affinity in HUVEC and A549 cells. So dual-anb3-ntegrin-NRP1-targeting paramagnetic liposome with an RGD-ATWLPPR CPPs was assumed to be a suitable molecular imaging system for tumor [139]. Tahara et al. developed a real-time in vivo imaging system (IVIS) to evaluate the interaction of chitosan or glycolchitosan modified liposomes in gastrointestinal tract after oral administration. An NIR dye indocyanine green (ICG) was used to study the dynamic behavior of the liposomes by quantitative measurement of the intensity of the fluorescence of ICG in tissue homogenates. Retention of nano-sized unilamellar vesicles was longer as compared to micro-sized multilamellar vesicles in the GIT. Surface modification of liposomes increased the retention of liposomes in the GIT and prevented the early excretion [140].

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Theranostic engineered liposomes

Theranostics is an interdisciplinary field which combines both therapeutics and diagnostics [141]. Among all the nanodelivery systems, liposomes drew the attention due to their physical and chemical properties. The strategies approached to prepare for the theranostic liposomes are shown in Fig. 4.13. The theranostic liposomes carry the therapeutics (chemotherapeutic drugs or biological moieties) and radiolabeled with diagnostic agents for diagnosis by SPECT/CT imaging as represented in Fig. 4.13a. Sometimes double radionuclei are labeled for both diagnosis and treatment like labeling with 64Cu/177Lu as represented in Fig. 4.13b [142]. The liposomes may be loaded with therapeutic radionuclide like 31I, 177Lu, or 186,188Re which are capable of emitting g radiation and can be detected by SPECT as represented by Fig. 4.13c [109]. The lipids which are chosen for bilayer formation of liposomes as theranostic imaging must be in gel state. The liposomal properties can be enhanced by addition of cholesterol and/or PEG are added to the lipids. The drugs and genetic material loaded for theranostic application can have different modalities [143]. Patel et al. reported the delivery of two drugs raloxifene and leuprolide for the treatment of endometriosis of uterus. Raloxifene and leuprolide were labeled with 99mTc prior to encapsulation into liposomes for intravaginal administration. Parenterally, both the drugs have poor bioavailability with significant side effects. The retention of encapsulated liposomes in the uterus was found to be longer than the nonencapsulated formulations and leuprolide was released slowly revealed by images of scintigraphy [144,145]. Duan et al. [141] formulated and evaluated novel pHsensitive long-circulating liposome-based theranostic systems for molecular imaging

Figure 4.13 General schemes for labeling of radiolabeled theranostic liposomes [109].

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and drug delivery. In this work, liposomal particles were generated using hydrogenated soy (HS) phosphatidylcholine, cholesteryl hemisuccinate (CHEM), polyethylene glycol (PEG) and diethylenetriamine pentaacetic acid-modified phosphatidyl ethanolamine with film hydration and extrusion methods. The liposome was labeled with 111 In. As compared to two formulations, i.e., 1% PEG-HS-CHEM and 4% PEGHS-CHEM, the later showed delayed release but higher stability in acidic microenvironment of cancer cells. Comparison between biodistribution of 4% PEG-HS-CHEM and 4% PEG-HS-Chol revealed that 4% PEG-HS-CHEM showed higher accumulation in spleen, liver, and kidneys [141]. Panikar et al. designed a peptide conjugated liposomes for targeted chemo-dynamic therapy of HER2 positive breast cancer. The ligand-targeted liposomes were encapsulated with doxorubicin for the chemotherapy and methylene blue attached NaYF4:Yb, Er were used for NIR activated imaging of tumor cells and the photo-excitation of methylene blue for photodynamic therapy. Target specificity of the liposomal formulation was studied by confocal imaging on SKBR-3 (HER2-positive) and MCF-7 (HER2-negative) breast cancer cell lines. The chemo-photodynamic therapy was found to be more efficacious than the chemo or photodynamic therapy alone as theranostics [146].

4.8

Challenges and limitations of engineered liposomes as nanotheranostics

Refrigeration is required for storage of liposomes, but can’t be stored in freezer as the phospholipid bilayers in the liposomes may be ruptured due to formation of ice crystals. Though oral route of administration of liposomes is more suitable but the marketed formulation of liposomes loaded with anticancer drugs are mostly designed for intravenous administration. For development of liposomal formulations, different key factors such as size, shape, charge, density of ligand, and the target cell features are considered carefully [147]. Small-sized liposomes show decreased interaction between ligand and the receptor. Spherical, cylindrical, and rod-shaped liposomes showed higher cellular uptake. Positively charged liposomes show enhanced cellular internalization as the electrostatic attraction is increased with negatively charged cell membrane. Optimal density of ligands on the surface of liposomes can increase the tumor cell uptake, whereas increased ligand density leads to agglomeration of liposomes. The optimum size of liposomes may affect the extent of cellular uptake by the tumor cells depending on variation in phenotype [148]. Similarly, the density and length of PEG chains must be optimized for liposomal formulations. PEG with longer chains like PEG5000 lacks linearity in aqueous medium and folds into globular structure like mushrooms. The nonlinearity of PEG may cause steric hindrance during ligand and receptor interaction, so careful consideration of binding affinity between the receptor and the ligand can address the challenge [149]. Other challenges associated with liposomes to cross the biological barriers like increased hydrostatic pressure, disturbed vasculature, and barrier to the binding site need to be accounted [150]. Various ligands used for surface engineering of liposomes caused increase in detection

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by immune system [151]. Ligands and serum proteins may interfere in targeting of liposomal formulations [152]. Stability of liposomes is a significant factor for eliciting optimum cytotoxicity in cancer chemotherapy. The release of drugs from liposomes is also another challenge which should be taken into consideration during designing the delivery system. Charged liposomes show some adverse reactions like polyvalent positively charged liposomes showed pulmonary toxicity due to reactive oxygen intermediates [153]. Scaling up the surface engineered liposomes in large scale is another challenge and some important factors need to be carefully considered like drug loading, entrapment efficiency, stability, reproducibility, and methods of sterilization. Diagnosis of cancer in the early stage is still a major challenge in the field of cancer research. Complete recovery of patient within a short span of time and minimum adverse effects is the most challenging requirement of the hour. Treatment of genetic causes of cancer research has been utilized the targeted delivery of chemotherapeutic drugs. Targeted delivery to cancer cells by either active targeting or passive targeting may enhance the efficacy of chemotherapy with recovery of patient at short time [154,155]. Another important challenge is the delivery of anticancer drug to brain using the liposomes. The liposomal formulation must be designed in such a way that it is able to cross the dual barrier of bloodebrain barrier and the barrier of the tumor. So, an efficient liposomal delivery system required to be designed targeting the gliomas [156,157]. Cost of commercially available liposomal formulations is too high, which is due to expensive raw material and scale up methods for production of liposomal formulation [16]. So modifications of the existing methods for cost effective production of liposomal formulation must be required.

4.9

Conclusion and future perspective

Understanding the specific mechanism by which a nanocarrier, such as liposomes, can access the intended region and impose therapeutic response could be crucial in tackling existing drug delivery and imaging issues. The additional properties of designed nanocarriers, such as liposomes, are dramatically affected by changes in physico-chemical attributes. The regulatory approval and quality monitoring of liposomes need to be provided considerable emphasis. Employing a proper surface engineering strategy for liposomes with ligands should be orchestrated without inactivating the ligands. An essential element to address is the tuning of ligand concentration on the surface of surface engineered liposomes with minimal fluctuation in circulation time. The methodologies presented in this chapter, which covers a wide range of techniques for surface engineering to construct and formulate liposome-based delivery systems with multiple functions in a single formulation such as stimuli sensitivity and receptor-specific targeting, which can bring an important functionality in addressing current hurdles such as multi-drug resistance (MDR). Collaboration among all associated professionals involved in the advancement of liposome technology is required to design liposome formulations with high therapeutic efficacy and minimal adverse effects.

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[112] Metselaar JM, Storm G. Liposomes in the treatment of inflammatory disorders. Expet Opin Drug Deliv 2005;2(3):465e76. [113] Mozhi A, Ahmad I, Okeke CI, Li C, Liang XJ. pH-sensitive polymeric micelles for the Co-delivery of proapoptotic peptide and anticancer drug for synergistic cancer therapy. RSC Adv 2017;7(21):12886e96. [114] Zhang Q, Lu L, Zhang L, Shi K, Cun X, Yang Y, et al. Dual-functionalized liposomal delivery system for solid tumors based on RGD and a pH-responsive antimicrobial peptide. Sci Rep 2016;6(1):19800. [115] Ding Y, Cui W, Sun D, Wang G, Hei Y, Meng S, et al. In vivo study of doxorubicinloaded cell-penetrating peptide-modified pH-sensitive liposomes: biocompatibility, biodistribution, and pharmacodynamics in BALB/c nude mice bearing human breast tumors. Drug Des Dev Ther 2017;11:3105e17. [116] Yang Z, Li J, Wang Z, Dong D, Qi X. Tumor-targeting dual peptides-modified cationic liposomes for delivery of siRNA and docetaxel to gliomas. Biomaterials 2014;35(19): 5226e39. [117] Lakkadwala S, Singh J. Dual functionalized 5-fluorouracil liposomes as highly efficient nanomedicine for glioblastoma treatment as assessed in an in vitro brain tumor model. J Pharmacol Sci 2018;107(11):2902e13. [118] Shi K, Long Y, Xu C, Wang Y, Qiu Y, Yu Q, et al. Liposomes combined an integrin avb3-specific vector with pH-responsible cell-penetrating property for highly effective antiglioma therapy through the bloodebrain barrier. ACS Appl Mater Interfaces 2015; 7(38):21442e54. [119] Yin S, Xia C, Wang Y, Wan D, Rao J, Tang X, et al. Dual receptor recognizing liposomes containing paclitaxel and hydroxychloroquine for primary and metastatic melanoma treatment via autophagy-dependent and independent pathways. J Contr Release 2018; 288:148e60. [120] Lee K, Shin S, Lee J, Ju E, Park Y, Hwang J, et al. A strategy for actualization of active targeting nanomedicine practically functioning in a living body. Biomaterials 2017;141: 136e48. [121] Deshpande P, Jhaveri A, Pattni B, Biswas S, Torchilin V. Transferrin and octaarginine modified dual-functional liposomes with improved cancer cell targeting and enhanced intracellular delivery for the treatment of ovarian cancer. Drug Deliv 2018;25(1):517e32. [122] Dai W, Yang T, Wang Y, Wang X, Wang J, Zhang X, et al. Peptide PHSCNK as an integrin a5b1 antagonist targets stealth liposomes to integrin-overexpressing melanoma. Nanomedicine 2012;8(7):1152e61. [123] Wang Y, Fu M, Liu J, Yang Y, Yu Y, Li J, Pan W, Fan L, Li G, Li X, Wang X. Inhibition of tumor metastasis by targeted daunorubicin and dioscin codelivery liposomes modified with PFV for the treatment of non-small-cell lung cancer. Int J Nanomed 2019;14: 4071e90. [124] Zhang L, Ren Y, Wang Y, He Y, Feng W, Song C. Pharmacokinetics, distribution and anti-tumor efficacy of liposomal mitoxantrone modified with a luteinizing hormonereleasing hormone receptor-specific peptide. Int J Nanomed 2018;13:1097e105. [125] Baek S, Lee K, Park Y, Oh D, Oh S, Kim K, et al. RNA aptamer-conjugated liposome as an efficient anticancer drug delivery vehicle targeting cancer cells in vivo. J Contr Release 2014;196:234e42. [126] Tian M, Song R, Wang T, Sun M, Liu Y, Chen X. Inducing sustained release and improving oral bioavailability of curcumin via chitosan derivatives-coated liposomes. Int J Biol Macromol 2018;120:702e10.

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[127] Seong J, Yun M, Park S. Surfactant-stable and pH-sensitive liposomes coated with Nsuccinyl-chitosan and chitooligosaccharide for delivery of quercetin. Carbohydr Polym 2018;181:659e67. [128] Lobatto ME, Binderup T, Robson PM, Giesen LF, Calcagno C, Witjes J, Fay F, Baxter S, Wessel CH, Eldib M, Bini J. Multimodal positron emission tomography imaging to quantify uptake of 89Zr-labeled liposomes in the atherosclerotic vessel wall. Bioconjugate Chem 2019;31(2):360e8. [129] Stendahl J, Sinusas A. Nanoparticles for cardiovascular imaging and therapeutic delivery, Part 2: radiolabeled probes. J Nucl Med 2015;56(11):1637e41. [130] Lee SG, Gangangari K, Kalidindi TM, Punzalan B, Larson SM, Pillarsetty NV. Copper64 labeled liposomes for imaging bone marrow. Nucl Med Biol 2016;43(12):781e7. [131] Cabrera M, Lecot N, Fernandez M, Gambini J, Porcal W, Cabral P. 99mTc stearyl 6(benzylidenehydrazinyl) nicotinamide liposomes as tumor permeability evaluation tracer. AAPS PharmSciTech 2021;22(3):115. [132] Fu P, Shen B, Zhao C, Tian G. Molecular imaging of MDM2 messenger RNA with 99mTc-labeled antisense oligonucleotides in experimental human breast cancer xenografts. J Nucl Med 2010;51(11):1805e12. [133] Kang C, Koo H, Lee S, Lee K, Oh Y, Choe Y. 64Cu-Labeled tetraiodothyroacetic acidconjugated liposomes for PET imaging of tumor angiogenesis. Nucl Med Biol 2013; 40(8):1018e24. [134] van der Geest T, Laverman P, Gerrits D, Franssen G, Metselaar J, Storm G, et al. Comparison of three remote radiolabelling methods for long-circulating liposomes. J Contr Release 2015;220:239e44. [135] Chen Q, Shang W, Zeng C, Wang K, Liang X, Chi C, et al. Theranostic imaging of liver cancer using targeted optical/MRI dual-modal probes. Oncotarget 2017;8(20):32741e51. [136] Bandekar A, Zhu C, Jindal R, Bruchertseifer F, Morgenstern A, Sofou S. Antieprostatespecific membrane antigen liposomes loaded with 225Ac for potential targeted antivascular a-particle therapy of cancer. J Nucl Med 2013;55(1):107e14. [137] Zhang K, Liu M, Tong X, Sun N, Zhou L, Cao Y, et al. Aptamer-modified temperaturesensitive liposomal contrast agent for magnetic resonance imaging. Biomacromolecules 2015;16(9):2618e23. [138] Ribeiro RS, Belderbos S, Danhier P, Gallo J, Manshian BB, Gallez B, Ba~ nobre M, De Cuyper M, Soenen SJ, Gsell W, Himmelreich U. Targeting tumor cells and neovascularization using RGD-functionalized magnetoliposomes. Int J Nanomed 2019;14: 5911e24. [139] Song Y, Li W, Meng S, Zhou W, Su B, Tang L, et al. Dual integrin avb 3 and NRP-1targeting paramagnetic liposome for tumor early detection in magnetic resonance imaging. Nanoscale Res Lett 2018;13(1):380. [140] Tahara K, Nishio M, Takeuchi H. Evaluation of liposomal behavior in the gastrointestinal tract after oral administration using real-time in vivo imaging. Drug Dev Ind Pharm 2018; 44(4):608e14. [141] Duan Y, Wei L, Petryk J, Ruddy T. Formulation, characterization and tissue distribution of a novel pH-sensitive long-circulating liposome-based theranostic suitable for molecular imaging and drug delivery. Int J Nanomed 2016;11:5697e708. [142] Ehlerding E, Goel S, Cai W. Cancer theranostics with 64Cu/177Lu-loaded liposomes. Eur J Nucl Med Mol Imag 2016;43(5):938e40. [143] Choi H, Lee Y, Hwang D, Lee D. Translational radionanomedicine: a clinical perspective. Eur J Nanomed 2016;8(2):71e84.

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Polymeric micelles for theranostic uses

5

Somasree Ray, Shalmoli Seth, Uttam Kumar Bhattacharyya and Malini Sen Gupta College of Technological Sciences, Asansol, West Bengal, India

5.1

Introduction

Tremendous efforts have been given for the development of site-specific drug delivery system such as liposomes, nanoparticles, and polymeric microspheres. Nowadays, polymeric micelles have been emerged as a novel carrier for the delivery of poorly water-soluble drug as micelle can solubilize these drugs in their inner core. A polymeric micelle is a macrolmolecular structure that is formed from synthetic block copolymers or graft polymers. Block copolymer having ampiphilic character form polymeric micelle in aqueous environment with microscopic size range [1,2]. They are spherical in shape having a core shell architecture where hydrophobic core acts as a reservoir for entrapment of hydrophobic drug, DNA, and protein molecules, whereas hydrophilic shells face biological media. Formed micelle consists of twophase structure with spherical inner core and an outer shell. Micelles are formed from copolymers having A-B diblock structure, where A (hydrophilic polymer shell) and B is hydrophobic polymers (core), respectively (Figs. 5.1 and 5.2). Multiblock

Figure 5.1 Structure of block copolymer micelle having A-B diblock structure with A (hydrophilic shell) and hydrophobic core B. Design and Applications of Theranostic Nanomedicines. https://doi.org/10.1016/B978-0-323-89953-6.00007-6 Copyright © 2023 Elsevier Ltd. All rights reserved.

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Figure 5.2 Polymeric micelle carrier system with inner shell and outer shell.

copolymers are also self-assembled to form micelle like poly (ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide) (A-B-A) and can be used as a potential drug delivery system. One of the attractive characteristics of polymeric micelle is their availability in low size range and has a propensity to escape scavenging by mononuclear phagocyte system [3]. Recently, the focus has been done on the use of polymeric micelle as a colloidal drug delivery system that can fulfill the goal of targeting drugs to a particular portion of the body. Particle size and surface properties of the micelle, modification of the chemical structure of core-forming block in polymer micelle increases the drug entrapment efficiency, enhances stability and specificity for a particular organ or tissue which ultimately improves the targeting efficiency. The design, size, and surface characteristics of the polymeric micelle are very important for the modulation of the delivery of drugs. One of the drawbacks associated with colloidal drug delivery system is the nonspecific uptake of the colloidal particles by reticuloendothelial systems and if the target site is present outside the blood compartment, i.e., observed in case of solid tumors the vehicle is required to have a capability of penetration through the vessel wall unless a sustained release mechanism is adopted [4,5]. In comparison to surfactant micelles, polymeric micelles are more stable as compared to surfactant micelles with low critical micelle concentration. The slow rate of dissociation of polymer micelle allows sustained release of drug for a long period.

5.2 5.2.1

Advantages and disadvantages of polymeric micelle Advantages

1) The main advantage of the polymeric micelle is that it can encapsulate a large amount of hydrophobic drug inside the inner core, at the same time, the micelle can maintain its solubility in water and prevent intermicellar aggregation of hydrophobic cores, presence of hydrophilic outer shell acts as a barrier for intermicellar aggregation. 2) Another advantage of the polymeric micelle is its small particle size (diameter range from 10 to 100 nm) with a substantial narrow particle size distribution, that narrow size distribution is ideal for circulation in the bloodstream. Due to its small particle size, polymeric micelles can

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be easily sterilized by filtration process having 0.45 mm or 0.22 mm pore size without micron-sized particle clogging. 3) Low toxicity of polymeric micelle is another advantage. Polymeric micelles possess a larger size than the critical filtration in the kidney. Polymeric micelle can evade renal filtration even if the molecular weight of the constituting block copolymer is lower than the critical molecular weight of renal filtration [4,6]. 4) After the release of the drug, the polymer chain dissociates and excretes through the renal route. If the molecular weight of the dissociated polymer chain is less than the critical value for renal filtration, it is completely excreted through the kidney. This is another advantage of polymeric micelle over nonbiodegradable polymeric drug delivery systems.

Different types of drugs, genes, and contrasting agents can be entrapped into the polymeric micelle. The hydrophobic drug can be easily entrapped into the micellar inner core either by different techniques like chemical conjugation of drug with inner core-forming block polymer or by physical method of entrapment due to hydrophobic interaction of drug and inner hydrophobic core. Polymeric micelle can also be formed due to ionic interactions of charged polymer chains. Formation of macromolecular assembly known as “polyion complex micelle” takes place in an aqueous medium due to electrostatic interactions of charged block polymers and polyethylene glycol chains [6,7]. Polyion complex micelle is completely water-soluble. One of the remarkable properties of polyion complex micelle is that it can act as a micro reservoir for charged compounds like DNA and enzyme and allow modification of their properties such as solubility and stability. The core of these types of micelle provides a unique field for the enzymatic reaction because the core forms a distinct phase from the outer phase which is an aqueous phase. Polymeric micelle prepared from polyethylene glycol-bpoly(lysine) block copolymers and poly (aspartic acid) homopolymers contains positively charged poly(lysine) chain and negatively charged poly (aspartic acid) chain. When negatively charged polypeptides are used in place of polyaspartic acid, the pharmacologically active macromolecules can be entrapped into polymeric micelle for protein, gene, and interfering RNA delivery. Metal ion chelate can also be entrapped into polymeric micelle using coordination bonds or ionic interactions (Table 5.1).

Table 5.1 Advantages and disadvantages of polymeric micelle as a drug delivery system.

Advantages 1) 2) 3) 4) 5)

Small particle size (diameter 10e100 nm) High water solubility Stable in nature A large number of drugs can be loaded Able to incorporate different types of chemical agents

Disadvantages 1) 2) 3) 4)

Difficulty arises in polymer synthesis and reproducibility on a large scale Immature technology of incorporation of drug in a physical manner Slower extravasation of polymeric carrier system than low molecular weight drugs Risk of chronic liver toxicity

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5.2.1.1

Disadvantages

Polymeric micelle has some disadvantages also as a drug delivery system: 1) The first and foremost disadvantage is the difficult synthetic procedure of the preparation of the polymeric chain. From the micellar structure, we have seen that AB block copolymer is one of the most common chemical structures of the polymeric micelle. Though the architecture of the structure of AB block copolymer is very common and simple, the synthetic procedure is much more difficult as compared to random polymers. 2) Another problem is related to the reproducibility of the synthetic procedure on a large industrial scale. Sometimes, it takes a lengthy procedure to prepare the polymer. 3) Immature technology for drug entrapment in a physical manner is another problem. The efficiency of drug incorporation depends on several factors. Sometimes it is easy to entrap drugs on a small laboratory scale, but it is found to be difficult to entrap drugs on a large industrial scale. 4) There is a difference in the extravasations mechanism between low molecular weight drugs and the polymeric carrier system. Due to this, slower extravasations of polymeric carrier system were observed than low molecular weight drugs. To compensate for slow extravasation, the polymeric carrier system should be present in blood circulation for a long period of time. 5) Drugs entrapped in the polymeric micellar carrier systems are metabolized in the liver at a slower rate than free drugs because the access of metabolic enzymes to the drug is obstructed as the drug is incorporated or conjugated in the polymeric micelle. 6) The toxic effect of free drugs can be decreased through metabolism within a short period whereas in the case of the polymeric carrier system toxic effects of the drug are observed for a long period.

5.3

Different types of polymer micelle as carrier systems used for the delivery of drugs

Different types of polymeric micelle systems used for the delivery of drugs: 1. Micelle forming polymer-drug conjugates 2. Polymeric micellar nanocontainers 3. Polyion complex micelle

5.3.1

Micelle forming polymer-drug conjugates

In this type of conjugates, the drug is incorporated within the micellar structure and hydrolyzable chemical bonds are present between the functional groups that is present in the polymer and the drug molecule (Fig. 5.2 aed). Drug block copolymer conjugates are formed between poly (ethylene oxide)-bpoly(ester) and drug due to the formation of a covalent bond between the reactive group of the drug and the terminal hydroxyl group present in the poly(ester) section (Fig. 5.3a). Poly (amino acid) block also forms drug-polymer conjugates with a large number of different types of drug molecules. The poly (amino acid) segment contains

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Hydrophilic block

Hydrophobic block

Hydrophilic block

Hydrophobic block

Drug

(A)

(B) Hydrophobic Spacer

Drug

Hydrophilic block

Hydrophobic block

(C)

Hydrophobic unit

Hydrophilic block

Drug

Drug

Hydrophobic block

(D) Drug

Figure 5.3 Different models for micelle-forming from drugeblock copolymer conjugates. 3A) covalent bond formation between the activated terminal hydroxyl group of the poly(ester) section and reactive groups present on the drug molecule 3B) to 3D)Conjugation of drug molecules to several sites of one polymeric chain.

several functional groups and provides different sites of attachment for the drug molecule (3B to 3D) in one polymeric chain. Again the presence of different types of functional groups are present on the poly(amino acid) chain allows the formation of conjugates at different points in the polymer.

5.3.2

Polymeric micellar nanoparticles

In this type of system either hydrogen or hydrophobic bonds are formed between block copolymer and drug and this helps insolubilization of drug present in polymeric micelles. Polymeric micelle nanocontainers can be prepared by direct incubation of the drug with block copolymers in the aqueous environment if the drug and block copolymer is water-soluble. But this method is not effective for most of the structures of drugs and block polymers and drug entrapment is low. Physical incorporation of the drug in micelle can be easily accomplished by different methods like dialysis method, o/w emulsion method, freeze-drying method, cosolvent evaporation method, solvent evaporation method (Fig. 5.4).

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Figure 5.4 Different physical methods of encapsulation of drug in polymeric micelles: (a) dialysis method; (b) oil/water emulsion method; (c) solvent evaporation method; (d) co-solvent evaporation; (e) freeze-drying method.

5.3.2.1

Dialysis method

In the dialysis method, both drug and polymer are dissolved in a water-miscible organic solvent (e.g. N, N dimethylformamide) followed by the dialysis of the solution against water [7e9]. Self-association of block copolymers are observed due to gradual replacement of organic solvent with water which is a nonsolvent for the core-forming block and this triggers the encapsulation of drug in assembled structure. The semipermeable membrane of the dialysis bag allows the removal of unentrapped drugs from the polymeric micelle. One of the drawbacks associated with this method is the incomplete removal of unentrapped drugs from polymeric micelle (Fig. 5.4a).

5.3.2.2

o/w emulsion method

In the o/w emulsion method drug is first dissolved in a water-miscible organic solvent like chloroform or methylene chloride and this organic phase is added to the aqueous phase with vigorous stirring. Polymer is dissolved either in the organic phase or aqueous phase under vigorous stirring. The organic phase is removed by evaporation. Doxorubicin was physically loaded in poly (ethylene glycol)-poly (beta benzyl Laspartate) block copolymer by o/w emulsion method and drug loading level was 15e20 %w/w [10]. In another study Block copolymer micelle of poly (ethylene oxide)-block-poly (B benzyl L aspartate) were prepared by oil in water emulsification method and doxorubicin was loaded into it. The resulting micelle acted as a drug depot for the slow release of doxorubicin even in the presence of 10%w/v serum albumin [11] (Fig. 5.4b).

Polymeric micelles for theranostic uses

5.3.2.3

115

Solvent evaporation method

In the solvent evaporation method involves the drug and the polymer are dissolved in a volatile organic solvent and subsequent evaporation of the organic solvent results in the formation of polymer film loaded with the drug. Reconstitution of the film in an aqueous phase with vigorous shaking forms micelle. Lavasanifar et al. [12] investigated the role of fatty acid substitution on the release profile of amphotericin B from micelle prepared from poly (ethylene oxide)-block-poly {N(6-hexyl stearate)L- aspartamide}. At 11%, 50%, 70% stearic acid substitution, poly (ethylene oxide)block-poly [N(6hexylstearate)-L-aspartamide} forms self-assembled micelle that can be used to entrap amphotericin B by a solvent evaporation method (Fig. 5.4c).

5.3.2.4

Cosolvent evaporation method

Here drugs and polymers are dissolved in suitable water miscible and volatile organic solvent (cosolvent). The addition of an aqueous phase which is nonsolvent for the core-forming block followed by the evaporation of the organic cosolvent triggered the formation of micelle. Shuai et al. [13] developed micellar carrier of poly (epsilon-caprolactone) and monomethoxy polyethylene glycol. Nanoscopic micelles are formed by self-assembly of the amphiphilic block copolymer and doxorubicin was encapsulated in the hydrophobic core. Hemolytic studies demonstrated that free doxorubicin was responsible for 11% hemolysis at 200 mg/mL, where no hemolysis was observed with doxorubicin-loaded micelle at same concentration of the drug. In another study, fenofibrate was incorporated in poly(ethylene glycol)-block-poly (epsilon-caprolactone) micelle [14,15], and micelles were prepared by the removal of a negative ACN water azeotrope under reduced pressure (Fig. 5.4d).

5.3.2.5

Freeze-drying method

Drug and polymer were dissolved in a freeze dryable suitable organic solvent like tert butanol. Then this solution is mixed with water and freeze-dried. Freeze-dried product is then reconstituted with isotonic aqueous media. The feasibility of the method depends on the solubility of block copolymers and drug in tert butanol. Some block copolymers like polyethylene oxide are not soluble in tert butanol. Hence this method is not suitable for poly ethylene oxide containing block polymers (Fig. 5.4e).

5.3.3

Polyion complex micelle

In this technique, drug is incorporated through electrostatic interaction between oppositely charged polymer and drug. Neutralization of the charge present on the surface of the core segment of block polymer allows self-assembly of the complex and stabilizes the complex within the micellar core.

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5.4

Design and Applications of Theranostic Nanomedicines

Mechanism of drug release from polymeric micelles

The rate of release of drug from polymeric micelle depends on the chemical structure of the micelle, physicochemical properties of the drug, and localization of entrapped drug in the polymeric micelle. Sometimes the basic chemical structure of the micelle forming block polymer is tailored to modify the rate of release of drug to get an instant release or sustained release of the drug. Hydrophobicity and rigidity of polymeric micellar core can be enhanced to modulate the penetration of water and free ions to the core of micelle-forming drug conjugates and polyion complex micelles. Low penetration of water into the micelle leads to the delayed release of the drug. Release of drug from micelle forming block copolymer and drug conjugates follows two major pathways: 1) Micellar dissociation and drug cleavage from the polymeric unimers 2) Cleavage of drug within the micelle followed by diffusion out of micellar carrier

Drug is released from micelle forming block copolymer drug conjugates by micellar dissociation but diffusion is the major mechanism of release of drug from micellar nano containers. Release of drug from polyion complex micelles depends on drug exchange with free ions and proteins present in physiological media. Method of entrapment of drug in polymeric micelles can be modified to improve drug entrapment efficiency, localization, and rate of release of drug from polymeric micelle. Addition of hydrophilic or stimulus responsive groups with core structure can be used to get instant release or pulsed release of drug. In vitro release of drug from polymeric micelle can be measured using different methods: 1) Dialysis method: Polymeric micellar solution is kept in the dialysis bag. Then dialysis bag is immersed into a beaker containing release medium and temperature is kept constant. At definite time intervals some amount of medium is removed and replaced by fresh medium. Amount of drug present in the medium is measured spectrophotometrically or suitable method. 2) Gel permeation chromatography: In this method aliquots of micelles were applied onto a size exclusion high performance liquid chromatography column equipped with guard column [16,17]. Phosphate buffer saline was used as a mobile phase which was filtered and degassed prior to use. The eluent was detected at suitable wavelength.

5.5 5.5.1

Pharmaceutical applications of polymeric micelle Use of polymeric micelle as a solubilizing agent for waterinsoluble drugs

Surfactant molecules assemble to form a micellar structure with a hydrophobic inner core. This relatively hydrophobic, nonpolar core provides a more suitable environment

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Figure 5.5 Model depicting incorporation of drug in micelle by physical encapsulation or chemical encapsulation. Circle represents drug molecules. (a) Chemical encapsulation: involves chemical conjugation of drug to the hydrophobic block of the surfactant; (b) Physical encapsulation: Drug preferentially partition into the core of micelle without any chemical tethering.

for poorly soluble compounds than the aqueous phase. An increase in solubility of poorly soluble compounds by this technique is known as solubilization. The drug can be incorporated into the micellar structure by physical encapsulation and chemical encapsulation methods (Fig. 5.5). In the case of chemical encapsulation, the drug is chemically conjugated with the core-forming block of the surfactant. This method allows higher drug loading and reduces the chances of premature release of drug by minimizing the drug release from the core through diffusion [9,13]. In most of the cases drug is inactive when it is bound chemically to the surfactant. The drug is released by either hydrolysis or enzymatic cleavage of the bond that is present between surfactant and drug. In the case of physical encapsulation, no chemical bond is present between drug and polymer, drug selectively partitions into the inner core of the micelle, and the rate of encapsulation depends on the interaction, compatibility of coreforming block, and drug. Unlike chemical encapsulation, the physically entrapped drug is released from the micelle by diffusion or degradation of the micellar structure. The drug can be entrapped in the surfactant micelle by different methods like dialysis, solvent evaporation, cosolvent evaporation.

5.5.2

Passive targeting of drug-using polymer micelle

Drug targeting attributes to the delivery of drugs to a specific organ or tissue where the pharmacological activity of the drug is required. The drug can be targeted to a specific site by two methods: (1) active targeting and (2) passive targeting. Active targeting means delivery of drug to a particular target area by using biologically suitable

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interactions like antigen-antibody binding or the use of suitable ligands. On the other hand, in the case of passive targeting, nanocarriers containing drugs remain in blood circulation for sufficiently long time in order to accumulate in the tissue. The physical and chemical properties of the drug delivery system increase the target/nontarget ratio of the amount delivered. After intravenous administration, traditional nanocarriers are cleared rapidly from the circulation by mononuclear phagocyte system and localize in spleen, liver, bone-marrow. A different serum protein are adsorbed on the particle surface and promotes interaction of traditional nanocarriers with the membrane receptors of monocytes and macrophages. By modifying the surface properties of nanocarriers the inherent defense mechanism of body can be overcome and drug can be targeted to specific tissue. Apart from stealth nanocarriers, polymeric micelle emerges as a potential carrier for the delivery of anticancer drugs as most of the anticancer drugs are hydrophobic in nature and the micellar core arranges an excellent environment for the incorporation of anticancer drugs. Nanoscopic size range and stealth properties help the micelle to pile up in tumor tissue to a greater extent than free drug due to enhance permeation and retention effect [18]. Attachment of ligands on the outer surface of the micelle helps micelle to target the specific cancer tissue whereas nanosize range facilitates deep penetration of micelle to the tumor tissue. Polymeric micelle shows enhanced systemic circulation time because of their small size and high molecular weight prevents excretion through renal route whereas hydrophilic shell reduces uptake of micelle by the mononuclear phagocytic system (Fig. 5.6). The tumor vessels are less permselective and leakier than normal vessels that helps the perivascular accumulation of colloidal carriers [19]. Several in vivo studies illustrates that the efficiency of anticancer drugs can be increased by incorporating them in the polymeric micelle. A hydrophobic anticancer drug, Adriamycin was conjugated with poly(ethylene oxide)-poly(aspartic acid) block copolymer, and the length of each copolymer segment varies from 1000 to 12,000 in molecular weight. This type

Figure 5.6 Accumulation of polymeric micelles in tumors.

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of micellar structure with hydrophobic inner core and hydrophilic outer shell can be utilized for the effective targeting of the anticancer drug [20]. The outer shell of the micelle is accountable for the interactions with several blood components like cells and proteins that alter the biodistribution and pharmacokinetic behavior of drugs. Thus, outer shell controls the in vivo release of the drug without affecting the inner core that is responsible for pharmacological activity (Fig. 5.7). Wakabayashi et al. [21] prepared polyion complex micelle conjugated with lactose for incorporating plasmid DNA as a targetable gene vector system. The lactose conjugated polyion complex micelle revealed substantially higher transfection efficiency compared to polyion complex micelle without lactose conjugation against HepG2 cells possessing asialoglycoprotein receptors recognizing beta-D-galactose residue. Kwon et al. [14] performed a biodistribution study of poly (ethylene oxideaspartate) block copolymer Adriamycin conjugates in marine colon adenocarcinoma in 26 tumor-bearing mice following intravenous administration. Enhanced accumulation of micelle forming drug copolymer conjugates was observed after 24 h (10% dose per gram of tumor) relative to free adriamycin (0.90% dose per gram of tumor). Drug polymer conjugates remain in blood circulation for a prolonged period without decomposition and reduced uptake by major organs of reticuloendothelial systems were observed; hence, the drugs are more efficiently delivered to the tumor cells than free adriamycin. In another study by Yokoyama et al., Adriamycin was bound to poly(aspartic acid) chain of poly(ethylene glycol)poly (aspartic acid) block copolymer via formation of amide bond between carboxyl groups of poly(aspartic acid) and amino groups of adriamycin. In vivo study showed that anticancer activity of micelle forming polymeric drug against P388 mouse leukemia was performed and micelle showed low toxicity profile compared with free Adriamycin. In another study, Adriamycin was loaded into polymeric micelle prepared from poly (ethylene glycol)-poly (aspartic acid) block copolymer by physical entrapment and chemical conjugation. Physically entrapped Adriamycin played a crucial role in Figure 5.7 Concept of formation of micelle.

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the case of in vivo delivery of drugs to solid tumors. Micelle containing a high amount of chemically conjugated and physically entrapped Adriamycin showed very high antitumor activity in vivo against murine C26 tumor, but micelle containing only chemically conjugated Adriamycin expressed negligible antitumor activity [16]. Nakanishi et al. [22] developed a novel type of polymeric micelle (NK911), that entraps only original doxorubicin and preclinical studies were carried out. The prepared micelle circulates in blood circulation for a prolonged period because of evading the reticuloendothelial system. Uptake due to the presence of hydrophilic polyethyleneglycol outer layer, due to enhanced permeability and retention effect, a micelle is accumulated in tumor tissue, doxorubicin is released from the inner core of micelle by diffusion ad showed higher activity than free doxorubicin in all tumor lines. Dauson et al. [23] performed a pharmacokinetic study of SP1049C, a novel anticancer formulation containing pluronic bond doxorubicin in patients with advanced stage of cancer. The objective of the study was to determine the maximum tolerated dose, toxicity profile, limiting toxicity, and pharmacokinetic profile. Doxorubicin content of starting dose was 5 mg m 2 and it was given as an intravenous infusion once every 3 weeks for up to six cycles. 26 patients were administered 78 courses at seven dose levels. Antitumor activity of SP1049C was observed in several patients with acute cases of Ewing’s sarcoma, carcinosarcoma, esophageal adenocarcinoma. The maximum tolerated dose was 70 mg m 2 after getting promising results, the phase II stage was planned. Phase II clinical trial was performed by Valle et al. [24]. Nonhematological and hematological toxicity was found in some patients and in some cases, a significant fall in left ventricular ejection fraction was observed. Febrile neutropaenia, mucostitis, and atopecia were observed. SP1049C showed promising action against advanced esophageal adenocarcinoma. Paclitaxel is a widely used anticancer drug that is hydrophobic. Cremophor EL is a widely used vehicle for the formation of poorly water-soluble drugs. However, cremophor is not an inert vehicle and when it is used in paclitaxel formation, inevitably the large amount of cremophor is coadministered in the body after IV administration of paclitaxel formulation. This large amount of cremophor leads to a severe hypersensitivity reaction and neurological toxicity, aggregation of erythrocytes, hyperlipidemia, peripheral neuropathy, depending on the dose of formulation and duration of infusion. To avoid inherent problems associated with the use of cremophor, new formulations were developed where cremophor is absent. Polymeric micellar carriers have received tremendous attention that can reduce toxicity and increase the efficacy of the injectable paclitaxel. Polymeric micellar formulation replaced the use of toxic solubilizing agent cremophor EL, in the commercial formulation of paclitaxel. Different types of new cremophor free formulations were developed that were under pre (clinical) development like cosolvent system (ethanol/tween80/pluronic L64), emulsion (triacetin), water-soluble polymers (e.g., polyethylene glycols), and nanocapsules [25].

5.5.3

Active targeting of drugs using polymeric micelle

Enhanced permeability and retention effect is considered for passive targeting of drug through polymeric micelle, but active targeting of drug to a particular region is also

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possible where polymeric micelle is decorated with some ligands like sugar, antibodies, peptides, bioadhesive polymers [26]. Surface-modified micelle enhanced the specificity of the micelle for a particular target organ. In a study by Yamamoto et al., amino acid and dipeptide were successfully attached on the outer surface of polymeric micelle by Schiff base formation followed by reductive amination to show the functionalization of the micelle as a successful drug carrier. Micelle prepared with peptidyl ligands had the dual role of exploring the action of surface charge on the pharmacokinetic action of the formulation as well as the modulated release of the drug where cellular peptidyl receptors play a crucial role in specificity toward target cells. Thiols are mainly used in biomedical fields to increase mucoadhesive properties of drug delivery systems and the formation of disulfide bridges stabilize Nano assemblies. Terminal thiol groups are very effective to react quantitatively and selectively with maleimides or thiols under normal physiological conditions and facilitate the chemical functionalization of polymers. Dufresne et al. [27] prepared asymmetric poly (ethylene glycol)-block-poly(2-(N, N-dimethylamino)ethyl methacrylate) copolymer containing thiol groups at the end of the poly (ethylene glycol) chain. The biotinylated copolymers are self-assembled with an oligonucleotide in aqueous media and form polyion complex micelles where the outer surface contains biotin groups. Generation of intermicellar disulfide bonds under oxidative conditions tends to the formation of a stimuli-sensitive micellar structure. Another novel approach to increase the efficacy of Polymeric micelle involves the use of external stimuli like temperature, an ultrasound that triggers the release of drugs. Thermoresponsive systems can be utilized to enhance drug release or vascular transport by a local change of temperature. Temperature-sensitive polymeric micelle was prepared using block copolymers of (poly(N-isopropyl acrylamide-b-butyl methacrylate) [28]. Adriamycin was loaded into the inner micellar core and outer shell of poly Nisopropyl acrylamide to stabilize and initiate micellar thermo response. Hydrophilicity of the outer shell that prevents inner core interaction with other biocomponents and other micelles can be suddenly switched to hydrophobic one at a specific site by local temperature increase beyond lower critical solution temperature (32.5 C). Reversible structural changes of micelles allow drug release upon heating and cooling thermal fluctuation through lower critical solution temperature. The development of pHresponsive polymeric micelle is another approach to localize the delivery of drugs to tumor tissue. pH-sensitive micelles are designed that responds only to the localized change in pH in our body and drug is released in that specific regions of the body. The pH of the interior of endosomes and interstitial space of solid tumors is more acidic than the pH of blood plasma (pH 7.4). pH-responsive micelles are stable in normal blood plasma but cleavage of pH-sensitive bonds takes place [29] under mildly acidic conditions. Doxorubicin, a common anticancer drug possesses two groups, suitable for covalent attachment to a particular carrier, these groups are keto groups, and amino groups. The primary amino group was suitable for ionic bond mediated entrapment of doxorubicin into poly (ethylene oxide)-block-poly (methacrylic acid) micelle [30]

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or covalent attachment of doxorubicin take place between poly (ethylene oxide)-blockpoly (aspartic acid) due to the presence of amide bond. Keto group of doxorubicin was used for hydrazone bond mediated entrapment of doxorubicin into polyethylene oxide-block-(aspartic hydrazide -co- b-benzyl aspartate) micelles [31]. Hruby et al. developed pH-sensitive polymeric micelles by self-assembly of amphiphilic deblock copolymers in aqueous solutions. The copolymers consist of a hydrophilic block of polyethylene oxide and the hydrophobic block contains covalently bound doxorubicin and they are bound to the carrier by pH-responsive hydrazone bond. Hence pHsensitive micelles can be used to target drugs to tumors, endosomal compartments, inflamed tissues with a lower pH than normal tissue.

5.6

Conclusion

Polymeric micelle is considered as an ideal carrier for the delivery of poorly soluble drugs because of its inherent advantages like small particle size, high solubility, easy sterilization procedure, and sustained release of drug from the micelle. Size of the micelle varies from between 10 and 100 nm, i.e., an ideal size range for extravasation and permeation of micelle into the tissue but it was not taken by reticulo endothelial system. Polymeric micelle can be used to deliver anticancer drugs to tumor cells and targeting of drug can be done by attaching a ligand or antibody to the core forming blocks. More work should be done on the interaction of polymeric micelles with plasma and cellular components, pharmacokinetics, and biodistribution of polymeric micelle.

References [1] Moffitt M, Zhang L, Khougaz K, Eisenberg A. Micellization of ionic block copolymers in three dimensions. In: Solvents and self-organization of polymers. Dordrecht: Springer; 1996. p. 53e72. [2] Tuzar Z, Kratochvil P. Block and graft copolymer micelles in solution. Adv Colloid Interface Sci 1976;6(3):201e32. [3] Kwon GS, Okano T. Polymeric micelles as new drug carriers. Adv Drug Deliv Rev 1996; 21(2):107e16. [4] Yokoyama M. Clinical applications of polymeric micelle carrier systems in chemotherapy and image diagnosis of solid tumors. J Exp Clin Med 2011;3(4):151e8. [5] Kataoka K, Kwon GS, Yokoyama M, Okano T, Sakurai Y. Biodistribution of micelleforming polymeredrug conjugates. Pharmaceut Res 1993;10(7):970e4. [6] Harada A, Kataoka K. Formation of polyion complex micelles in an aqueous milieu from a pair of oppositely-charged block copolymers with poly (ethylene glycol) segments. Macromolecules 1995;28(15):5294e9. [7] Rapoport NY, Herron JN, Pitt WG, Pitina L. Micellar delivery of doxorubicin and its paramagnetic analog, ruboxyl, to HL-60 cells: effect of micelle structure and ultrasound on the intracellular drug uptake. J Contr Release 1999;58(2):153e62.

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[8] Batrakova EV, Li S, Miller DW, Kabanov AV. Pluronic P85 increases permeability of a broad spectrum of drugs in polarized BBMEC and Caco-2 cell monolayers. Pharmaceut Res 1999;16(9):1366e72. [9] Kwon GS, Naito M, Yokoyama M, Okano T, Sakurai Y, Kataoka K. Physical entrapment of adriamycin in AB block copolymer micelles. Pharmaceut Res 1995;12(2):192e5. [10] Kataoka K, Matsumoto T, Yokoyama M, Okano T, Sakurai Y, Fukushima S, Okamoto K, Kwon GS. Doxorubicin-loaded poly (ethylene glycol)epoly (b-benzyl-l-aspartate) copolymer micelles: their pharmaceutical characteristics and biological significance. J Contr Release 2000;64(1e3):143e53. [11] Kwon G, Naito M, Yokoyama M, Okano T, Sakurai Y, Kataoka K. Block copolymer micelles for drug delivery: loading and release of doxorubicin. J Contr Release 1997; 48(2e3):195e201. [12] Lavasanifar A, Samuel J, Kwon GS. The effect of fatty acid substitution on the in vitro release of amphotericin B from micelles composed of poly (ethylene oxide)-block-poly (Nhexyl stearate-L-aspartamide). J Contr Release 2002;79(1e3):165e72. [13] Shuai X, Ai H, Nasongkla N, Kim S, Gao J. Micellar carriers based on block copolymers of poly (ε-caprolactone) and poly (ethylene glycol) for doxorubicin delivery. J Contr Release 2004;98(3):415e22. [14] Kwon G, Suwa S, Yokoyama M, Okano T, Sakurai Y, Kataoka K. Enhanced tumor accumulation and prolonged circulation times of micelle-forming poly (ethylene oxideaspartate) block copolymer-adriamycin conjugates. J Contr Release 1994;29(1e2):17e23. [15] Jette KK, Law D, Schmitt EA, Kwon GS. Preparation and drug loading of poly(ethylene glycol)-block-poly(ε-caprolactone) micelles through the evaporation of a cosolvent azeotrope. Pharmaceut Res 2004;21:1184e91. [16] Yokoyama M, Fukushima S, Uehara R, Okamoto K, Kataoka K, Sakurai Y, Okano T. Characterization of physical entrapment and chemical conjugation of adriamycin in polymeric micelles and their design for in vivo delivery to a solid tumor. J Contr Release 1998;50(1e3):79e92. [17] Li Y, Kwon GS. Methotrexate esters of poly(ethylene oxide)-block-poly(2-hydroxyethylL-aspartamide). Part I: effects of the level of methotrexate conjugation on the stability of micelles and on drug release. Pharm Res 2000;17(5):607e11. [18] Matsumura Y, Maeda H. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res 1986;46(12 Part 1):6387e92. [19] Yuan F, Dellian M, Fukumura D, Leunig M, Berk DA, Torchilin VP, Jain RK. Vascular permeability in a human tumor xenograft: molecular size dependence and cutoff size. Cancer Res 1995;55(17):3752e6. [20] Yokoyama M, Kwon GS, Okano T, Sakurai Y, Seto T, Kataoka K. Preparation of micelleforming polymer-drug conjugates. Bioconjugate Chem 1992;3(4):295e301. [21] Wakebayashi D, Nishiyama N, Yamasaki Y, Itaka K, Kanayama N, Harada A, Nagasaki Y, Kataoka K. Lactose-conjugated polyion complex micelles incorporating plasmid DNA as a targetable gene vector system: their preparation and gene transfecting efficiency against cultured HepG2 cells. J Contr Release 2004;95(3):653e64. [22] Nakanishi T, Fukushima S, Okamoto K, Suzuki M, Matsumura Y, Yokoyama M, Okano T, Sakurai Y, Kataoka K. Development of the polymer micelle carrier system for doxorubicin. J Contr Release 2001;74(1e3):295e302. [23] Danson S, Ferry D, Alakhov V, Margison J, Kerr D, Jowle D, Brampton M, Halbert G, Ranson M. Phase I dose escalation and pharmacokinetic study of pluronic polymer-bound

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doxorubicin (SP1049C) in patients with advanced cancer. Br J Cancer 2004;90(11): 2085e91. Valle JW, Lawrance J, Brewer J, Clayton A, Corrie P, Alakhov V, Ranson M. A phase II, window study of SP1049C as first-line therapy in inoperable metastatic adenocarcinoma of the oesophagus. J Clin Oncol 2004;22(14_Suppl. l):4195. Gelderblom H, Verweij J, Nooter K, Sparreboom A, Cremophor EL. The drawbacks and advantages of vehicle selection for drug formulation. Eur J Cancer 2001;37(13):1590e8. Yamamoto Y, Nagasaki Y, Kato M, Kataoka K. Surface charge modulation of poly (ethylene glycol)epoly (d, l-lactide) block copolymer micelles: conjugation of charged peptides. Colloids Surf B Biointerfaces 1999;16(1e4):135e46. Dufresne MH, Gauthier MA, Leroux JC. Thiol-functionalized polymeric micelles: from molecular recognition to improved mucoadhesion. Bioconjugate Chem 2005;16(4): 1027e33. Chung JE, Yokoyama M, Yamato M, Aoyagi T, Sakurai Y, Okano T. Thermo-responsive drug delivery from polymeric micelles constructed using block copolymers of poly(Nisopropylacrylamide) and poly(butylmethacrylate). J Contr Release 1999;62(1e2): 115e27. Hruby M, Konak C, Ulbrich K. Polymeric micellar pH-sensitive drug delivery system for doxorubicin. J Contr Release 2005;103(1):137e48. Bronich TK, Nehls A, Eisenberg A, Kabanov VA, Kabanov AV. Novel drug delivery systems based on the complexes of block ionomers and surfactants of opposite charge. Colloids Surf B Biointerfaces 1999;16(1e4):243e51. Bae YS, Fukushima S, Harada A, Kataoka K. pH responsive drug-loaded polymeric micelles: intracellular drug release correlated with in vitro cytotoxicity on human small cell lung cancer SBC-3. In: Proceedings of the winter symposium and 11th international symposium on recent advances in drug delivery systems, Salt Lake City, Utah, U.S.A., March 3rde6th, 2003; 2003.

Dendrimers: an effective drug delivery and therapeutic approach

6

Bapi Gorain 1, 4 , Hira Choudhury 2 , Anroop B. Nair 3 and Bandar E. Al-Dhubiab 3 1 School of Pharmacy, Faculty of Health and Medical Science, Taylor’s University, Subang Jaya, Selangor, Malaysia; 2Department of Pharmaceutical Technology, School of Pharmacy, International Medical University, Kuala Lumpur, Malaysia; 3Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa, Saudi Arabia; 4 Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra, Ranchi, India

6.1

Introduction

Introduction of nanotechnology in the drug delivery has brought to us a potential platform to achieve enhanced efficacy of the therapeutic molecules with reduced toxicity through achieving more target oriented delivery of the delivery tools. Thus, the concept of “old wine in a new bottle” has brought to us a novel arena of research with lots of scope in effective drug delivery, where nanotechnology plays the superiority in achieving the goal when compared to the conventional therapies [1e3]. The safer and effective deliveries of nanotechnology-based products are known to overcome the limitations of the pharmacokinetic parameters of the conventionally available treatment strategies [4,5]. Several nano-meter-based delivery tools have been introduced in their research by a number of researchers worldwide, including polymeric nanoparticles, solid-lipid nanoparticles, liposome, micelle, quantum dots, nanoemulsion, niosomes, dendrimers, etc. Such deliveries are fabricated using specific techniques with biodegradable and biocompatible components with definite payloads to achieve sustained release of the entrapped therapeutics. In addition, passive and active targeting of the therapeutics within the biosystem allows target specific delivery of the therapeutics to exert safer deliveries [1,6,7]. Among the explored nanocarriers, dendrimer has gained emerging popularities because of the unique characteristics of the products, such as nanometer-based sizes of the particles with low polydispersity index, high density, low viscosity, ability to entrap hydrophobic components within the void spaces, possibility to adorn the surface groups with target specific ligands, fabrication in star like structures, etc. [8e10]. Promising outcomes of the recent dendrimer-based researches are providing the resources of novel investigations toward the fabrication of dendrimers for the improvement of solubility of poorly soluble agents, in the application of diagnosis of diseases as imaging agents (magnetic resonance imaging, X-ray, and radiotherapy), therapeutic improvement of several therapeutic agents in a wide

Design and Applications of Theranostic Nanomedicines. https://doi.org/10.1016/B978-0-323-89953-6.00002-7 Copyright © 2023 Elsevier Ltd. All rights reserved.

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variety of diseases and also in theranostic application, for diagnostic and therapeutic approach using the same tool [11e24]. The theoretical concept of the tree-like polymeric three-dimensional configuration was initialized by Flory in the year 1941 [24,25]. Later, the first synthesized tree-like branched structure was brought by Buhleier et al.; however, such preparation was of low generations [26]. Finally, in the late 1970s, the first full-embodied synthesis of dendrimer was successfully done by Donald A. Tomalia [27,28]. These dendritic structures are well-defined hyperbranched structures with tree-like appearance in their three-dimensional configuration. These structures are spherical in appearance within the nanometric size range ( 2-hydroxyethyl methacrylate > methyl methacrylate. During preparation of nanohydrogel, some portion of the polymers, solvents, surfactants, and initiators remained unreacted and that is responsible for some toxicity. Extensive purification and washing of the final product can minimize this problem. Suitable analytical technique can be

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used to detect the residual amount of monomer, solvent present in the prepared formulation. Use of natural polymers for the preparation of nanogels can minimize the toxicity problems related to nanohydrogel. Entirely aqueous based methods were used for the preparation of chitosan nanogels surface decorated with sodium alginate for the delivery of silver sulfadiazine in burn infections. Surface modification of positively charged chitosan nanoparticles with polyanions produces nanogels with enhanced stability and cytocompatibility [81]. Ouchida et al. [82] developed nasal vaccine delivery system based on cationic natural polymer pullulan for infectious respiratory diseases. Oral and nasal mucosal vaccines showed effective immunization methods that will produce antigen specific immune response at mucosal surfaces. Nasal vaccine will produce antigen specific immune responses in the systemic compartment as well as in the mucosal sites of the upper and lower respiratory tracts. These natural polymers are nontoxic, easily available, less expensive than synthetic counterparts. One of the additional advantages of natural polymer-based nanohydrogels is that they do not require organic solvents for their processing.

10.6

Conclusion

In recent years, smart nanocarriers bring the novel strategy to deliver the drug to the target tissue. These smart multi stimuli responsive nanocarriers respond to the internal stimuli like pH, redox as well as external stimuli like light, magnet, and temperature can deliver the active cargo at the target site with controllable kinetics. Redox sensitive nanogels deliver the chemo therapeutic drug to cancer cells due to existence of difference of redox potential between normal cells and cancer tissues and minimizing the adverse effect of anticancer drugs to normal tissues. Sometimes to increase the efficacy of nanohydrogel and to protect the nanocarrier from the degradation in biological tissue surface of nanocarriers are modified with hydrophilic polymers. Triggered release is observed as the result of the structural changes caused by the stimuli like eCOOH group, eNH2 group, coumarin group resulting in swelling and collapsing of nanogel. Stimuli-responsive nanocarriers showed widespread application not only for the targeted delivery of drugs but also for probing, sensing, , diagnosis, and it can be combined with antibodies for tumor immunothe`rapy.

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[4] Onaca O, Enea R, Hughes DW, Meier W. Stimuli-responsive polymersomes as nanocarriers for drug and gene delivery. Macromol. Biosci. 2009;9(2):129e39. [5] Bawa P, Pillay V, Choonara YE, Toit LCD. Stimuli-responsive polymers and their applications in drug delivery. Biomed. Mater. 2009;4:022001e15. [6] Oishi M, Sumitani S, Nagasaki Y. On off regulation of 19F magnetic resonance signals based on pH-sensitive PEGylated nanogels for potential tumor-specific smart 19F MRI probes. Bioconjugate Chem. 2007;18(5):1379e82. [7] Peng HS, Stolwijk JA, Sun LN, Wegener J, Wolfbeis OS. A nanogel for ratiometric fluorescent sensing of intracellular pH values. Angew. Chem. 2010;122(25):4342e5. [8] Hasegawa U, Shin-ichiro MN, Kaul SC, Hirano T, Akiyoshi K. Nanogel-quantum dot hybrid nanoparticles for live cell imaging. Biochem. Biophys. Res. Commun. 2005;331(4): 917e21. [9] Hayashi C, Hasegawa U, Saita Y, Hemmi H, Hayata T, Nakashima K, Ezura Y, Amagasa T, Akiyoshi K, Noda M. Osteoblastic bone formation is induced by using nanogel-crosslinking hydrogel as novel scaffold for bone growth factor. J. Cell Physiol. 2009;220(1):1e7. [10] Richtering W, Pich A. The special behaviours of responsive coreeshell nanogels. Soft Matter 2012;8(45):11423e30. [11] Wong JE, Richtering W. Layer-by-layer assembly on stimuli-responsive microgels. Curr. Opin. Colloid Interface Sci. 2008;13(6):403e12. [12] Schmid AJ, Dubbert J, Rudov AA, Pedersen JS, Lindner P, Karg M, Potemkin II, Richtering W. Multi-shell hollow nanogels with responsive shell permeability. Sci. Rep. 2016;6(1):1e3. [13] Li X, Yang B, Zhang S, Jia X, Hu Z. Facile synthesis of hairy microparticle-/nanoparticlesupported MacMillan and its application to DielseAlder reaction in water. Colloid Polym. Sci. 2017;295(4):573e82. [14] Gref R, Amiel C, Molinard K, Daoud-Mahammed S, Sébille B, Gillet B, Beloeil JC, Ringard C, Rosilio V, Poupaert J, Couvreur P. New self-assembled nanogels based on hosteguest interactions: characterization and drug loading. J. Contr. Release 2006;111(3): 316e24. [15] Sabir F, Asad MI, Qindeel M, Afzal I, Dar MJ, Shah KU, Zeb A, Khan GM, Ahmed N, Din FU. Polymeric nanogels as versatile nanoplatforms for biomedical applications. J. Nanomater. 2019;2019. [16] Akiyoshi K, Kang EC, Kurumada S, Sunamoto J, Principi T, Winnik FM. Controlled association of amphiphilic polymers in water: thermosensitive nanoparticles formed by self-assembly of hydrophobically modified pullulans and poly (N-isopropylacrylamides). Macromolecules 2000;33(9):3244e9. [17] Sim T, Lim C, Hoang NH, Oh KT. Recent advance of pH-sensitive nanocarriers targeting solid tumors. J. Pharm. Inv. 2017;47(5):383e94. [18] Zangabad PS, Mirkiani S, Shahsavari S, Masoudi B, Masroor M, Hamed H, Jafari Z, Taghipour YD, Hashemi H, Karimi M, Hamblin MR. Stimulus-responsive liposomes as smart nanoplatforms for drug delivery applications. Nanotechnol. Rev. 2018;7(1):95e122. [19] Duan C, Gao J, Zhang D, Jia L, Liu Y, Zheng D, Liu G, Tian X, Wang F, Zhang Q. Galactose-decorated pH-responsive nanogels for hepatoma-targeted delivery of oridonin. Biomacromolecules 2011;12(12):4335e43. [20] Madhusudana Rao K, Krishna Rao KS, Ramanjaneyulu G, Ha CS. Curcumin encapsulated pH sensitive gelatin based interpenetrating polymeric network nanogels for anti cancer drug delivery. Int. J. Pharm. 2015;478:788e95.

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Shilpi Rawat, Anjali Pal, Rashmi Choubey, Jaya Bajpai and A.K. Bajpai Bose Memorial Research Lab, Department of Chemistry, Government Autonomous Science College Jabalpur, Madhya Pradesh, India

11.1

Introduction

An interdisciplinary integration of core subjects like physical, chemical, biological, and engineering sciences has emerged as an independent avenue of nanotechnology that deals with challenging materials with dimension lying in the range from 1 to 100 nm with minimum 1-dimension [1e3]. Moreover, it also speaks to the extent to control, employ matter within level of individual molecules and atoms [4]. In this manner, nanotechnology’s suggestion for improving noninvasive delivery of drugs may assist in synthesizing unique, efficient, and better pharmaceutical formulations to improve the transport of bioactive pharmaceutical agents across the bloodebrain barrier (BBB) [5,6]. Several researchers have done investigation of nanostructures based drug release systems, inclusive of nanoparticles, carbon nanotubes, micelles, liposomes, and dendrimers, which have the tendency to provide the specified amount of sedate to the brain [7]. The brain is known to be the most vital organ of human body. Therefore, it is mandatory to adopt certain measures for its safety from the infection due to environmental and immigrant objects, which can result in adjustments in internal and external number of neuron cells and consequently lead to disabilities in nerve conduction and disorder inside the frame manipulate forms [8]. Brain is the most complicated and fragile part in the human body, and is cautiously protected from numerous toxic substances through barriers which block the access of drugs (e.g., antineoplastic agents) and central nervous system (CNS) active drugs into the brain, i.e., blood-cerebrospinal fluid barrier (BCFB) and the BBB. In addition, different efflux transporters proteins operating on the BBB keep away from the segment of medication into the CNS. In the recent studies by Tucker et al. [9], Gaillard et al. [10], Mandava et al. [11], Leonor Pinzon- Daza et al. [12], Fig. 11.1 depicts certain strategies for crossing of BBBs. However, numerous other genuine brain disorders don’t react to conventional therapeutic molecules, HIV infection of the brain Alzheimer’s disease, brain cancer, brain and spinal cord injury, stroke/neuroprotection, amyotrophic lateral sclerosis (ALS), Huntington’s disease various ataxia reducing disorder and in-born genetical errors in childhood affects the brain badly [8].

Design and Applications of Theranostic Nanomedicines. https://doi.org/10.1016/B978-0-323-89953-6.00015-5 Copyright © 2023 Elsevier Ltd. All rights reserved.

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Figure 11.1 Schematic representation of blood brain crossing by nanocarriers.

On the other hand, numerous approaches have been attaining in brain-targeted studies. Brain and CNS disruption stay the world’s driving source of mental and physical disability, demanding more hospitals visits and prolonged care in comparison to other diseases [13]. There are only a few drug delivery systems for the brain, which are highly safe and efficacious. If pharmacological techniques are enhanced, the invasive treatments will be less required. So, diverse ligands appended to BBB-centered nanocarriers (LABTNs) have emerged as the focal point of drug targeting to brain. The delivery of drugs into the entire BBB is the rate determining step in medical research pertaining to brain. There are three basic classes of endogenous transportation at BBB that are used for brain targeting viz., (1) active efflux transport, (2) receptormediated transport, and (3) carrier-mediated vehicle [14]. Several new methods involving nanocarriers such as carbon nanotubes, liposomes, dendrimers, micelles, and engineered nanoparticles utilizing covalent and noncovalent strategies to affix appropriate ligands are intensely researched to attain brain drug delivery. This chapter also presents in brief the structural and physiological descriptions of the BBB and discusses the widely used nanocarriers for drug delivery purposes. The intense efforts to transform preclinical to concrete clinical application is really a worth from the point of view of economic viability.

11.2

Bloodebrain barrier

The selectivity of BBB is very high and, moreover, the structural and biochemical barriers allow brain’s nutrition to pass through and protects against environmental and negative external factors. It coupled the description of the extrinsic environment with the indications of the intrinsic environment to perform certain task. With the entire specific tasks that take place at the neuronal level, it is paramount that the

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chemical environment in which these cells function is strictly regulated. The term BBB is acquainted with the unique characteristics of the microvascular system of the CNS. If one examines and looks at the CNS, it can be found that the CNS encompasses brain and spinal cord and two significant barriers to blood and brain which are built up of endothelial cells of the brain’s microvessels, and blood and cerebrospinal fluid barriers in the epithelial layer. The arachnoid matter includes the choroid plexus, brain’s ventricles, and the outer surface of the brain [15]. The notion of BBB was established almost a century ago. When the pigment is injected into the blood vessels of the animals, it, of course, affects all of the tissues, except the brain. When the cerebrospinal fluid was injected with a dye, it goes into stained brain and spinal cord, and not to other tissues. It was therefore suggested that something was blocking the colors entering the brain from the blood and was referred to as BBB. Fig. 11.2 shows the structure of BBB and its cellular components. It is therefore known that the BBB is comprised of endothelial cells layer that line inner surface of the capillaries in the brain.

11.2.1 What is BBB? The major characteristic of BBB is its distinctive structure, high efficiency, and by way of the interaction between the cellular and the cellular components. The primary role of the BBB is to provide excellent conditions for smooth operation of the neural network [16]. In addition, it increases the efficiency of nerve cells by allowing glucose transport [17]. This process permits for the movement of other molecules through the passive diffusion, and the selective and efficient transport of various bioactive species like organic and inorganic charged species, nutrients, and high molecular weight

Figure 11.2 Structure of BBB and its cellular components. Reproduced from Panche, A., Chandra, S., Sanjay Harke, D., Alzheimer’s and current therapeutics: A review, Asian J. Pharmaceut. Clinical Res., 8(3), 2015.

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compounds. The molecules that are soluble in lipids have the potential to freely diffuse through the capillary of the endothelial membrane, and it can passively cross the BBB, that may be said to be unreliable, especially for fat-soluble compounds with polar molecules, and ions of small size. Although the lipophilicity of itself is not determined by the permeability of membrane to molecules [18]. The BBB could be imagined as an impermeable membrane that selectively controls the transport of large and small molecules into the microenvironment of neurons. Obstacle cells actively transport metabolic products using certain transport proteins. This is achieved by several cellular transport channels that are spread along the membrane.

11.2.1.1 Cellular transport channels 1. 2. 3. 4. 5.

amino acid transporters glucose transporter nucleoside and nucleotide transporters monocarboxylate transporters ion transporters (Naþ/Kþ-ATPase pumps)

11.2.1.2 Essential features of the BBB A significant feature on which the BBB functions is its low and selective permeability to molecules which forms the basis of its characteristic biological function. The BBB limits the free exchange of material between the blood and the brain. It provides essential nutrients to the brain and the production of metabolites from the brain to the blood. 1. An important function of the BBB is to check the spread of solute and entrance of germs and hydrophilic molecules in the spinal fluid in the blood. On the other hand, the BBB mediates the passage of hydrophobic molecules such as oxygen, carbon dioxide, and hormones [19]. 2. The function of obstetric cells is to allow transport of metabolic products like glucose across the BBB with the help of some transport proteins. 3. The BBB also checks the passing of immune defenses, like marking of antibodies, immune cells, and molecules, within the CNS, which protects the brain from any damage caused by immune events. 4. It delivering essential nutrients to the brain and releasing metabolites from the brain to the bloodstream, removing toxins, or unwanted substances from the brain into the bloodstream to prevent brain function. 5. The regulation of endocrine function. However, in pathophysiological conditions, such as being hit tumor, infection, ischemia, or other factors, b-b-b is sensitive to damage, which leads to increased permeability and loss of barrier function. 6. It regulating hormonal functions. However, under pathophysiological conditions, such as tumor damage, inflammation, ischemia, or other factors, the BBB is at risk of injury, causing an increase in penetration and loss of “barrier” function. 7. It plays a pivotal role in managing homeostasis in the brain and implements controls that are protected from the entry and production of molecules [20].

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11.2.1.3 Cells of the BBB 11.2.1.3.1

Endothelial cells

Endothelial cells are the first barrier that makes up the BBB. Endothelial cells (ECs) shape the walls of blood vessels found in simple epithelial cells that are derived from mesodermally simple squamous epithelial cells. The strong interaction between cells tightens the gap between endothelial cells, and there is no pore structure on the surface of the cell membrane. Other tissues have unique properties compared to EC which permit them to rigidly control the transport of molecules, ions and cells, among the brain and the blood. The CNS EC is held in place by a solid junction (TJ), which places a significant limit on the flow of solvents. This strong paracellular and transcellular barrier forms a vascular cell, consisting of lumenal and abdominal components, such as that action between blood and brain are often tightly controlled by normal cellular transport. Endothelial cells not only maintain BBB integrity but also act as a barrier to toxins and pathogens [21].

11.2.1.3.2

Astrocytes

Astrocytes are also known as glial cells, which assist to carry and shield the neurons through the regulation of the neurotransmitter and an ion concentration to maintain a homeostatic balance in the central nervous system microenvelo, on the basis of the change in synaptic transmission, and regulation of the immune system and its interaction with endothelial cells, on the basis of their prediction end-feet on the basolateral side of the brain, the blood vessels [22]. Astrocytes form a bridge to neuronal signaling, and the vasculature of the central nervous system. Astrocytes are mixed up in the maintenance of the integrity of the BBB is mainly due to the release of active substances, in particular of the growth factor in this process. In addition, there is a complex relationship between astrocytes and blood vessels in the brain, which plays a decisive role in sustaining the BBB functions.

11.2.1.3.3

Pericytes

Pericytes are involved in the vascular smooth muscle cells, a major cellular components of the postcapillary venule, and the blood vessels. It is closely related to endothelial cells, N-cadherin, gap junction, and tight junction, outside of the blood vessels, as well as the shares in the same basement membrane of endothelial cells. Pericytes are involved in a number of important functions, while in a stroke, including the direction of blood flow and BBB permeability, in addition to the repair of the neurovascular unit [23].

11.2.1.3.4

Basement membrane

The basement membrane contains Type IV collagen, laminin, and fibronectin. The extracellular matrix as a key component of the basement membrane is composed of molecules, which are synthesized and secreted into the extracellular space. Fibronectin binds to the basement membrane into the surrounding tissue, and the extracellular matrix, thus suggesting a prime role to maintain BBB functions. The basement membrane may be influenced by matrix metalloproteinases (MMPs). The degradation of the

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basement membrane components results in enhancing the permeability of the BBB leading to swelling, bleeding, or even death [24].

11.2.1.3.5

Neurons

The nerve cells remain in the vicinity of blood vessels, to make a connection with the endocetic endfeet in the BBB. The nerve cells are rarely higher than in the 8e20 mm in the blood vessels of the brain, and it is estimated that each neuron has capillary. In the vicinity of the EC, this permits the neurons to respond to an alternating environment, and in particular, in relation to balance the ion. The nerve cells perform an important role in controlling the regulation of microvascular penetration, blood flow, interaction with the extrinsic source, and the liberation of substances promote angiogenesis. The neurons help to increase the endothelial cells of the brain, culture, and assist in the synthesis of proteins in synthesis and localization.

11.2.2

Physiological properties of BBB

The brain has broad network of blood vessels in the brain tissue which consist of arterial and venous. However, most actions take place at the level of capillaries. The central nervous system consists of neurons and glial cells in vertebrates. However, neurons are not regenerative cells; therefore, it is important to maintain homeostasis to protect neurons. The BBB is a barrier to solute exchange between the brain and blood to store this microenological substance, which was discovered and named by biologist Ehrlich and Goldman in the late 19th century. BBB is a series of physiological factors that may require to be activated (metabolic enzymes, transporters, TJs) or inhibited in CNS ECs. The CNS vasculature is the main gateway that controls access to blood-borne molecules in the brain, which is why it acts as a channel between the brain and external effects. This immune barrier, which allows only the transport of selected cells from all parenchyma of the brain and endothelial cells (ECs), is understood as BBB [25]. The neurovascular unit (NVU) helps in substrate formation of BBB; a cellular association formed by a close affiliation of the activity of ECs, astrocytes, pericytes, and sustained by other CNS cell types [26]. ECs in the CNS block cell transport by interlocking proteins forming hard pathways which are tight junction (TJs), allowing the transport of metabolites and nutrients only by compactly controlled passage and restricting entry of unwanted products by inhibited efflux carriers and transcytosis.

11.2.2.1 Regulation of the BBB formation and homeostasis BBB is a potent compound that converts molecules between blood and brain in response to homeostatic correction in disease and health. Throughout life, the strength of the BBB is in keeping with the ever-changing changes in various body regions. The physical variability of BBB resilience under healthy conditions, and therefore the mechanisms that control how BBB structures tend to revive brain homeostasis are poorly understood. In this regard, focuses on the present knowledge of BBB’s dynamic adaptation to respond to physical exchanges that include factors like growth,

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pregnancy, sleep, aging, environmental pressures and dietary changes are mandatory. Exploring these powerful BBB rules could open up new avenues to understand how systemic variables can change in dissimilar neuropathological regions, thus revealing current therapeutic approaches. By embryonic stages, the fetus is exposed to a host of harmful substances that circulate through the mother’s bloodstream through the placenta, which develops a barrier between the blood and the placenta [27]. However, the BBB is earlier made up of embryonic levels and offers additional safety to the developing CNS. It is a critical phase of time when the body, including the mind, changes into adulthood. In the course of puberty, follicle stimulating hormone and luteinizing hormone increase, thus causing sex steroids to be produced (testosterone and estradiol). Sex hormones trigger synaptogenesis and spinal repair regulating the growth of brain, resulting in sexual cohesion in other regions of the brain during adolescence. Steroid hormones can cross the BBB one by one, because of their small size and lipid concentration. High radiology (magnetic resonance imaging) has shown a progressive age increase in BBB stiffness. Similarly, BBB degeneration appears as the first biomarker of Alzheimer’s disease.

11.2.2.2 Regulation of barrier properties during angiogenesis Angiogenesis contains cell proliferation, vessel proliferation, anastomosis formation, pruning and reconstruction, the detection of endothelial quiescence Antigenic and differential function of endothelial progenitors determine the mechanisms of EPCsupported angiogenesis. The process of vascularization in the brain mainly occurs via angiogenesis and therefore the different cell communities involved in angiogenic events results in an increased growth of vessel, strengthening the cellular matrix, to establish microvessels and its maturity. barrier and access to delivery function. Angiogenesis constantly supports growing tissues; however, many blood vessels become quiescent during growth. Quiescent vessels regenerate only under certain conditions in adults, such as in the uterus and ovary, in the placenta in the course of pregnancy, and in skeletal muscle for supporting muscles growth caused by exercise. After injury, the angiogenesis is regenerated to promote the process of tissue repair by increasing the supply of vascular, but this kind of response could be harmful, for example, in the case of ocular pathologies like proliferative diabetic retinopathy or a “wet” type of age-related macular degeneration, in which tissue ischemia results in the formation of ectopic and leaky arteries. In addition, tumor growth can also be promoted by tumor angiogenesis, thereby augmenting the progression of cancer. In these disorders, neoangiogenesis often results in the formation of abnormal vessels with enlarged blood vessels. It is also known that the vascular stiffness is advantageous after severe tissue damage by the introduction of coagulation factors, antibodies, and cytokines, chronic exposure may result in pathological tissue edema. It is also well recognized that angiopoietin is a potent angiogenic factor during the fetal development of vessel. The deficiency of Ang-1 also results in the occurrence of embryonic nerve damage in the (CNS) as well as in other organs of the body due to improper interaction of the upper matrix of cells and supporting cells [28].

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11.2.2.3 Regulation of the BBB by pericytes Pericyte cells are mural cells they come in the walls of the arteries under the smooth vascular muscle cell series. In spite of the fact that these cells were discovered more than 100 years ago, pericytes rarely attracted attention since they are considered as endothelial cells which support endothelial cells. It has been recently established that pericytes provide physical support to endothelial cells as well as play critical roles in vessel function. In fact, pericytes form contacts that focus on endothelial cells at those cites which are often coined as nail contacts. In these contacts, the endothelial cells are connected via strong connections, gap and adhesions. Pericyte installation varies between numerous vessels. The ratio of pericyte to endothelial cells varies in the range from 1:100 in skeletal muscle to 1:1 in the retina. In general, vessels in the CNS show superior pericyte coverage, thus emphasizing the significance of pericytes in the formation and maintenance of CNS vasculature.

11.2.2.4 Regulation of the BBB by astrocytes Astrocytes are also produced by ACE-1 (angiotensin-converting enzyme-1), that alters angiotensin I to angiotensin II and activates angiotensin type 1 (At1) receptors that are shown by the BBB ECs. Angiotensin II strengthens vessels, and, in the CNS, activation of AT1 inhibits the entry of BBB and strengthens protein synthesis by their synthesis by their uptake into lipid rafts. Perivascular cells, including astrocyte, secrete Ang1 (angiopoietins), participate in the network process of BBB differentiation by activating angiogenesis, thus reducing depressive-induced endothelial proliferation. This occurs with high regulation of junctional protein expression. Unlike Ang-2, it is well known for being involved in the premature stages of the mechanism of eliminating BBB damage in injuries and diseases. Interestingly, when known BBB compromise factors such as vascular endothelial growth factor (VEGF) are linked to Ang1, barrier reliability is enhanced and security features are developed. Astrocytes also secrete angiogenic substances, thus promoting the growth of blood vessel. During development, VEGF is essential for the survival of embryonic blood vessels, formation, and regeneration.

11.2.3

Crossing the BBB

BBB offers numerous selective pathways for transferring nutrients to the brain. From structural point of view, the BBB is known to be the most selective barrier that keeps apart the circulating blood from the fluid at the exterior of the brain cells within the CNS. The BBB is made up of endothelial cells in the brain that are connected by strong sides. The BBB permits the permeation of water, other gases, and lipid-soluble molecules by inconsistent circulation, and the selective transport of glucose and amino acids, which are quite essential for neural function. In addition, it inhibits penetration of potential lipophilic neurotoxins in the form of an active P-glycoprotein. Active mobility conducts movement compared to the concentration gradient and demands ATP hydrolysis. The movement between cells is called cell proliferation. Paracellular transport is used to move material through the epithelium by the space between cells.

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11.2.3.1 Passive permeability For drugs to be freely filled with cerebral endothelium, an important requirement is the hydrophobicity of the molecule [29]. However, lipophilic molecules must have a molecular weight of less than 600 Da for penetrating into membrane [30]. In addition, speculation about energy penetration involves the molecule’s ability to combine with hydrogen ions. Thus, the replacement of hydrogen-binding groups by groups with no affinity of these ions increases the molecular lipophilicity. However, these substances can reduce the time it binds to plasma due to the rapid dissolution of highly lipophilic molecules and the slow melting of these substances into body fluids.

11.2.3.2 Carrier-mediated transport Carriers are membrane-bound that transport molecules which are much smaller than the endothelial cell, which are used to facilitate the transfer of nutrients such as hexose, nucleoside, purine base, amino acids to the brain. It is worth mentioning here that at least eight elements transport systems have been identified having similar group structures [31]. The mode of transport is the preferred substrate and the level of transport depends on the level of activity of the supervisor, and may be influenced by competing and noncompetitive inhibitors as shown in Fig. 11.3.

11.2.3.3 Active efflux transport Study of BBB detection methods can prove to be supporting for drugs targeting (e.g., Paclitaxel) in the brain and achieving the anticipated CNS drug effect or reducing BBB drug infiltration to reduce CNS side effects. Within the CNS, multiple efflux pathways influence drug concentration in the brain. Organic anion operators are also present in the BBB and prevent binding of certain drugs and molecules to the brain. These systems are known as efflux transporters and include P-glycoprotein (P-gp), which is a glycosylated cell-derived component that is superior to large ATP-binding cassette (ABC) carrier family. P-gp is often coined as multidrug resistance protein (MRP) Figure 11.3 Schematic representation of carriermediated transport.

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and takes active part in the removal of drugs from the brain parenchyma, such as chemotherapeutics, antibiotics, ion channel modulators, and immunosuppressants. Other MRPs are expressed in brain microvessels, including BCRP and anticancer members of the organic anion transporter polypeptide (OATP) family, which regulate the efflux of anionic chemicals. All of these carriers are able to work in tandem, reducing the intake of many drugs in the brain and increasing their efflux from the brain [32].

11.2.3.4 Receptor-mediated transport This is a process initiated by endocytosis of the ligand-receptor complex as shown in Fig. 11.4. Thereafter, it is involved in an endosomal chamber that can be transported to lysosomes or next to the cytoplasm for exocytosis. This type of transportation is power and temperature dependent. Receptors are able to transport large molecules such as proteins and small particles. To date, several receptors have been shown to live on BBB, including insulin, transferrin, growth factors such as insulin (IGF), leptin, and lipoprotein having low density [33].

11.2.3.5 Adsorption-mediated transport The surface of the plasma membrane of brain capillaries is poorly charged at body pH because of the presence of proteoglycans, mucopolysaccharides, and sulfate- and sialic

Figure 11.4 Schematic representation of receptor-mediated transport. Reproduced from Grant, B.D., Sato, M., Intracellular trafficking. In: Edited by James M. Kramer and Donald G. Moerman. Last revised March 10, 2005. Published January 21, 2006. WormBook, Ed. The C. elegans Research Community, WormBook, doi/10.1895/ wormbook.1.77.1, http://www.wormbook.org. Copyright: © 2006.

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acid containing glycoproteins and glycolipids. Adsorption occurs as a result of electrostatic interactions between a well-charged peptide amount and a poorly charged region in the plasma skin area. The type of transport is pure and undefined and occurs at a very low level under physical conditions. As a result of these structures, adsorption transport has been widely investigated for improving delivery of peptides and proteins to the brain [34]. Various bioactive molecules such as protamine, polylysine, glylysine, glycated albin, histone, and avidin enter the BBB through the process of adsorption [35].

11.3

Ligand appended nanocarriers

Active targeting process of drug delivery encompasses assimilation of targeting modules, e.g., on the surfaces of nanocarriers are present ligands or antibodies which are specific for certain cell types in the body. To facilitate active targeting, a huge quantum of biological ligands have been reported and studied. Such kind of biological ligands most commonly attach to some definite receptors present on the surface of the target cells, thus increasing the cellular uptake of drug loaded NPs which result in therapeutic effectiveness [36].The nanocarriers offer high surface to volume ratio that allow multiple ligands to find specific sites on the surfaces [37]. Therefore, the various types of ligands include polysaccharides, proteins, peptides, nucleic acids, and low molecular weight molecules appended to BBB-targeted nanocarriers (LABTNs) are now the center of delivering drug to the brain. Moreover, on the nanocarriers surface the multiple ligand addition can be considered for a multivalent binding to target cells. In addition, increasing affinity and the molecular signals may be triggered by the cellular receptors accumulation. Such ligand-loaded nanocarriers are advantageous for treating small metastases and circulating cancer cells that are not easy to be treated by traditional drugs and nanocarriers. On the other hand, ligand-loaded nanocarriers could be utilized to overcome vascular barriers that prevent accumulation and extravasation in contrarily impermeable tissues, e.g., the brain [38,39]. Various techniques were developed for enhancing drug delivery to the brain: (1) chemical agent mediated delivery systems, e.g., lipid-mediated transport; (2) biological delivery systems, include refurbishment of pharmaceuticals to cross the BBB through specified endogenous transporters present inside the capillary endothelium of the brain; (3) interference of the BBB, such as by adjustment of tight junctions; (4) the utilization of molecular Trojan horses, for example, peptidomimetic monoclonal antibodies for large molecule transports (e.g., RNA interference drugs, recombinant proteins, antibodies) through the BBB; and (5) particulate systems of drug-carrier [40].

11.3.1 Types of nanocarriers Active-targeting ligands used in drug delivery comprises folates, antibodies, peptides aptamers, and transferring as shown in Fig. 11.5.

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Figure 11.5 Depiction of active targeting of drug carrying nanoparticles by biological ligands. Reproduced from Yoo, J., Park, C., Yi, G., Lee, D., Koo, H. Active Targeting Strategies Using Biological Ligands for Nanoparticle Drug Delivery Systems.

11.3.1.1 Folate Folate-binding proteins have high affinity membrane also known as folate receptor (FR) developed by Gu et al. [41]. FR is most familiar to be overexposed in macrophages and solid cells of tumor, which develops them as captivating targets for various nanoparticles through receptor-mediated endocytosis [42,43]. Nanoparticle attached to an FR was found by Yoo and his colleagues. The copolymers of poly (D, L-lactic-coglycolic acid) (PLGA) and polyethylene glycol (PEG) were used by them to form micelles. Biodegradation, after carrier drug delivery, PLGA facilitate PEG, which leads to increment in the particles retention time. The studies conducted by Yi showed the effectiveness of FR as a target for the ailment of inflammation related diseases [44]. As the macrophages show great number of folate receptors, they contribute importantly to inflammatory diseases. These FRpositive activated macrophages offer selective targeting and reveal as a systematic route for the diagnosis and treatment of inflammatory diseases. Qi et al. and Yang et al. attempted similar methods wherein active folate receptor targeting in macrophages was applied to confirm drug delivery at specific inflammation site [45,46].

11.3.1.2 Transferrin Transferrin (Tf) is a type of glycoprotein which binds iron and helps in its transport inside the body [47]. These receptors are greatly formulate in specific tissues and cells

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and can be targeted by Tf-modified nanoparticles. The studies by Sahoo and colleagues suggested for materials that initiate the process of drug delivery with the help of copolymerized poly (vinyl alcohol) (PVA) and PLGA. Sahoo and Labhasetwar demonstrated that the transfer of conjugated nanoparticles suppresses cell proliferation and growth of the tumor and at the same time increase cellular uptake [48]. The potential of these conjugated nanoparticles is particularly based on their absorption capacity mostly by receptor-mediated based endocytosis [49].

11.3.1.3 Aptamers Aptamers are a type of small nucleic acid (DNA or RNA) that consists of various nucleotides. The aptamers are biodegradable, smaller in size, highly sensitive, and having immunogenicity, which makes them good nanocarriers for active ligands targeting [50]. Duo et al. utilized aptamer of AS-1411 type for targeting mesoporous nanoparticles of silica carrying CX-5461 to the nucleus of cancerous cells [51]. The aptamer AS-1411 G-rich DNA particularly observe nucleolin, which is a type of protein that is uncontrolled in several cancerous cell lines.

11.3.1.4 Antibodies Antibodies are the biological ligands, having the longest history of targeting specific receptors. The antibodies possess a size of nearly tens of kilodaltons, and moreover, they are highly specific and compatibility that they bind with larger ligands with greater ease and specificity [52]. In general, most of the antibodies are used in targeting as well as for therapeutic purposes. However, the antibodies larger in size restricted their accumulation and consequently density on the nanoparticles surfaces during moderation. Roncato et al. mentioned application of antiepidermal growth factor receptor (EGFR) antibody (cetuximab) in antibody-guided avidin-nucleic-acid nanoassemblies (ANANAS) in cancer [53]. Active targeting of different types of tumors depend on the antibodies characteristics. This specialty mainly lies because of their tendency to differentiate healthy and unhealthy cells in almost all kind of cancers but specifically in the case of colorectal cancer [54].

11.3.1.5 Peptides Peptides are one the targeting ligand which have various advantage among all such as good stability, low production costs, and because of their small size easily attached nanoparticles surfaces with greater density [55]. Steichen et al. suggested that the peptides can also be employed for targeting those drugs that are used in the treatment of cancers [56]. Similar to antibodies, peptides have capabilities to disrupt interaction of tumor cells that takes place on it and terminate cellular proliferation. The phage library of combinatorial type has been widely utilized to secure ligands of protein [56]. The ligands formed by this method having the length of 10e15 amino acids and able to identify and bind to high affinity tumors [41] (Table 11.1).

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Table 11.1 Various ligands used for active targeting of nanoparticles drug delivery systems [57]. Example of ligands

Type

Peptides

Transferrin, antibodies RGD, IL4RPep-1

Aptamers

GBI-10, AS-1411

Polysaccharides

Hyaluronic acid

Small molecules

Folate, phenylboronic acid, anisamide

Proteins

Advantage or disadvantage Highly specific, low stability, large size Easily fabricated, small size and divisible by peptidase Highly specific, small size, high cost and cleavable by nuclease Utilized as an overexpressed receptors in liver tissue and polymer backbone of nanoparticles Smaller in size and low cost

Reproduced from Yoo, J., Park, C., Yi, G., Lee, D., Koo, H. Active Targeting Strategies Using Biological Ligands for Nanoparticle Drug Delivery Systems.

11.3.2

Preparation methods

In the drug delivery process to brain, the drug across the BBB in the brain parenchyma is one of the biggest challenges. LABTNs are specified tools to facilitate the process; the small size enables BBB penetration, proteins binding and its stabilization, and lysosomal cells to escape after endocytosis [29]. The functionalization of surface of nanocarriers (NCs) is the most important step toward nanoscale drug delivery systems. Conjugation of ligands that target the BBB on the colloidal carrier surface, either by covalent or noncovalent linkage, is known to enhance the selectivity level for brain tumors, and the progress for the anticancer drug delivery across the BBB for the therapy of brain tumors is to depend on the advancement of targeted transport-increasing nanocarriers. Drug release by the carriers should follow transvascular transport and extravasation, and the dosages of therapeutic drug must be achieved. There are two approaches that are currently applied for synthesizing NCs and to prepare them to target the BBB: one is covalent and the second is noncovalent coupling, respectively.

11.3.2.1 Covalent coupling The coupling process is most commonly applied to couple covalent proteins to NCs, thus including MAb and peptides as represented in Fig. 11.6. In order to enable conjugation, PEGylated liposomes were functionalized with the help of bifunctional PEG which at one end consists of a hydrophobic lipid anchor and a cross-linking agent at another end. The cross-linking agent might be amine reactive (e.g., derivatives of N-hydroxysuccinimide group (NHS)) or it could be thiol reactive (e.g., derivatives of maleimide and pyridyl dithio-propionylamine). The phospholipids were most commonly used as lipid part for e.g., dipalmitoylphosphatidylethanolamine (DPPE) or DSPE. Bifunctional PEG was imported into preformed liposomes or lipid

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Figure 11.6 Represent the process of covalent-coupling.

nanocarriers (LNCs) using either a preinsertion method or a postinsertion method. To functionalize the nanoparticles surface, sulfhydryl and terminal amine reactive groups were attached to copolymers [58]. The subsequently functionalized nanoparticles by their primary amine or sulfhydryl groups they are covalently coupled to proteins. Amine groups are found on the amino acids side chain such as residues of arginine, lysine, and glutamine and at amine terminal. Sulfhydryls are present in residues of cysteine or in the Fab fragments of hinge region, or can be inserted into the structure of protein in various ways through the carbohydrates or the primary amine groups. Other techniques have been prepared for the association of site-directing biomolecules to the surface of NC.

11.3.2.2 Noncovalent coupling There are mainly two techniques have been produced for noncovalent conjugation of ligands to NCs that are avidin/biotin strategy. In the first technique, into the structure of ligand the Straptavidin (SA) or avidins are inserted. With the help of N-maleimidobenzoyl-N-hydroxysuccinimide the avidin or SA become active and then react with the sulfhydryl group which leads to the development of a thio-ether bond. With the help of electrostatic interactions SA has been linked with cationic SLN. Ligands attached to SA/avidin were directly coupled to biotinylated NCs. The second method is related with biotinylation of ligand. Most commonly NHSPEG-biotin is utilized to inherit molecules of biotin on protein-carrying primary amine groups. Then avidin or SA incubated biotinylated NCs for the coupling was finally attached to biotinylated ligands [59]. The modification of the surface of nanoparticles in which the drug is encapsulated, mixed, entrapped, or covalently attached is possible. Through the linkers, drugs were linked to their vector carriers. The formation of chemical bonds attached the drugs to its nanocarriers is also a possible interest for the particular acquaintance of the treating agents. Hence, the prerequisite methods of synthesis and the fabrication of appropriate and biocompatible linkers for the conjugation must be developed.

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Physicochemical properties

Designing highly efficient NCs that fulfill all the conditions to design a drug delivery process is a complicated method. The key requirement for the precise development of these nanomaterials to serve as a platform for sustained-release drug-delivery depending on their shape, size, biocompatibility, constitution, and surface charge. The physico-chemical properties of nanomaterials affect the binding, accumulation, and interaction of cells, resulting in healing or toxic effects [60,61]. It is therefore of fundamental importance to develop the nanomaterials in such a way that they maximize their usefulness for biomedical applications.

11.3.3.1 Size and shape of the nanomaterials The nanomaterials size and shape determine the range of their accumulation of tumor and in vivo distribution. The size of the nanomaterials will also affect the cellular uptake of drugs and the interaction with some of the tissues for medicinal purposes. In addition, the nanomaterials size and shape influence drug loading and release, as well as stability.

11.3.3.2 Surface charge of nanoparticles The surface charge of nanomaterials determines its biomedical effectiveness and appropriateness. The surface charges that are present on the nanoparticles are important factors to head-on the interactions of nano-bio interface. In addition, the nanoparticles in vivo biodistribution indicates that at the tumor sites the particles that are negatively charged are assembled more precisely [62]. Generally, positively charged nanomaterials can efficiently internalize on cell membranes, because of the negative charge present on the cell surface. It is also evident that the distribution of nanomaterials in the cell is strongly determined by the surface charge they consist, which must be designed to ignore unwanted uptake from the normal cells in order to achieve an effect specific to the target without any negative effects on normal cells.

11.3.3.3 Surface chemistry of nanoparticles Surface chemistry of nanomaterials is essentially required to design for its applications in biomedical field; it also helps in decreasing the toxicity and enhancing the stability of nanomaterials.

11.4

Applications of ligand appended nanocarriers

It is well recognized that the shells of NP ligand plays a key role in designing nanotherapeutic probes controlling their toxic effects, pharmacokinetics, and efficacy. A significant aspect is that the ligands must coat the nanoparticles surfaces to enhance

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colloidal stabilization as well as their sealing from specific molecules which are present in the critical biological environments. Furthermore, the intended ligands must add potential to targeting ability of nanoparticles to enable them to perform their biological roles. In the first instance, one has to be precise over minimizing the aggregation of nanoparticles for in vivo applications which may be achieved by judicious selection of the ligands. It is known that the size of NPs greatly determine their in vivo density. For instance, if NPs are intended for targeting the tumors, their size must be tuned in such a way that they can easily penetrate the tumors. It is, therefore, clear that unwanted aggregation of NPs will result in hampering of tumor targeting. Interestingly, whereas very small-sized NPs are likely to undergo slight leakage into blood stream, the aggregated NPs may pose physiological problems in their clearance without targeting the diseased sites. It is, therefore, extremely important to allow proper coating of NPs surface with pertinent ligands for their better applications in biomedical areas. At the same time, however, it is important that the ligands must be biodegradable, biocompatible, and have excellent excretion for biomedical applications. It is likewise really worth citing that for the applications in the biomedical field, selection of the ligand must also consider the biodegradability/excretion and biocompatibility of the ligands as general requirement for medicinal use. Like NPs, nanocomposites are taken unexpectedly via means of the (RES) and are especially disbursed into an amount of (60%e90%) in liver and (2%e10%) in spleen and to a small portion in the bone marrow. From the bone marrow, the phagocytic cells are derived constituted by RES. Although it is useful for targeting the brain, most effective a restricted amount of NCs attain the brain due to their immoderate uptake via way means of RES after IV injection [63]. A little bit of surface improvement of nanocomposites might be applied to extend stay time in blood, to lessen nonspecific arrangement and in LABTNs case, to target tissue or specific cell-surface antigens through a targeting ligand (e.g., molecules of aptamer, antibody fragments, peptide). The first procedure after IV administration results in the NCs uptake through the MPS is the appearance of opsonization. Opsonin especially encompass supplement proteins, apolipoproteins, immunoglobulins (Igs), and fibroprotein interaction with unique monocytes and tissue macrophages receptor membrane ensuing in identification and phagocytosis. The hydrophobic surfaces have been proven to encourage proteins absorption and negative surfaces seemed to be supplement system activator synthesizing NCs with its main focus on ligands may be favorable to the improvement of goal-directed carrier systems. The NCs have an advantage of getting entry in the brain through variety of aforementioned mechanism controlled by the ligands nature. NCs enhanced with surface functionalized material can easily assist the drug delivery to the brain via a unique phenomenon [64]. The design of LABTNs as multifunctional structures is major task and few specimens of their synthesis and application have been studied by researchers Bedureau and Juillerat-Jeanneret [65,66]. There are certain examples of LABTNs which are capable to ferry sedate across the BBB to CNs tumors are reported and a number of vital traits of such equipment had been defined via chemical derivization and encapsulation into polymeric particles has been evaluated as opportunity reinforcing the

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selectivity of drug [67]. LABTNs are usually imputed into cells through fluid e segment endocytosis, phagocytosis, or receptor-mediated endocytosis. The modification on the surface of NC can be executed to boom cellular uptake and delivery tendency of the bioactivities in various compartments of cell. For BBB transporters, NCs have been adorned with ligands [68]. A team of Lai et al. have studied various techniques to deliver therapies to the brain through liposomes. The notable progression of liposomal delivery system of brain coupled with selected ligands along with good specificity and less immunogenicity to help in the regulation of liposomes design through appropriate choice of the unique homing tool and delivery mechanism [69].

11.5

Underlying challenges and future prospects

Rapid advancement in BBB research has resulted in a better understanding of BBB morphology and physiology. However, some fundamental queries relevant to the standard human progress of the BBB remain unanswered. A significant challenge is drug targeting and its delivery, as it gets restricted by the BBB, which protects the brain against many foreign substances. For advanced and effectual therapy of neurodegenerative disease, stroke, and brain cancer, which are common diseases, interestingly, new approaches for the amplified transit of the BBB need to evolve. Nanotechnology-based strategies are currently studied, including micelles, liposomes, nanoparticles, dendrimers, and carbon nanotubes as nanocarriers control the BBB and transport the drug of required quantity to the targeted site. Moreover the study is required to acknowledge and convey the mechanism of intersection of BBB and build the ability of techniques of brain delivery through nanotechnology. Despite tremendous improvement in drug delivery to the brain, several loopholes are being explored to treat neurological diseases and brain tumors. With the rapid increment in the aging population and the overwhelming number. of patients with neurological disorders worldwide, more targeted NCs are needed for safe and effective administration through the BBB. This chapter is a sum up of a nanoparticulate process with significant potential as a drug carrier. These processes can convert less soluble, imperfectly absorbed, and labile biologically active agents into promising available drugs. New possibilities have been predicted by nanoparticulate drug delivery systems with the treatment of acute and chronic brain disease. Generally, drug transport to the brain/CNs has been significantly improved recently via the rational layout of nanoparticulate drug delivery system based primarily on polymers referred to as LABTNs. As already mentioned, the limiting factor in treating brain diseases is the release of bioactive substances to the brain by BBB. A luminal bloode brain carrier system acknowledges drug carrier or drug together with a molecule is imperative. Several methods have assessed drug colligation to blood to brain transporters, which need the drug to impersonate the endogenous ligand as several transporters (e.g., glucose transporters) are incredibly selective. The chemical entity that helps move the bioactive agents across the BBB is representative for transporting drugs to the brain. In a futuristic way, researchers are trying to explain the restricted

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access of bioactive ingredients to the brain chemical moderation of the biologically active substances (i.e., the prodrug method) through lipid-mediated transport or the use of blocking systems. In the coming years, more knowledge about the mechanism of the BBB disorder could assist formulated schemes for BBB protection and prevention and for treating BBB-related pathologies.

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[39] Xiong K, Wei W, Jin Y, Wang S, Zhao D, Wang S, Gao X, Qiao C, Yue H, Ma G, Xie HY. Biomimetic immuno-magnetosomes for high-performance enrichment of circulating tumor cells. Adv Mater 2016;28:7929e35. [40] Patel MM, Goyal BR, Bhadada SV, Bhatt JS, Amin AF. Getting into the brain: approaches to enhance brain drug delivery. CNS Drugs 2009;23(1):35e58. [41] Gu FX, Karnik R, Wang AZ, Alexis F, Levy-Nissenbaum E, Hong S, et al. Targeted nanoparticles for cancer therapy. Nano Today 2007;2:14e21. [42] Nogueira E, Gomes AC, Preto A, Cavaco-Paulo A. Folate-targeted nanoparticles for rheumatoid arthritis therapy. Nanomed Nanotechnol Biol Med 2016;12:1113e26. [43] Son J, Yang SM, Yi G, Roh YJ, Park H, Park JM, Choi M-G, Koo H. Folate-modified PLGA nanoparticles for tumor-targeted delivery of pheophorbide a in vivo. Biochem Biophys Res Commun 2018;498:523e8. [44] Yi Y-S. Folate receptor-targeted diagnostics and therapeutics for inflammatory diseases. Immune Network 2016;16(6):337. [45] Qi R, Majoros I, Misra AC, Koch AE, Campbell P, Marotte H, et al. Folate receptortargeted dendrimer-methotrexate conjugate for inflammatory arthritis. J Biomed Nanotechnol 2015;11(8):1431e41. [46] Yang M, Ding J, Zhang Y, Chang F, Wang J, Gao Z, et al. Activated macrophage-targeted dextranemethotrexate/folate conjugate prevents deterioration of collagen-induced arthritis in mice. J Mater Chem B 2016;4(12):2102e13. [47] Schieber C, Bestetti A, Lim JP, Ryan AD, Nguyen T-L, Eldridge R, White AR, Gleeson PA, Donnelly PS, Williams SJ, Mulvaney P. Conjugation of transferrin to azidemodified CdSe/ZnS coreeshell quantum dots using cyclooctyne click chemistry. Angew Chem Int Ed 2012;51:10523e7. [48] Sahoo SK, Labhasetwar V. Enhanced antiproliferative activity of transferrin conjugated paclitaxel-loaded nanoparticles is mediated via sustained intracellular drug retention. Mol Pharm 2005;2:373e83. [49] Sahoo SK, Ma W, Labhasetwar V. Efficacy of transferring conjugated paclitaxel-loaded nanoparticles in a murine model of prostate cancer. Int J Cancer 2004;112:335e40. [50] Jo H, Ban C. Aptamerenanoparticle complexes as powerful diagnostic and therapeutic tools. Exp Mol Med 2016;48. [51] Duo Y, Yang M, Du Z, Feng C, Xing C, Wu Y, Xie Z, Zhang F, Huang L, Zeng X, Chen H. CX-5461-loaded nucleolus-targeting nanoplatform for cancer therapy through induction of pro-death autophagy. Acta Biomater 2018;79:317e30. [52] Kim KS, Kim J, Kim DH, Hwang HS, Na K. Multifunctional trastuzumab-chlorin e6 conjugate for the treatment of HER2-positive human breast cancer. Biomater. Sci. 2018;6: 1217e26. [53] Roncato F, Rruga F, Porcu E, Casarin E, Ronca R, Maccarinelli F, Realdon N, Basso G, Alon R, Viola G, Morpurgo M. Improvement and extension of anti-EGFR targeting in breast cancer therapy by integration with the Avidin-Nucleic-Acid-Nano-Assemblies. Nat Commun 2018;9. [54] Dhar M, Sepkovic DW, Hirani V, Magnusson RP, Lasker JM. Omega oxidation of 3hydroxy fatty acids by the human CYP4F gene subfamily enzyme CYP4F11. J Lipid Res 2008;49:612e24. [55] Chen K, Conti PS. Target-specific delivery of peptide-based probes for PET imaging. Adv Drug Deliv Rev 2010;62:1005e22. [56] Steichen SD, Caldorera-Moore M, Peppas NA. A review of current nanoparticle and targeting moieties for the delivery of cancer therapeutics. Eur. J. pharm Sci. 2013;48:416e27.

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[57] Yoo J, Park C, Yi G, Lee D, Koo H. Active targeting strategies using biological ligands for nanoparticle drug delivery systems. Cancers 2019;11:640. [58] Tessmar J, Mikos A, Gopferich A. Amine-reactive biodegradable diblock copolymers. Biomacromolecules 2002;3:194e200. [59] Pedersen N, Hansen S, Heydenreich AV, Kristensen HG, Poulsen HS. Solid lipid nanoparticles can effectively bind DNA, streptavidin and biotinylated ligands. Eur J Pharm Biopharm 2006;62:155e62. [60] Navya PN, Daima HK. Rational engineering of physicochemical properties of nanomaterials for biomedical applications with nanotoxicological perspectives. Nano Converg 2016;3(1):1. [61] Kaphle A, Nagraju NP, Daima HK. Contemporary developments in nanobiotechnology: applications, toxicity, sustainability and future perspective. In: Dhawan A, editor. Nanobiotechnology: human health and the environment. Boca Raton, FL: CRC Press; 2018. p. 1e34. [62] He C, et al. Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials 2010;31(13):3657e66. [63] Chavanpatil MD, Khdair A, Panyam J. Nanoparticles for cellular drug delivery: mechanisms and factors influencing delivery. J Nanosci Nanotechnol 2006;6:9e10. [64] Patel P, Acharya S, Acharya N. Potential of surface functionalized nanoparticles for improved therapy of refractory central nervous system disorders. J Pharm Res 2011;4(4): 1093e9. [65] Beduneau A, Saulnier P, Benoit JP. Active targeting of brain tumors using nanocarriers. Biomaterials 2007;28(33):4947e67. [66] Juillerat-Jeanneret L. Critical analysis of cancer therapy using nanomaterials. In: Kumar Challa SSR, editor. Nanomaterials for cancer therapy and diagnosis. Hoboken, NJ: WileyVCH; 2006. p. 199e232. [67] Rabinow BE. Nanosuspensions in drug delivery. Nat Rev Drug Discov 2005;3:785e96. [68] Juillerat-Jeanneret L. The targeted delivery of cancer drugs across the bloodebrain barrier: chemical modifications of drugs or drug-nanoparticles? Drug Discov Today 2008;13: 1099e106. [69] Lai F, Fadda AM, Sinico C. Liposomes for brain delivery. Expet Opin Drug Deliv 2013; 10(7):1003e22.

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Suman Mallik 1 and Shalmoli Seth 2 1 Narayana Super Speciality Hospital, Kolkata, West Bengal, India; 2Gupta College of Technological Sciences, Asansol, West Bengal, India

12.1

Introduction

Since the past few decades, advances in science and technology are being played a significant role in the diagnosis and treatment of cancer, yet there is high chances of mortality worldwide [1]. Cancer is one of the third largest causes of mortality worldwide and brain cancers is regarded as most dangerous and obstructive type of central nervous system (CNS) disease [2]. Brain cancer comprising both malignant and nonmalignant are most common among the age group of 0e19 years and average rate of sufferers of 5.57 per 1,000,000 population [3,4]. Above all, it has been observed that the rate of survival of patients with CNS cancer is only 33.3% [5]. Brain cancers are widely divided into two types: primary and metastatic neoplasm [6]. Primary neoplasm is mainly called “glioma” as it originates from “glia” or its respective forerunner [7]. These malignant gliomas are heterogeneous in nature and have high chances of recurrence within few months after surgery and chemotherapy [8]. Due to aforementioned facts, the personalized medicines having theranostic approach are gaining attention in the recent times [9]. Theranostic nanomedicines allow an effective detection and target specific treatment of the malignant brain tumors. These are more efficacious than current brain cancer treatment strategies as they provide site specific drug delivery, high blood brain barrier (BBB) penetration, greater bio distribution of drugs, effective action against metastatic neoplasm and above all effective modality for treatment monitoring [10e13]. Thus, these nanotheranostic agents allow the physician to monitor the type of treatment and dosing pattern for individual patients in order to present the dose dumping and harmful side effects [14].

12.2

Glioblastoma

Gliomas are most common primary brain tumor derived from neurological progenitor cell. Glioblastomas consist of 48% of all primary malignant brain tumors [15]. Morphologically, they are traditionally classified as astrocytic, oligodendroglial, or ependymal tumors. Degrees of malignancy or aggressiveness of the tumors are defined by Grade I to IV. In the last 2 decades, there have been immense progress in understanding of gliomas in terms of understanding natural histories, transcriptomic and

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epigenetic profiling and availability modern more effective and less toxic treatment options. Computer tomography or CT scan is considered as most common initial assessment modality for any neurodeficit, but the gold standard is magnetic resonance imaging (MRI). Gliomas are generally seen as well defined, sometimes infiltrating hypo intense in T1 image and hyper intense in T2 image with or without perilesional oedema. Low grade gliomas do not enhance with contrast but most of the high grade gliomas including glioblastomas usually show enhancement with gadolinium (contrast used in MRI). Glioblastoma shows high choline and creatinine ratio in spectroscopic images and hyperperfusion in perfusion scan. Primary modality of most of the gliomas is surgery. Surgery includes maximum safe resection to achieve desired control of the tumor with preservation of neurological function. Based on tumor histology, grade, genetic classification adjuvant radiotherapy, and chemotherapy are considered. For Grade I gliomas (pilocytic astrocytoma) mainstay of treatment is surgery alone. Adjuvant radiotherapy required in some cases and radical radiotherapy (where radiotherapy is considered as sole modality) is considered when surgery is not feasible. Grade II gliomas require some form of adjuvant radiotherapy. A large randomized trial has shown that there is overall survival benefit in addition to radiotherapy in diffuse gliomas or oligodendroglioma. EORTC trial has shown benefit of addition of systemic chemotherapy in terms of disease free survival. Grade III and Grade IV gliomas are treated with adjuvant chemoradiation (partial brain radiotherapy along with concurrent chemotherapy with temozolomide) followed by adjuvant chemotherapy. A landmark trial on Grade IV gliomas was published in 2005 which has made chemoradiation with temozolomide as a standard of care. As per said protocol, partial brain is treated with conformal radiotherapy to dose of 60 Gy in 30 fractions over 6 weeks along with oral temozolomide 75 mg/ m2 during radiotherapy followed by adjuvant chemotherapy with the same agent temozolomide 150 mg/m2 [D1 to D5] for first cycle and 200 mg/m2 for rest cycles (repeated every 28 days). Median overall survival was 14.6 in those patients who received radiotherapy and temozolomide compared to 12.1 months in those patients who received radiotherapy alone [16]. Antimitotic activity also been proved with TTF (tumor treating field), a technique where alternating electrical fields of low intensity (1e3 V/cm) and intermediate frequency (100e300 kHz) are applied over scalp in addition of standard therapy in gliblastom. This disrupts DNA repair, alter cell permeability and causes immunological responses to achieve anti mitotic effect in gliomas [17]. In the aforementioned study TTF was applied over scalp during adjuvamt temazolamide. Tumor treating fields have shown its benefits in terms of improvement of overall survival to 6.7 months compared to 4.0 months with temozolomide alone. In spite of all the modalities of treatment, outcome of glioblastoma is dismal with median survival of 14.6 months. The low recovery rate in case of gliobastoma is due to its complicated anatomy, multiple chemotherapeutic drug resistance, very limited surgical facilities, and most predominantly very poor penetration capabilities of therapeutic agents on the tumor side. Furthermore, the complex features of brain and presence of various heterogeneous barriers like BBB and blood brain tumor barrier (BBTB) serves as a major obstacle for the treatment of glioblastoma.

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309

Blood brain barrier (BBB)

The brain being the most crucial and versatile organ of the body is constituted about 2% of body weight and 20% of total blood supply in human body. The blood vessel so present in brain behaves differently than normal blood vessels and restricts the entry and exit of different molecules to brain. The aforementioned phenomenon is due to the presence of a protective barrier, i.e., BBB. This BBB restricts the entry of all potential drugs to brain due to the presence of tight junctions, various immunological barriers and metabolic enzymatic activity. Among several strategies to deliver drug to brain, nanoparticles-based active targeting strategies (Table 12.1) are gaining importance in the recent age for the treatment of several kinds of brain cancers. Nanoparticles ranging 10e800 nm are widely being as biomedicines for early diagnosis and treatment of various brain malignancies [24]. Nanoparticles are advantageous over the conventional strategies as they provide site specific and prolonged drug delivery, improved drug solubility, alleviated cytotoxicity, and ameliorate drug pharmacokinetic profiles. Active BBB targeting strategies include absorptive mediated transcytosis, transporter mediated transcytosis, and receptor mediated transcytosis. Nanoparticles along with active BBB targeting strategies (Fig. 12.1) give an enhanced permeability of anticancer drugs for the treatment of brain cancer.

12.4

Blood brain tumor barrier (BBTB)

BBTB is situated in-between brain tumor material and endothelial cells-established microvessels, prevents the entry of hydrophilic drugs that are responsible for treating gliobastoma. The targeting strategies to treat various malignant tumors are mainly focused on the receptor mediated transport across BBTB. Various receptors like integrin and epidermal growth factor receptors (EGFR) found on the tumor microenvironment, which helps to deliver anticancer drugs loaded nanoparticles for the treatment of brain cancer [25]. In a work, Zan et al. prepared polyethylene glycol (PEG)-polylactic acid (PLA) micelles and conjugated it with cyclic RGD (arginine-glycine-aspartic acid) to target chemotherapeutic agents in intracranial glioma cells. The obtained results showed an effective targeting of drugs for the treatment of glioblastoma [26]. Similarly, Tsutsui et al. prepared bionanocapsules and conjugated it with antihuman epidermal growth factor receptor (EGFR). The results so obtained from the study showed successful receptor mediated delivery of bionanocapsule across BBTB [27].

12.5

Nanotheranostics

As the word suggests, nanotheranostics are nanosized particles possessing both therapeutic and diagnostic properties (Fig. 12.2) in single system [28]. Nanotheranostics are

Active strategies

310

Table 12.1 Nanoparticles based active BBB targeting strategies.

Examples

References

Absorptive mediated transcytosis

An interactivity between oppositely charged moieties (i.e., interaction among positively charged protein moieties and negatively charged membrane facet permits entry across BBB) (Fig. 12.1)

[18,19]

Transported mediated transcytosis

Substrate-choosy transport (i.e., substance mimicking endogenous substrates are only transported across BBB) (Fig. 12.1)

Receptor mediated transcytosis

Ligand-specific, structure-assisted transport (i.e., substances binds to specific receptors and permeated across BBB) (Fig. 12.1)

1. Lu et al. prepared polyglycolated nanoparticles and conjugated it with positively charged bovin serum albumin (BSA). On further evaluation, it was seen that positively charged BSA-conjugated nanoparticles showed higher permeability (7.76 times more) than negatively charged BSAconjugated nanoparticles proving the absorptive mediated transport mechanism. 2. Du et al. prepared liposomal formulation and conjugated it with positively charged protein (wheat germ agglutinin) and exemplified an enhanced absorptive mediated transport. 1. Umezawa et al. prepared liposomes and included a mannose derivative into it and conveyed BBB permeability through glucose transporter mediated transcytosis mechanism. 1. Zhang et al. prepared polyphosphoester micelles loaded with paclitaxel and conjugated it with transferrin. The prepared formulation were evaluated and the obtained results showed an effective transferrin receptor mediated transport of paclitaxel into brain. 2. Other low density lipoproteins showed higher transcytosis capabilities due to active receptormediated transport mechanisms.

[20]

[21e23]

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Mechanism

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Figure 12.1 Various active strategies by which nanoparticles cross the BBB.

Figure 12.2 Various applications of nanotheranostic agents.

gaining importance in medical applications due to their structural framework, multiple imaging prospective, site specificity, improved drug delivery, increased therapeutic efficacy, most predominantly decreased adverse effects, and cytotoxicity [29,30]. Due to their nanosized structural framework, theranostic nanoparticles can easily be tailored and conjugated with various imaging agents, hence proving an enhanced imaging and therapeutic potency. Literature review shows that various physicochemical properties of theranostic nanoparticles such as size shape and surface charge influence the efficacy of the nanoparticles as cancer theranostics (Table 12.2) [38]. Due to several drawbacks of conventional treatment strategies to treat CNS malignancy and gliomas, nanotheranostic agents are gaining importance in recent times. Various types of nanotheranostic agents that are used in the treatment of glioma and CNS malignancy are presented in Fig. 12.3.

Physicochemical properties

312

Table 12.2 Various physicochemical properties of nanotheranostic agents.

Advantages

Examples

Reference

Size (nanometer size range)

• Easy surface moderation • Higher target specificity • Increased capacity of cellular uptake

[31,32]

Shape (spherical, rod, and hollow shape)

• Increased cellular uptake (spherical > rod > hollow) • Easy transport in the biological fluid

Surface charge

• Enhanced cellular absorption (greater uptake of positively charged particles than negatively charged or neutral nanoparticles) • Enhanced diffusion rate (neutral nanoparticles have greater rate of diffusion than others)

Shan et al. prepared gold nanoparticles (AuNPs) of different sizes. They showed that smaller the size of the nanoparticles greater was the cellular uptake and cell internalization. Bartczak et al. prepared AuNPs different shapes. From the result so obtained it was seen that the spherical particles had a much greater cellular uptake while the hollow particles had the lowest cellular uptake. Chan et al. prepared and evaluated AuNPs of spherical and rod shape. The obtained results showed that the cellular uptake of spherical particles was found greater than the rod shaped particles. Tang et al. prepared nanoparticles with different surface charge and dispersed in cultural media. The obtained results showed that the positively charged nanoparticles were better absorbed than negative or neutral charged particles. Graf et al. prepared silica nanoparticles with different surface charge and evaluated. The obtained results showed that the positively charged nanoparticles showed a greater cellular uptake and better cellular internalization.

[33e35]

Design and Applications of Theranostic Nanomedicines

[8,36,37]

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Figure 12.3 Various types of nanotheranostic agents that are used in the treatment of glioma and CNS malignancy.

12.5.1 Gold nanoparticles (AuNPs) AuNPs are gaining importance in cancer therapeutics because of their unique characteristics like inherent properties of gold core, biocompatibility, ability to alter, and modify the structure and functions with most predominantly minimal cytotoxic effects [39]. Along with the aforementioned characteristics, AgNPs possess intrinsic imaging characteristics, diagnostic property, and light scattering ability, which makes AgNPs as promising candidates in cancer diagnostic and therapy. In a research, Hainfeld et al. prepared AgNPs of 11 mm size and administered intravenously to mice in order to find out imaging and radiotherapeutic characteristics. The images of the brain received from the microcomputed tomography showed localized gold uptake in brain gliomas. The gold uptake was estimated when compared with normal cells produced a 19:1 tumor to normal brain ratio. It was further observed that mice with administered AgNPs and radiotherapy showed a greater long-term tumor-free survivance (average 53%) than mice with only radiotherapy (average 9%). Hence, AgNPs can be effectively used as diagnostic agents and therapeutic agents treat brain gliomas [40]. In another study, Melancon et al. formulated gold nanoshells and coated it with super paramagnetic iron oxide in order to provide a novel image-guided laser ablation to treat head and neck cancers. The prepared nanoparticles were conjugated with C225 monoclonal antibody and evaluated. It was observed that the selective binding of C225-SIIO@AuNPs took place in mice carrying A431 tumor cells and further caused destruction of A431 tumor cells by laser ablation. From the study, it was proved that AuNPs can be used for targeted and site specific therapy which in turn elevate the thermal ablation efficacies and decrease thermal damage of normal cells [41]. Similarly, Heo et al. prepared paclitaxel (PTX) loaded AuNPs and coated it with PEG, biotin, and b cyclodextrin to evaluate the site-specific

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theranostic effect in cancer therapy. The release of PTX from the prepared NPs was due to presence of intercellular glutathione (GSH). Furthermore, confocal laser scanning microscopy and fluorescence activated cell shorting (FACS) showed that these tailored AuNPs possessed distinct interaction with Hela, A549, and MG63 cancer cells when compared to normal NIH3T3 cells. The results showed these tailored AuNPs were effective effectively against the cancer cells. These tailored AuNPs plays can be used in both diagnosis and treatment of cancer. In addition, other studies have been carried out in order to confirm the theranostic behavior of the AuNPs [42]. Sun et al. prepared gold and super paramagnetic iron oxide-loaded micelles and coated it with polyethylene glycolepolycaprolactone (PEG-PCL). The prepared micelles were administered to gh2ax DNA in gliobastoma cell lines and the damage caused to the gh2ax DNA was quantified. First, these micelles were administered to mouse model inoculated with gliboblastoma tumors and images of tumor lining were obtained by computed tomography (CT) and magnetic resonance imaging (MRI). The images obtained from MRI were far better than that of the images obtained from CT. Furthermore, when radiosensitizing capability of micelles when applied in association with radiotherapy, the damage of gh2ax DNA was doubled [43]. Thus, the obtained results suggested that these developed micelles possessed the thermostatic characteristics.

12.5.2

Quantum dots (QDs)

QDs are nanosized inorganic semiconductors synthesized mainly from Group IIIeV and IIeVI periodic table elements [44]. More precisely, QDs display quantum confined circumstance associated with a drastic change in the behavior of the electrons present mainly at the borders of Bohr radiuses and are mainly of size less than 10 mm. This exceptionally small size of the QDs makes it early interactions with the biological structures [45]. Due to the aforementioned facts, QDs are widely being used as nanomedicines. Moreover, QDs depending on the composition and structure can emit light of varying wavelengths. Thus, QDs can also be used as nanodiagnostic agents. In a research, Zang et al. prepared CdSe and ZAIS QDs having different emission wavelengths and characteristic fluorescence lifetime. On evaluation, it was found out that the prepared QDs showed specific spectral imaging from green CdSe and ZAIS as well as red CdSe and ZAIS QDs. On further evaluation with fluorescence lifetime imaging microscopy, the obtained images showed considerable differences than fluorescence lifetime. Thus, it is proved that QDs can be used in nanotheranostic treatment procedure [46]. Similarly, Tian et al. prepared doxorubicin (DOX)-loaded liposomal QDs hybrid by including CdSe/ZnS TOPO-capped into the dieteroxyl phosphotidylcholin (DSPC) and egg phosphotidylcholin bilayers. On performing atomic force microscopy (AFM), it was observed that the assimilation of QDs within the membrane has occurred due to hydrophobic self-association method. Carboxyfluorescence was incorporated for optical purpose. It was observed that, due to the presence of QDs, the carboxyfluorescence release was aided in the formulation containing egg phosphotidylcholin while carboxyfluorescence release was minimal in formulation

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containing DSPC, thus proving greater stability. Osmotic gradient technique was further used to load DOX into the liposomal-QD hybrids. On further evaluation, it was seen that the formulation containing 97% drug encapsulation efficiency and fluorescence microscopy showed the presence of QD (green emitting), thus proving the presence of both DOX (drug) and QDs in a single system [47]. From the aforementioned fact, it can be suggested that the prepared formulation can provide both diagnostic activity due to the presence of QDs and therapeutic activity due to the presence of DOX.

12.5.3 Magnetic nanoparticles Nowadays, magnetic nanoparticles are widely being used in various biomedical systems and most prominently hyperthermia, MRI guided drug delivery [48e50]. Moreover, magnetic nanoparticles are also playing an important role in theranostic applications in the treatment of glioma. In a study, Chertok et al. prepared nanoparticles made out of iron oxide for MRI guided magnetic targeting and delivery of chemotherapeutics to treat brain tumors. On evaluating the nanoparticles, it was found out that the nanoparticles possess 10 mm hydrodynamic diameter and 94 emu/g Fe saturation magnetization. Further, in vivo studies were carried out on rats bearing orthotopic 9L gliosarcomas. The experimental rats were administered with the formulation intravenously under super paramagnetic magnetic field density. On obtaining the MR images, it was sure that there was an increase (fivefold) in vulnerability of glioma cells to the prepared magnetic nanoparticles than normal tumor and TSI (target selectivity index) of prepared nanoparticles were 3.6-fold more in glioma than normal brain [30]. These prepared magnetic nanoparticles can be used for both imaging and targeting gliosarcomas. Recently, several formulation/magnetic nanoparticles are being prepared and going through clinical trials. Nanotherm, which is recently within Phase-I, Phase-II clinical trial are nanoparticles of size range 10e15 mm formed by colloidal dispersion of FeOnanoparticles in magnetic fluid and core were over layered with aminosalicylates. This formulation can be used in treatment of brain tumor, esophageal cancer, prostate cancer, etc. The mechanism, by which Nanotherm works, is by converting magnetic energy into heat. Furthermore, the energy conversion (i.e., magnetic to thermal) can be calculated by density distribution, which can be further estimated by CT [51].

12.5.4 Mesosporous silica nanoparticles (MSNs) The unique characteristics of MSNs, i.e., distinct surface area, adaptive pore size and width, flexible functionalization, etc., make MSNs as useful candidates for nanotheranostic treatment for brain cancers [52]. It was experimentally seen that MSNs naturally possess a large surface area and easily operable pore volumes. Due to these characteristics, MSNs are very convenient to incorporate a wide variety of chemotherapeutics [53]. However, MSNs-based drug delivery systems are not able to untimely controlled release of drugs [54]. This is because of the fact that the drug once incorporated into

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the MSNs along with other materials become immovable due to porous capping nature [55]. These porous capping materials block the pores present in MSNs and can only open on application of external stimuli such as temperature, pH, redox potential, and light enzymes. These aforementioned characteristics of tailored (with capping agent/targeting ligands) MSNs can be considered as good candidates for target specific and controlled release of anticancer therapeutic agents [54].

12.5.5

Solid lipid nanoparticles (SLNs)

Lipid nanoparticles are more preferable in crossing the BBB, which in turn is advantageous for the delivery of hydrophilic drugs. In a research study by Jain et al., temozolamide-loaded transferrin conjugated SLNs were synthesized by ethanol injection method for delivering anticancer drug to brain. The average size and drug entrapment efficiency of the conjugated SLNs were 249  2.6 nm and 64.21  2.27%, respectively. In vitro cytotoxicity studies were carried out on human cancer cell lines, which resulted in significant tumor inhibitory effect. Furthermore, fluorescence studies showed higher/greater uptake of transferrin-SLNs in brain tissues [56]. In another study, SLNs were prepared to transport camptothecin across BBB. Cytotoxicity study was performed against microphage and glioma human cell lines. Cell death was observed with less maximal inhibitory concentration (IC50), which proved antitumor activity of prepared SLNs. Along with this, in vivo biodistribution study was performed in rats and a significant accumulation of drug was observed in brain, which confirmed that the prepared formulation can be used in the treatment of cancer [57].

12.5.6

Dendrimers

The word dendrimer is derived from two Greek words “Dendron” meaning branch and “Meros” meaning part. Therefore, dendrimers are defined as a class of hyper branched polymers, which in turn leads to formation of globular structure with high surface density and low molecular volume [58]. Dendrimers are also known as “cascade polymer” or “arboroles” [59]. The literature suggests that dendrimers are widely used in anticancer drug delivery and treatment. Because of its physicochemical characteristics, the uses of dendrimers in anticancer therapy are more advantageous than conventional polymeric micelles. For example, Malik et al. prepared ciplastin loaded dendrimers and evaluated to determine its antineoplastic effect [60]. The obtained results showed a greater bioavailability, greater dose tolerability and prolonged period of the experimental animals. In another study, Wiener et al. prepared dendrimers and conjugated it with magnetic resonance imaging contrast agents [61]. The prepared formulation showed huge proton relaxation and greater molecular relaxation rate. The result showed that the prepared dendrimer had a relaxation rate higher than (six times) that of free Gd(III)-DTPA complex. On performing in vivo study on rabbits, the MRI images so obtained showed outstanding image of blood vessels proving the imaging characteristics of dendrimers. These dendrimers can be used for imaging, drug delivery, and therapeutic purpose in the treatment of cancers.

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12.5.7 Liposomes Liposomes are phospholipid bilayer vesicles, which can entrap both hydrophilic and lipophilic molecules in the aqueous core or lipid membrane respectively. Furthermore, liposomes possess unique characteristics like high drug loading efficiency, biocompatibility, biodegradability, high BBB penetration, prolonged and sustained release kinetic and very good biodistribution [9,62]. Due to the aforementioned characteristics, a wide variety of drugs and imaging agents can be easily incorporated into liposomes [63,64]. Kostarelos et al. developed DOX-loaded liposomal formulation and tagged it with ICG (indocyanin green) dye as the fluorescent marker. The prepared liposomal formulation was characterized and the optoacoustic tomographic images so obtained showed both diagnostic and therapeutic properties of the liposomes [64]. In another study, Li et al. developed DOX-loaded liposomal formulation and tagged with MRI contrast agent, a fluorescent dye and radioactive ions. The images so obtained were compared and proved an efficacious multimodal imaging [65]. Similarly, Zhou et al. formulated PTX-loaded liposomes and tagged with MRI contrast agent and fluorophore. The results of in vitro study, in vivo study as well as fluorescent and magnetic resonance images indicated an effective uptake of the liposomes by tumor in the experimental animals. The overall results suggested of allowing a multimodal imaging together by these liposomes with an effective therapeutic activity [66].

12.6

Conclusion

Glioblastoma, a primary form of brain malignancy, is well-known as one of the fatal diseases. The BBB and BBTB, as the main physiological barriers, heterogeneity, and the invasive characteristics of glioblastoma lead to an insufficient concentration of chemotherapeutics at the tumor site, restricting the existing treatments for brain cancers. These complexities can be surmounted using nanotheranostics demonstrating a prospect in cancer therapeutics and diagnostics. Accordingly, in the recent years, nanotheranostics lead to direct advancements in the management of brain cancers because of their smaller sizes, surface functionalizations and, more recently, the prospect to co-develop therapeutics as well as diagnostics in a single nanostructure system. In the upcoming years, the global perception for the uses of nanotheranostics is optimistic to diagnose and treat CNS malignancy and gliomas.

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Abul Kalam Azad 1 , Ng Yen Ping 2 , Md Saquib Hasnain 3 and Amit Kumar Nayak 4 1 Faculty of Pharmacy, Pharmaceutical Technology Unit, AIMST University, Kedah, Malaysia; 2 Faculty of Pharmacy, Clinical Pharmacy Unit, AIMST University, Kedah, Malaysia; 3 Department of Pharmacy, Palamau Institute of Pharmacy, Chianki, Daltonganj, Jharkhand, India; 4Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Jharpokharia, Mayurbhanj, Odisha, India

13.1

Introduction

Researchers all around the world are working tirelessly to develop successful targeted nanotheranostics for cancer diagnosis and treatment [1e3]. In recent years, nanomedicine researchers have facilitated the production of “smart” nanocarriers or nanotheranostics, which comprise nanovehicles that simultaneously transport medications and act as imaging agents in a single system [4,5]. This technology improves the monitoring of drug biodistribution. It also offers flexibility in analyzing target site accumulation of nanomedicines, allows the quantification and visualization of triggered drug release from nanomedicines [6]. The tailored nanomedicines-based chemotherapeutic interventions available today have been effective in enhancing the patient compliances. The uses of nanomaterials have generated interest in their potential, such as the incorporation of radionuclides with conventionally used nanomaterials to impart new traits for the purpose of cancer diagnosis and treatment [7,8]. Researchers have made the uses of nanoparticles to emit ionizing radiation in therapeutic settings for the purposes of diagnostics as well as theranostics [9,10]. The success of nanoparticle-mediated radionuclide therapy is linked to their ability to provide the targeted administration of ionizing radiation for a set amount of time for curative or palliative treatments as well as in a theragnostic approach. The composition of nanomedicines may differ from one another. Various nanomedicines can be classified as biodegradable polymers (i.e., chitosan, dextran, phospholipids, polylactic acid, polylactic-co-glycolide, etc.), carbon-based (i.e., graphene, nanotubes etc.), metallic (i.e., metal oxides, sulfides, etc.), and semiconductors (i.e., quantum dots [QDs]) [11,12]. Because of unique physicochemical features, theranostics are being investigated not only as drug-delivery nanocarriers but also as synthetic scaffolding for imaging probes used in cancer diagnosis [1,3]. The applications and biopharmaceutical performances of these nano-based systems are highly influenced by features like chemical compositions (such as metallic, polymeric, lipid-based and carbon-based) (Fig. 13.1), surface characteristics (such as charge and hydrophobicity), physical characteristics (such as size, shape, and stiffness), and surface functionalizations with

Design and Applications of Theranostic Nanomedicines. https://doi.org/10.1016/B978-0-323-89953-6.00004-0 Copyright © 2023 Elsevier Ltd. All rights reserved.

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Figure 13.1 Different types of nanotheranostics used for cancer diagnosis and therapy. (i) metallic nanotheranostics, (ii) polymeric nanotheranostics, (iii) lipid-based nanotheranostics, and (iv) carbon-based nanotheranostics. MRI, magnetic resonance imaging; PET, positron emission tomography; PDT, photodynamic therapy. From Misra R, Acharya S. Smart nanotheranostic hydrogels for on-demand cancer management. Drug Discovery Today 2021;26(2):344e359, with permission, Copyright © 2020 Elsevier Ltd.

specific targeting ligands and functional groups [13]. These qualities may add nanomedicine an appealing approach to the nanotheranostic systems for cancer diagnosis and treatment. There are several nanomedicines that have been approved for cancer treatment in the commercial market while others are in different phases of clinical trials [14]. Doxil/Caelyx and Abraxane are the most renowned clinically approved nanomedicines

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for the treatment of cancer [15,16]. Abraxane is an albumin-based nanoparticles (NPs) of paclitaxel, which has been approved for treatment of metastatic breast cancer. Meanwhile, Doxil is the liposomal formulation of doxorubicin clinically approved to control Kaposi’s sarcoma. It is also used in the treatment of refractory breast and ovarian cancer. Finally, iron oxide NPs, Resovist and Feridex/Endorem have received clinical approval to be used as nanodiagnostics, specifically for imaging lesions in liver/spleen [16]. Approved nanomedicines for chemotherapeutics and imaging purposes in cancer diagnosis and therapy are listed in Table 13.1 [10]. It is worth noting that most of the listed nanodiagnostics and nanotherapeutics are organic-based NPs. These NPs are highly preferred for their high biocompatibility and low toxicity. At the same time, FDA has approved inorganic nanomedicines, such as Resovist and Feridex/Endorem, Aurimune (colloidal gold platform), and Auro-Lase (contains gold-coated silica NPs), which have gone through different phases of clinical studies [17]. Cancer nanomedicine research has grown at an exponential rate over the past year and ongoing research in cancer nanodiagnostics and nanotherapeutics is promising. New findings could reshape cancer treatment strategy while taking patient compliance into account. Table 13.2 lists the different nanomedicine formulations studied for therapeutics, diagnostics, and cancer theranostics, and summarizes in vitro and in vivo outcomes [10].

13.2

Nanomedicines as cancer theranostics

Nanotheranostics is a concept that reflects on the design of NPs, which allow them to diagnose, treat, and monitor therapeutic response concurrently in a single integrated system [38]. Theranostics merge the abilities of diagnostic as well as therapeutic abilities in a single system (Fig. 13.2) [39]. The introduction of multifaceted NPs is expected to minimize risks and costs, subsequently, which elevate therapeutics and diagnostics into new heights. In this regard, new polymerization and emulsifying techniques allow the production of NPs with hydrophilic and hydrophobic surfaces. This produces NPs payload with various active compounds, like a contrasting agent with a hydrophilic feature or a therapeutic agent with hydrophobic nature. The possibilities of nanomedicine application are dependent on the functional nanoplatforms, which combines therapeutic elements and multimodal imaging capability. Coordinating detecting capability and therapeutic interventions help to overcome the hindrance of adaption and cancer heterogeneity [40]. Theranostics allows NPs to diagnose, treat and monitor therapy response simultaneously in a single integrated system [38]. Novel polymerization and emulsifying techniques produce NPs with hydrophilic and hydrophobic surfaces, allowing their payload to contain a variety of active compounds (i.e., a contrasting agent of hydrophilic nature while a therapeutic agent of hydrophobic nature and vice versa). Moreover, this coordination of detecting capability with therapeutic interventions is critical in overcoming the obstacles of cancer heterogeneity as well as adaptation, leading to lower minimize risks and costs in cancer treatment [40]. For instance, NPs with

Table 13.1 Some of the cancer nanomedicines (approved or in clinical trial stages), their characteristics, and indications. 326

Cancer nanomedicines DaunoXome DepoCyt

Doxil Marqibo Mepact

Oncoprex (Genprex) Oncaspar Eligard Genexol Opaxio

Halaven E7389LF Abraxane

Daunorubicin citrate encapsulated liposomes (