Biomedical Applications of Functionalized Nanomaterials: Concepts, Development and Clinical Translation

By Bruno Sarmento and Jose Das Neves

Biomedical Applications of Functionalized Nanomaterials: Concepts, Development and Clinical Translation presents a concise overview of the most promising nanomaterials functionalized with ligands for biomedical applications. The first section focuses on current strategies for identifying biological targets and screening of ligand to optimize anchoring to nanomaterials, providing the foundation for the remaining parts. Section Two covers specific applications of functionalized nanomaterials in therapy and diagnostics, highlighting current practice and addressing major challenges, in particular, case studies of successfully developed and marketed functionalized nanomaterials. The final section focuses on regulatory issues and clinical translation, providing a legal framework for their use in biomedicine.

This book is an important reference source for worldwide drug and medical devices policymakers, biomaterials scientists and regulatory bodies.

• Provides an overview of the methodologies for biological target identification and ligand screening
• Includes case studies showing the development of functionalized nanomaterials and their biomedical applications
• Highlights the importance of functionalized nanomaterials for drug delivery, diagnostics and regenerative medicine applications

• Language: English
• Publisher: Elsevier Science
• Release date: Mar 1, 2018
• ISBN: 9780323508797

Book preview

Biomedical Applications of Functionalized Nanomaterials

Concepts, Development and Clinical Translation

Editors

Bruno Sarmento

José das Neves

Table of Contents

Cover image

Title page

Copyright

List of Contributors

Preface

Chapter 1. From the Magic Bullet to Advanced Nanomaterials for Active Targeting in Diagnostics and Therapeutics

1. Paul Ehrlich and the Magic Bullet

2. Passive Versus Active Targeting in Cancer as Model

3. Emerging Challenges and Perspectives

Section I. Ligand Selection and Functionalization of Nanomaterials

Chapter 2. Conjugation Chemistry Principles and Surface Functionalization of Nanomaterials

1. Conjugation Chemistry in the Context of Biomedical Nanomaterials

2. Conjugation Chemistry Principles

3. Self-Assembled Monolayers as a Powerful Tool for the Design of Surface-Engineered Nanomaterials

4. Challenges in (Bio)conjugation

Chapter 3. Phage Display Technology for Selection of Antibody Fragments

1. Introduction

2. Antibody Phage Display Libraries

3. Selection and Screening of Antibody Phage Display Libraries

4. Antibody Engineering

5. Conclusions and Future Perspectives

Chapter 4. Ribosome Display Technology for Selecting Peptide and Protein Ligands

1. Introduction

2. Emergence of In Vitro Display Technologies

3. Basic Principles and Features of Ribosome Display Technology

4. Selection of Peptides Using Ribosome Display Technology

5. Selection of Antibody Fragments Using Ribosome Display Technology

6. Selection of Proteins Using Ribosome Display Technology

7. Conclusions and Future Perspectives

Chapter 5. Engineered Protein Variants for Bioconjugation

1. Introduction

2. Bioconjugation on Natural Amino Acids

3. Bioconjugation on Unnatural Amino Acids

4. Affinity-Induced Bioconjugation

5. Conclusions and Future Perspectives

Chapter 6. Bioengineered Approaches for Site Orientation of Peptide-Based Ligands of Nanomaterials

1. Introduction

2. Control of Peptide Structure and Functionality

3. Impact of Bond Strength and Linker Length on Bioconjugation

4. Impact of Ligand Density on the Targeting Efficiency of Nanoconjugates

5. Protein Corona Effect and Minimization of Nonspecific Interactions

6. Conclusion and Future Perspectives

Chapter 7. Nanozymes for Biomedical Sensing Applications: From In Vitro to Living Systems

1. Introduction

2. Nanozymes for In Vitro Sensing

3. Nanozyme for Sensing in Living Systems

4. Conclusions and Perspectives

Abbreviations

Chapter 8. Systematic Evolution of Ligands by Exponential Enrichment for Aptamer Selection

1. Introduction

2. Potential Aptamer Targets

3. Advantages of Aptamers

4. Random Oligonucleotide Libraries

5. Systematic Evolution of Ligands by Exponential Enrichment

6. Sequencing of the Enriched Aptamer Pools

7. Evaluation of Aptamer-Binding Kinetics

8. Post–Systematic Evolution of Ligands by Exponential Enrichment Modifications

9. Conclusion

Section II. Specific Applications of Functionalized Nanomaterials in Therapy and Diagnostics

Chapter 9. Graphene-Based Nanomaterials in Bioimaging

1. Introduction

2. Synthesis of Graphene-Based Nanomaterials

3. Surface Functionalization of Graphene-Based Nanomaterials

4. Graphene-Based Nanomaterials in Bioimaging

5. Prospects and Challenges

6. Conclusions

Chapter 10. Functionalized Transition Metal Dichalcogenide-Based Nanomaterials for Biomedical Applications

1. Introduction

2. Basic Properties of Transition Metal Dichalcogenides

3. Synthesis of Two-Dimensional Transition Metal Dichalcogenides

4. Functionalization of Transition Metal Dichalcogenides for Biomedical Applications

5. Conclusion and Outlook

Chapter 11. Intracellular Targeting Using Surface-Modified Gold Nanoparticles

1. Introduction

2. Nuclear Targeting of Gold Nanoparticles

3. Structure of the Nuclear Pore Complex

4. Mechanism of Nuclear Entry and Transport

5. Different Surface Functionalizing Strategies for Nuclear Targeting of Nanoparticles

6. Imaging Techniques for Probing Nuclear Targeting

7. Gold-Based Nanostructurmes for Improved Cancer Therapeutics

8. Radiation Therapy

9. Anticancer Drug Delivery

10. Conclusions and Future Direction

Chapter 12. Multifunctional Magnetic Nanoparticles for Theranostic Applications

1. Introduction

2. Iron Oxide Nanoparticles: Magnetic Properties and Chemical Synthesis

3. Surface Modification Routes for the Preparation of Multifunctional Fe3O4 Magnetic Nanoparticles

4. Organic-Modified Magnetic Nanoparticles for Biomedical Applications

5. Concluding Remarks and Perspectives

Chapter 13. Combinatorial Approach in Rationale Design of Polymeric Nanomedicines for Cancer

1. Introduction

2. Challenges in Cancer Therapy and Motivation for Nanomedicines

3. Challenges With Developing Nanomedicines

4. Synthetic Approaches for Combinatorial Design

5. Illustrative Applications of Combinatorially Designed Polymeric Nanosystems

6. Microfluidic Technologies in Nanoparticle Formulation, Scale-Up, and Screening

7. Conclusions and Future Perspective

Chapter 14. Functional Moieties for Intracellular Traffic of Nanomaterials

1. Introduction

2. Intracellular Delivery: Barriers and Challenges

3. Intracellular Delivery by Nanomaterials

4. Nanomaterial-Based Strategies to Target Subcellular Organelles

5. Conclusions and Future Perspectives

Chapter 15. Functionalized Polymeric Nanostructures for Mucosal Drug Delivery

1. Introduction

2. Mucosal Barriers

3. Models for Studying Mucosal Drug Delivery

4. Formulation Strategies to Improve Mucosal Delivery

Chapter 16. Biofunctionalized Mesoporous Silica Nanomaterials for Targeted Drug Delivery

1. Introduction

2. Mesoporous Silica Nanoparticles: From Fabrication to Applications

3. Future Perspectives

Chapter 17. Nanoparticle-Mediated RNA Interference for Cancer Therapy

1. RNA Interference and Cancer Therapy

2. Systemic Delivery of RNA Interference Effectors to Tumors

3. Tumor Microenvironment as a Factor Influencing Nanoparticle-Mediated Delivery of RNA Interference Effectors

4. Noninvasive Pharmacokinetic Analysis of RNA Interference Effectors

5. Undesirable Effects Caused by RNA Interference Effectors and Delivery Vehicles

6. The Accelerated Blood Clearance Phenomenon of Delivery Vehicles

7. Therapeutic Studies of Anticancer RNA Interference Effectors Formulated in Nanoparticles

8. Concluding Remarks

Chapter 18. Biomolecular Therapeutics for HIV

1. Introduction

2. Aptamer-SIRNA Nanoparticles for Targeted Anti-HIV Therapeutics

3. Anti-HIV Vectors

4. Engineering HIV Resistance With Genome Editing

5. Chimeric Antigen Receptor T Cells

6. Concluding Remarks

Chapter 19. Self-Assembled Peptide and Protein Nanofibers for Biomedical Applications

1. Introduction

2. Classes of Self-Assembling Peptide and Protein Nanofibers

3. Applications of Self-Assembling Peptide and Protein Nanofibers for Biomedicine

4. Future Perspectives

Chapter 20. Peptide-Modified Hydrogels for Therapeutic Vascularization

1. Introduction

2. Hydrogels in Vascular Tissue Engineering

3. Biofunctionalization of Hydrogels With Angiogenic Peptides

4. Conclusions and Future Perspectives

Section III. Manufacturing, Regulatory Challenges and Clinical Testing of Functionalized Nanomaterial-based Products

Chapter 21. Manufacturing and Safety Guidelines for Manufactured Functionalized Nanomaterials in Pharmaceutics

1. Introduction

2. Manufactured Nanomaterials in Pharmaceutics

3. Physicochemical Characterization of Nanomaterials

4. Critical Quality Attributes and Quality Control

5. Pharmacological Evaluation

6. Biopharmaceutical Characterization

7. Conclusion

Abbreviations

Chapter 22. Regulation of Biomedical Applications of Functionalized Nanomaterials in the European Union

1. Overview of European Union Legislation and Procedural Framework

2. Medicinal Products Developed with Nanotechnology

3. Scientific Guidance

4. Medical Devices

5. International Convergence on Nanomedicines

6. Conclusions and Next Steps

Chapter 23. Translational Exploration and Clinical Testing of Silica–Gold Nanoparticles in Development of Multifunctional Nanoplatform for Theranostics of Atherosclerosis

1. Introduction

2. Silica–Gold Nanoparticles for Imaging and Therapy of Atherosclerosis

3. Future of Nanomedical Applications for Imaging and Therapy of Atherosclerosis

4. Conclusion

5. Future Perspectives

Abbreviations

Index

Copyright

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List of Contributors

Mansoor M. Amiji,     Northeastern University, Boston, MA, United States

Marco Araújo,     Universidade do Porto, Porto, Portugal

Tomohiro Asai

University of Shizuoka, Shizuoka, Japan

University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Svetlana Avvakumova,     Università di Milano-Bicocca, Milano, Italy

Cristina C. Barrias,     Universidade do Porto, Porto, Portugal

Hikmet Budak

Sabanci University, Istanbul, Turkey

Montana State University, Bozeman, MT, United States

John C. Burnett,     Beckman Research Institute of City of Hope, Duarte, CA, United States

Maria T. Cambria,     Università di Catania, Catania, Italy

Wen Cao,     Nanjing University, Nanjing, China

Bárbara Carreira,     Universidade de Lisboa, Lisbon, Portugal

Devika B. Chithrani

University of Victoria, Victoria, BC, Canada

Ryerson University, Toronto, ON, Canada

St Michaels’s Hospital, Toronto, ON, Canada

Miriam Colombo,     Università di Milano-Bicocca, Milano, Italy

Guglielmo G. Condorelli,     Università di Catania, Catania, Italy

João Conniot,     Universidade de Lisboa, Lisbon, Portugal

Noemi S. Csaba,     University of Santiago de Compostela, Santiago de Compostela, Spain

Falk Ehmann,     European Medicines Agency, London, United Kingdom

Tália Feijão,     Universidade do Porto, Porto, Portugal

Cláudia S.M. Fernandes,     Universidade Nova de Lisboa, Caparica, Portugal

Fabrícia Saba Ferreira,     National Health Surveillance Agency (ANVISA), Brasília, Brazil

Helena F. Florindo,     Universidade de Lisboa, Lisbon, Portugal

Sidónio C. Freitas,     Universidad Cooperativa de Colombia – Sede Medellín, Medellín, Colombia

Elisabetta Galbiati,     Università di Milano-Bicocca, Milano, Italy

Rogério S. Gaspar,     Universidade de Lisboa, Lisbon, Portugal

Maria Gonzalez-Pajuelo,     FairJourney Biologics, Porto, Portugal

Leaf Huang,     University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Peng Huang,     Shenzhen University, Shenzhen, China

Yan Huang,     Shenzhen University, Shenzhen, China

Gregory A. Hudalla,     University of Florida, Gainesville, FL, United States

Babar Hussain,     Sabanci University, Istanbul, Turkey

Olga Iranzo,     CNRS, Aix-Marseille Université, Marseille, France

Christine Janas,     Goethe University, Frankfurt am Main, Germany

Alexander N. Kharlamov

De Haar Research Foundation, Rotterdam, The Netherlands

De Haar Research Foundation, New York, NY, United States

Priyadarshi Kumar

A∗STAR (Agency for Science Technology and Research), Singapore, Singapore

Indian Institute of Science Education and Research, Pune, India

Hasan Kurt,     Istanbul Medipol University, Istanbul, Turkey

Victoria Leiro,     Universidade do Porto, Porto, Portugal

Jing Lin,     Shenzhen University, Shenzhen, China

Shichao Lin,     Nanjing University, Nanjing, China

Shasha Li,     Beckman Research Institute of City of Hope, Duarte, CA, United States

Zibiao Li,     A∗STAR (Agency for Science Technology and Research), Singapore, Singapore

M. Victoria Lozano,     University of Castilla-La Mancha (UCLM), Albacete, Spain

Maria Cristina L. Martins,     Universidade do Porto, Porto, Portugal

Ana I. Matos,     Universidade de Lisboa, Lisbon, Portugal

Serena Mazzucchelli,     Università di Milano, Milano, Italy

Liane I.F. Moura,     Universidade de Lisboa, Lisbon, Portugal

Faheem Muhammad,     Nanjing University, Nanjing, China

Naoto Oku,     University of Shizuoka, Shizuoka, Japan

Paula Parreira,     Universidade do Porto, Porto, Portugal

Ana Paula Pêgo

Universidade do Porto, Porto, Portugal

Universidad Cooperativa de Colombia – Sede Medellín, Medellín, Colombia

Carina Peres,     Universidade de Lisboa, Lisbon, Portugal

Ruben Pita,     European Medicines Agency, London, United Kingdom

Davide Prosperi,     Università di Milano-Bicocca, Milano, Italy

Antti Rahikkala,     University of Helsinki, Helsinki, Finland

Ana C.A. Roque,     Universidade Nova de Lisboa, Caparica, Portugal

Jessica M. Rosenholm,     Åbo Akademi University, Turku, Finland

Rany Rotem,     Università di Milano-Bicocca, Milano, Italy

Vanessa Sainz,     Universidade de Lisboa, Lisbon, Portugal

Manuel J. Santander-Ortega,     University of Castilla-La Mancha (UCLM), Albacete, Spain

Hélder A. Santos,     University of Helsinki, Helsinki, Finland

Dillon T. Seroski,     University of Florida, Gainesville, FL, United States

Ana L. Silva,     Universidade de Lisboa, Lisbon, Portugal

Liana C. Silva,     Universidade de Lisboa, Lisbon, Portugal

Amit Singh,     AllExcel Inc., West Haven, CT, United States

Alejandro Sosnik,     Technion-Israel Institute of Technology, Haifa, Israel

Daniela Teixeira,     FairJourney Biologics, Porto, Portugal

Gonçalo D.G. Teixeira

Universidade Nova de Lisboa, Caparica, Portugal

CNRS, Aix-Marseille Université, Marseille, France

René Thürmer,     BfArM – Federal Institute for Drugs and Medical Devices, Bonn, Germany

Lungile N. Thwala

University of Santiago de Compostela, Santiago de Compostela, Spain

Wildlife Pharmaceuticals (Pty) Ltd., White River, South Africa

Ana L. Torres,     Universidade do Porto, Porto, Portugal

Cristina Tudisco,     Università di Catania, Catania, Italy

Fernanda Pires Vieira,     National Health Surveillance Agency (ANVISA), Brasília, Brazil

Matthias G. Wacker

Fraunhofer-Institute for Molecular Biology and Applied Ecology, Frankfurt am Main, Germany

Goethe University, Frankfurt am Main, Germany

Akira Wada,     RIKEN Center for Life Science Technologies, Yokohama, Japan

Hui Wei,     Nanjing University, Nanjing, China

Swee Liang Wong,     A∗STAR (Agency for Science Technology and Research), Singapore, Singapore

Jiangjiexing Wu,     Nanjing University, Nanjing, China

Jia Yao,     Nanjing University, Nanjing, China

Meral Yüce,     Sabanci University Nanotechnology Research and Application Centre, Istanbul, Turkey

Preface

"There’s Plenty of Room at the Bottom," the famous 1959 speech by Richard Feynman that is often quoted as the prelude to nanotechnology, provides an incredible voyage into the dimensions and possibilities at the nanoscale (and below!) (Feynman, 1960). The emergence of this new and incredibly wide field has led to an astonishing amount of fascinating developments in science and technology that would not otherwise allow us to live the wonders of modern days. Even now, the excitement and fresh possibilities do not seem to be fading as attention and investment are being set forward for continuing research and translation efforts. Among all the potential and actual applications of nanotechnology, the biomedical arena has been capitalizing on insights into cell and molecular biology, health and disease mechanisms, and processing and characterization of (nano)materials, to name a few, to develop novel diagnostic and therapeutic tools. The manipulation of materials at the nanoscale and use of nanomaterials are indeed frequent sources of innovation for health care-related products that have propelled in many ways nanomedicine into current (and hopefully prospective) clinical practice. In tandem with the simpler questions of scale and following on the principles already recognized in the original speech by Dr. Feynman, the use of nanomaterials for biomedical applications is now increasingly focused on providing function, often in multiple and complementary ways, for the rational design of precisely engineered systems (Araújo et al., 2017). Indeed, a wide array of advanced functional nanomaterials have been and are unceasingly being proposed, and a few are already set to start helping health-care professionals and, more importantly, patients.

This book aims at providing a concise and up-to-date overview of the field of nanomaterials functionalized with diverse ligands, namely focusing on the most promising ones for biomedical applications. It starts with an introduction on the developments in the subject, from an historical perspective. The first section will be largely devoted to available strategies for identifying biological targets and screening of ligands regarding the optimization of anchoring to nanomaterials. Although standing as an individual section on its own, it provides the ground basis for the following content of the book. Specific applications of functionalized nanomaterials in therapy and diagnostics will be covered in second section. This part of the book conveys, in particular, practice-oriented contributions and is expected to address objective questions of the scientific community. In particular, extensive emphasis on case studies of successfully developed and some already marketed functionalized nanomaterials is provided. Finally, third section focuses on manufacturing, safety assessment, regulatory issues, and clinical translation pertinent to the subject of nanomaterials and nanomedicine, tentatively making of this book an indispensable compendium for worldwide drug and medical device policy makers and regulatory bodies.

Research and development of nanomaterials, namely of those specifically functionalized for biomedical applications, has come a long way but many exciting opportunities and challenges remain. We intend with this book to provide academic, industrial, and health-care scientists interested on target drug delivery systems, tissue engineering and regenerative medicine, applied nanomaterial research and bioactive materials with a comprehensive, reference compendium that, above all, may be integrated into their daily practice, and continuing scientific efforts. Also, we envision that it may help researchers and professionals whose main topic is related with nanomedicine and personalized therapeutics to get familiarized with and exploit the synergic effect between functionalized nanomaterials and biomedical applications.

Last but not least, we would like to express our deepest gratitude to all the scientists who accepted to share their valuable knowledge and expertise in this book. This is truly their book that we were honored to aid in its conception, organization, and overall edition. Finally, a word of appreciation is due to Elsevier and all of its staff for believing in our work and helping in the making of this book.

Bruno Sarmento, and José das Neves

July 2017

Porto, Portugal

References

Araújo F, das Neves J, Martins J.P, Granja P.L, Santos H.A, Sarmento B. Functionalized materials for multistage platforms in the oral delivery of biopharmaceuticals. Prog. Mater. Sci. 2017;89:306–344.

Feynman R.P. There’s plenty of room at the bottom. Eng. Sci. 1960;23:22–36 Available at:. http://calteches.library.caltech.edu/1976/1/1960Bottom.pdf.

Chapter 1

From the Magic Bullet to Advanced Nanomaterials for Active Targeting in Diagnostics and Therapeutics

Alejandro Sosnik     Technion-Israel Institute of Technology, Haifa, Israel

Abstract

Paul Ehrlich, recipient of the Nobel Prize in Medicine in 1908 for his fundamental contributions to the understanding of the immune system, introduced the visionary concept of magic bullet, referring to an ideal therapeutic agent that selectively targets a pathogen, a cancer cell, or a toxin at sufficiently low concentrations that prevent any harm to the healthy cells of the patient. Many years later and primarily motivated by the urgent need to improve the diagnosis and chemotherapy of cancer, the conceptual revolution introduced by Ehrlich became the moto of drug designers and it was also embraced by the nanomedicine field, paving the way for the design of a plethora of innovative nanomaterials that owing to their small size and uniquely fine-tuned shape and surface properties target specific cell populations, tissues and organs by different passive and active pathways. This chapter will overview the most outstanding hallmarks in this thrilling way to realizing his pioneering vision with focus on the developments done in cancer, a disease that owing to its broad incidence worldwide became the flagship of the nanomedicine field.

Keywords

Albumin; EPR; Folate receptors; Lectin-like receptor; Magic bullet; Pathogens

Chapter Outline

1. Paul Ehrlich and the Magic Bullet

2. Passive Versus Active Targeting in Cancer as Model

2.1 Sugars

2.2 Transferrin and Lactoferrin

2.3 Folic Acid

2.4 Hyaluronic Acid

2.5 Antibodies

2.6 Aptamers

3. Emerging Challenges and Perspectives

Acknowledgments

References

1. Paul Ehrlich and the Magic Bullet

Paul Ehrlich, recipient of the Nobel Prize in Medicine in 1908 for his fundamental contributions to the understanding of the immune system, introduced the visionary concept of magic bullet (magische kugel in German) compounds more than one century ago (Winau et al., 2004; Schwartz, 2004). By magic bullet, he referred to an ideal therapeutic agent that selectively targets a pathogen, a cancer cell, or a toxin at sufficiently low concentrations that prevent any harm to the healthy cells of the patient. His research focused on the treatment of parasitic and bacterial infections and in the late 1900s led to the development of diamidodioxyarsenobenzol (also known as arsphenamine, Ehrlich 606, or Salvarsan), the first active agent for the treatment of syphilis, a bacterial infection caused by the spirochete Treponema pallidum (Winau et al., 2004). Intriguingly, the chemical structure of this drug remained under debate for almost 100  years and mass spectroscopy studies published in 2005 revealed that in fact it likely is the mixture of two small arsenic rings, a trimer (Fig. 1.1A) and a pentamer (Fig. 1.1B), and not the originally proposed noncyclic molecule (Fig. 1.1C) (Lloyd et al., 2005).

Figure 1.1  Proposed chemical structures of Salvarsan. Recent research revealed that (A) and (B) are more likely than the originally proposed (C).

Many years later and primarily motivated by the urgent need to improve the diagnosis and chemotherapy of cancer, the conceptual revolution introduced by Ehrlich that became the moto of drug designers was also embraced by the nanomedicine field (the application of nanotechnology tools in diagnosis, prophylaxis, and therapy of disease) and it paved the way for the design of a plethora of innovative nanomaterials that owing to their small size and uniquely fine-tuned shape and surface properties target specific cell populations by different passive and active pathways (Zhao, 2005; Datta et al., 2016). This chapter will overview the most outstanding hallmarks in the thrilling and, at the same time, struggling way of nanomedicine to realizing Ehrlich’s pioneering vision with special focus on cancer, a disease that owing to its broad incidence and high mortality rates worldwide led to remarkable breakthroughs that improved the efficacy of the diagnosis and the chemotherapy.

2. Passive Versus Active Targeting in Cancer as Model

The rationale behind the magic bullet was to make selective the interaction between the diagnostic and therapeutic agent with molecular or cellular structures of the pathogen and thus to minimize toxic effects on the healthy cells of the host. This could be clearly exemplified for antibiotics where the different families target pathways that are exclusive in bacteria without interacting with counterpart ones (e.g., protein synthesis) in the host (Fig. 1.2) (Coates et al., 2002; Lewis, 2013).

Antiviral (De Clercq, 2007), antiprotozoal (Horn and Duraisingh, 2014), and antifungal (Roemer and Krysan, 2014) drugs also inhibit mechanisms that are vital for the growth and proliferation of the pathogen with minimal or no effect on eukaryotic cells. At the same time, it is worth remarking that regardless of their specificity, these drugs are accompanied by side effects that might range from negligible to severe.

Figure 1.2  Main targets of antibacterial drugs in bacteria: cell wall synthesis, DNA gyrase, metabolic enzymes, DNA-directed RNA polymerase, and protein synthesis. In the case of protein synthesis, aminoglycosides and tetracyclines target the 30S RNA, and macrolides, chloramphenicol, and clindamycin inhibit 50S RNA. Reproduced from Coates, A., Hu, Y., Bax, R., Page, C., 2002. The future challenges facing the development of new antimicrobial drugs. Nat. Rev. Drug Discov. 1, 895–910 with permission of Nature Publishing Group.

Cancer puts together different pathologies associated with abnormal and uncontrolled cell growth and displays the potential to spread to other body sites. Cancers claim 8.2  million lives every year, and with an expected increase of 70% of the cases in the next two decades, it represents one of the leading causes of death worldwide (Cancer - World Health Organization). Tremendous progresses have been made in the chemotherapy of cancer from the use of arsenicals in the early 1900s to molecular-targeting drugs that capitalize on the overexpression of specific receptors by tumor cells such as the tyrosine kinase inhibitors introduced in the mid-2000s (DeVita and Chu, 2008); note that Ehrlich also coined the term chemotherapy for the use of chemicals to treat disease. Regretfully, the specificity of anticancer drugs remains elusive and they display serious short- and long-term side effects that in many cases preclude the continuation of the treatment (Ahmad et al., 2016). Moreover, diagnosis in advanced stages of the disease and development of resistance reduces dramatically the therapeutic repertoire and the chance of cure (Zhou et al., 2017). Thus, investigation of more sensitive diagnostic tools became a field of equal impact as an effective chemotherapy (Kasahara and Tsukada, 2004). In this scenario, nanotechnology emerged as a phenomenal toolbox to make diagnosis more sensitive and efficacious and to overcome main pharmacokinetics, pharmacodynamics, and toxicological disadvantages of anticancer drugs through to the modification of fundamental features such as aqueous solubility and physicochemical stability in the biological milieu and pharmacokinetic parameters (e.g., increased biodistribution in the tumor with respect to off-target tissues and organs) (Heath and Davis, 2008; Ferrari, 2005; Schroeder et al., 2012). Another beneficial effect of nanomedicines would pertain to the ability to overcome resistance mechanisms such as efflux transporters of the adenosine triphosphate (ATP)-binding cassette superfamily, which reduce the effective intracellular concentration of the drug in the target cells (Sosnik, 2013). The ability to manipulate the matter at the atomic and molecular level and the invention of cutting-edge characterization methods (e.g., scanning tunnel microscope) led to the emergence of nanoscience and nanotechnology. More recently, the application of these tools to medicine gave birth to the field of nanomedicine and led to a revolution in the capabilities to diagnose and treat disease. For instance, the term nanomedicine was probably used for the first time in the book "Unbounding the Future: The Nanotechnology Revolution" authored by Drexler et al. (1991) and published by Morrow in 1991. First reports on the synthesis of nanoparticles for drug delivery date from the 1960s (Kreuter, 2007) and it was only in 1995 that an intravenous liposomal formulation of the anthracycline antibiotic doxorubicin commercialized as Doxil or Caelyx (Janssen) or the generic Myocet (Teva Pharmaceuticals) formally became the first US Food and Drug Administration (FDA)-approved nanomedicine (Barenholz, 2012). The most advantageous feature of this pioneering nanopharmaceutical was the ability to increase the accumulation of the cargo in highly vascularized tumors by the so-called enhanced permeability and retention (EPR) effect, a passive targeting pathway that relies on the presence of vascular imperfections (fenestrations) at the nanometer scale range in the endothelium that enables the extravasation of sufficiently small nanomaterials to the tumoral stroma and their increased accumulation with respect to the free drug (Fig. 1.3) (Fang et al., 2011). This phenomenon is accompanied by a lack of lymphatic drainage that disfavors clearance. Liposomal doxorubicin also reduces the exposure of cardiac muscle to the drug and its cardiotoxicity, confirming that the biodistribution is governed by the nanocarrier (Safra et al., 2000).

However, in nonsolid (e.g., leukemia) or poorly vascularized tumors (e.g., bladder carcinoma), this mechanism cannot be exploited (Prabhakar et al., 2013).

A similar principle of increased vascular permeability has been investigated in more recent years for the treatment of inflammatory diseases, among them infections (Fig. 1.4) (Azzopardi et al., 2013; Nehoff et al., 2014).

Regardless of the remarkable breakthrough achieved with the EPR effect, the benefits of nanomedicines remained relatively limited because the increased accumulation in the tumor stroma did not ensure significantly higher intracellular delivery of the chemotherapy. Thus, the further modification of the nanomaterial surface with specific ligands that selectively bind cellular structures (e.g., receptors) overexpressed in the diseased (e.g., cancer) cells was attempted to favor the internalization of the drug-loaded nanocarrier by diverse endocytic pathways, a strategy known as active targeting (Fig. 1.3) (Ferrari, 2005; Byrne et al., 2008). A paradigmatic example of a nanomedicine involving both passive and active targeting is albumin-bound paclitaxel (nab-paclitaxel, Abraxane, Celgene Corp.) used in the therapy of metastatic breast, ovarian, and non–small cell lung cancer (Fig. 1.5) (Desai, 2012).

Figure 1.3  Nanomaterials extravasate into the tumor stroma through the fenestrations of the endothelium, a passive targeting pathway known as enhanced permeability and retention effect. Then, modification of the nanomaterial surface with specific ligands is exploited to make the cellular uptake more selective by an active targeting pathway. Reproduced from Ferrari, M., 2005. Cancer nanotechnology: opportunities and challenges. Nat. Rev. Cancer 5, 161–171 with permission of Nature Publishing Group.

This nanopharmaceutical product utilizes albumin transport pathways, including the glycoprotein 60 albumin receptor and subsequent caveolae-mediated endothelial transcytosis across the endothelium of the blood-tumor barrier and interaction with albumin-binding proteins in the tumor parenchyma such as secreted protein acidic and rich in cysteine (Fig. 1.6) (Desai, 2012; Hawkins et al., 2008; Yardle, 2013).

So far, nab-paclitaxel remains a one-of-a-kind example of actively targeted nanomedicine that has been approved by the FDA in 2005 and by the European Medicines Agency in 2008 for metastatic breast cancer. Numerous attempts to translate actively targeted nanomedicines to the clinics failed in different stages or were abandoned because of economic considerations, while few products are still undergoing preclinical and early clinical trials (Xu et al., 2015).

Figure 1.4  Effect of inflammation on the development of the enhanced permeability and retention (EPR) effect in inflammatory tissue. Inflammatory tissue will release a range of mediators that will induce the EPR effect. Inflammation will cause the vessel to dilate resulting in a higher blood flow. Furthermore, the contraction of endothelial cells will allow the penetration of nanoparticles into the tissue. The major difference between inflammatory tissue and tumor tissues in relation to macromolecular targeting is the presence of a functional lymphatic system in inflammation. Retention of nanomedicine in this case can be attributed to macrophage uptake. Reproduced from Nehoff, H., Parayath, N.N., Domanovitch, L., Taurin, S., Greish, K., 2014. Nanomedicine for drug targeting: strategies beyond the enhanced permeability and retention effect. Int. J. Nanomed. 9, 2539–2555 with permission of Dove Press.

An issue that remains elusive and controversial around active targeting is related to the fact that upon intravenous administration, nanoparticles usually undergo adsorption of plasma proteins (a process known as opsonization) such as albumins, fibronectins, complement proteins, immunoglobulins, and apolipoproteins, and thus the surface ligands could be partly or completely masked, precluding their direct interaction and binding to the target (Nie, 2010). Although that in vitro studies of protein adsorption are often conducted to characterize this interaction, their ability to predict the performance in the complex in vivo environment is very low. Another constraint of active targeting with nanomedicines resides in the common use of expensive ligands (e.g., antibodies) and complex synthetic and purification pathways and production processes that are feasible in a laboratory scale, though that make scalability under an industrial setting cost-inviable and/or that have a strong impact on the final cost of the medication (Muthu and Wilson, 2012; Hare et al., 2017; Landesman-Milo and Peer, 2016). At the same time, there exists strong experimental evidence that if the nanocarrier is designed properly and using technologies that are more easily scaled up (e.g., spray drying) (Sosnik and Seremeta, 2015), active targeting might breakthrough the treatment of disease, especially considering the possibility of dramatically improving the efficacy of old (and usually cheaper) anticancer drugs as opposed to more innovative and expensive ones of controversial medical benefit (Siddiqui and Rajkumar, 2012).

Figure 1.5  (A) Scheme of the nab-paclitaxel structure and (B) cryo-TEM microphotograph showing the spherical morphology of the nanoparticle. TEM , transmission electron microscope. Reproduced from Desai, N., 2012. Challenges in development of nanoparticle-based therapeutics. AAPS J. 14, 282–295 with permission of Springer.

Figure 1.6  Two routes of albumin to reach tumors. The left half of the figure shows albumin metabolism of a growing tumor, whereas the right half reflects the cytotoxic effect on tumor cells from the uptake of albumin-bound paclitaxel. Albumin and albumin-bound paclitaxel are hypothesized to reach the tumor stroma by both transcytosis and the enhanced permeability and retention effect. Reproduced from Yardle, D.A., 2013. nab-Paclitaxel mechanisms of action and delivery. J. Control. Release 170, 365–372 with permission of Elsevier.

Another crucial issue to consider is that there exists strong evidence that active targeting can take place only when the ligand and the target are at a distance <0.5  nm (Bae and Park, 2011). In this framework, it relies on the primary accumulation of the nanomedicine in the tumor by EPR effect, and thus it faces fundamental challenges such the variability of the effect among tumors and individuals (Harrington et al., 2001), relatively high pressure in the tumor stroma (Stohrer et al., 2000), nonhomogeneous vascular permeability in different areas of the tumor (Yuan et al., 1995), and heterogeneity of the overexpression of specific receptors among cancers and even in the same cancer (Rao et al., 2014). Moreover, the development of appropriate and clinically relevant animal models to assess the performance of the nanomedicine is another critical stage (Lammers et al., 2011).

In this chapter, the most popular ligands used for the surface decoration of nanocarriers in active drug targeting will be overviewed. The focus on cancer should not be understood as an attempt to neglect the contributions made in, for example, malaria, systemic mycoses, the infection by human immunodeficiency virus, and inflammatory gastrointestinal maladies but as an unbiased expression of how the field has progressed in the last 20  years and the clinical relevance of these developments; a search for nanomedicine in Scopus for the period 1997–2016 rendered 9860 articles, while more than 3700 were on cancer, representing almost 40%. The impact of cancer nanomedicine is even more noticeable in scientific meetings; here most sessions and oral presentations are on this specific niche. It is also worth pointing out that one of the most fundamental features for a ligand to be feasible is the availability of reactive functional group that enables easy conjugation and that do not play a key role in the binding to the receptor. Otherwise, its targeting capacity could be jeopardized.

2.1. Sugars

There exists a broad spectrum of transmembrane proteins that recognize sugars and sugar clusters. For example, macrophages are a phagocytic cell type of the immune system that expresses a receptor known as lectin-like receptor (LLR). LLRs recognize and bind sugar clusters in the wall of bacteria (Taylor et al., 2005). Another example is the asialoglycoprotein (ASP) receptor in hepatocytes. In this context, dextrans and glucose polymers have been tested for the targeted delivery to the liver and it was found that after being modified with galactose and mannose, dextran binds to hepatocytes and Kupffer cells, which are specialized cells in the liver as well (Cuestas et al., 2010). Dendritic cells (DCs) are a key player of the immune system. DCs bind the mucus owing to the expression of sugar receptors with a strong affinity to a number of different sugars (e.g., N-acetyl galactosamine, L-fucose, and D-mannose) (Uwatoku et al., 2001). Finally, different tumors (e.g., breast) including many pediatric ones are sugar-avid and upregulate glucose transporters known as glucose receptor (GLUT) (Macheda et al., 2005). For example, a peculiarity of rhabdomyosarcoma is the overexpression of glucose receptors 1, 3, and 4 (Ito et al., 2000). A similar phenomenon is observed in Ewing sarcoma (McCarville et al., 2005). In advance, the main sugar transporters will be briefly overviewed.

LLRs are a type of protein that binds to the cell membrane and has high affinity for saccharides. They consist of a large family of lectins that can be divided according to their structural similarities and different functionalities and which play an important role in the immune system (Zelensky and Gready, 2005). LLRs functionality includes recognition of pathogen-associated molecular patterns and their phagocytosis and recognition of endogenous ligands and mediated cell–cell interaction, either between the white blood cells themselves or with the endothelium. In addition, they recognize carbohydrate structures and form multimeric complexes that contribute to increasing the ligand binding (Mitchell et al., 2001). Moreover, they are involved in cell adhesion and migration. However, LLRs do not discriminate well between self- and non-self sugars. The pathogen recognition ability by soluble lectins such as the macrophage cell surface mannose receptor is influenced by the characteristics of the terminal monosaccharide residue at the surface of the cells. The glycosylation of LLR ligands affects the binding ability and influences the development, survival, migration, and the reactivity of the immune cells (Daniels et al., 2002). Thus, the adhesive and the pathogen-binding properties of the leukocytes may greatly differ (Cambi and Figdor, 2003).

ASP receptors are lectins with an exposed galactose residue on removal of the terminal sialic acid from the glycoprotein and therefore rapidly cleared from the systemic circulation (Spiess, 1990). The functional ASP comprises two subunits, each one has a C-terminal carbohydrate recognition domain that interacts with matching galactose residues in the ligand glycan and results in the high affinity and specificity of the binding (Meier et al., 2000). They belong to the C-type classification of lectins that are characterized by Ca²+ requirement, are extracellularly located, are disulfide bonds-dependent for activity, and recognize a broad spectrum of specific sugar residues. ASP is a prototype to receptors that are able to penetrate the cell membrane via receptor-mediated endocytosis, an energy-dependent process. They are most commonly found in the liver; hence an active site for cell–cell interaction is provided on the hepatocyte membrane (Stockert, 1995).

Glucose is a key fuel in mammals and is obtained from ingested disaccharides and polysaccharides and it is also synthesized from other substrates in different organs. Regulation and control of the glucose concentration in the blood for cerebral metabolism and its delivery to the peripheral tissue for storage and utilization are of immense importance (Wood and Trayhurn, 2003). However, the plasma membrane is impermeable to polar molecules such as glucose and thus its uptake is accomplished by membrane-associated carrier proteins that are able to bind and transfer it across the lipid bilayer. Glucose is also a vital nutrient in diseased tissues, i.e., tumors, where its uptake is a fundamental mechanism for maintaining the accelerated metabolism and growth (Rown, 2000). GLUTs are one of these carrier proteins (Mueckler, 1992). There exist 14 GLUTs and the expression in tissues is dependent on the specific role of the glucose metabolism (McCarville et al., 2005). The GLUT family may be divided into three groups. Class I comprises the well-characterized GLUT1–GLUT4, while the fructose transporter GLUT5 and GLUT7, GLU9, and GLUT11 belong to Class II. Finally, Class III gathers five recently discovered proteins GLUT6, GLUT8, GLUT10, GLUT12, and H+ myo-inositol transporter. Each one of the GLUT types has its own affinity toward glucose and other sugars. GLUT1, GLU3, and GLU4 present the highest affinity for glucose. In the case of cancer, there is an increased glucose transport toward the tumor. Thus, targeting the specific GLUT could provide more efficacious detection and treatment methods for the disease (Macheda et al., 2005). Moreover, the right design would allow to discern between different cell types in the same organ. For example, hepatocytes versus Kupffer cells in the liver display receptors that are selective for glucose and galactose, respectively (Cuestas et al., 2010). Thus, this could be capitalized on to reduce toxicity in off-target cells.

Because of the diversity of sugar receptors and possible substrates, the glycosylation of drug-loaded nanocarriers with different types of simple sugars and glycans has been extensively and systematically investigated to improve the treatment of disease with special interest in cancer by exploiting the expression or overexpression of a variety of transmembrane sugar receptors in specific cells types (Bojarova and Kren, 2016). This work included the development of innovative methods for the synthesis of glycosylated biomaterials (Fig. 1.7) (Macheda et al., 2005; Glisoni and Sosnik, 2014; Bukchin, 2016). One of the advantages of this approach is the relatively high versatility of the chemistry and the nonimmunogenicity of the ligands.

However, as previously mentioned, these efforts remain to date untranslated into the clinics.

2.2. Transferrin and Lactoferrin

The transferrin receptor (TfR1 or CD71) is ubiquitously expressed in most normal tissues and plays a key role in the cell internalization of iron ions by endocytosis of the TfR-iron complex (Daniels et al., 2006). The expression of another member of this family, TfR2, is mainly restricted to hepatocytes. TfR1 is overexpressed in cancerous cells, and thus its levels have been used to correlate disease state and progression (Daniels et al., 2006, 2012). Thus, active targeting of antineoplastic drugs to cancer cells has been attempted by the conjugation of active agents or the surface modification of drug-loaded nanocarriers with the natural Tf or TfR monoclonal antibodies or its fragments (e.g., single-chain variable fragments of immunoglobulin) and short peptides that contain the active domains of the protein (Fig. 1.8) (Daniels et al., 2012). The main concerns of this strategy are the blockage of the interaction of the Tf-conjugates with the receptor by native Tf and the possible cytotoxicity on TfR2-expressing hepatocytes because of the nonselective nature of the interaction of Tf with both receptors. Because of the high expression of TfR in the blood–brain barrier, Tf-modified nanocarriers were also utilized for active targeting to the central nervous system, though because of limitations, peptide shuttles that resemble its structure were successfully designed (Oller-Salvia et al., 2016). Another strategy is the antagonizing of the receptor function by means of monoclonal antibodies (Crépin et al., 2010). For a detailed description of TfR targeting, the readers are recommended to read the review of Daniels et al. (2012).

Figure 1.7  (A) Generation of glycans for carbohydrate libraries. Glycans prepared by synthetic or enzymatic methods and by genetic engineering or from natural sources. LG , leaving group; PG , protecting group. (B) Synthetic pathway of the microwave-assisted ring-opening conjugation of gluconolactone (b) to the terminal -OH group of Pluronic F127 (a) to render a glucosylated derivative of the synthetic poly(ethylene oxide)- b -poly(propylene oxide)- b -poly(ethylene oxide) triblock (c). (A) Reproduced from Bojarova, P., Kren, V., 2016. Sugared biomaterial binding lectins: achievements and perspectives. Biomater. Sci. 4, 1142–1160 with permission of the Royal Society of Chemical and (B) reproduced from Glisoni, R.J., Sosnik, A., 2014. Novel poly(ethylene oxide)-co-poly(propylene oxide) copolymer-glucose conjugate by the microwave-assisted ring opening of a sugar lactone. Macromol. Biosci. 14, 1639–1651 with permission of Springer.

Figure 1.8  Examples of strategies for targeting therapeutic agents via transferrin receptor (TfR) to malignant cells. Targeting can be mediated by its natural ligand Tf, a specific peptide, monoclonal antibodies, or single-chain antibody fragments specific for the extracellular domain of the TfR. The therapeutic agent can be delivered conjugated to the compound or enclosed in a carrier. Targeting the TfR has been an option to deliver chemotherapeutic drugs, therapeutic proteins, genes in vectors, oligonucleotides, or radionuclides. Reproduced from Daniels, T.R., Bernabeu, E., Rodríguez, J.A., Patel, S., Kozman, M., Chiappetta, D.A., Holler, E., Ljubimova, J.Y., Helguera, G., Penichet, M.L., 2012. The transferrin receptor and the targeted delivery of therapeutic agents against cancer. Biochim. Biophys. Acta 1820, 291–317 with permission of Elsevier.

Lactoferrin or lactotransferrin (Lf) is a Tf analog found in bovine and human milk and other secretion fluids (e.g., saliva), which is also involved in the iron metabolism (Lönnerdal and Iyer, 1995). Lf receptors exist in the small intestine, the monocytes/macrophages system, and the blood–brain barrier, though Lf can also interact with other receptors such as low-density lipoprotein receptor-related protein receptors and the ASP receptors in the liver (Wei et al., 2012). The use of Lf for active targeting of chemotherapy in cancer has been focused on the imagining and therapy of tumors of the central nervous system (e.g., glioma) (Xu et al., 2017; Fang et al., 2016; Tomitaka et al., 2015; Lim et al., 2015) and the liver (Wei et al., 2015) owing to the fact that LfR is overexpressed in these tissues and not specifically in any cancer type. Incipient research has been also conducted in lung cancers such as bronchogenic carcinoma because LfR is expressed on the apical side of bronchial epithelial cells (Pandey et al., 2015; Kurmi et al., 2011). Two drug delivery systems employing this targeting approach are under clinical trials. PEGylated polymeric nanoparticles loaded with αRRM2 siRNA (CALAA-01, Calando Pharmaceuticals) is in Phase I for the treatment of solid tumors (Davis, 2009; Zuckerman and Davis, 2015). Another technology platform of transferrin-conjugated nanocarriers was developed by Mebiopharm (Mebiopharm); its liposomal oxaliplatin (MBP-426) is in Phase II for gastric and esophageal cancer (Sankhala et al., 2009), while other drug delivery systems are in preclinical phase (Mebiopharm).

2.3. Folic Acid

Folate receptors (FRs) bind folic acid (also known as vitamin B9) and its reduced forms and they are involved in the intracellular delivery of tetrahydrofolate (Wibowo et al., 2013). In humans, there are three functional isoforms, namely hFRα, hFRβ, and hFRγ (Elnakat and Ratnam, 2004). hFRα is overexpressed in a broad variety of cancers, among them adenocarcinomas of uterus, ovary, breast, cervix, kidney and colon and testicular choriocarcinoma, ependymal brain tumors, malignant pleural mesothelioma, and nonfunctioning pituitary adenocarcinoma, while hFRβ in leukemias and activated macrophages (Wibowo et al., 2013; Low et al., 2007). Conversely, the expression in healthy tissues is limited to the placenta, lung, kidneys, and choroid plexus (Parker et al., 2005). In fact, since the pioneering publication by Leamon and Low in 1991, the active targeting of hFRα became a Trojan horse in the race toward a more effective diagnosis and chemotherapy of cancer and inflammatory diseases (Fig. 1.9) (Low et al., 2007), reaching clinical stages more than a decade ago with drug–folate conjugates (Parker et al., 2005; Paulos et al., 2004) and more recently with folate–vaccine and folate–monoclonal antibody conjugates (Cheung et al., 2016). Conversely, the clinical evaluation of folate-modified nanomedicines loaded with anticancer drugs has not been extended to clinics yet and it mainly remained limited to the targeting of metallic nanoparticles for cancer diagnosis.

Figure 1.9  Folate receptor (FR)-mediated endocytosis of a folic acid–drug conjugate. Folate conjugates bind FR with high affinity and are subsequently internalized into endosomes that can reduce disulfide bonds. Within the endosome, a folate–disulfide–drug conjugate is released from the FR and the prodrug is reduced to liberate the parent drug cargo. Because the pH of FR-containing endosomes is only mildly acidic, acid-labile linkers do not release the attached drug as efficiently. A similar pathway could be exploited for the delivery of drug-loaded nanocarriers. Reproduced from Low, P.S., Henne, W.A., Doorneweerd, D.D., 2007. Discovery and development of folic-acid-based receptor targeting for imaging and therapy of cancer and inflammatory diseases. Acc. Chem. Res. 41, 120–129 with permission of the American Chemical Society.

Currently, several products for the treatment of cancer, inflammation, and polycystic kidney disease are in the pipeline of Endocyte, Inc. and under different stages of advanced preclinical or early clinical research (Endocyte Inc.).

2.4. Hyaluronic Acid

Cluster of differentiation-44 (CD44) is a glycoprotein ubiquitously expressed on the surface of a broad spectrum of mammalian cells (e.g., epithelial cells) and that plays a key role in cell–cell interactions (Ponta et al., 2003). The receptor is overexpressed in solid tumors, among them pancreas, breast, and lung cancer, and thus, different research groups have dedicated efforts to actively target it and by doing so to improve the efficacy of the chemotherapy (Mattheolabakis et al., 2015; Rao et al., 2016; Cadete and Alonso, 2016). Hyaluronic acid is an anionic nonsulfated glycosaminoglycan component of the extracellular matrix, and because of its biocompatibility and nonimmunogenicity, it is profusely used in drug delivery and tissue engineering applications. In prostate and breast cancer, hyaluronic acid is used as marker of tumor progression and malignancy. Hyaluronic acid strongly binds the CD44 receptor and it became a very popular approach to target different diagnostic and therapeutic nanomedicines to CD44+ cells with special interest in breast cancer and for the delivery of doxorubicin or paclitaxel (Table 1.1) (Dosio et al., 2016).

2.5. Antibodies

Antigen-antibody reactions are highly specific and selective, and thus the conjugation of full antibodies or their fragments has attracted major attention to actively target drugs, biologicals, and diagnostic and therapeutic nanomaterials (Fay and Scott, 2011). Since the pioneering and enlightening works on the design and performance of immunoliposomes in the early 1980s by Huang et al. (Huang et al., 1983; Connor and Huang, 1985) to date, a profuse research has been published in the field with special interest in cancer imaging and chemotherapy (Kozlowska et al., 2009; Cheng and Allen, 2010; Mastrobattista et al., 1999). However, immunoliposomes were found to increase the extent of cellular uptake, though not of tumor localization in vivo (Kirpotin et al., 2006; Bartlett et al., 2007). There exist a number of antibody-targeted nanomedicines for the chemotherapy of cancer in Phase I clinical trials (Table 1.2), though one of the main limitations of this strategy is the cost of the product that in many cases becomes prohibitive (van der Meel et al., 2013).

2.6. Aptamers

Aptamers are single-stranded RNA or DNA oligonucleotides or oligopeptides (5–20 amino acids) that bind the target molecule (e.g., gene or protein) with high affinity and selectivity (Weihong et al., 2011; Reverdatto et al., 2015). They are selected from a random pool of molecules initially containing as much as 10¹³–10¹⁶ sequences through an in vitro combinatorial selection process that in the case of oligonucleotide aptamers is known as systematic evolution of ligands by exponential enrichment (SELEX) (Weihong et al., 2011).

The conjugation of aptamers to nanomaterials of different nature has been recently explored as an innovative, safe, and cost-viable strategy to actively target diagnostic and therapeutic agents (Table 1.3) (Weihong et al., 2011). Owing to their small size and high stability, their manipulation is simpler than antibodies, enabling the fine-tuning of the modification extent. In this framework, they have shown promising results in the targeting of tumor cells and transport of small molecules (e.g., proteins, drugs, siRNA) through the microvasculature or the tumor stroma. It is important to stress that regardless of encouraging preliminary results, often works employ nanomaterials that have not been approved as biomaterials (e.g., carbon nanotubes). The company BIND Biosciences founded in 2007 has developed a series of targeted nanopharmaceuticals with RNA and peptide aptamers as targeting ligand, among them BIND-014, a delivery system of docetaxel-loaded polymeric nanoparticles that target the prostate-specific membrane antigen and reached Phase I clinical trials (Hrkach et al., 2012; Biosciences, 2013). This aptamer-targeted nanomedicine was the first to ever reach clinical trials. Intriguingly, aptamers also display anticancer and antiviral activity, and thus the targeting features can be combined with therapeutic ones.

Table 1.1

Reproduced with modifications from Dosio, F., Arpicco, S., Stella, B., Fattal, E., 2016. Hyaluronic acid for anticancer drug and nucleic acid delivery. Adv. Drug Deliv. Rev. 97, 204–236 with permission of Elsevier.

Table 1.2

Reproduced with modifications from van der Meel, R., Vehmeijer, L.J.C., Kok, R.J., Storm, G., van Gaal, E.V.B., 2013. Ligand-targeted particulate nanomedicines undergoing clinical evaluation: current status. Adv. Drug Deliv. Rev. 65, 1284–1298 with permission of Elsevier. The clinical status of the different products has been updated with information available from the corresponding companies.

Table 1.3

EGFR, epidermal growth factor receptor; PSMA, prostate-specific membrane antigen; SELEX, systematic evolution of ligands by exponential enrichment.

Reproduced from Weihong, T., Wang, H., Chen, Y., Zhang, Y., Zhu, H., Yang, C., Yang, R., 2011. Molecular aptamers for drug delivery. Trends Biotechnol. 29, 634–640 with permission of Elsevier.

3. Emerging Challenges and Perspectives

The emergence and progress of nanomedicine was motivated by the interest to overcome serious drawbacks in the chemotherapy of cancer, where efficacy remains elusive and relapse and metastasis are common outcomes. These phenomena originate in the intratumor heterogeneity owing to genetic and nongenetic determinants that include the properties of the tumor microenvironment (Kreso and Dick, 2014) and the presence of cell populations that, as in the case of tumor-associated macrophages, facilitate neoplastic transformation, tumor immune evasion, and the subsequent metastatic cascade (Williams et al., 2016). For example, there is solid evidence that cancer stem cells (CSCs), a cell population that displays self-renewal and differentiation potential, are less sensitive to conventional chemotherapy and that they might play a fundamental clinical role by being involved in the development of resistance and metastasis (He et al., 2016; Geng et al., 2014). Because of this, the design of innovative strategies that target CSCs and tumor-associated macrophages has emerged in recent years as a new approach to improve the treatment of the disease (Williams et al., 2016). These strategies capitalize on the experience previously gained in the field and utilize similar nanocarriers and targeting ligands, as exemplified in Table 1.4 for breast CSCs (He et al., 2016). At the same time, it is remarkable how incipient works at the interface of nanoscience and nanotechnology and cancer that emerged several decades ago and, in some way, hoarded the field for many years, fueled the exploration of such a diversity of innovative nanodrug delivery systems for the optimization of an immense spectrum of acute and chronic congenital and acquired diseases, including poverty-related infections, cardiovascular diseases, and diabetes. The growth and diversification of the nanomedicine field has been uncountable, though it is worth pointing out that, at the same time, it has not been smooth and deprived of hurdles. The perception and the confidence of the public in the potential benefits of nanomedicine are often in jeopardy mainly because of overestimated and unrealized expectations about these benefits and ethical concerns (Sechi et al., 2014; Satalkar et al., 2016a,b). To affront this, more rigorous, robust, validated, and harmonized production and characterization protocols than those usually reported in the scientific literature and that facilitate the standardized evaluation of the performance and the nanotoxicology in more reliable models of disease need to be established. Finally, scalability and cost-viability are two aspects that cannot be neglected anymore, and for this, the use of processes that utilize equipment that complies with the regulations of the pharmaceutical industry will be critical to maximize our capacity to successfully crystallize Ehrlich’s centennial magic bullets vision.

Table 1.4

Reproduced with modifications from He, L., Gu, J., Lim, L.Y., Yuan, Z-X., Mo, J., 2016. Nanomedicine-mediated therapies to target breast CSCs. Front. Pharmacol. 7, a313.

Acknowledgments

The Sosnik’s laboratory is funded by the European Union’s Seventh Framework Programme (grant #612765-MC-NANOTAR), the Phyllis and Joseph Gurwin Fund for Scientific Advancement, the Niedersächsisches Ministerium für Wissenschaft und Kultur and VolkswagenStiftung, the Israel Science Foundation (grant #269/15), EuroNanoMed-II Cure2DIPG, and the Teva National Network of Excellence in Neuroscience Research.

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