Nanobiomaterials in Drug Delivery: Applications of Nanobiomaterials

China

Preface of the series

Ecaterina Andronescu

Department of Science and Engineering of Oxide Materials and Nanomaterials, Faculty of Applied Chemistry and Materials Science, University Politehnica of Bucharest, Bucharest, Romania

The era of nanosized materials is now considered the center of the evolution of future tools and emerging technologies with wide applications in industry, research, health, and beyond. Despite recent scientific progress, biological applications of nanomaterials are far from being depleted and current knowledge is limited by the poor access to significant data, but also by widespread and usually unfounded speculation. Although exhaustive, the current literature is difficult to reach and understand because of the specificity and strict focuses of researchers investigating different applications of nanomaterials.

In this context, the scientific series entitled Applications of Nanobiomaterials was motivated by the desire of the Editor, Alexandru Mihai Grumezescu, and others to bring together comprehensive, up-to-date and relevant findings on the field of biological applications of nanostructured materials, to promote the knowledge and expand our vision regarding future perspectives. Even though the approached domain is quite specific and research-oriented, this multivolume set is easily intelligible for a wide audience including: under-graduate and post-graduate students, engineers, researchers, academic staff, pharmaceutical companies, biomedical sector and industrial biotechnologies. However, some basic knowledge of the field of materials science (nanobiomaterials, pharmaceutical industry, products for medicinal treatments, nanoarchitectonics for delivery of biological active molecules and release, bone implants and stomatology) and engineering is a requisite for understanding technical aspects.

The selected authors of each chapter are outstanding specialists in the field of nanobiomaterials, who have made impressive contributions in a specific area of research or applied area within the scope of this book.

Each of the 11 volumes of the series contains 15 chapters, addressing the most relevant and recent matters on the field of the volume.

The first volume, Fabrication and Self-Assembly of Nanobiomaterials, introduces the reader to the amazing field of nanostructured materials and offers interesting information regarding the fabrication and assembly of these nanosized structures. In Volume II, entitled Engineering of Nanobiomaterials, readers can easily find the most commonly investigated methods and approaches for obtaining tailored nanomaterials for a particular application, especially those with a great deal of significance in the biomedical field. In the following step, readers will discover the importance and the ways of modifying the surface of nanostructured materials to obtain bioactive materials, by reading Volume III, Surface Chemistry of Nanobiomaterials. Starting with Volume IV Nanobiomaterials in Hard Tissue Engineering and Volume V Nanobiomaterials in Soft Tissue Engineering the biomedical applications of engineered nanomaterials are revealed and discussed, focusing on one of the most impacted fields, tissue engineering. Volume VI, Nanobiomaterials in Antimicrobial Therapy, highlights the potential of different nanostructured materials to be utilized in the development of novel efficient antimicrobial approaches to fight the global crisis of antibiotic inefficiency and emerging infectious diseases caused by resistant pathogens. Volume VII moves on to another key biomedical domain—cancer therapy. This volume, Nanobiomaterials in Cancer Therapy, describes current issues of cancer therapy and discusses the most relevant findings regarding the impact of nanobiomaterials in cancer management. Medical Imaging represents the focus of Volume VIII, while Volume IX deals with applications of Nanobiomaterials in Drug Delivery. Volume X, entitled Nanobiomaterials in Galenic Formulations and Cosmetics, refers to the perspectives highlighted by the utilization of nanosized functional biomaterials in the development of improved drugs and active principles for different biomedical industries. Finally, Volume XI is dedicated to the impact of Nanobiomaterials in Dentistry, which currently represents one of the most investigated and controversial domains related to the biomedical applications of nanostructured materials.

Due to their specific organization, each volume can be treated individually or as a part of this comprehensive series, which aims to bring a significant contribution to the field of research and biomedical applications of nanosized engineered materials.

Preface

Alexandru Mihai Grumezescu

http://grumezescu.com/

Department of Science and Engineering of Oxide Materials and Nanomaterials, University Politehnica of Bucharest, Bucharest, Romania

Department of Biomaterials and Medical Devices, Faculty of Medical Engineering, University Politehnica of Bucharest, Bucharest, Romania

About the Series (Volumes I–XI)

The increased production of nanosized materials with applications in the biomedical field by using biomimetic and bio-inspired processes and formulations has recently led to a new concept, termed nanobiotechnology. This complex research brings together significant knowledge from physical, chemical, biological, and technological sciences in an applicative field.

Medical applications of nanobiomaterials range from the development of adequate scaffolds for tissue engineering to therapeutic nanostructures, such as targeted drug-delivery systems. The purpose of this multivolume set entitled Applications of Nanobiomaterials is to offer a broad, updated, and interdisciplinary point of view regarding the applications of these materials of the future medicine, starting with their fabrication, specific engineering, and characterization but also discussing about their impact in tissue engineering, antimicrobial and cancer therapies, and also the development of different medical and cosmetic use products. These books bring together the work of outstanding contributors who have significantly enhanced the basic knowledge and applicative concepts of this research field in their respective disciplines.

The multivolume set Applications of Nanobiomaterials contains 165 chapters, organized in 11 volumes, which are ready to present a novel and up-to-date approach related to this intriguing domain. Each chapter has been carefully composed and illustrated to highlight the relevance of nanobiomaterials on most biomedical fields, revealing recent applications on each specific domain. The whole set represents a great material for the academic community, starting with undergraduate and postgraduate students, researchers, engineers, and medical doctors, but also pharmaceutical companies and innovative biotechnologies.

These 11 volumes cover all relevant aspects related to the Applications of Nanobiomaterials as it follows:

Volume I: Fabrication and Self-Assembly of Nanobiomaterials

Volume II: Engineering of Nanobiomaterials

Volume III: Surface Chemistry of Nanobiomaterials

Volume IV: Nanobiomaterials in Hard Tissue Engineering

Volume V: Nanobiomaterials in Soft Tissue Engineering

Volume VI: Nanobiomaterials in Antimicrobial Therapy

Volume VII: Nanobiomaterials in Cancer Therapy

Volume VIII: Nanobiomaterials in Medical Imaging

Volume IX: Nanobiomaterials in Drug Delivery

Volume X: Nanobiomaterials in Galenic Formulations and Cosmetics

Volume XI: Nanobiomaterials in Dentistry.

About Volume IX

Volume IX, entitled Nanobiomaterials in Drug Delivery, represents an up-to-date book discussing current efforts of researchers made in the area of drug delivery, which have led to successful formulations using nanobiomaterials.

State-of-the-art chapters regarding drug delivery and drug targeting at the molecular and cellular levels are extensively presented and approach the following aspects: (1) design of various nanobiomaterials; (2) encapsulation of bioactive molecules with various properties (hydrophilic, hydrophobic, or amphiphilic character); (3) protein and peptide delivery; (4) recent developments and applications of various nanotechnologies in drug delivery; and (5) contribution of modern nanosystems including niosomes, polymeric nanoparticles, colloidal nanocomposites, hydrogels, polymeric micelles, dendrimers, aptamers, capsosomes, nanoneedles, molecularly imprinted polymers, stimuli-responsive polymers, therapeutic polymers (polymer–drug conjugates), polymeric artificial cells to the rapid development of nanobiotechnology for diagnosis (imaging), drug delivery and targeting of extremely serious disorders, such as cancer, Parkinson and Alzheimer diseases, chronic inflammations, ocular dysfunctions, microbial/viral infections, or as possible treatments for different neurodegenerative disorders.

Volume IX contains 15 chapters, prepared by outstanding international researchers from Canada, the United States of America, Brazil, the United Kingdom, Spain, Belgium, Poland, Romania, Russia, Turkey, Iran, India, and China.

In Chapter 1, entitled Nanobiomaterials and Drug Delivery, Mehdi Rajabi et al. give an overview regarding the design and characterization of various nanomaterials for drug delivery to treat cancer.

Magdalena Markowicz-Piasecka et al., in Chapter 2, Dendrimers in Drug Delivery, present an up-to-date review regarding the encapsulation of bioactive hydrophilic, hydrophobic, or amphiphilic molecules into dendritic structures, which act as efficient carriers. The authors highlight the potential properties of dendrimers that could be utilized as drug vehicles, including (1) an extended life time of the drug in the circulatory system; (2) protection of the drug from the external environment; (3) increasing the stability of the active compound; and (4) efficient tissue targeting.

Chapter 3, Lipid Nanoparticles as Non-Viral Vectors for siRNA Delivery: Concepts and Applications, prepared by Raquel Petrilli et al., discusses the efforts that have been carried out to deliver siRNA loaded in lipid nanoparticles, highlighting the advantages, disadvantages, applications, clinical relevance, and future perspectives.

In Chapter 4, Nanobiomaterials: Novel Nanoplatform for Protein and Peptide Delivery, Surbhi Dubey et al. present new insights regarding the impact of nanoparticles in protein and peptide delivery.

Amit Kumar Mittal et al., in Chapter 5, Current Status and Future Prospects of Nanobiomaterials in Drug Delivery, highlight the applications of engineered nanomaterials used to identify specific cells to deliver the drugs at the affected sites. New types of nanobiomaterials used for the treatment of microbial infections, cancer detection and diagnosis, cardiovascular, and other types of diseases are extensively discussed in this chapter.

In Chapter 6, Magnetoanisotropic Biodegradable Nanocomposites for Controlled Drug Release, Alexey Leonodovich Iordanskii et al. outline the design of the novel therapeutic systems for controlled drug release based on poly(3-hydroxybutyrate), chitosan, with encapsulated magnetic nanoparticles and active drugs. Encapsulation of impermeable nanoparticles and their aggregates in magnetic nanocomposites impacts on drug diffusivities as a result of tortuosity enhancing, free volume ratio decrease, and magnetostriction.

In Chapter 7, Nanomaterials in Drug Delivery: Existing Scenario and Potential Scope, Habibur Rahman et al. introduce recent developments and applications of current nanotechnologies in drug delivery. Various nanoscale formulations for drug delivery are described with special emphasis on nanoparticulate drug-delivery systems.

Chapter 8, Natural and Synthetic Polymers for Drug Delivery and Targeting, prepared by George Dan Mogoşanu et al., highlights the contribution of modern nanosystems including niosomes, polymeric nanoparticles, colloidal nanocomposite hydrogels, polymeric micelles, dendrimers, aptamers, capsosomes, nanoneedles, molecularly imprinted polymers, stimuli-responsive polymers, therapeutic polymers (polymer–drug conjugates), polymeric artificial cells to the rapid development of nanobiotechnology for diagnosis (imaging), drug delivery, and targeting of extremely serious disorders, such as cancer, Parkinson and Alzheimer diseases, chronic inflammations, ocular dysfunctions, and microbial/viral infections.

Chapter 9, Magnetically Based Nanocarriers for Drug Delivery, prepared by Emir Baki Denkbaş et al., discusses the potential advantages and applications of a magnetic targeting method by targeting the magnetic nanoparticles carrying therapeutic agents to target organs or tissues by intravenous injection of nanoparticles with the manipulation of a magnetic field.

Palazzo Claudio et al., in Chapter 10, Drug-Delivery Nanocarriers to Cross the Blood–Brain Barrier, describe polymeric nanocarriers, solid lipid nanocarriers, and liposomes developed to deliver drugs otherwise not able to pass the blood–brain barrier, due to their physicochemical characteristics.

Chapter 11, entitled Nanotechnology-Based Drug-Delivery Systems Releasing Growth Factors to the CNS: Focusing on Neurodegenerative Disorders, by Oihane Gartziandia et al., reveals novel opportunities to formulate growth factors using a wide variety of biodegradable nanocarriers as possible treatments for the different neurodegenerative disorders.

Xin Zhao et al., in Chapter 12, Bionanofibers in Drug Delivery, give recent information about nanofibers fabricated by different techniques as well as their applications in drug delivery and tissue regeneration.

Indu Pal Kaur et al., in Chapter 13, Nanobiomaterials as Gene-Delivery Vehicles, provide a critical update on the applications of lipidic and polymeric nanobiomaterials/carriers, for effective delivery of gene-based drug molecules.

Chapter 14, Nanobiomaterials Set to Revolutionize Drug-Delivery Systems for the Treatment of Diabetes: State-of-the-Art, prepared by Abolfazl Yazdanpanah et al., describes the recent advances and future prospects of using nanobiomaterials for innovative drug-delivery systems in diabetes.

Ye Tian et al., in Chapter 15, Chitosan and its Derivatives-Based Nano-formulations in Drug Delivery, reviews chitosan- and chitosan-derivative-based nano-formulations for the delivery of various classes of drugs including poorly soluble molecules, proteins, peptides, genes, and vaccines.

Chapter 1

Nanobiomaterials in drug delivery

Mehdi Rajabi, Mathangi Srinivasan and Shaker A. Mousa,    The Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Rensselaer, NY, USA

Abstract

In recent years many diverse scientific strategies have been developed for cancer therapy. One of the most unique research areas is nanotechnology that is related to synthesis, manipulation of nanomaterials, and their application in cancer diagnosis and therapy. Multifunctional nanoparticles can be prepared for different biomedical applications that can carry and deliver small molecules (dyes or chemotherapeutic drugs) to target, detect, and treat. A well-established nanomaterial can enhance the efficiency of drug delivery to reach pathological areas by decreasing their toxicity and side effects. Small molecules can be conjugated to nanomaterials using different chemical linkages that can be released by biodegradation and self-regulation of nanomaterials. In this chapter, we introduce the design and characterization of various nanomaterials for drug delivery to treat cancer in vitro and in vivo.

Keywords

Nanotechnology; nanomaterials; drug delivery; targeting; imaging; chemotherapy; multifunctional nanoparticle; bioconjugation chemistry

Abbreviations

AFM atomic force microscope

AMD age-related macular degeneration

anti-VEGF antivascular endothelial growth factor

cisplatin cis-diamminedichloroplatinum (II)

CT computed tomography

CuAAC copper-catalyzed azide–alkyne 1,3-cycloaddition

DOX doxorubicin

DSS disuccinimidyl suberate

EGFR epidermal growth factor receptor

EGFRvIII epidermal growth factor receptor variant III

EMCH.TFA 3,3′-N-[ε-maleimidocaproic acid] hydrazide and its trifluoroacetic acid salt

EPR enhanced permeability and retention

GBM glioblastoma multiforme

GC gas chromatography

GNPs gold nanoparticles

GPC gel permeation chromatography

HPLC high-performance liquid chromatography

IONP iron oxide nanoparticle

IR infrared spectroscopy

LC-MS liquid chromatography-mass spectroscopy

LC-SPDP succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate

MALDI-TOF matrix-assisted laser desorption ionization time-of-flight

MRI magnetic resonance imaging

MS mass spectrometry

mSPIO monocrystalline superparamagnetic iron oxide

NHS N-hydroxysuccinimide ester

NMR nuclear magnetic resonance

OI optical imaging

PEG polyethylene glycol

PEI polyethyleneimine

PET positron emission tomography

PLA polylactide

PLGA poly(lactic-co-glycolic acid)

PXL paclitaxel

QDs quantum dots

RGD arginine-glycine-aspartate

SEC size exclusion chromatography

SELEX systematic evolution of ligands by exponential enrichment

SIA N-succinimidyl iodoacetate

SMCC succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate

SPDP N-succinimidyl-3-(2-pyridyldithio)-propionate

SPECT single-photon emission computed tomography

SPION superparamagnetic iron oxide nanomaterial

SPM scanning probe microscopy

TEM transmission electron microscope

US ultrasound

UV–vis ultraviolet–visible

VCR vincristine

VLB vinblastine

XRD X-ray diffraction

XPS X-ray photoelectron spectroscopy

1.1 Introduction

Cancer is still the leading cause of death around the world, despite the many diagnostic procedures and treatments developed over the past 30 years (Rajabi et al., 2010, 2011, 2012). Nanotechnology has contributed to significant advances in cancer treatment by targeting, detecting, and destroying cancer tumors with minimal effect on normal tissues. The field of nanotechnology involves different disciplines, such as computing, engineering (electrical/mechanical/chemical/textile), material sciences, physics, chemistry, agriculture, food sciences, and biology (Figure 1.1) (Moghimi et al., 2005; Shu et al., 2014).

Figure 1.1 Multidisciplinary applications of nanotechnology. The various disciplines including computing, engineering (electrical/mechanical/chemical/textile), material sciences, physics, chemistry, agriculture, food sciences, and biology of cancer involve the field of nanotechnology.

A nanotechnology-based delivery system offers many advantages to traditional chemotherapy treatment regimens (Sahoo and Labhasetwar, 2003), including delivery of a higher dose of drugs to the tumor and improved solubility of poorly soluble drugs. It also offers a protective shell that can shield the drug or active agent from unfavorable environments (such as the stomach and lysosome, which are highly acidic) or from the high levels of disruptive enzymes and proteases in the bloodstream, increased half-lives of the drug in the bloodstream, controlled release of the drugs, targeted delivery to the tumor site, and using tumor-specific ligands and delivery of multiple drugs or active agents simultaneously (Couvreur, 2013; Bharali and Mousa, 2010).

There are two essential issues in developing nanomaterials for cancer therapy: recognition of the tumor and ability of the nanomaterials to reach the tumor site (binding affinity). A well-established and -designed nanomaterial can convey unique advantages for cancer treatment, such as enhancing the efficiency of drugs delivered to pathological areas by decreasing the toxicity and side effects of the drugs (Angeli et al., 2008; Peer et al., 2007). Well-designed nanomaterials should be able to carry a high concentration of chemotherapeutic agents or imaging agents as well as targeting moieties to the site of tumors, which are associated with blood vessels.

Nanomaterials can be designed, synthesized, and manipulated in the size range of 1–1000 nm and can be classified into essential categories including organic dendrimers (Huang et al., 2010; Menjoge et al., 2010); polymer micelles (Blanco et al., 2009; Torchilin, 2007); liposomes (Barenholz, 2001; Elbayoumi and Torchilin, 2010; Lian and Ho, 2001; Puri et al., 2009); ferritins (Keyes et al., 2011; Kim et al., 2011, 2012; MaHam et al., 2009; Uchida et al., 2006); inorganic quantum dots (QDs) (Bera et al., 2010; Biju et al., 2010; Cheki et al., 2013; Larson et al., 2003; Walling et al., 2009; Xing and Rao, 2008; Yong et al., 2007; Yu et al., 2006); gold nanoparticles (GNPs) (Alkilany and Murphy, 2010; Cai et al., 2008; Chithrani et al., 2006; Corti et al., 2002; Daniel and Astruc, 2004; Jain et al., 2012); superparamagnetic iron oxide nanomaterials (SPIONs) (Kim et al., 2001; Laurent and Mahmoudi, 2011; Laurent et al., 2011; Qiao et al., 2009; Weinstein et al., 2010); and paramagnetic lanthanide ions (Chen et al., 2010a, 2013; Nyk et al., 2008; Zhan et al., 2011) (Figure 1.2).

Figure 1.2 Two essential categories of nanomaterial, including organic (dendrimers, micelles, liposomes, and ferritins) and inorganic (quantum dots (QDs), gold, superparamagnetic iron oxide (SPIO), and paramagnetic lanthanide ions).

1.1.1 Parameters of Delivery of Nanoparticles into Biological Systems

A nanoparticle-based drug-delivery system needs to be based on multiple parameters including the nanoparticles’ size, surface charge, and surface chemistry, which will differ depending on the types of drugs to be delivered and the cancer that is being treated (De Jong and Borm, 2008). A comprehensive understanding of the biological interactions that take place inside an animal or human system from when the nanoparticle is injected to when it reaches the site of drug delivery is required to tailor the nanoparticles for effective drug delivery. Once nanoparticles enter the biological system, drug delivery can be divided into two important stages: accumulation of the nanoparticle system at the tumor site, and entry into the cancer cells to release the drug into the preferred cellular compartment (nuclear or cytosolic).

Upon administration into the circulatory system, the nanoparticles will be treated as a foreign body and will be subjected to clearance mechanisms such as the host immune system (e.g., the mononuclear phagocytes, opsonization, and complement activation) and clearance through the kidneys and the liver (Nie, 2010). While one of the advantages of using a nanoparticulate system is that it can eventually be cleared by the host, the particles need to be in the circulation long enough to identify the tumor site and accumulate there to deliver the drug. Effective drug delivery is therefore a balance between the rate of clearance of the nanoparticle by the host and the properties of the nanoparticle system that cause tumor-specific accumulation. This preferential accumulation depends on a number of factors such as size, surface charge, and the functionalities that are present on the surface of the nanoparticle encapsulating the drug (Longmire et al., 2008).

Nanoparticles have an inherent ability to preferentially accumulate at tumor sites. This is called passive targeting and is due to a physiological effect of the tumor microenvironment called the enhanced permeability and retention (EPR) effect (Maeda, 2001). The blood vessels that surround the tumor microenvironment are generally leakier than the tight endothelial junctions seen in normal vasculature, enabling the nanoparticles to easily enter and accumulate at the tumor site. Insufficient lymphatic drainage in the tumor area further increases the accumulation (Maeda et al., 2013). Based on tumor type, the openings between the blood vessels are usually between 100 and 800 nm. Experimental trials with different tumor models have shown that nanoparticles with a size range of 30–200 nm have the best tumor retention capability (Jain and Stylianopoulos, 2010). It was also seen that spherical nanoparticles could more effectively extravasate into the tumor interstitium because they maintained a laminar flow pattern, compared to rod-shaped or bar-shaped particles, because their flow patterns in the blood were more unstable (Decuzzi et al., 2005).

Passive targeting still has multiple limitations, such as uneven distribution inside the solid tumor and entrapment within the tumor, which can be overcome by active targeting—this involves the molecular recognition of the tumor by the nanoparticles (Bertrand et al., 2014). Tumor-specific ligands, such as antibodies or small molecules, can be conjugated to the surface of the nanoparticle, which can then form high-affinity bonds with the receptors that are expressed on the tumor surface. This also aids in efficient receptor-mediated endocytosis and internalization of the nanoparticle into the cell for release of the drug (Byrne et al., 2008). Nanoparticles that have these molecular recognition signals are called multifunctional nanoparticles and will be discussed further in later sections of this chapter (Bao et al., 2013).

Passive and active targeting, therefore, act as complementary processes in delivering the drug into the tumor. Passive targeting ensures the accumulation of the nanoparticle drug from the bloodstream into the tumor microenvironment, while active targeting then enhances the retention and internalization of the drug by the tumor cells.

1.1.2 Intratumor Drug Release

A number of factors influence the internalization and processing of the nanoparticle containing the drug inside the tumor cell that ultimately results in drug release and its cytotoxic effect on the cell. Internalization into the cell happens through the various endocytic pathways (Oh and Park, 2014). Larger particles (250 nm or more) are internalized by phagocytosis. Nanoparticles are first opsonized by immunoglobulin, serum proteins, or by complement components. These opsonized complexes then bind to the cell surface, causing the formation of extensions of the plasma membrane, which engulfs the particles to form an intracellular vesicle called the phagosome that later fuses with the lysosome (Aderem and Underhill, 1999). The acidic environment of the lysosome might be detrimental to the drug carried by the nanoparticle, which should be considered. Slightly smaller particles (about 100 nm) are taken in by a similar process called pinocytosis, where the nanoparticle along with the extracellular fluid that it floats in is internalized in a vesicle (Wang et al., 2011). Internalization of smaller nanoparticles (around 100 nm and smaller) is brought about by clathrin-mediated endocytosis. Nanoparticles that bind to ligand-specific receptors on the tumor cell surface are internalized by the clathrin proteins that polymerize to the receptor on the cytosolic side of the cell and form a vesicle surrounding the receptor–ligand–nanoparticle complex. The clathrin coat is shed inside the cell, with the vesicle moving toward the destination of the receptor (Doherty and McMahon, 2009). Another process of internalizing smaller nanoparticles (less than 100 nm) is caveolae-mediated endocytosis. Caveolin proteins on the cytosolic side bind to the receptor that is bound to the nanoparticle–ligand complex and form a flask-shaped vesicle that is then endocytosed. Caveolin-coated vesicles bypass the lysosomes and therefore prevent degradation of the cargo enclosed inside. Instead, they move along the microtubules to the endoplasmic reticulum from where they are released into the cytosol (Parton and Simons, 2007).

While size is an important factor contributing to the type of endocytic pathway that the nanoparticle–drug complex is internalized through, there are other characteristics that influence how the nanoparticles are brought into the tumor cell (Kuhn et al., 2014). Surface charge is one such factor. The cell membrane is negatively charged; therefore cationic nanoparticles could rapidly enter the cell due to a strong electrostatic interaction with the cell (Coulman et al., 2009). Anionic nanoparticles interact with the positively charged area of the receptor proteins that they bind to be internalized (Yeung et al., 2008). The hydrophobicity of a nanoparticle also enhances internalization by the tumor cell because it has a higher affinity toward the cell membrane. Hydrophilic polymers such as polyethylene glycol (PEG) (Lankveld et al., 2011) and dextran (Moore et al., 2000) interfere with the interaction between the nanoparticle and the lipid bilayer of the cell; however, they are commonly used in synthesizing nanoparticles because this interference prevents nonspecific binding and degradation and increases the circulation time of the nanoparticle in the bloodstream. The presence of a ligand that specifically recognizes a receptor protein on the tumor cell surface greatly increases the internalization and retention of the nanoparticle. It is therefore a combination of multiple factors that need to be considered during the synthesis of the nanoparticle–drug complex that determines the mode of internalization into the tumor cell for drug release.

1.1.3 Biological Clearance

Nanoparticles in the bloodstream are considered foreign objects and are subjected to the various ways of clearance from the body such as the immune system, and organs such as kidneys and liver (Moghimi et al., 2001). For effective tumor uptake, the nanoparticles need to overcome these forces of clearance that actively work to expel them from the body. The mononuclear phagocytic system consists of the phagocytic cells, such as macrophages, Kupffer cells, and monocytes that are present in organs such as the liver, spleen, and bone marrow that can engulf nanoparticles and cause degradation (Song et al., 2014). The phagocytes recognize the nanoparticles by the presence of the opsonins such as the immunoglobulin proteins, complement proteins, albumins, fibronectin, and apolipoproteins that are present in the blood serum. Once the nanoparticles have been opsonized and engulfed, they are subjected to enzymatic degradation, with the segments that are not degraded sent to organs such as the liver and spleen. The Kupffer cells and hepatocytes in the liver are involved in cleaning the segments of the nanoparticles (Johnston et al., 2010). Nanoparticles larger than 250 nm preferentially also accumulate and are cleared by the spleen (Lee and Cheng, 2013). Various methods have been utilized to reduce clearance of the nanoparticles before they have delivered their drug cargo. Nanoparticles that are charged are opsonized more rapidly than neutral particles (Longmire et al., 2008). So by neutralizing the surface charge, opsonization can be minimized. Additionally, coating nanoparticles with molecules such as PEG results in lower opsonization, which increases the plasma circulation time of nanoparticles. Renal clearance is a size-based filtration process with the kidneys clearing out nanoparticles that are less than 8 nm in size (Choi et al., 2002).

1.1.4 Toxicity of Nanoparticles

The other side to clearance of the nanoparticle before drug delivery is the harmful toxicity that is associated with the nanoparticle itself. Nanoparticles as a whole, or the products of degradation of these nanoparticles, have been known to cause symptoms such as epidermal and dermal changes and sensitization, neurotoxicity, developmental toxicity, cardiotoxicity, and genotoxicity (Love et al., 2012). It is also notable that studies monitoring the long-term effects of these nanoparticles are still underway and data are still being collected on the toxic effects of certain nanoparticles. Biodegradable nanopolymers, such as polylactide (PLA), polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), and gelatin, can be completely broken down and have been widely used to encapsulate many anticancer drugs to minimize toxicity (Panyam and Labhasetwar, 2003).

The synthesis of nanoparticles that can effectively deliver drugs to the target tissue is a complex process that is influenced by multiple characteristics that all need to be balanced against each other. As discussed in this section, a feature that might be advantageous in one therapeutic setting may well be a deterrent in another. It is therefore imperative to comprehensively understand the environment of the tumor, the type of tumor cells and the mode of internalization, the preferred intracellular target of the drug, and the clearance mechanisms that are involved in the chosen therapeutic setting for drug delivery through nanoparticle systems.

1.2 Preparation and Characterization of Nanomaterials

1.2.1 Preparation

Human cells are 10,000–20,000 nm in diameter (with plasma membrane of 6 nm), while nanomaterials can be generated in 1–1000 nm in diameter from a wide variety of materials such as polymeric forms of amino acids, synthetic organic polymers, lipids, inorganic salts, oligonucleotides, etc. (Figure 1.3a). There are three general structural architectures that nanomaterials usually fall into: (I) shell-type nanoparticles, such as liposomes, where an aqueous compartment is surrounded by a shell bilayer of phospholipids; these can also be used as a promising and robust platform for delivery of therapeutic or imaging agents (Mandal et al., 2013); (II) nanomaterial impregnated with therapeutic or imaging agents that can be modified with targeting molecules by chemical reactions through a linker (Brigger et al., 2002); and (III) nanoparticles based on metal cores that can be modified through a linker-targeting ligand system (Huang et al., 2011) (Figure 1.3b).

Figure 1.3 Nanoparticle size and structural architectures. (a) Comparison of human cell size with nanoparticle size. (b) Three general structural architectures that nanomaterials can fall into: (I) shell-type nanoparticles, such as liposomes; (II) impregnated nanomaterials with therapeutic or imaging agents that can be modified with targeting molecules by chemical reactions through a linker; (III) nanoparticles based on metal cores that can be modified through a linker-targeting ligand system.

Recently, developments in nanotechnology have allowed the introduction of several types of nanomaterials capable of carrying small molecules to target cells. The general and multifunctional classes of known nanomaterials used in biotechnology include fullerenes (buckyballs and carbon tubes), liquid crystals (composed of organic liquid crystal materials that mimic biomolecules like proteins or lipids), liposomes (lipid-based liquid crystals), nanoshells (core-shells), QDs (nanosized semiconductors that can emit light), superparamagnetic molecules (molecules that are attracted to a magnetic field), dendrimers (nanomaterials with multiple molecular hooks on their surfaces), nanorods (semiconducting materials as imaging and contrast agents), and polymers.

The first nanotechnology drug-delivery system for cancer chemotherapy was a liposome used as a closed bilayer phospholipid system made of a lipid surrounding a water core that was used for encapsulation of the anthracycline antibiotic doxorubicin, which is an effective anticancer agent for leukemia, solid tumors (Treat et al., 1990), breast, and ovarian cancer cells (Forssen and Tokes, 1983; Straubinger et al., 1988).

1.2.2 Characterization

1.2.2.1 Physicochemical characterization

Physical and chemical properties of nanomaterials affect their interactions with biological systems with regard to cellular uptake, receptor binding, and target cell access. This is very important to better understand the toxicology (Oberdörster, 2010; Oberdörster et al., 2005) and medical applications (Aggarwal et al., 2009; Treuel et al., 2013) of nanomaterials. There are many varieties of nanomaterials currently being studied and developed for biomedical applications, and since each class of nanomaterials has a different structure, they have different physical properties such as size, shape, molecular weight, surface chemistry, purity, identity, functional capability, stability, and solubility. Therefore, various strategies, tools, and analytical instruments are required for characterizing the physicochemical properties of nanomaterials. These properties can be measured with different spectroscopic techniques such as ultraviolet–visible (UV–vis) spectrophotometry, mass spectrometry (MS), nuclear magnetic resonance (NMR), infrared spectroscopy (IR), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), and other analytical and chromatographic methods, including gas chromatography (GC), high-performance liquid chromatography (HPLC), size exclusion chromatography (SEC), liquid chromatograph interfaced to mass selective detector (LC-MS), and gel permeation chromatography (GPC). Scanning probe microscopy (SPM) can be used for size and structure topology of nanomaterials. For structural, magnetic, electronic, and thermal properties of nanomaterials, scanning tunneling microscopy, electric field gradient microscopy, scanning thermal microscopy, magnetic field microscopy, transmission electron microscope (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and atomic force microscope (AFM) can be employed (Hughes et al., 2008; Lin et al., 2014; Podila and Brown, 2013; Rao and Biswas, 2009). Using these analytical and spectroscopic methods provides the necessary physicochemical information of synthesized nanomaterials that will be very useful in structural analysis of the nanomaterials with target molecules (Lin et al., 2014).

1.3 Nanotechnology-Assisted Formulating of Poorly Water-Soluble Compounds

It is known that more than 40% of bioactive small compounds identified through combinatorial screening methods are poorly water-soluble, which causes many problems in formulation of these molecules. For those bioactive molecules that need to pass through cell membranes, the molecule generally must possess some lipophilic or hydrophobic properties. In order to overcome this issue, researchers usually convert the poorly water-soluble small molecules to the salt form, which increases their water solubility while retaining biological activity. However, an alternative approach for enhancement of solubility for nanoparticle production is to use a formulation approach that will not allow the molecule to be abandoned early on in its development process so that the product is not launched with suboptimal properties including poor bioavailability, lack of fed/fasted equivalence, lack of optimal dosing, presence of extra excipients that pose limitations with respect to dose escalation, and ultimately, poor patient compliance (Merisko-Liversidge and Liversidge, 2008). Solubility of bioactive molecules is very important and is one of the key factors in the evaluation of their bioactivity and their affinity and interaction with the relevant biological target. Therefore, nanometer-sized particles can be used in order to increase the bioavailability of the poorly water-soluble molecules that are attributed to dissolution-rate kinetics, which is directly proportional to the surface area of the drug according to the Noyes–Whitney model for dissolution kinetics (Jinno et al., 2006; Langguth et al., 2005; Noyes and Whitney, 1897; Wu et al., 2004). On the other hand, sometimes nanoparticle formulations have been used to reduce bioavailability effects, if it depends on the nutritional state of the subject or is not dose-proportional. The formulations system for these compounds consists of water, drug, and safe excipients to improve bioavailability and enhance drug exposure for oral and parenteral dosage forms.

1.4 Biodegradable Polymers Used in Controlled Drug Delivery

The pharmaceutical applications of nanomaterials based on polymers are becoming increasingly important in the field of drug delivery and cancer therapy. Polymers can be obtained from natural sources like protein-based polymers (collagen, albumin, gelatin) and polysaccharides (alginate, cyclodextrin, chitosan, dextran, agarose, hyaluronic acid, starch, cellulose). Polymers can also be chemically synthesized as biodegradable (polyester, polyanhydride, polyamides, phosphorous-based polymer, polycyanoacrylates, polyurethanes, polyorthoester, polyacetals, etc.) and nonbiodegradable (cellulose derivative, silicons). There are different synthetic biodegradable polymers currently being developed and utilized as drug-delivery systems or as suitable structures for tissue engineering because of their known biocompatibility and biodegradability (Gavasane, 2014; Hubbell, 1998; Nair and Laurencin, 2007, 2006; Tian et al., 2012). There are many advantages of biodegradable polymers as drug carriers, but the five most important advantages of biodegradable polymers over other carriers are sustained delivery of drug, localized delivery of drug, stabilization of drug, release rate (which is less dependent on the drug properties), and steady release rate with time (Gavasane, 2014).

1.5 Multifunctional Nanomaterials for Cancer Therapy

In order to enhance the delivery efficiency of therapeutic and diagnostic agents, the use of nanomaterials has been extensively increased for applications in both experimental and clinical trials. Developing new multifunctional nanomaterials is attractive because they can carry diverse functionalities (targeting, therapeutic, and imaging modules), all in one nanomaterial that enhances their ability to achieve effective therapeutic treatments and will make them highly suitable for targeted therapeutic delivery in different clinical areas (Figure 1.4) (Montet et al., 2006; Srinivasan et al., 2015). Several agents with diagnostic, targeting or therapeutic properties can be incorporated into nanoparticles by modification of the surface of nanoparticles. Targeting molecules will increase the binding affinity of a nanoparticle to target a cell and then the nanoparticle will be internalized into the cell through endosome. The presence of imaging agents is very important to trace the nanoparticle using different imaging machines. In the following sections, surface modifications of nanoparticle systems to provide multimodal functionalities using different chemical and enzymatic reactions both in vitro and/or in vivo will be discussed (Bae et al., 2011; Das et al., 2009; Jinhao et al., 2009; Pan et al., 2008).

Figure 1.4 A well-designed multifunctional nanoparticle should be capable of carrying diverse functionalities (targeting, therapeutic, and imaging modules), all in one nanomaterial. DOTA, 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid; RGD, arginine glycine aspartic acid.

1.5.1 Targeting

Molecularly targeted therapy is one of the major modalities for cancer treatment by physically targeting cancer cells to block cancer cell growth without any uptake to normal cells. Two important strategies are passive and active targeting, and they are required in the design of nanomaterials to achieve effective drug delivery. By employing these two strategies, drugs should be able to reach the desired tumor sites with minimal loss to their activity in the blood circulation and without harmful effects to healthy tissues (Figure 1.5).

Figure 1.5 Passive and active targeting are two important strategies that are required in the design of nanomaterials to achieve effective drug delivery.

Passive targeting is a size-dependent process and is based on the pharmacokinetics of nanomaterials. It can be realized by enhanced permeability in tumor cells through the enhanced permeability and retention (EPR) effect that allows penetration of macromolecules up to 400 nm into the surrounding tumor region. Passive delivery can be employed by modification of the nanomaterials’ surface using different hydrophilic spacers such as poloxamer, polyethylene oxide (PEO), PEG, lauryl ethers, and polysorbate (Soppimath et al., 2001). These linkers enable nanomaterials to avoid uptake by the mononuclear phagocyte system and thereby improve the circulation time in blood (Shakesheff et al., 1997).

Active targeting relies on molecular interactions between therapeutic nanomaterials and receptors on cell membranes, thereby delivering the drug to the exact site of action, which can potentially increase the efficacy and reduce the toxicity of therapeutic agents. In active delivery strategy, bioactive targeting ligands can be classified into four types including small bioactive molecules (vitamins like biotin, folate, galactose, mannose, and glucose), peptides (like L-arginine glycine L-aspartic acid (RGD)), proteins (transferrin and antibodies), and oligonucleotides (aptamers).

Among vitamins, biotin (also known as vitamin H or coenzyme R) is a growth promoter of cells. It has been used as an adequate targeting agent in targeted drug-delivery systems and can be taken up preferentially by cancer cells (Chen et al., 2010b; Taheri et al., 2011; Yang et al., 2009). Biotin also plays a key role in intermediary metabolism, energy production, cell differentiation, cell proliferation in the retina, and as a coenzyme for different carboxylases in pathways of gluconeogenesis, fatty acid biosynthesis, and catabolism of various odd-chain fatty acids, branched chain amino acids; it is also involved in the regulation of gene expression of specific membrane transporters and in the regulation of the intracellular cGMP. It has been observed that biotin is significantly overexpressed in some cancerous tumors, more than in normal tissue and, due to its high content in cancerous tumors, the cancer cells proliferate rapidly. These cancer cells often overexpress biotin-specific receptors on the cell surface. In order to increase the uptake of anticancer drugs in cancer cells, researchers have tried to conjugate biotin to small bioactive molecules (therapeutic agents like drugs, siRNA, and miRNA). For the application of biotin-conjugated nanoparticles to biological systems, whether the biotin moiety can guide the nanoparticle preferentially to biotin receptor-positive or biotin receptor-negative tumor cells has been investigated. In this regard A549 and WI38 cells can be used, because they are known to be biotin receptor-positive and -negative cells, respectively (Chen et al., 2010b; Heo et al., 2012; Maiti et al., 2013).

Folic acid or folate, also known as vitamin B, is important for the production and maintenance of new cells, particularly during both pregnancy and infancy when cell growth is extremely rapid. Folate receptor is overexpressed on the surface of many human cancer cells, particularly the KB carcinoma cell line and has high affinity to folate–nanoparticle conjugates, triggering cellular uptake via endocytosis (Chen et al., 2009; Hilgenbrink and Low, 2005; Leamon and Low, 2001; Low et al., 2008; Lu and Low, 2012; Toffoli et al., 1997).

Carbohydrate agents can be used as an important class of targeting moiety in various drug-delivery systems (Guillén et al., 2010; Jones, 1994; Kim and Nie, 2005; Lepenies et al., 2013; Pashov et al., 2005; Sinha et al., 2006). The surfaces of cells coated by carbohydrate ligands affect tumor cell interactions by carrying the required information for cell–cell recognition (Bozzaro, 1985; Brandley and Schnaar, 1986; Bucior and Burger, 2004). For example, carbohydrate conjugated to lectin (sugar-specific receptors) is an important and classical model to target cell surface carbohydrates, and this ligand–carbohydrate interaction can be designed using nanoparticles containing carbohydrate moieties that are bound to lectins via direct lectin targeting or reverse lectin targeting. Therefore, drug-delivery systems that have been developed based on this novel interaction between carbohydrates and lectins can be studied for the development of smart nanocarrier molecules for drug delivery (Gorelik et al., 2001; Minko, 2004; Raz et al., 1986; Smart, 2004; Yamazaki et al., 2000).

Peptides are an important class of targeting agent that can be used as targeting moieties and in line with this concept, peptide ligands containing arginine-glycine-aspartate (RGD) displayed a strong affinity and selectivity to the αvβ3 integrin (Danhier et al., 2012b; Li et al., 2004). RGD is a tumor marker for αvβ3 and other integrins that are highly expressed on the surface of angiogenic blood vessels (Gao et al., 2004; Wang et al., 2010a,b; Wang and Guo, 2013; Yu et al., 2010). One of the important RGD compounds that has entered phase II clinical trials as an antiangiogenic agent is the cyclic pentapeptide c[-RGDf(NMe)V-] (Cilengitide) developed by Kessler and colleagues (Dechantsreiter et al., 1999). However, RGD is also able to bind to other integrins such as α5β1 and α4β1, but these are not specific to cancer cells and that may limit their application (Peer et al., 2007). There are several other peptides that have been identified as potential tools to target tumor cells. For example, amino acid sequence (YSA; YSAYPDSVPMMS) is a peptide identified by phage display that selectively targets EphA2 and that effectively delivers anticancer agents to prostate cancer tumors (Koolpe et al., 2002; Mitra et al., 2010). Two peptides, CREKA (contains a cysteine residue) and LyP-1 (CGQKRTRGC as a cyclic peptide with a disulfide bond), can be specifically used for targeting cancer cells and lymphatic vessels upon injection in mice bearing MDA-MB-435 tumors (Karmali et al., 2009; Laakkonen et al., 2002; Wykosky and Debinski, 2008). By modification of adenovirus vector with PEG-linked CGKRK, a specific interaction of the CGKRK peptide with a receptor at the cell surface has been observed, enabling efficient internalization of CGKRK-linked adenovirus (Yao et al., 2012).

Aptamers are oligonucleotides with 15–40 bases that bind to various molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. They can be engineered through an in vitro repeated procedure called SELEX (systematic evolution of ligands by exponential enrichment), which can be selected from pool libraries of random oligonucleotides containing between 1×10¹³ and 1×10¹⁵ members (Lee et al., 2006; Tuerk and Gold, 1990; Wilson and Szostak, 1999). In general there are two SELEX types for discovery of aptamers: DNA SELEX and RNA SELEX. For DNA SELEX, first the double-stranded DNA is prepared by PCR techniques and then a library of single-stranded DNA is prepared by separation from that DNA double strand. For RNA SELEX, first the double-stranded DNA templates are transcripted in vitro using recombinant T7 RNA polymerase and then the library of single-stranded RNA molecules is prepared. These aptamers, based on both DNA or RNA, showed high affinity to target a wide range of protein families including proteases, kinases, cell-surface receptors, cytokines, and cell-adhesion molecules. They have different therapeutic applications in the various stages of cancer progression, such as prevention of thrombosis, angiogenesis, neovascularization, and metastasis. For example, aptamers that can be used for prevention of tumor development can target several proteins, including receptor tyrosine kinase RETC634Y, substance P, mucin 1, epidermal growth factor receptor variant III (EGFRvIII), ghrelin, HER3 (ERBB3), cytotoxic T-lymphocyte-associated protein 4, cytohesin 2, E2F transcription factor, gonadotropin-releasing hormone 1, αvβ3 integrin, and tenascin C.

One of the FDA-approved aptamers for the treatment of all types of neovascular age-related macular degeneration (AMD) is pegaptanib sodium (Macugen®), an antivascular endothelial growth factor (anti-VEGF) RNA aptamer (Ng et al., 2006). AS1411 is another antinucleolin aptamer in phase II clinical development with a 26-base guanine-rich oligodeoxynucleotide with potential affinity to nucleolin, a nucleolar phosphoprotein that is overexpressed on the surface of various cancer cells (Bates et al., 2009; Mongelard and Bouvet, 2010).

An antibody, a large Y-shape protein produced by plasma cells, can be used to identify and recognize cancer cells. It has been reported that EGFRvIII antibody-conjugated iron oxide nanoparticles (IONPs) can bind to the epidermal growth factor receptor (EGFR) deletion mutant, a receptor that presents on human glioblastoma multiforme (GBM) cells (Gao et al., 2004; Hadjipanayis et al., 2010). Transferrin is a plasma glycoprotein that controls the level of free iron and the regulation of cell growth. Transferrin is extensively employed as a cancer-targeting ligand for the delivery of antitumor drugs due to overexpression of transferrin receptor on the surface of different fast-growing cancer cells (Figure 1.6) (Gaspar et al., 2012; Qian et al., 2002; Suzuki et al., 2008; Yhee et al., 2013).

Figure 1.6 Bioactive targeting moieties can be classified into four types including small bioactive molecules (biotin, folate, galactose, mannose, and glucose), peptides (like L-arginine glycine L-aspartic acid (RGD)), proteins (transferrin and antibodies), and oligonucleotides (aptamers).

1.5.2 Imaging

Several traditional imaging techniques, such as ultrasound (US), magnetic resonance imaging (MRI), optical imaging (OI), computed tomography (CT), and radionuclide imaging (positron emission tomography (PET), single-photon emission computed tomography (SPECT)) have been established for various experimental and clinical applications during recent decades. These imaging techniques have been extensively applied in small-animal imaging, preclinical and clinical imaging, as well as in diagnosis and treatment (Massoud and Gambhir, 2003; Michalet et al., 2005).

The use of nanomaterials in molecular cancer imaging provides new opportunities for the development of novel imaging agents and techniques in cancer imaging. Design and synthesis of imaging probes at the scale of nanometers allows better-controlled biodistribution, sensitivity, enhancement, spatial, and temporal information, that could be ultimately translated to clinical advantages, including earlier diagnosis and real-time assessment of disease progression. Novel, multifunctional nanosized probes can be used in various imaging techniques such as US, PET/SPECT, and MRI (Gao et al., 2004; Larson et al., 2003; Walling et al., 2009).

IONPs as contrast agents can be used for T2 and T1 measurements in MRI molecular imaging. IONPs can be produced with monodispersity and uniform crystallinity using thermal decomposition methods and subsequently these hydrophobic IONPs can be coated with silica, phospholipids, or amphiphilic polymers. In terms of various particle sizes, IONPs can be classified into three types: VSOP (diameter <10 nm), USPIO (diameter ~20 nm), and SPIO (diameter >30 nm) (Gupta and Gupta, 2005; Kiessling et al., 2010; Lin et al., 2010; Wang et al., 2001).

Gold nanoparticles have been well studied over the past decade and have wide applications in nanomedicine, including as nanoshells, nanocages, nanorods, and surface-enhanced Raman scattering nanoparticles (Kiessling et al., 2010). Kim et al. reported and prepared a multifunctional magnetic gold nanoshell by coating silica spheres with gold nanoshells embedded with Fe3O4 nanoparticles. These multifunctional magnetic gold nanoshells can be used as a platform for MRI and photothermal therapy of breast cancers, and MRI along with NIR radiation can be used for selectively killing breast cancer cells (Kim et al., 2006; Park et al., 2008).

Quantum dots (QDs) are nanocrystals made of semiconductor materials that are able to exhibit quantum mechanical properties that can be readily stabilized with surface modifications and conjugated with different targeting ligands. Modified QDs can be used as molecular imaging probes for MRI and fluorescence microscopy. To that aim, QDs were PEGylated and coated with paramagnetic ligands and subsequently functionalized by RGD peptides (Mulder et al., 2006).

1.5.3 Chemotherapy

The use of unique nanomaterials in drug delivery has several advantages, including increase of drug stability, solubility, and enhancement of drug half-lives in the blood circulation with fewer effects to normal and healthy cells. One type of nanomaterial that has been extensively used for delivering anticancer drugs is liposomes as a phospholipid vesicle with a bilayered membrane structure. For example, PEGylated liposome can encapsulate doxorubicin for chemotherapy and be surface-functionalized by gadolinium or fluorophores for imaging. Other important nanocarriers in chemotherapy are biodegradable polymers such as poly(lactic-co-glycolic acid) (PLGA), polylactide (PLA), polyethyleneimine (PEI), polylysine, and cyclodextrin. Another important class of multifunctional nanomaterial for chemotherapy is dendrimers because they have a network of branching chemical bonds around an inner core. Among the various polymers developed to formulate polymeric nanomaterials, PLGA is one of the most successfully developed biodegradable polymers and has attracted considerable attention due to its attractive properties such as biocompatibility, biodegradability, and well-described formulations methods. It is also attractive because of its properties for protection of the drug from degradation, the possibility of sustained release, and the possibility for surface modification. Finally, it is approved by the FDA for use in drug-delivery systems (Danhier et al., 2012a; Gentile et al., 2014; Muthu, 2009).

Some of these nanoparticle-based drugs have been approved by the FDA for cancer therapy. Figure 1.7 shows the chemical structure of some anticancer drugs, including doxorubicin (DOX), paclitaxel (PXL), docetaxel, cis-diaminedichloroplatinum (II) (cisplatin), cytarabine, vincristine (VCR), vinblastine (VLB), vinorelbine (microtubule inhibitor), camptothecine, lurtotecan, and irinotecan that can be conjugated or encapsulated in nanomaterials and increase their water solubility and bioavailability. For example, Doxil® (for DOX), a PEGylated liposomal system, and Abraxane® (for PXL), albumin-bound paclitaxel nanomaterials, have been used for the treatment of patients with late-stage (metastatic) pancreatic and breast cancers, with a half-life about 100 times longer than that of the free drugs (Allen and Martin, 2004; Desai et al., 2006; Gabizon et al., 2003; Miele et al., 2009; Sharpe et al., 2002).

Figure 1.7 Examples of the chemical structure of some anticancer drugs such as doxorubicin (DOX), paclitaxel (PXL), docetaxel, cis-diamminedichloroplatinum (II) (cisplatin), cytarabine, vincristine (VCR), vinblastine (VLB), vinorelbine (microtubule inhibitor), camptothecine, lurtotecan, and irinotecan that can be conjugated or encapsulated in nanomaterials and thereby increase their water solubility and bioavailability.

1.6 Chemical Conjugation of Nanomaterials

Multivalency of the multifunctional nanomaterials allows for a combination of targeting moieties (e.g., chemical ligand and aptamer), imaging agents (e.g., fluorophore and radionucleolide), and therapeutic agents (e.g., drug, siRNA, and miRNA), all in one nanoparticle. For this purpose, biological or chemical small molecules can be incorporated into nanomaterials using enzymatic or chemical reactions. A well-designed nanoparticle construct consists of three components: nanoparticle with proper functional group, a spacer (linker), and small molecules. Nanomaterials with reactive groups, such as hydroxyl, aldehyde, alkyne, amine, thiol, and azide, etc., provide appropriate functionalized nanomaterials that can react with small molecules (drugs, targeting ligands, or a dye) (Figure 1.8).

Figure 1.8 A well-designed nanoparticle construct consists of three components: nanoparticle with proper functional group, a spacer (linker), and small molecules. Nanomaterials with reactive groups, such as hydroxyl, aldehyde, alkyne, amine, thiol, and azide, etc., provide appropriate functionalized nanomaterials that can react with small molecules (drugs, targeting ligands, or a dye).

The linker separates the nanoparticle from ligands while altering the hydrophobicity or hydrophilicity of the nanomaterials and can also control the binding orientations of ligands. For bioconjugation, chemists use a variety of linker molecules depending on their applications. Figure 1.9 shows some important linkers that are used in bioconjugation of small molecules to nanomaterials such as succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC), N-succinimidyl iodoacetate (SIA), N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP), succinimidyl 6-(3-[2-pyridyldithio]-propionamido) hexanoate (LC-SPDP), heterobifunctional PEG molecules (NHS-[PEG]n-MAL), 6-azido-hexanoic acid, disuccinimidyl suberate (DSS), NHS-Azide Staudinger Ligation Reagents, 3,3′-N-[ε-maleimidocaproic acid] hydrazide and its trifluoroacetic acid salt (EMCH.TFA) (Figure 1.9).

Figure 1.9 Some important linkers that are used in bioconjugation of small molecules to nanomaterials such as SMCC, sulfo-SMCC, SIA, SPDP, LC-SPDP, NHS-[PEG]n-MAL, DSS, and EMCH.

Pharmacokinetics and biodistribution profiles of drugs will be changed after their conjugation to nanomaterials, which improves the efficacy of the nanomaterials’ therapeutics and produces physicochemical changes that favor drug release at the target site. These physicochemical changes include physical signals such as electric field, temperature, ultrasound, and magnetic field and chemical signals such as pH, redox potential, ionic strength, and enzymatic activities.

1.6.1 pH-Sensitive Linkages Used for Bioconjugation of Nanomaterials

Among physicochemical change signals, pH gradients have been extensively used for design and synthesis of new nanomaterials (Gao et al., 2010; Li et al., 2010; Mano, 2008; Medeiros et al., 2011; Shen et al., 2008; Zha et al., 2011). Figure 1.10 shows examples of some common chemical pH-sensitive reactions used in conjugation chemistry and their hydrolytic products (Gao et al., 2010; Shen et al., 2008). For example, the reaction of aldehyde-modified nanoparticles with various reactive groups such as amino, hydrazide, and aminoxy groups form imines (Schiff base), hydrazones, and oximes, respectively, and are able to release the small molecules via a pH-controlled manner (Figure 1.10a–c). The Schiff-base linkage can be hydrolyzed at acidic pH conditions (Gu et al., 2008; Wang et al., 2008). In addition, one of the advantages of the imine bond is that it can form in aqueous solution (Figure 1.10a) (Ahmad et al., 2006; Wang et al., 2010a). Hydrazones can be made from aldehydes (RHO) or ketones (R1–CO–R2) and hydrazides (R–NH–NH2) under very mild conditions like aqueous solutions. The formation of a hydrazone bond can occur even in vivo from separate fragments that self-assemble under physiological conditions (Figure 1.10b). Hydrazones are much more stable than imines due to delocalization of the π-electrons and become a good, stable candidate for application in drug-delivery systems (Kale and Torchilin, 2007). Oxime linkages were confirmed to be an interesting bond for the design of pH-sensitive polymeric drug-delivery systems. The oximization reaction by the carbonyl group of an aldehyde or ketone with alkoxyamines (R–O–NH2) affords the oxime ethers in mild conditions, which will make this conjugation ideal for a vast number of applications (Figure 1.10c) (Miranda et al., 2007; Sohma and Kent, 2009). Vinyl ether can be hydrolyzed through a rate-determining proton transfer step to the β-carbon of the vinyl ether, followed by rapid hydration and rapid decomposition of a hemiacetal intermediate (Figure 1.10d) (Li and Lee, 2010; Shin et al., 2012; Xu et al., 2008). Esterification can occur by reaction of carboxylic acids with hydroxyl group of ligands (Figure 1.10e) (Sengupta et al., 2005; Tong and Cheng, 2009). β-Thiopropionate linkage (3-sulfanylpropionyl linkage) can be prepared by reaction of an acrylate-functional block nanoparticle with a thiol group of ligands via thiol-ene click chemistry. β-Thiopropionate linkage is a pH-sensitive linkage that can be easily cleaved at the pH corresponding to an intracellular endosomal compartment (pH 5.0–6.5) (Figure 1.10f) (Oishi et al., 2005). Various pH-sensitive nanomaterials have been developed based on acetal-containing amphiphilic polymers for loading and triggered release of poorly water-soluble anticancer drugs, including doxorubicin and paclitaxel (Gillies and Fréchet, 2005; Gillies et al., 2004; Oishi et al., 2005). Gu et al. reported that acetal-linked paclitaxel prodrug nanomaterials can effectively overcome multidrug resistance, which is probably due to effective cellular uptake via the endocytosis pathway as well as fast paclitaxel release in the mildly acidic endo/lysosomal compartments. These acetal-based delivery systems have several advantages, such as fast degradation under endo/lysosomal pH conditions, absence of acidic degradation products, and versatile preparation and applications (Gu et al., 2013). This acetal linkage can be prepared by reaction of vinyl ether-modified nanoparticles with the hydroxyl group of the ligands (Figure