Polymeric Nanomaterials in Nanotherapeutics

By Elsevier Science

Polymeric Nanomaterials in Nanotherapeutics describes how polymeric nanosensors and nanorobotics are used for biomedical instrumentation, surgery, diagnosis and targeted drug delivery for cancer, pharmacokinetics, monitoring of diabetes and healthcare. Key areas of coverage include drug administration and formulations for targeted delivery and release of active agents (drug molecules) to non-healthy tissues and cells. The book demonstrates how these are applied to dental work, wound healing, cancer, cardiovascular diseases, neurodegenerative disorders, infectious diseases, chronic inflammatory diseases, metabolic diseases, and more. Methods of administration discussed include oral, dental, topical and transdermal, pulmonary and nasal, ocular, vaginal, and brain drug delivery and targeting.

Drug delivery topics treated in several subchapters includes materials for active targeting and cases study of polymeric nanomaterials in clinical trials. The toxicity and regulatory status of therapeutic polymeric nanomaterials are also examined. The book gives a broad perspective on the topic for researchers, postgraduate students and professionals in the biomaterials, biotechnology, and biomedical fields.

• Shows how the properties of polymeric nanomaterials can be used to create more efficient medical treatments/therapies
• Demonstrates the potential and range of applications of polymeric nanomaterials in disease prevention, diagnosis, drug development, and for improving treatment outcomes
• Accurately explains how nanotherapeutics can help in solving problems in the field through the latest technologies and formulations

• Language: English
• Publisher: Elsevier Science
• Release date: Oct 26, 2018
• ISBN: 9780128139332

Book preview

Polymeric Nanomaterials In Nanotherapeutics

Edited by

Cornelia Vasile

Physical Chemistry of Polymers Department, Romanian Academy, P. Poni Institute of Macromolecular Chemistry, Iasi, Romania

Micro & Nano Technologies Series

Table of Contents

Cover image

Title page

Copyright

List of Contributors

Chapter 1. Polymeric Nanomaterials: Recent Developments, Properties and Medical Applications

Abstract

1.1 Introduction

1.2 Types of Nanomedicines/Nanotherapeutics and Nanoformulations

1.3 Stealth Strategies to Improve Therapeutic Efficacy of Drug Nanocarriers

1.4 Nanomaterials: Physicochemical Properties and Risk Assessment

1.5 Applications

1.6 States of Some Nanotherapeutics

1.7 Challenges and Future Trends

References

Further Reading

Chapter 2. Responsive Polymeric Nanotherapeutics

Abstract

Acknowledgments

2.1 Introduction

2.2 External Stimuli-Sensitive Nanosystems

2.3 Internal Stimuli-Sensitive Nanosystems: Bioresponsive Nanotherapeutics

2.4 Responsive Dendrimers

2.5 Dual-Responsive Nanotherapeutics

2.6 Functionalization of Nanoparticles to Create Stimuli Responsiveness

2.7 Conclusion

References

Chapter 3. Nanorobots With Applications in Medicine

Abstract

3.1 Introduction

3.2 Design of Nanorobots

3.3 Classification of the Nanorobotic Systems

3.4 Medical Applications of Nanorobots

3.5 Conclusions

References

Chapter 4. Polymeric Nanobiosensors

Abstract

4.1 Introduction

4.2 Polymeric Materials Used in Biosensors

4.3 Conclusions and Future Trends

References

Chapter 5. Nanomaterials Derived From Phosphorus-Containing Polymers: Diversity of Structures and Applications

Abstract

5.1 Introduction

5.2 Synthetic Approaches to Phosphorus-Containing Polymers

5.3 Nanomaterials Derived From Phosphorus-Containing Polymers

5.4 Biorelated Properties

5.5 Nanotherapeutic Applications

5.6 Conclusion

References

Further Reading

Chapter 6. Nucleic Acids–based Bionanomaterials for Drug and Gene Therapy

Abstract

Acknowledgment

6.1 Introduction

6.2 Nanotechnology Applications in Nucleic Acid Delivery and Gene Therapies

6.3 Strategies for Obtaining Nucleic Acids–based Nanotherapeutics

6.4 U.S. Food & Drug Administration–Approved Cellular and Gene Therapy Products

6.5 Conclusions and Future Trends

References

Further Reading

Chapter 7. Electrospun Polymeric Nanostructures With Applications in Nanomedicine

Abstract

7.1 Introduction

7.2 Electrospinning Applications

7.3 Applications of Aligned Nanofibers

7.4 Applications of the Electrospun Nanofibers as Tubular Scaffolds and Neural Tissue Engineering

7.5 Conclusions and Future Trends

References

Further Reading

Chapter 8. Nanocoatings: Preparation, Properties, and Biomedical Applications

Abstract

8.1 Introduction

8.2 Definition and Preparation Methods of Nanocoatings

8.3 Application of Nanocoatings in Biomedical Fields

8.4 Conclusion and Future Trends

References

Further Reading

Chapter 9. Functionalization of Polymer Materials for Medical Applications Using Chitosan Nanolayers

Abstract

Acknowledgments

9.1 Introduction

9.2 Chemical Structure of Chitosan

9.3 Chitosan Nanoparticles

9.4 Mechanisms of Chitosan’s Antimicrobial Action

9.5 Impact of Chitosan and Chitosan Nanoparticle Composites for Various Advanced Applications

9.6 Future Trends and Conclusions

References

Chapter 10. Magnetic Polymeric Nanocomposites

Abstract

10.1 Introduction

10.2 Magnetic Functionality at the Nanometer Scale

10.3 Magnetic Nanostructure Synthesis Techniques

10.4 Polymer Matrix: Evolution and Structure/Property Relationship of Nanocomposites

10.5 Current Strategies for Conception and Structural Optimization of Magnetic Polymeric Nanocomposites

10.6 Specific Requirements for Medical Applications

10.7 Medical Applications of Magnetic Polymeric Nanocomposites

10.8 Conclusions

References

Further Reading

Chapter 11. Nanogels Containing Polysaccharides for Bioapplications

Abstract

11.1 Introduction to Polysaccharide Nanogels

11.2 Nanogels Based on Homopolysaccharides

11.3 Nanogels Based on Heteropolysaccharides

11.4 Some Data on Clinical Trials of Nanogels Containing Polysaccharides

11.5 Conclusions and Future Perspectives

References

Chapter 12. Nanomaterials in Tissue Engineering

Abstract

12.1 Introduction

12.2 Nanomaterials for Soft Tissues

12.3 Nanomaterials for Soft–Hard Tissue Interfaces

12.4 Nanomaterials for Hard Tissues

12.5 Conclusions and Future Trends

References

Chapter 13. Nanoscaled Dispersed Systems Used in Drug-Delivery Applications

Abstract

Acknowledgments

13.1 Introduction

13.2 General Features of Nanodispersed Systems

13.3 Micro-/Nanoemulsions Used for Drug-Delivery Systems

13.4 Polymeric Nanodispersions for Drug-Delivery Systems

13.5 Applications of Pharmaceutical Nanodispersions as Drug-Delivery Systems

13.6 Conclusions and Future Perspectives

References

Further Reading

Chapter 14. Biological Applications of Nanoparticles in Optical Microscopy

Abstract

14.1 Introduction

14.2 Fluorescent Dots

14.3 Upconversion Nanoparticles

14.4 Metallic Nanoparticles

14.5 Nonfluorescent Nanoparticles

14.6 Nanoparticles as Carriers

14.7 Conclusions and Future Perspectives

References

Chapter 15. Regulatory Status of Therapeutic Polymeric Nanomaterials

Abstract

15.1 Introduction

15.2 Regulatory Agencies and Definitions of Nanotechnology

15.3 Regulatory Agencies and Issues for Nanotechnology and Nanomaterials

15.4 Regulations Concerning the Characterization of Nanomaterials

15.5 Regulations Concerning the Assessment of the Nanotechnology Risk for Human Health

15.6 Nanomedicine Safety Issues

15.7 Toxicity, Safety, and Risk Assessment With Polymeric Nanoparticles

15.8 Conclusions

References

Further Reading

Abbreviations

Index

Copyright

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List of Contributors

Mihaela Cristina Baican,     Grigore T. Popa University of Medicine and Pharmacy, Iasi, Romania

Matej Bračič,     Faculty of Mechanical Engineering, Institute of Engineering Materials and Design, University of Maribor, Maribor, Slovenia

Catalina Natalia Cheaburu-Yilmaz

Department of Physical Chemistry of Polymers, Petru Poni Institute of Macromolecular Chemistry, Iasi, Romania

Faculty of Pharmacy, Department of Pharmaceutical Technology, Ege University, Izmir, Turkey

Aurica P. Chiriac,     Laboratory of Inorganic Polymers, Romanian Academy, Petru Poni Institute of Macromolecular Chemistry, Iasi, Romania

Sandra Madalina Constantin,     Pharmaceutical Chemistry Department, Grigore T. Popa University of Medicine and Pharmacy, Iasi, Romania

Andreia Corciova,     Control of Drugs Department, Medicine and Pharmacy University, Iasi, Romania

Alina Diaconu,     Laboratory of Inorganic Polymers, Romanian Academy, Petru Poni Institute of Macromolecular Chemistry, Iasi, Romania

Hatice Yesim Karasulu,     Faculty of Pharmacy, Department of Pharmaceutical Technology, Ege University, Izmir, Turkey

Neli Koseva,     Institute of Polymers, Bulgarian Academy of Sciences, Sofia, Bulgaria

Anca Margineanu,     Advanced Light Microscopy, Max Delbrück Centrum, Berlin, Germany

Violeta Mitova,     Institute of Polymers, Bulgarian Academy of Sciences, Sofia, Bulgaria

Bogdanel Silvestru Munteanu,     Faculty of Physics, Al. I. Cuza University of Iasi, Iasi, Romania

Iordana Neamtu,     Laboratory of Inorganic Polymers, Romanian Academy, Petru Poni Institute of Macromolecular Chemistry, Iasi, Romania

Manuela Tatiana Nistor,     Romanian Academy, Petru Poni Institute of Macromolecular Chemistry, Iasi, Romania

Loredana Elena Nita,     Laboratory of Inorganic Polymers, Romanian Academy, Petru Poni Institute of Macromolecular Chemistry, Iasi, Romania

Daniela Pamfil,     Physical Chemistry of Polymers Department, Romanian Academy, Petru Poni Institute of Macromolecular Chemistry, Iasi, Romania

Zdenka Peršin,     Faculty of Mechanical Engineering, Institute of Engineering Materials and Design, University of Maribor, Maribor, Slovenia

Lenuţa Profire,     Pharmaceutical Chemistry Department, Grigore T. Popa University of Medicine and Pharmacy, Iasi, Romania

Tijana Ristić,     Health Care Department, Tosama d.o.o., Production of Medical Supplies, Domžale, Slovenia

Alina Gabriela Rusu,     Laboratory of Inorganic Polymers, Romanian Academy, Petru Poni Institute of Macromolecular Chemistry, Iasi, Romania

Olivera Šauperl,     Faculty of Mechanical Engineering, Institute of Engineering Materials and Design, University of Maribor, Maribor, Slovenia

Elena Stoleru,     Physical Chemistry of Polymers Department, Romanian Academy, Petru Poni Institute of Macromolecular Chemistry, Iasi, Romania

Simona Strnad,     Faculty of Mechanical Engineering, Institute of Engineering Materials and Design, University of Maribor, Maribor, Slovenia

Zornica Todorova,     Institute of Polymers, Bulgarian Academy of Sciences, Sofia, Bulgaria

Ivelina Tsacheva,     Institute of Polymers, Bulgarian Academy of Sciences, Sofia, Bulgaria

Cornelia Vasile,     Physical Chemistry of Polymers Department, Romanian Academy, Petru Poni Institute of Macromolecular Chemistry, Iasi, Romania

Ali Yorgancioglu,     Faculty of Engineering, Leather Engineering Department, Ege University, İzmir, Turkey

Onur Yilmaz,     Faculty of Engineering, Leather Engineering Department, Ege University, İzmir, Turkey

Lidija Fras Zemljič,     Faculty of Mechanical Engineering, Institute of Engineering Materials and Design, University of Maribor, Maribor, Slovenia

Chapter 1

Polymeric Nanomaterials

Recent Developments, Properties and Medical Applications

Cornelia Vasile,    Physical Chemistry of Polymers Department, Romanian Academy, Petru Poni Institute of Macromolecular Chemistry, Iasi, Romania

Abstract

The unique physicochemical properties of polymeric nanomaterials (nanoscale size, large surface area to mass ratio, and high reactivity) individualize them in many application fields due to the specific features they offered to systems. Their use in nanomedicine has greatly changed the therapeutic and diagnostic modalities because they are precisely engineered materials at a molecular level. This chapter offers a general view on polymeric nanomaterials, including classification, properties, and a short methodology of characterization, applications, and the state of various nanotherapeutics. Selected types used in the medical field are described in subsequent chapters.

Keywords

Nanotherapeutics; nanomedicine; polymer; polymeric nanomaterials; diagnostic; properties; sheathed technologies

1.1 Introduction

Nanomaterials: in October 2011, the European Commission (EC) recommended to define nanomaterials as natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate/agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range of 1–100 nm and the specific surface area/volume of the material is greater than 60 m²/cm³ (EC, 2011; Kreyling et al., 2010). The Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR) proposed a limit of 0.15% of nanoparticles below 100 nm for the definition of nanomaterials (SCENIHR, 2010). Nanomaterials can be classified as zero-dimensional, one-dimensional, two-dimensional, or three-dimensional. Nanomaterials possess unique physical (ultrasmall size, large surface area to mass ratio, high surface energy, optical, electrical, magnetic, etc.), chemical (high reactivity), and biological properties, which are different from bulk materials of the same composition. By their characteristics nanomaterials are able to modify the fundamental properties of therapeutic and diagnostic agents and other materials.

Nanotechnology is defined as the science and engineering involved in the design, synthesis, characterization, and application of materials and devices whose smallest functional organization in at least one dimension is on the nanometer scale (10−9 m). That means it controls, programs, and manipulates matter on the nanometer length scale, that is, at the level of atoms, molecules, and supramolecular structures (i.e., molecular precision).

Nanotechnology applied in biotechnology is called nanobiotechnology (Torchilin, 2014).

Nanomedicine is considered as a subdiscipline within nanotechnology or nanosciences applied in medical sciences and helps in the prevention and treatment of various diseases. Diagnostic uses include for monitoring, repair, construction, and control of human biological systems at the molecular level, using engineered nanodevices and nanostructures (Sahoo et al., 2007). It combines nanotechnology with pharmaceutical and biomedical sciences. Nanomedicine goals are of developing novel therapeutic and diagnostic modalities and imaging agents with higher efficacy and improved safety and toxicological profiles. As a refinement of molecular medicine, nanomedicine integrates advances in genomics and proteomics, facilitating the development of personalized medicine (Jain, 2008; Zhang et al., 2007a).

The benefits of nanomedicine include: effective and less toxic therapeutic interventions, simplified therapeutic procedures, targeted drug delivery, accelerating the healing process, improved patient compliance and quality of patient’s life, reducing the frequency of dosage, minimally invasive method of administration, improved therapeutic outcomes, reducing adverse drug effects, personalized therapy, etc. From an economic point of view the benefits consist of an overall reduction in healthcare costs (e.g., by increasing the drug efficacy, reducing the duration of in-patient care stay, reducing personal healthcare costs, and the effective treatment of expensive major diseases), improving the quality of healthcare services, improved use of costly (bio)pharmaceuticals (e.g., low-dose formulation, improved drug solubility/stability, controlled drug release, improved pharmacokinetic profile, targeted drug delivery).

The integration of diagnostics with therapeutics facilitates the development of personalized medicine, that is, prescription of specific therapeutics best suited for an individual. Nanomedicine uses biomaterials, such as hard tissue implants, bone substitute materials, dental restoratives, soft tissue implants, and antimicrobial materials, drug carriers, etc. (Huber et al., 2009; Wagner et al., 2006c).

The number of academic papers using the term nanomedicine has increased exponentially since 2000 (Web of Science) from only a few to over a thousand. The use of nanoparticles (NPs) addresses two of the most important health challenges facing society: cancer treatment and the need for new antimicrobials.

Nanomedicines are defined as nanomaterials for specific diagnostic or therapeutic purposes (Kostarelos, 2006), as therapeutic or imaging agents. Nanomedicines control the in vivo biodistribution, improve targeting, enhance the efficacy, and reduce toxicity of a drug or biologic. It is known that the physiological and pathological processes at the cell level occur on a nanoscale. Nanoscale devices can readily interact with biomolecules (such as enzymes and receptors) on both the surface of the cell and inside the cell. Nanoscale devices are 100–10,000 times smaller than human cells. Therefore, nanoparticles can detect disease at the microlevel, provide detailed information on the progression of disease, and deliver treatment.

The terms nanopharmaceuticals and nanotherapeutics have been introduced, while colloidal systems are redefined as nanosystems, and colloidal drug-delivery systems are called nanodrug-delivery systems.

Nanotherapeutics, including polymeric ones, refers to the use of nanomedicines in areas of drug delivery and therapy conferring additional and unique properties to the drug (Hafner et al., 2014) with regard to bioavailability enhancement (Fakes et al., 2009; Lammers et al., 2012), reduced acute/systemic toxicity (Ando et al., 2011; Rom et al., 2013), or improved therapeutic efficiency by targeting compounds to a specific site of action (Low et al., 2011; Martinez et al., 2014). NPs have improved the bioavailability of drugs compared to their free form, such as cyclosporine (119% of free form) (Italia et al., 2007), estradiol (1014%) (Mittal et al., 2007), doxorubicin (DOX) (363%) (Grama et al., 2011), amphotericin B (793%) (Grama et al., 2011), curcumin (2583%) (Grama et al., 2011), (2200%) (Tsai et al., 2011), (1560%) (Khalil et al., 2013), and lutein (Chen et al., 2016). Nanotherapeutics tools are used to improve drug solubility/diffusivity of poorly water-soluble drugs (including micelles (Pepic et al., 2010) and nanocrystals (Junghanns and Müller, 2008)) to guide drugs to the desired location of action with increased precision (drug targeting (Crielaard et al., 2012;Zhang et al., 2012a)), to control drug release (nanoparticles (Hafner et al., 2009, 2011; Vasile et al., 2015a,b) and liposomes (Pavelic et al., 2005), and/or to enhance transport across biological barriers (micelles (Pepic et al., 2013)), prolong the half-life of drug systemic circulation by reducing immunogenicity, decreased degradation or physiologic clearance rates, and improve a drug’s therapeutic index and release characteristics (inherent characteristics also for nanomaterials). The release of drugs occurs at a sustained rate or in an environmentally responsive manner and thus lowers the frequency of administration. They are very promising for the treatment of cancer, diabetes, pain, asthma, allergy, bacterial infections, and so on (Brannon-Peppas and Blanchette, 2004; Kawasaki and Player, 2005). Polymeric nanotherapeutics also have the ability to improve pharmacokinetics of therapeutic cargoes: delivering drugs in a targeted manner to minimize systemic side effects, multidrug codelivery (two or more drugs simultaneously for combination therapy to generate a synergistic effect), and suppressing drug resistance, protecting DNA and other labile agents from degradation in endolysosomes (Panyam et al., 2002; Panyam and Labhasetwar, 2003), and can be functionalized with targeting ligands for active targeted delivery (Fassas et al., 2003; Prabhu et al., 2015; Raghuvanshi et al., 2002), stimuli-responsive triggers, combinatorial drug encapsulation, temporally controlled drug release, and enhanced drug safety and efficacy (Hu et al., 2014a). These properties usually cannot be achieved with conventional dosage forms. The terms of nanomedicines and nanotherapeutics are sometimes interchanged.

1.2 Types of Nanomedicines/Nanotherapeutics and Nanoformulations

Nanoparticles, devices, or systems are made from a vast range of materials and forms, including polymers (polymeric nanoparticles, micelles, or dendrimers), lipids (liposomes), viruses (viral nanoparticles), and even organometallic compounds (nanotubes), metals colloids (gold, silver), metal oxides (e.g., titanium dioxide (TiO2), silicon dioxide (SiO2)), inorganic materials (carbon nanotubes (Kam et al., 2005) quantum dots (QDs)), nanocrystals, nanoshells and nanowires, nanorods, nanopores, nanospheres, nanobelts, nanorings, nanocaps, fullerenes (bucky-balls) (Rao and Kumar, 2013).

The following are current types of nanomedicines/nanoformulations (Torchilin, 2005): liposomes, solid lipid nanoparticles, micelles, nanoemulsions, nanogels, polymer conjugates, drug nanocrystals, nanosuspensions, polymeric nanosystems for integrated image-guided cancer therapy, polysaccharide-based nanocarriers for drug delivery, dendrimers (Kukowska-Latallo et al., 2005), layer-by-layer as smart polyelectrolyte multilayer capsules and coatings, magnetic nanoparticles, inorganic nanopreparations, silica-based nanoparticles for biomedical imaging and drug delivery, nanobased drug carriers, nanobeads and micronanoprojection vaccines (Wong, 2016), carbon nanotubes, core–shell nanoparticles, tumor-targeting gold nanoparticles, silver nanoparticles as antibacterial and antiviral agents, magnetic nanoparticles, QDs, gene delivery, organelle-targeted nanocarriers, bacteriophage-targeted and molecular imaging, stimuli-sensitive nanostructured systems, biomimetic systems, and nanoreactors. Almost all types of nanoparticles include polymeric (Cho et al., 2007) nanostructures found both as individual nanomedicines/nanotherapeutics or they are used to modify the properties of other materials on surfaces or in bulk.

Today, the most common polymeric nanoparticle platforms include: liposomes and polymeric drugs, polymer–drug conjugates, polymer–protein conjugates, polymeric nanoparticles, micelles, nanoshells, dendrimers, engineered viral nanoparticles, albumin-based nanoparticles, polysaccharide-based nanoparticles, polymersomes, polyplexes, or interpolyelectrolyte complexes (a complex formed by interaction of a polycation and an anionic oligonucleotide or plasmid) for DNA delivery, polymer–lipid hybrid systems, polymeric nonviral vectors, and inorganic (metallic, ceramic) nanoparticles modified with polymers (Fig. 1.1 and Table 1.1) (Prabhu et al., 2015; Seiler et al., 2007). Targeted drug and gene-delivery systems have been developed by Osaka University in Jain et al. (2003). On the same track, SWRI (South West Research Institute) developed nanocapsules, which deliver drug, antibiotics, and vaccine at the specific site without any side effects (Saravana and Vijaylakshmi, 2006).

Figure 1.1 Schematic illustration of polymeric therapeutic nanoparticles or nanotherapeutics-containing polymers, that is, polymeric nanoparticles in which drugs are conjugated to or encapsulated in polymers; polymeric micelles, dendrimers, nanomaterials with inherent antimicrobial properties, and nanoparticle-based antimicrobial drug-delivery systems. The red dots represent hydrophilic drugs and the blue dots represent hydrophobic drugs. Adapted from Cho, K. Wang, X. Nie, S. (Georgia) Chen, Z., Shin, D.M., 2008. Therapeutic nanoparticles for drug delivery in cancer. Clin. Cancer Res. 14(5), 1310–1316; Duncan, R., 2003. The dawning era of polymer therapeutics. Nat. Rev. Drug Discov. 2, 347–360; Hall, J.B., Dobrovolskaia, M.A. Patri A.K., McNeil S.E., 2007. Characterization of nanoparticles for therapeutics. Nanomedicine (Lond.) 2(6), 789–803; Neamtu, I., Rusu, A.G., Diaconu, A., Nita, L.E., Chiriac, A.P., 2017. Basic concepts and recent advances in nanogels as carriers for medical applications. Drug Deliv. 24(1), 539–557. https://doi.org/10.1080/10717544.2016.1276232; Vasile, C., Nistor, M.T., Cojocariu, A.M., 2015b. Nano-sized polymeric drug carrier systems. In: Arias, J.L. (Ed.), Nanotechnology and Drug Delivery, Vol. 1: Nanoplatforms in Drug Delivery. CRC Press, Boca Raton, FL, pp. 81–141 (Chapter 3); Zhang, L. et al., 2007a. Co-delivery of hydrophobic and hydrophilic drugs from nanoparticle–aptamer bioconjugates. Chem. Med. Chem. 2, 1268–1271; Zhu, X., Radovic-Moreno, A.F., Wu, J., Langer, R., Shi J., 2014. Nanomedicine in the management of microbial infection—overview and perspectives. Nano Today 9(4), 478–498. https://doi.org/10.1016/j.nantod.2014.06.003.

Table 1.1

PEO, polyethylene oxide; PGA, poly(L-glutamate); HPMA, N-(2-hydroxypropyl)-methacrylamide copolymer; PEG, polyethylene glycol; PAA, poly(L-aspartate); PLA, poly(L-lactide); PAMAM, poly(amidoamine); PTX, paclitaxel; DOX, doxorubicin; MTX, methotrexate; PK, pharmacokinetics; EPR, enhanced permeability and retention; HSP, heat shock protein; CPMV, cowpea mosaic virus; PEI, polyethyleneimine.

1.2.1 Polymer Nanotherapeutics

Polymer therapeutics include the family of compounds and the drug-delivery technologies mentioned above, using water-soluble polymers as a common core component.

1.2.1.1 Polymers as drugs

Polymeric drugs are defined as polymers that are active pharmaceutical ingredients (Dhal et al., 2006; Duncan, 2003). It is well- known that the natural polymers extracted from plants, animals, or seaweeds (polyanions and polysulfates) possess antiviral and antitumor activity. Modified polysaccharides, synthetic polypeptides, and some synthetic polymers are already used as drugs. These therapeutic agents have high molecular weight and functionality and are able to selectively recognize, sequester, and remove low-molecular-weight and macromolecular disease-causing species in the intestinal fluid. The advantages of their use compared with traditional small-molecule drug products include long-term safety profiles, polyvalent binding interactions, they are able to sequester bile acids, phosphate, and iron ions, to bind toxins, viruses, and bacteria as well as polymeric enzyme inhibitors and fat binders as antiobesity agents. Functional polymers treat autoimmune disease and sickle cell anemia. Sodium polyethylene sulfonate, a new drug with heparin-like properties, exhibits antilipemic and anticoagulant activities in man (Kuo et al., 1958).

1.2.1.2 Polymeric nanosystems

Safe and nontoxic nanoparticle building blocks are constituted from synthetic or natural biocompatible and biodegradable polymers, such as: polyethylene glycol (PEG), poly(D,L-lactide-co-glycolide) (PLGA) (Kapoor et al., 2015), poly(lactic acid) (PLA), poly(glutamic acid) (PGA), poly(caprolactone) (PCL), N-(2-hydroxypropyl)-methacrylate copolymers (HPMA), polystyrene–maleic anhydride copolymer, and poly(amino acids) (PAAs) (Li, 2002).

The polymer molecular weight, polydispersity, architecture, and conjugation chemistry have a significant impact on their safety and efficacy (Duncan, 2011).

PEG is nontoxic and nonimmunogenic, making it suitable for clinical applications. PEG is a highly hydrated flexible polymer chain that reduces plasma protein adsorption and biofouling of nanoparticles, while reducing renal clearance of relatively smaller drug molecules, and thus prolongs drug circulation half-life (Davis, 2002). Nanoparticle PEGylation (Jokerst et al., 2011), that is, the addition of PEG to the NP surface, provides a hydration layer and steric barrier surrounding the polymeric core, reduces nonspecific binding of serum proteins to the particles, thereby prolonging the circulation and retention time, decreasing the proteolysis and the renal excretion, and shielding antigenic determinants from immune detection without obstructing the substrate-interaction site, reducing clearance by cells of the mononuclear phagocytic system (MPS). PEG has been widely used to enhance the pharmacokinetics of various nanoparticle formulations.

Naturally occurring polymers, such as albumin, polysaccharide, chitosan, heparin, and virus-based nanoparticles have been studied for the delivery of oligonucleotides, DNA, and protein, as well as drugs. Albumin-based polymers represent another class of nanoparticle platforms comprised of biopolymers and their self-assemblies. These nanoparticles have peculiar therapeutic potential because of their specific biological characteristics. If small-molecule drugs are conjugated with human serum albumin (Wosikowski et al., 2003; Xie et al., 2006) or a polysaccharide such as chitosan (Chavanpatil et al., 2007; Hyung Park et al., 2006), their stability and biodistribution can be significantly improved.

1.2.1.3 Polymeric nanoparticles

Polymeric nanoparticles are nanosized solid particles that consist of natural or synthetic polymers. Two types of nanoparticles can be distinguished: (1) nanospheres, which are matrix systems where the drug is uniformly dispersed; and (2) nanocapsules, which are reservoir systems where the drug is located in the core surrounded by a polymer membrane. Depending on the method of preparation, the drugs are either physically entrapped within the nanoparticles or covalently conjugated or adsorbed to the constitutive polymers of the polymer matrix (Rawat et al., 2006).

Polymeric nanoparticles are able to control drug release either by diffusion through polymer matrix or by matrix degradation. They have been investigated as drug-delivery systems for the site-specific targeting of tumors and for the transport of drugs across biological barriers, particularly the blood–brain barrier.

PLGA- and PLA-based nanoparticles and microparticles have been investigated as a nonviral gene-delivery system because of their sustained-release characteristics, biocompatibility, biodegradability, and ability to protect DNA from degradation in endolysosomes. It was established that smaller particle size and uniform size distribution are important to enhance the nanoparticle-mediated gene expression. PLGA nanoparticles exhibit greater gene transfection than those formulated using PLA polymer in breast cancer (MCF-7) and prostate cancer cell lines (PC-3), because of higher DNA release from PLGA nanoparticles, especially those with high molecular weight. A slow release of DNA from nanoparticles localized inside the cells was found (Prabha and Labhasetwar, 2004a,b). High-molecular-weight PLGA nanoparticles had a higher DNA loading and higher gene expression than those formulated with low-molecular-weight PLGA (Prabha and Labhasetwar, 2004b).

1.2.1.4 Polymer–drug conjugates

Nanoparticles with a hydrodynamic size of 5–200 nm are conjugated to existing drugs in order to change the pharmacokinetic and/or pharmacodynamic properties (Duncan, 2006; Hu et al., 2013). The nanoparticle/drug conjugates achieve their effects through passive targeting, that is, a nonspecific accumulation in diseased target tissue (usually solid tumors). The conjugation of small-molecule drugs to polymeric nanocarriers can reduce undesirable adverse effects. Polymer–drug conjugates prolong the in vivo circulation time from several minutes to several hours and also reduce cellular uptake to the endocytic route. It is known that NPs show efficient drug delivery to the tumor microenvironment owing to the enhanced permeability and retention (EPR) phenomenon. This enhances the passive delivery of drugs to tissues with leaky blood vessels, such as tumors and atherosclerotic plaques (Deguchi et al., 2006; Tanaka et al., 2004). Many polymers have been proposed as drug-delivery carriers, but only a few with linear architecture have been accepted into clinical practice (Davis, 2002).

1.2.1.5 Polymeric micelles

The functional properties of micelles are based on amphiphilic block copolymers, which assemble to form a nanosized core/shell structure in aqueous media (Fig. 1.1F) tailored for controlled delivery of hydrophobic drugs. The hydrophobic core region serves as a reservoir for hydrophobic drugs, whereas the hydrophilic shell region stabilizes the hydrophobic core that is polar enough to allow dissolution in aqueous solution (Pepic et al., 2013). The micelles are in the size range of 20–80 nm (Torchilin, 2007). Micellar structure provides an ideal drug-delivery nanocarrier. Its hydrophobic core is capable of carrying pharmaceuticals, especially poorly soluble drugs, with high loading capacity (5%–25% weight). Its hydrophilic shell provides not only a steric protection for the micelle, thereby increasing its stability in blood, but also functional groups suitable for further micelle modification. PEG is the shell-forming polymer of choice, because it is nontoxic and already approved by the US Food and Drug Administration (US FDA) for use in drug products. Additionally, PEG limits micelle interactions with other micelles (which could lead to aggregation) and proteins (opsonins) (Lu and Park, 2013). Each polymeric micelle can carry more drugs due to its considerably larger size and can release these drugs in a more regulated manner in comparison with polymer–drug conjugates. The polymeric micelle systems can also be used to codeliver two or more drugs with similar or different water solubility for combination therapy, or to simultaneously deliver two or more therapeutic modalities, such as radiation agents and drugs (Zhang et al., 2007b). The encapsulated drugs can be released through the surface or bulk erosion of the biodegradable polymers, diffusion of the drug through the polymer matrix, or polymer swelling followed by drug diffusion. Moreover, external conditions, such as change of pH and temperature, can also trigger drug release from polymeric micelles (Farokhzad and Langer, 2006; Gu et al., 2007). The use of block copolymers as the amphiphiles has led to lower critical micelle concentration and thus higher stability in blood in comparison to traditional surfactant-based micelles or liposomes (Oerlemans et al., 2010). The surface modification of the micelles with ligands such as antibodies, peptides, nucleic acids, aptamers, antibody/antibody fragments, small molecules, carbohydrates, and small molecules can differentially target their delivery and uptake by a subset of cells, which will further increase their specificity and efficacy, and reduce their systemic toxicity (Farokhzad et al., 2006; Fonseca et al., 2003; Schnyder et al., 2005). Poly(D,L-lactic acid), poly(D,L-glycolic acid), poly(ε-caprolactone), and their copolymers at various molar ratios, diblocked or multiblocked with poly(ethylene glycol) are the most commonly used biodegradable polymers to form micelles for drug delivery and controlled release, and have been extensively studied (Farokhzad et al., 2006; Kabanov et al., 2002; Torchilin, 2007). Through careful design of the hydrophobic/hydrophilic balance in the amphiphilic copolymer, the size and morphology of the assembled micelles can be controlled. Such particles are appropriate candidates for intravenous (i.v.) administration (Adams et al., 2003). The drug can be loaded into a polymeric micelle in two ways: physical encapsulation (Batrakova et al., 1996) or chemical covalent attachment (Nakanishi et al., 2001). Multifunctional polymeric micelles containing targeting ligands and imaging and therapeutic agents have been developed (Nasongkla et al., 2006).

The first polymeric micelle formulation of paclitaxel, Genexol-PM (PEG-poly(D,L-lactide)-paclitaxel), is a cremophor-free polymeric micelle-formulated paclitaxel tested in patients with advanced refractory malignancies (Kim et al., 2004).

Biodegradable polymeric micelles with 10–200 nm size are promising drug-delivery nanocarriers and have shown remarkable therapeutic potential.

1.2.1.6 Nanogels

Nanogels result from physical or chemical crosslinking of a variety of synthetic or natural polymers or their combination (Neamtu et al., 2017). They are constituted by spherical particles but different shapes can also be prepared, with even a core–shell morphology being reported (Kamerlin and Elvingson, 2016). Most nanogels are hydrophilic, showing the ability to swell in water (hydrogels), a good shape stability in various media, high loading capacity for drugs, proteins, and DNA, and some are stimuli responsive (Richtering and Pich, 2012).

1.2.1.7 Dendrimers

A dendrimer is a synthetic polymeric macromolecule of nanometer dimensions, composed of multiple highly branched monomers that emerge radially from the central core (Fig. 1.1M). Their monodisperse size, modifiable surface functionality, multivalency, water solubility, and available internal cavity make them attractive for drug delivery (McCarthy et al., 2005; Svenson and Tomalia, 2005). Dendrimers are globular, consisting, as already described, of a core and multiple layers with active terminal groups. These layers are comprised of repeating units and each layer is called a generation. The core of a dendrimer is denoted as generation zero. The specific molecular structure of dendrimers enables them to carry various drugs through covalent conjugation or electrostatic adsorption onto their multivalent surfaces. Moreover, they are easily modifiable by simultaneous conjugation with several molecules, such as imaging contrast agents, targeting ligands, or therapeutic drugs, yielding a dendrimer-based multifunctional system (Svenson and Tomalia, 2005). Alternatively, dendrimers can be loaded with drugs using the cavities in their cores through hydrophobic interactions, hydrogen bond, or chemical linkage (Bhadra et al., 2006; Dutta et al., 2007; Kukowska-Latallo et al., 2005; Morgan et al., 2006). Polyamidoamine-based G5 dendrimer has a diameter of about 5 nm and more than 100 functional primary amines on the surface (Uppuluri et al., 1998). By attaching folate as the targeting molecule and methotrexate as the therapeutic agent, the G5 dendrimer was about 10 times more effective and had less systemic toxicity than methotrexate alone in prohibiting tumor growth (Dutta et al., 2007). Polyamidoamine (PAMAMs) dendrimers are the most frequently used and characterized dendrimers for gene delivery. The dendrimer–DNA complex retains genes by electrostatic interactions between the negatively charged phosphate groups on the DNA backbone and the positively charged amino groups on the polymer (D’Emanuele and Attwood, 2005; Maksimenko et al., 2003). Dendrimers form extremely small particles being effective as DNA conjugates (Al-Jamal et al., 2005; Dufes et al., 2005), which have shown improved transfection. They can be used as a scaffold, and are conjugated with cisplatin (Malik et al., 1999). VivaGel, a poly-L-lysine dendrimer, is in a phase I trial as a drug for genital herpes and HIV infection. Dendrimers can also recognize cancer cells.

1.2.1.8 Nanoemulsions

Nanoemulsions are biphasic dispersions of two immiscible liquids: either water-in-oil (W/O) or oil-in-water (O/W) droplets stabilized by an amphiphilic surfactant being a colloidal particulate system in the submicron size range (Fig. 1.1M). The nanoemulsions (Singh et al., 2017; Tiwari and Amiji, 2006) have also shown therapeutic potential. NB-001, a nanoemulsion-based therapeutic product, entered its phase II trial in 2007 as a topical treatment for genital herpes infection.

1.2.1.9 Polyplexes

Polyplexes are polymeric systems containing condensed and/or complexed gene or siRNA through electrostatic interactions between cationic groups of the polymer and the negatively charged nucleic acids. They protect nucleic acids from enzymatic degradation and enable cargo release to tumor sites (Prabhu et al., 2015). Some polymers used in polyplexes summarized in Table 1.1, are poly-L-lysine-based vector, phosphorylcholine-modified polyethyleneimine (PEI) PEGylated PEI-based polyplexes, PEGylated poly(dimethylaminomethyl methacrylate) containing folate, PEI-grafted-α,β-poly(N-3-hydroxypropyl)-DL-aspartamide, galactose-modified trimethyl chitosan-cysteine-based polymeric vectors, cationic (oligoethanamino)-amide-based polymers were conjugated with folic acid for targeted delivery of siRNA in human cervical carcinoma cells, etc.

1.2.1.10 Polymersomes

Polymersomes are self-assembled polymer vesicles of synthetic amphiphilic block copolymers consisting of discrete hydrophilic and hydrophobic blocks, an architecture similar to that of liposomes (vesicles derived from phospholipids). However, the polymersomes possess greater stability, storage capability, and prolonged circulation time than liposomes but similar activity in retarding tumor growth. Polymer vesicles can efficiently encapsulate DOX in their aqueous center (Levine et al., 2008; Prabhu et al., 2015). Both targeted polymersomes and nontargeted polymersomes have been developed.

1.2.1.11 Viruses

Viruses can be regarded as living nanoparticles with a core–shell structure. The core contains infectious agents that can control the transcription and translation machinery of the host cells. The shell is comprised of various proteins or proteins embedded in lipid membranes. Virus-based nanoparticles have been extensively used as gene-delivery vehicles due to their high gene transfection efficiency (Everts et al., 2006; Raja et al., 2003) (Fig. 1.1K).

Nanoparticles are revolutionizing the diagnosis and treatment of bacterial infections, especially those caused by multidrug-resistant (MDR) strains. Metallic and organic nanoparticles have been found to synergize the killing effect of antimicrobial agents (Zaidi et al., 2017).

1.2.1.12 Multifunctional nanoparticles

It is known that NPs show efficient drug delivery to the tumor microenvironment owing to the EPR phenomenon. This is because certain nanoparticles can permeate the leaky vasculature surrounding tumors and areas of inflammation. Many anticancer drug strategies are based on this passive targeting mechanism which led to concentrated drugs in tumors of soft tissue and epithelial cell origin. The accumulation of nanomedicines within the tumor microenvironment is explained by the increased permeability of blood vessels combined with poor lymphatic drainage or transport (Duncan and Sat, 1998). The preferential delivery of drugs to tumors makes lower dosages effective and reduces the undesirable, often pernicious side effects of chemotherapeutics.

1.2.1.13 Multiplex nanoparticles

Multiplex nanoparticles are capable of detecting malignant cells (active targeting moiety), visualizing their location in the body (real-time in vivo imaging), killing the cancer cells with minimal side effects by sparing normal cells (active targeting and controlled drug release or photothermal ablation), and monitoring treatment effects in real time (Fig. 1.1 and Table 1.1). Multifunctional nanoparticles (Fig. 1.1J) are able to carry one or more therapeutic agents, to offer the biomolecular targeting through one or more conjugated antibodies or other recognition agents to give imaging signal amplification, by way of co-encapsulated contrast agents.

A nanoparticle can also serve as a scaffold for the attachment of chemical moieties that perform a variety of medical functions. Ligands (e.g., proteins, antibodies, small molecules) for particular cellular receptors can be attached/immobilized to a nanoparticle surface and facilitate active targeting. As an example, the hydrophilic molecules, such as PEG, can be bound to a nanoparticle surface to increase solubility and biocompatibility. Image contrast agents, such as chelated gadolinium, are conjugated to nanoparticles for diagnostics. The resulting complex nanoparticle therapeutic is a multifunctional entity very different to the contained drugs (Fig. 1.1J).

1.2.1.14 Stimuli-responsive nanomaterials

Stimuli-responsive nanomaterials contain at least one fragment of a responsive polymeric chain, which can be actuated by an appropriate endogenous or exogenous stimulus. In this manner the interactions with the surroundings are controllable by their responsiveness, assuring a better control of activities in various fields and especially in drug delivery.

1.3 Stealth Strategies to Improve Therapeutic Efficacy of Drug Nanocarriers

Nanocarriers strongly interact with the surrounding environment, as endothelium vessels, cells, and blood proteins as specific blood circulating components, opsonins which include complement proteins such as C3, C4, and C5, laminin, fibronectin, C-reactive protein, collagen type I, and immunoglobulins. Surface opsonization promotes the removal of nanoparticles from the circulation within seconds to minutes through the MPS or reticuloendothelial system (RES), and by Kupffer cells, specialized phagocytic macrophages located in the liver, lining the walls of the sinusoids. The anatomical and pathophysiological differences between solid tumors and normal tissues consist mainly of EPR. The angiogenesis leads to high vascular density in solid tumors, so large gaps exist between endothelial cells in tumor blood vessels (Fang et al., 2011). In order to exploit these peculiarities of tumor tissues, and prevent their capture by organs, the nanocarriers need prolonged circulation in the bloodstream, ideally over 6 h. This can be achieved by the design of nanoparticles with special surface characteristics by stealth functionalization as a polymeric coating (Fig. 1.2). Stealth functionality enables prolonged pharmacokinetics and improved biodistribution of the particles. Hydrophilic and neutrally charged particles undergo less opsonization than the hydrophobic and charged ones. Surface coating with hydrophilic polymers prevents the opsonization process. Their capacity to repel proteins depends on composition, molecular weight, density on the carrier surface, thickness of the coating, conformation, flexibility, and architecture of the amphiphilic copolymer chains (multiblock or diblock copolymers, linear or branched). The mushroom and brush configurations are most adequate for stealth properties (Papisov, 1998). A protective polymer coating can be physically or chemically bonded to the nanocarrier surface (Liu et al., 2017). PEG as a mushroom or a brush-like conformation confers some of these characteristics to nanoparticle surfaces which are influenced by its length and surface density on nanoparticle surfaces (Beletsi et al., 2005; Gref et al., 2000; Mosqueira et al., 2001; Perry et al., 2012; Salmaso and Caliceti 2013). Higher surface coverage with PEG increases the circulation time of nanoparticles, and PEG with a molecular weight of 5000 Da provides the ideal surface coating for stealth functionalization (Perry et al., 2012; Wang and Ho, 2010). However, some immunological implications of PEG have been reported (Knop et al., 2010) concerning the induction of PEG-specific antibodies (Ma et al., 2010; Tagami et al., 2011; Xu et al., 2010), and increased phagocytosis by the RES and finally it results in an accelerated blood clearance phenomenon (Armstrong et al., 2007; Sroda et al., 2005; Wang et al., 2007). Therefore, other hydrophilic synthetic and biological polymers have been studied for stealth functionalization, such as those presented in Fig. 1.2.

Figure 1.2 Examples of nanoparticle stealth functionalization of (A) polymeric nanoparticles; (B) inorganic nanoparticles with hydrophilic polymers. Man, Manose. Adapted from Hu et al. (2014b) and Liu et al. (2017).

1.3.1 Synthetic Polymers

Synthetic polymers include PEG-based amphiphilic block copolymers such as poloxamers (pluronics PEO–PPO–PEO triblock copolymers), poloxamines (tetronics ((PEO–PPO)2–N–CH2–CH2–N–(PPO-PEO)2)), polysorbates, polyvinyl pyrrolidone, polyvinylalcohol (PVA), and polyacrylamide (Adams et al., 2003). These polymers can be physically adsorbed on the nanocarrier surface through the hydrophobic PPO fraction. Biodegradable nanoparticles with PEG covalently bound to the surface have been produced using PEG derivatives of poly(lactic acid), poly(lactic acid-co-glycolic acid), or poly(alkylcyanoacrylates).

Poly(N-(2-hydroxypropyl)methacrylamide) (HPMA) shows good hydrophilicity, biocompatibility, and lack of immunogenicity (Kopecek et al., 2000), and it also bears multiple reactive sites. Upon conjugation with hydrophobic drug molecules, HPMA–drug conjugates as unimolecular micelles via a self-assembly process improved the pharmacokinetic profiles (Chytil et al., 2008; Greco and Vicent, 2009; Paul et al., 2007). Multiple therapeutic compounds can also be covalently attached (Duncan, 2009; Vicent et al., 2009) to develop polymeric nanocarriers. HPMA has been applied to stabilize polycation–DNA micellar complexes (Oupicky et al., 1999). Hydrophilic HPMA shells have been grafted onto gene-delivery vesicles to minimize plasma protein interactions and to prolong the circulation time (Konak et al., 2008). Core–shell structured, 100–150 nm nanoparticles, consisting of HPMA–PCL and HPMA–poly(D,L-lactide) (PDLLA), have been prepared using both A–B–A triblock copolymers and star-shaped block copolymers (Kang and Leroux, 2004; Lele and Leroux, 2002). They have been tested as nanocarriers using indometacin and paclitaxel as model drugs.

Zwitterionic betaine-based polymers bear hydrophobic function, which allows anchoring on the particle surface. Poly(carboxybetaine) (PCB) is more chemically stable than PEG and its interactions with proteins are lower (Kirk et al., 2013; Li et al., 2012; Yang et al., 2012), therefore the biofilm formation is reduced (Cheng et al., 2009; Jiang and Cao, 2010; Mi and Jiang, 2012; Shao et al., 2010). They show enhanced bioactivity due to their superhydrophilic nature and also display a higher substrate binding affinity, binding water through electrostatic interactions. PCB was used to coat a variety of nanoparticles, including silica (Yang et al., 2009), gold (Yang et al., 2009), iron oxide (Zhang et al., 2010), PLGA, liposomes (Cao et al., 2012), and hydrogel nanoparticles (Cao et al., 2012; Cheng et al., 2010; Salmaso and Caliceti, 2013; Zhang et al., 2011). PCB helps to improve the particles’ colloidal stability in protein solutions. PCB-grafted PLGA nanoparticles have been prepared using PCB–PLGA block copolymers (Cao et al., 2010). PCB-grafted and PEG-grafted poly(acrylic acid)-b-poly(lactide) nanoparticles reveal comparable in vivo survival in the bloodstream (Zhang et al., 2012b).

Other synthetic polymers have been explored as alternative stealth coatings for nanocarriers such as PVA (Takeuchi et al., 2001), PLGA (Menon et al., 2012), poly(oxazoline) (Woodle et al., 1994), poly(4-acryloylmorpholine) (PAcM) (Ishihara et al., 2010; Torchilin et al., 1995), poly(N,N-dimethylacrylamide) (PDAAm) (Ishihara et al., 2010), and poly(N-vinyl-2-pyrrolidone) (Ishihara et al., 2010), which extended the nanoparticle residence time in the circulation with a nonaccelerated blood clearance phenomenon.

1.3.2 Biopolymers

Poly(amino acid)s, PAAs: The stealth functionality and excellent biodegradability of PAAs are the reasons for the development of PAA-grafted polymeric nanoparticles, including block copolymers. Generally, a hydrophilic peptide sequence is conjugated with a hydrophobic polymer such as PLA for the self-assembly of PAA-grafted nanoparticles (Romberg et al., 2007). Liposomes coated with poly(hydroxyethyl-L-asparagine) showed superior survival in blood as compared with PEG-coated liposomes. A peptide coating was also found to outperform PEGylated liposomes, showing also a pH-triggered DOX release (Ranalli et al., 2017).

Polysaccharides are frequently used to coat nanoparticles owing to their excellent biodegradability and low immunogenicity (Kean and Thanou, 2010). Dextran (Dex), polysialic acid (PSA), hyaluronic acid (HA), chitosan (CH), and heparin are the most commonly used natural polysaccharides. The brush-like structure of heparin and dextran serves to protect the nanoparticles from in vivo clearance. Covalent linking of heparin and dextran to a poly(methyl methacrylate)-based nanoparticle significantly prolonged the circulation half-life of the particles from 3 min to several hours. The water-soluble chitosan reduced the phagocytic uptake efficiency and retarded the blood clearance of PLA nanoparticles. The nanoparticle functionalization with both PEG and chitosan yielded a circulation half-life of 63 h, much longer than that of the PEGylated particles. Conjugation of several protein therapeutics with PSA has prolonged their pharmacokinetic profiles (Fernandes and Gregoriadis, 1997; Jain et al., 2003; Muhlenhoff et al., 1998; Rutishauser, 1998). Sialic acid has been applied to PLGA nanoparticles for drug-delivery applications and has been shown to prolong particle residence within the brain (Bondioli et al., 2010; Tosi et al., 2010). A combination of two kinds of polymers is also used (Liu et al., 2017). Mannose-functionalized porous silicon NPs escaped capture by the spleen, and had higher blood retention. The highest efficiency of the stealth behavior was observed with PEGylated nanoparticles anchored with mannose, which was excreted in urine after 5 h.

1.3.3 Biologically Inspired Stealth Strategies

Proteins and glycans need to be attached to the particles in a nondisruptive and regio-selective manner. Techniques to prepare biologically inspired polymeric nanoparticles can be classified into a bottom-up approach based on protein conjugations and a top-down approach based on cell membrane coating. Incorporation of sialylated glycolipids and glycoproteins showed an extended circulation time in the blood.

Polymeric nanoparticle tagged with molecular self-markers is a bottom-up approach consisted of attaching marker-of-self molecules to nanoparticles. The integrin-associated protein CD47, found in cancer cells and viruses, is considered as a marker-of-self because it can protect cells against macrophage uptake through an inhibitory action via SIRPα binding (Oldenborg et al., 2000). The CD47 and its derivative peptide were attached to polystyrene nanoparticles using mixed 160 nm streptavidin-coated polystyrene beads to enhance their delivery and inhibit phagocytic clearance. By streptavidin–biotin coupling a proper orientation at a controllable density and also a nanoparticle camouflage were achieved (Rodriguez et al., 2013).

Polymeric nanoparticles camouflaged in cellular membranes is a top-down approach to coat and camouflage polymeric nanoparticles from immune clearance. A coextrusion process was applied to coat sub-100 nm PLGA nanoparticles with natural membranes derived from red blood cells (RBCs) (Hu et al., 2011).

The RBC membrane-coated nanoparticles (RBC–NPs) exhibited a core–shell structure, in which the RBC membrane formed a single bilayer around the polymeric core, providing a very good control (Feng et al., 2013; Hu et al., 2011; Ramakrishnaiah et al., 2013). The proteins were oriented almost exclusively in the right-side-out fashion, with the extracellular portion displayed on the particle surfaces. This right-side-out orientation was due to both the electrostatic repulsion between the negatively charged PLGA nanoparticles and the negatively charged sialyl moieties residing on the exoplasmic side of the membranes and to the stabilization effect by the exoplasmic glycans. Because of this orientation in membrane coating, macrophage uptake of the RBC–NPs was significantly impeded in vitro. The RBC–NPs possessed longer blood survival as compared to an analogous PEGylated formulation. The DOX-loaded RBC–NPs were shown to be efficient against a leukemia cell line (Aryal et al., 2013). The membrane-camouflage stealth strategy allows nanoparticle interaction with other biomolecules and provides novel therapeutic interventions against pathogenic factors that interact with cellular surfaces.

1.4 Nanomaterials: Physicochemical Properties and Risk Assessment

Characterization of the polymeric nanoparticles destined for medical applications includes physicochemical characterization, sterility and pyrogenicity (capacity to cause fever) assessment, biodistribution (absorption, distribution, metabolism, and excretion—ADME), and toxicity characterization, which includes both in vitro tests and in vivo animal studies. In vitro assays are performed for hemolysis, thrombogenicity, complement activation, and interaction with the immune system through phagocytosis, while in vivo studies are made by using multicomponent diagnostic labels.

The unique physico-chemical properties and biological activity of nanomaterials/nanotherapeutics intimately depend on: particle size, shape, surface chemistry, surface charge and aggregation/agglomeration state, density of targeting ligands, as well as tumor/disease type, characteristics, and microenvironment, which determine their performances related to the degree of protein adsorption, cellular uptake, biodistribution patterns, and the clearance mechanisms.

• Nanoparticle characterization and the development of protocols for this often requires concerted interdisciplinary efforts of material scientists, chemists, biologists, and clinicians, with the support of private industry, government, and regulatory agencies. Standardized methods are being developing by Food and Drugs Administration (FDA), the International Standards Organization (ISO)], the American National Standards Institute (ANSI), the American Society for Testing and Materials (ASTM), etc. Three independent scientific committees provide their expertise to the EC: the Scientific Committee for Consumer Safety (SCCS), Scientific Committee on Health, Environmental and Emerging Risks (SCHEER), and the Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR). The SCENIHR developed a scientific basis for the definition of nanomaterial by identifying particle characteristics relevant to safety assessment (see Chapter 15).

The protocols are different depending on the studied system. For soluble small-molecule drugs, measurement of the molecular weight and spectral characteristics are enough, while physicochemical characterization of a multipart nanoparticle includes assessment of the individual parts, the stoichiometry, and connections between the parts and the chemical stability of those connections.

On the other hand, any variations in the manufacturing process and in the formulation may result in a generic product with different physicochemical properties (e.g., size, size distribution, surface properties, drug loading and release profile, aggregation status, and stability), which could lead to different biopharmaceutical profiles with a significant impact on patient safety and efficacy. Different physico- chemical properties can result in different ratios of free to nanoparticle-incorporated/associated drug, pharmacological effects, specific cell–nanotherapeutic interactions, distributions, target organ uptake, immunological effects, and toxicities. To assess these differences, approaches more complex than the simple plasma concentration measurement are required. It is generally considered that the regulatory approach established for similar biological medicinal products (biosimilars) should be adopted for follow-on nanotherapeutic products (nanosimilars), because such an approach includes the stepwise comparison of their quality, safety, and efficacy (Borchard et al., 2012). Differences in tissue distribution and toxicological profiles have been observed among nanoparticle iron formulations that have different carbohydrate coatings (European Medicines Agency, 2011).

1.4.1 Size, Shape, and Morphology of Nanoparticles

The three main features of NPs are: (1) particle size; (2) particle shape; and (3) surface properties.

1. Particle size plays a key role in clearance of these materials from the body. Therefore the desired clearance mechanism can be a factor in the nanomedicine design. Small particles (<10 nm) are cleared via the kidneys, while the larger particles (>10 nm) are cleared through the liver and the mononuclear–phagocyte system. NPs with at least 50 nm in diameter avoid renal clearance, but have to be smaller than 300 nm to diffuse through the tumor interstitium in sufficient amounts to achieve a therapeutic effect. Therefore, the size of nanoparticles used in a drug-delivery system should be large enough to prevent their rapid leakage into blood capillaries but small enough to escape capture by fixed macrophages lodged in the RES (liver and spleen) (Moghimi et al., 2001). Fortunately, the size is tunable and NPs fate in the organism can be controlled by adjusting their size and surface characteristics (Rolfe et al., 2014; Fox et al., 2009).

The size of the sinusoid in the spleen and fenestra of the Kupffer cells in the liver varies from 150 to 200 nm (Wisse et al., 1996) and the size of the gap junction between endothelial cells of the leaky tumor vasculature may vary from 100 to 600 nm (Yuan et al., 1995). Consequently, the size of nanoparticles should be up to 100 nm to reach tumor tissues by passing through these two particular vascular structures. The size similarity of nanoparticles to biological moieties imparts many of their unique nano medical properties (McNeil, 2005).

The colloid size and size distribution cannot be measured accurately with a single technique; different methods based on different principles have to be combined. Field flow fractionation, high-performance liquid chromatography/size-exclusion chromatography, analytical ultracentrifugation, differential centrifugal sedimentation, and dynamic light scattering (DLS) are used.

2. Particle shape has gained considerable attention since it was found that nonspherical NPs could reduce phagocytosis by macrophages, thus exhibiting longer in vivo circulation time. The relationship of various chemical, electrostatic, and morphological factors is also important.

3. Scanning electron microscopy (SEM), atomic force microscopy, or transmission electron microscopy (TEM) are very useful to assess the particle size, shape, morphology, and some surface characteristics. Size characterization by TEM and DLS and localization in tissue by chemical composition detection using energy dispersive X-ray (EDX) spectroscopy are recommended. TEM uses more powerful electron beams than SEM, it has higher resolution, and provides greater detail at the atomic scale as the crystal structure and granularity of a sample.

Electron microscopies (EMs) cannot be applied for soft particles, such as biological compounds (e.g., liposomes and proteins) and engineered polymers (e.g., dendrimers) without application of the heavy-metal staining procedures. These techniques also cannot image the surface groups (e.g., PEG, targeting antibodies, or drugs) without the use of cryogenic methods and sometimes they do not give information on size in the physiologically relevant conditions. Sample preparation, the use of high vacuum in TEM, and thin sample sections and drying might alter the physicochemical state of the nanoparticle or introduce artifacts.

DLS, also known as photon correlation spectroscopy, provides particle sizes in solution. DLS is very sensitive to soft flexible biological molecules, such as polymers, proteins, and antibodies, because they cause significant frictional drag, which can influence the rate of the particle’s motion under Brownian diffusion dramatically, but information about the shape of particles cannot be obtained. The measurements under physiological conditions that resemble or mimic the physical state closely in vivo are often necessary, as the size distribution at physiological pH and ionic strength might differ from the distribution in water or the dry state. Isolation of nanomaterials or some components from biological fluids is also important. Capillary electrophoresis is a useful technique for such separation (Chan et al., 2007). Examination of the nanoparticles in cells, tissues, and organs after exposure (in vivo animal studies), postmortem evaluation is very useful. EM images of tissues provide information on nanoparticle disposition into cells, tissues, and organs; however, it is difficult to maintain the integrity of the multifunctional nanoparticle through the various stages of metabolism.

1.4.2 Composition

The elemental composition of a nanoparticle usually does not change in the biological environment (Powers et al., 2006). Examination of the tissues simultaneously by EM microscopy and spectroscopy (auger electron spectroscopy, electron energy loss spectroscopy, X-ray photoelectron spectroscopy and EDX) offers a map of nanoparticle distributions (mainly inorganic).

1.4.3 Surface Characteristics

In addition to their size, the surface characteristics of the nanoparticles are also important factors determining their life span and fate during circulation relating to their capture by macrophages. Surface properties influence the EPR effect as the nature of the surface is the first aspect relevant to contact. Nanoparticles should ideally have a hydrophilic surface to escape macrophage capture (Moghimi and Szebeni, 2003). This can be achieved in two ways: coating the surface of nanoparticles with a hydrophilic polymer, such as PEG, or nanoparticles can be formed from block copolymers with hydrophilic and hydrophobic domains (Harris et al., 2001). Chemical modifications of the particle surface can influence the distribution, uptake, and reactivity of the colloids and are of major importance for risk assessment.

The targeting of an active compound at the affected organ (tumor) can be passive by EPR and active using a targeting ligand or antibody as a targeting moiety, a polymer, or lipid as a carrier, and an active chemotherapeutic drug (Allen, 2002). As mentioned above, the microenvironment surrounding tumor cells is different from that of normal cells. Tumor cells use glycolysis to obtain extra energy, resulting in an acidic environment. Cancer cells express and release unique enzymes, such as matrix metalloproteinases, which are implicated in their movement and survival mechanisms (Deryugina and Quigley, 2006). Taking into account these particularities of the tumor microenvironment, the surface characteristics of the nanoparticles used as carriers were modified using various strategies and ligands to improve the efficacy of anticancer (antineoplastic or cytotoxic) chemotherapy drugs (Bies et al., 2004; Cho et al., 2007; Farokhzad et al., 2004, 2006; Hicke et al., 2006; Mansour et al., 2003; Minko, 2004; Sahoo and Labhasetwar, 2005; Seymour et al., 2002).

The surface characteristics of nanomaterials can be analyzed by using a number of spectroscopic methods such as solid-state nuclear magnetic resonance spectroscopy or Fourier transform infrared spectroscopy. More specific for colloids than for solids is the surface charge, which has an influence on the interaction of the nanoparticle itself with biological surfaces such as membranes (Torrano et al., 2013), mucus, or plasma proteins (Shah et al., 2012). It can be assessed by determining the zeta potential applying laser Doppler electrophoresis. The zeta potential and the surface charge are highly dependent on the environmental conditions, including ionic strength, pH, and medium components. By measuring the zeta potential as a function of the pH value, a more detailed statement on the surface charge profile of particles can be achieved (Wacker et al., 2011).

1.4.4 Sterility and Pyrogenicity

The sterility can be realized either in synthesis or different sterilization methods should be applied, such as: gamma irradiation, filtration, heat/steam, ethylene oxide, formaldehyde, hydrogen peroxide, plasma, or UV irradiation. The nanoparticles of low melting temperature and/or containing proteins or antibodies are destroyed by autoclaving, while polymeric materials are affected by a high dose of gamma irradiation. This means each nanomaterial should be sterilized by a particular method (Dobrovolskaia, 2017).

The US FDA recommends two types of pyrogen tests: a limulus amebocyte lysate (LAL)-based assay (United States Pharmacopeia 1) sensitive to even picogram quantities of endotoxin, and the so-called rabbit pyrogen test. The last is most frequently used because most nanoparticles have the potential to interfere with the LAL assay. Other tests have been proposed at: https://ntp.niehs.nih.gov/iccvam/docs/pyrogen/brd/pyrobrd2008.pdf (last accessed October 10, 2017).

1.4.5 Biodistribution (Absorption, Distribution, Metabolism, and Excretion)

Biodistribution (ADME) and toxicity characterization involve both in vitro tests and in vivo animal studies. In vitro experiments provide an initial cost-effective assessment of the toxicity and efficacy of a nanomaterial-based therapeutic and they give information on the design of animal studies. In vitro studies allow the elucidation of biochemical mechanisms—under controlled conditions not achievable by in vivo studies.

In vitro tests must be carefully selected because many nanoparticles lead to particular effects (aggregation or protein adsorption, gold colloids scatter light, and QDs have very large molar-extinction coefficients) invalidating in vitro tests assays (Patri et al., 2006).

The biocompatibility of nanomaterials with blood can be evaluated by in vitro tests using a variety of cell-based in vitro protocols. Cell viability is assessed following various characteristics of the cells (Dobrovolskaia and McNeil, 2007; Mickuviene et al., 2004). Hemolysis (damage to RBCs), complement activation, thrombogenicity (tendency of a material to cause blood clotting), and phagocytosis, which can be predictive of particle accumulation in organs of the RES system and immune response are also required (ASTM, 1998) to assess biocompatibility, mainly indicating toxicity. The complement activation results in the production of anaphylatoxins which are small, cytokine-like molecules causing anaphylactic shock and organ failure at high concentrations. Complement activation by nanoparticles is influenced by particle surface charge and hydrophobicity (Chanan-Khan et al., 2003; Vonarbourg et al., 2006). The nanoparticle thrombicity is examined initially in vitro through induced platelet aggregation and effects on blood coagulation by biochemical tests (Jiao et al., 2002; Koziara et al., 2005). Phagocytosis involves detection and quantification of particles inside cellular compartments. It is well known that cancer chemotherapies are highly toxic and use relatively insoluble drugs. Toxic solvents are necessary to deliver them by parenteral administration (such as Cremophor for paclitaxel) raising other toxicity situations. Therefore, dose reduction is necessary, but this limits the therapy efficacy. Chemotherapy nonspecificity of traditional drug delivery is also a problem because the accumulation of drug in tumors affects normal cells.

Nanotherapeutics as oncologic drugs solve such situations. As delivery vectors they accumulate at higher concentrations in tumors via the EPR effect. Being larger than free drugs therefore they do not permeate normal capillaries, and readily leak out of tumor vessels. The conjugation of specific molecular tags to the nanoparticle surface enhances drug accumulation in the tumor and so decreasing normal tissue exposure.

1.4.6 In Vitro Stability and Degradation

In the area of medicinal products, the US FDA and the European Medicines Agency (EMA) demand extensive characterization of drugs, excipients, and their degradation products including