Nanoscale Fabrication, Optimization, Scale-up and Biological Aspects of Pharmaceutical Nanotechnology

Argentina

Series Preface: Pharmaceutical Nanotechnology

Alina M. Holban, University of Bucharest, Bucharest, Romania

Due to its immense applicative potential, nanotechnology is considered the leading technology of the 21st century. The science and engineering of nanometer-sized materials is currently employed for the development of numerous scientific, industrial, ecological, and technological fields. Biology, medicine, chemistry, pharmacy, agriculture, food industry, and material science are the main fields which have benefited from the great technological progress developed in nanoscience.

In the pharmaceutical field, nanotechnology has revolutionized traditional drug-design concept and the art of drug delivery. The idea of a highly specific nanoscale drug for the targeted therapy of diseases is now considered a feasible treatment for severe health conditions.

Some scientists believe that the pharmaceutical domain has been reborn by the important contribution of nanotechnology. The field of pharmaceutical nanotechnology has the potential to offer innovative solutions for all diagnosis, therapy, and prophylaxis domains. Application of nanotechnology tools in pharmaceutical research and design is likely to result in moving the industry from a blockbuster drug model to personalized medicine. The current main focus of clinicians is to treat patients individually, not their general diagnosed diseases, which are usually difficult to diagnose or incorrectly diagnosed. There are compelling applications in the pharmaceutical industry where suitable nanotechnology tools can be successfully utilized. By designing and modifying drugs at nanoscale, pharmaceutical nanotechnology could be useful not only for the development of completely new therapeutic solutions, but also to add value to existing products. This possibility opens perspectives of success for pharmaceutical companies in existing markets, but also for new markets.

Scientists have manifested an impressive interest on the field of pharmaceutical nanotechnology research in recent years. However, we face today a true dilemma of data unavailability, due to the multitude of existing information which can be highly inaccurate and contradictory. This is because of the lack of an efficient model for sorting the plethora of nanotechnology tools and information that exists, and strategically correlate those with potential opportunities into different segments of pharmaceutical research and design.

This series is trying to cover the most relevant aspects regarding the great progress of nanotechnology in the pharmaceutical field and to highlight the currently emerging trend of pharmaceutical nanotechnology towards the personalized medicine concept.

The 10 volumes of this series are structured to wisely offer relevant information regarding basic concepts and also to reveal the newest approaches and perspectives in pharmaceutical nanotechnology.

Nanoscale Fabrication, Optimization, Scale-up and Biological Aspects of Pharmaceutical Nanotechnology, introduces the readers into the amazing field of nanoscale design. Also, this volume facilitate understanding of the biological requirements of nanostructured pharmaceutical formulations for advanced drugs.

In Design and Development of New Nanocarriers, the most recent progress made on the field of nano-delivery is discussed. Modern nanostructured drug carriers employ innovative solutions for the detection and treatment of various diseases in a personalized and efficient manner.

Design of Nanostructures for Theranostics Applications, highlights the impressive impact of nanotechnology in the development of combined diagnosis and therapy concept: theranostics.

Design of Nanostructures for Antimicrobial, Antioxidant and Nutraceutical Applications, offers a dynamic solution for immune modulation, treatment of diseases by natural-based products and infection control, while employing nanostructured solutions to achieve top results.

Nanostructures for the Engineering of Cells, Tissues and Organs: From Design to Applications, is a highly investigated and debated field; tissue engineering, is dissected through this volume. Here is shown how nanotechnology has advanced research and applications in the manipulation and engineering of cells and tissues in vitro.

Organic Materials as Smart Nanocarriers for Drug Delivery, deals with the specific world of organic nanomaterials, revealing their wide applications, types, and advantages in drug delivery.

In the volume entitled: Inorganic Frameworks as Smart Nanomedicines, the main focus is to discuss the variety and properties of inorganic nanostructures for therapy and drug delivery in the context of improved personalized medicine.

Lipid Nanocarriers for Drug Targeting, deals with recently developed lipid nanostructures and the advances made in drug targeting.

Drug Targeting and Stimuli-Sensitive Drug Delivery Systems, dissects smart stimuli-responsive nanosystems employed to specifically detect various biochemical conditions and control the release of drugs.

Fullerens, Graphenes and Nanotubes: A Pharmaceutical Approach, reveals major findings made on widely applied drug-design nanosystems, namely fullerens, graphenes and nanotubes. The impact of these nanostructures in pharmaceutical research is highlighted.

All 10 volumes are nicely illustrated and chapters are organized into a logical manner to be accessible to a wide audience. The series is a valuable resource of new and comprehensive scientific proof on the intriguing and emerging field of pharmaceutical nanotechnology, which could be of a great use for scientists, engineers, pharmaceutical representatives, clinicians, and any non-specialist interested user.

Preface

Alexandru M. Grumezescu, University Politehnica of Bucharest, Bucharest, Romania

The aim of this book is to give an overview on the progress made in the last years in the field of pharmaceutical nanotechnology. Special attention is assigned to the fabrication, optimization, scale-up, and biological aspects of pharmaceutical nanotechnology. Nanoparticle preparation methods have been marked by following aspects: (1) need for less toxic reagents; (2) simplification of the procedure to allow economic scale-up; (3) optimization to improve yield and entrapment efficiency; and (4) science and regulatory aspects of nanotechnology-based drug products.

The book entitled Nanoscale Fabrication, Optimization, Scale-up and Biological Aspects of Pharmaceutical Nanotechnology contains 16 chapters, prepared by outstanding researchers from United States, Mexico, India, Japan, Turkey, Finland, Saudi Arabia, Serbia, Argentina, UAE, Bulgaria, and Pakistan.

Chapter 1, entitled Fabrication of polymeric core–shell nanostructures, prepared by Emmanuel O. Akala and Simeon K. Adesina, gives an historical perspective on nanomedicine: from the first generation nanoparticles (developed for liver targeting) to the fourth generation dubbed theranostics (multifunctional nanoscale devices containing a combination of diagnosis agents and a therapeutic agent, and even a reporter of therapeutic efficacy in the same nanodevice package).

Chapter 2, The emulsification-diffusion method to obtain polymeric nanoparticles: two decades of research, prepared by Elizabeth Piñón-Segundo et al., focused on the study of the pharmaceutical impact of the emulsification-diffusion method since it was introduced. The authors describe the critical preparative variables, constitutive materials, formation mechanisms, and performance of the emulsification-diffusion method. The second part of the chapter includes an extensive collection of different types of drug-loaded nanoparticles obtained by this method. Finally, some novel innovations and applications of the method have also been compiled.

Chapter 3, Tools and techniques for the optimized synthesis, reproducibility and scale-up of desired nanoparticles from plant derived material and their role in pharmaceutical properties, prepared by Nadia Saleh and Zubaida Yousaf, presents an up to date overview about the optimized techniques and tool to produce desired nanostructures; the ways to increase the reproducibility, stability and scale-up of targeted result; factors affecting the morphology and chemistry of nanoparticles and variation in pharmaceutical properties due to change in nanostructures; and the issues regarding risks in the production of nanoparticles and their toxicity and predictability.

Chapter 4, Scale-up of biopharmaceuticals production, prepared by Nagesh K. Tripathi and Ambuj Shrivastava, present different production hosts, process development, fermentation process, scale-up, challenges in the scale-up of biopharmaceuticals production, purification of biopharmaceuticals and recent developments on scale-up of biopharmaceutical production.

Chapter 5, Physicochemical and morphological characterization of pharmaceutical nanocarriers and mathematical modeling of drug encapsulation/release mass transfer processes, prepared by Zvezdelina Yaneva and Nedyalka Georgieva, give an up to date overview about rational characterization strategy of pharmaceutical nanocarriers and mathematical modeling, which allows the prediction and simulation of the drug-carrier systems behavior, enables the determination of significant process parameters, and determines the effect of various system variables on the mechanism, thus outlining the probable host–guest interactions and establishing the optimal system conditions.

Chapter 6, Biopharmaceutics and pharmacokinetics of multifunctional nanoparticles, prepared by Hemant K.S. Yadav et al., illustrates the natural mechanism of absorption, distribution, metabolism and clearance for the various kinds of nanoparticles, and the multiple properties and features influencing the safety and efficacy of these delivery systems.

Chapter 7, Technological delivery systems to improve biopharmaceutical properties, prepared by Claudia Garnero et al., focuses on the design of systems with promising properties based on the intermolecular interactions of the components. It considers the formulation and manufacturing techniques of the delivery systems, as well as the factors that may affect the drug and the pharmaceutical form of the final product. Authors described the crucial role of innovative systems, including binary and multicomponent complexes with cyclodextrins, microemulsions, solid self-microemulsifying drug delivery systems and solid lipid nanoparticles. The authors also highlight the feasibility of these to improve biopharmaceutical properties.

Chapter 8, From physicochemically stable nanocarriers to targeted delivery: In vivo pharmacokinetic, pharmacodynamic and biodistribution studies, prepared by Ivana Pantelić et al., offers a review of diverse pharmacokinetic, pharmacodynamic and biodistribution studies with various types of nanostructured drug delivery systems. Special focus is placed on two groups of nanocarriers: those targeting the brain, that always prove to be an additional challenge; and those intended for topical application, in which case in vivo studies need to discern dermal from transdermal drug delivery.

Chapter 9, Sterile dosage forms-loaded nanosystems for parenteral, nasal, pulmonary and ocular administration, prepared by Tarek A. Ahmed, discussed the composition, characteristic, and different types of these sterile dosage forms. In-process and release specifications of these preparations and their applications via invasive and noninvasive routes were also discussed.

Chapter 10, Quantitative characterization of targeted nanoparticulate formulations for prediction of clinical efficacy, prepared by Melvin E. Klegerman, explores the different aspects of targeted nanoparticulate formulations, while presenting an approach to calculating ligand-target binding force. Consequently, a practical, targeted, nanoparticle-binding force parameter allows more effective examination of factors preventing clinical development of this important nanotechnology.

Chapter 11, Analytical tools for reliable in vitro and in vivo performance testing of drug nanocrystals, prepared by Annika Tuomela et al. gives an up to date overview about drug nanocrystals and their benefits for drug delivery purposes, the techniques to produce drug nanocrystals, and the most important applications for drug delivery.

Chapter 12, Application of affinity purification of drug target proteins with practical magnetic nanoparticles to drug development, prepared by Takumi Ito et al. discusses recently developed nanoaffinity particles (SG beads and FG beads), examples of target isolations and target-based new drug development.

Chapter 13, Molecularly imprinted polymers as a tool for biomolecule separation, prepared by Müge Andaç et al., presents the recent developments of molecularly imprinted materials in affinity separation and purification systems. Also, some significant applications are highlighted for further investigation.

In Chapter 14, Detection of DNA damage induced by nanomaterials, prepared by Burcu Doğan-Topal et al., the molecular damage induced by various nanomaterials was assessed in terms of DNA damage products, types, and detections techniques.

Chapter 15, Pharmacological usage of a selective inhibitor of 3-mercaptopyruvate sulfurtransferase to control H2S and polysulfide generation, prepared by Yusuke Suwanai and Noriyuki Nagahara, introduces the molecular properties, catalytic properties, physiological functions, and 3-mercaptopyruvate sulfurtransferase (MST) inhibitors, which selectively inhibit persulfurated MST and propose the pharmacological usage of an enzyme inhibitor.

Chapter 16, Nanotechnology-based drug products: Science and regulatory considerations, prepared by Ziyaur Rahman et al. presents an overview about the safety evaluation of nanotechnology-based drug products, which include environment and toxicological assessment. In terms of pharmaceutical quality, particle size characterization is a major challenge as there is no consensus on sizing technique. To ensure consistency in product quality and hence clinical performance, it is also important to understand the product and process variables that can affect the quality of products, especially particle size. The quality by design approach provides a mean to link material attributes and process variables to critical quality attributes of nanomedicines. This chapter provides science and regulatory aspects of nanotechnology-based drug products.

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Chapter 1

Fabrication of polymeric core-shell nanostructures

Emmanuel O. Akala and Simeon K. Adesina,    Howard University, Washington, DC, United States

Abstract

This chapter gives an historical perspectives on nanomedicine: from the first generation nanoparticles—suitable for liver targeting—to the fourth generation dubbed theranostic (multifunctional nanoscale devices which allow for a combination of a diagnostic agent with a therapeutic agent, and a reporter of therapeutic efficacy in the same nanodevice package). The rationale for the development of polymeric core-shell (corona) nanostrurcture is articulated. Materials—natural and synthetic polymers—for the fabrication of polymeric core-shell nanostructures are discussed, together with methods of fabrication and examples. Emphasis was placed on in-situ dispersion polymerization at ambient temperature extensively investigated in our laboratory. Applications of quality by design approach in the fabrication of core-shell nanostructures are presented: by carefully selecting which combinations of the formulation and process variables to evaluate, the properties of core-shell nanostructures can be optimized using statistical design of experiments, followed by computer numerical and graphical optimizations.

Keywords

Core-shell nanostructures; natural-polymers; synthetic-polymers; fabrication-methods; computer-optimization

Chapter Outline

1.1 Introduction 2

1.1.1 Definition and Historical Perspectives on Nanotechnology/Nanomedicine 2

1.1.2 The Rationale for the Development of Polymeric Core-Shell Nanoparticles 3

1.2 Materials for the Fabrication of Polymeric Core-Shell Nanostructures 11

1.2.1 Natural Polymers 12

1.2.2 Synthetic Polymers 12

1.2.3 Materials for the Metallic Core of Core-Shell Nanoparticles 13

1.3 Methods for the Fabrication of Polymeric Core-Shell Nanostructures 14

1.3.1 Methods for Preparation of Nanoparticles From Preformed Polymers 15

1.3.2 In-Situ Polymerization 17

1.4 Dispersion Polymerization the Fabrication of Core-Shell Nanoparticles 21

1.4.1 Mechanism of Dispersion Polymerization 22

1.4.2 Factors Influencing Properties of Particles Produced by Free Radical Dispersion Polymerization 23

1.4.3 In-Situ Dispersion Polymerization at Ambient Temperature Involving Redox Initiator System for the Fabrication of Core-Shell Nanoparticles 27

1.4.4 Examples of Nanoparticles From Our Laboratory Fabricated by Free Radical Dispersion Polymerization at Ambient Temperature Involving Redox Initiator System 27

References 40

Further Reading 49

1.1 Introduction

1.1.1 Definition and Historical Perspectives on Nanotechnology/Nanomedicine

Nanoparticles are submicron (<1 μm) colloidal systems which can be fabricated from varied and diverse materials in a variety of compositions, including quantum dots (QDs), polymers, gold, paramagnetic iron, etc. Over the past few decades, there has been considerable interest in developing nanoparticles as effective drug delivery devices. Thus, the awareness of the usefulness of nanotechnology in novel drug delivery systems has existed before the recent upsurge of interest in nanotechnology all over the world. This is exemplified by Peter Paul Speiser, who developed the first nanoparticles which can be used for targeted drug therapy at the end of the 1960s (Kreuter, 2007; Krukemeyer et al., 2015). The advent of new and sophisticated tools, such as atomic and electron microscopes, has allowed scientists to gain in depth understanding of nanostructured substances. Recent advances in the field of nanotechnology have made nanoparticles very promising in the delivery and targeting of bioactive agents, drug discovery and diagnostics. In fact, nanotechnology is one of the key technologies of the 21st century (Krukemeyer et al., 2015).

The progress in the development of nanoparticles for biomedical applications has moved from the first generation nanoparticles—mainly suitable for liver targeting, as they are captured in the liver by the reticuloendothelial system (RES), also known as mononuclear phagocyte system (MPS); into the second generation, stealth nanoparticles: the nanoparticle surface is decorated or tagged with water soluble polymers, especially polyethylene glycol (PEG) for long systemic circulation and passive targeting (sequestration of the nanoparticles into the leaky vasculature of the tumor blood vessels, followed by their retention—enhanced permeability and retention (EPR) effect); to the third generation nanoparticles, with targeting moiety. The nanoparticle surface is decorated with a ligand specific for the antigen or receptor expressed on the surface of the tumor cells (Hillareau and Couvreur, 2006; Akala, 2010; Ogunwuyi et al., 2015). Fourth generation nanoparticles have been dubbed theranostic: multifunctional nanoparticles which allow for a combination of diagnostic agent with a therapeutic agent and a reporter of therapeutic efficacy in the same nanodevice package (Kelkar and Reineke, 2011).

The evolution of polymeric nanoparticles reflects their advantages. Polymeric nanoparticles can help to increase the stability of bioactive agents. They can be used to provide targeted (cellular/tissue) delivery of drugs, to improve oral bioavailability, to sustain drug/gene effect in target tissue, to solubilize drugs for intravascular delivery, and to improve the stability of therapeutic agents against enzymatic degradation (nucleases and proteases). Due to their sub-cellular and sub-micron size, nanoparticles can penetrate deep into tissues through fine capillaries, cross the fenestration present in the epithelial lining (e.g., liver), and are generally taken up efficiently by cells, thereby allowing the efficient delivery of therapeutic agents to target sites in the body. By also modulating polymer characteristics (e.g., via crosslinking), it is possible to control the release of a therapeutic agent from nanoparticles to achieve desired therapeutic level in the target tissue for the required duration for optimal therapeutic efficacy. Further, nanoparticles can be delivered to distant target sites either by localized delivery using a catheter-based approach with a minimal invasive procedure, or they can be conjugated to a biospecific ligand, which would direct them to the target tissue or organ. Nanoparticles with appropriate ligands attached to their surfaces are also useful for the delivery of bioactive agents after binding to target cellular receptors by a mechanism called receptor-mediated endocytosis. Another characteristic function of nanoparticles is their ability to deliver drugs across several biological barriers to the target site. Drug delivery to the brain for a wide variety of drugs, such as antineoplastics and anti-HIV drugs, is markedly hindered because they have great difficulty in crossing the blood-brain barrier (BBB). The application of nanoparticles to brain delivery is a promising way of overcoming this barrier. It was recently demonstrated that poly(butylcyanoacrylate) nanoparticles coated with polysorbate-80, are effective in transporting the hexapeptide dalargin and other agents into the brain (Hillareau and Couvreur, 2006; Bro and Hunziker, 2007).

1.1.2 The Rationale for the Development of Polymeric Core-Shell Nanoparticles

Polymeric nanoparticles can be broadly classified into two: nanocapsules, which are reservoir or vesicular systems in which a liquid or semisolid drug-loaded core is surrounded by a polymeric membrane; or nanospheres which are matrix systems in which the drug is uniformly dispersed throughout a solid polymer matrix (core). In both systems, the nanoparticle surface (corona) may be tagged with polymers, especially hydrophilic polymers (Vauthier and Bouchemal, 2009; Mishra and Patel, 2010; Rao and Geckeler, 2011).

1.1.2.1 Drug solubility enhancement

Nanoparticles may be used to enhance the solubility of poorly water soluble drugs (especially anticancer drugs) (Yanasarn et al., 2009). Paclitaxel and docetaxel are poorly water soluble. Their incorporation into poly(lactide) or polycaprolactone hydrophobic nanoparticle core circumvented this problem (Adesina et al., 2014a; Ogunwuyi et al., 2015). This was also exemplified by Abraxane for injectable suspension (palclitaxel albumin-bound nanoparticles) (Sparreboom, 2005).

1.1.2.2 Combination drug therapy and modulation of the pharmacokinetics of the drugs

Combination chemotherapy is regarded as the standard of care against many cancer types, or other diseases, in order to increase the chance of therapeutic success. It is generally acknowledged that, through proper drug combinations, treatments can promote synergistic actions, improve target selectivity, and deter the development of drug resistance, especially in cancer drug treatment. However, the belief is that the current combinatorial approach—despite being a clinical standard—leaves much room for improvement. Though in vitro cellular studies have resulted in many leads for combinatorial regimens, their clinical results are often met with little improvement in efficacy and, at times, generate higher toxicity. One major factor that separates in vitro success from impressive clinical outcomes is the different pharmacokinetics profiles among different drugs. Upon systemic administration, drugs undergo distinctive physiological fates and non-uniform distribution. Thus, it is a major clinical challenge to predict and control the therapeutic drug mixtures that reach the diseased cells and tissues (biophase) (Delbaldo et al., 2004; Hu and Zhang, 2012). Synchronization of the disposition (pharmacokinetics) of drugs encapsulated in the core of nanoparticles facilitates therapeutic success. It is believed that the coordinated delivery of paclitaxel and other anticancer drugs encapsulated in the nanoparticle core will show more favorable pharmacokinetics when the drugs arrive at the biophase in the cancer cell simultaneously, following receptor mediated endocytosis. Varying biodistribution/pharmacokinetics of combination drugs through cocktail administration has been attributed to their ineffectiveness in the clinic (Hu and Zhang, 2012).

1.1.2.3 Site-specific drug delivery via passive/active targeting

The core-shell nanoparticles can be made multifunctional, with the multifunctional therapeutic nanoparticles having a unique ability to provide site-specific drug delivery (e.g., tumor targeting).

1.1.2.3.1 Passive targeting using stealth corona-core nanoparticles: EPR effect

The unique pathophysiological characteristics of tumor vessels enable macromolecules, including nanoparticles, to selectively accumulate in tumor tissues. Fast-growing cancer cells demand the recruitment of new vessels (neovascularization) or rerouting of existing vessels near the tumor mass to supply them with oxygen and nutrients (Maeda et al., 2000; Iyer, 2006; Greish, 2007). This phenomenon results in abnormalities of tumor vasculature, namely: hypervascularization, aberrant vascular architecture, and extensive production of vascular permeability factors stimulating extravasation within tumor tissues (Peppas and Blanchette, 2004; Iyer et al. 2006). The imbalance of angiogenic regulators, such as growth factors and matrix metalloproteinases, makes tumor vessels highly disorganized and dilated with numerous pores. These tumor vessels also show enlarged gap junctions between endothelial cells (Peppas & Blanchette, 2004; Danhier et al., 2010). This hyperpermeability allows the passage of macromolecules and drug delivery systems such as nanoparticles through the blood vessels that supply tumors, leading to entrapment and accumulation of the drug carriers for prolonged periods as a result of deficient lymphatic drainage in the tumor bed (Danhier et al., 2010; Taurina et al., 2012). This phenomenon is defined as EPR effect. The EPR effect (passive targeting) is now becoming important in cancer-targeting drug design used as a guiding principle by developers of nanocarriers.

It is known that the macrophages of the mononuclear phagocytic system (MPS) in the liver have the ability to remove unprotected nanoparticles from the bloodstream immediately following intravenous administration, rendering them ineffective as site-specific drug delivery devices (Gref et al., 2000). However, the macrophages cannot directly identify the nanoparticles themselves, but rather recognize specific opsonin proteins bound to the surface of the particles by a process called opsonization (Donald et al., 2006). Surface treatments that interfere with the binding of opsonin proteins to the particle surface as a means of imparting stealth, or MPS-avoidance characteristics to nanoparticles have been developed; they allow nanoparticles to bypass recognition by the MPS and increase their blood circulation half-life (Donald et al., 2006). Water soluble polymers, especially polyethylene glycol because of its biocompatibility, are used for the surface treatment of nanoparticles (attachment to the corona) to discourage opsonization. Other materials have been investigated such as long hydrophilic polymer chains and non-ionic surfactants: polysaccharides, polyacrylamide, poly (vinyl alcohol), poly (N-vinyl-2-pyrrolidone), PEG, and PEG-containing copolymers such as poloxamers, poloxamines, polysorbates, and PEG copolymers. It has been reported that the most effective and most commonly used are the PEG and PEG-containing copolymers because they are very flexible, highly hydrophilic, and are capable of shielding hydrophobic or charged particles from blood proteins. Furthermore, they are neutral in charge; the effect of electrostatic interactions is reduced (Donald et al., 2006). The molecular weight of PEG is important, as surface PEG chain molecular weight of 2000 or greater is required to achieve increased MPS-avoidance characteristics, due to the loss in flexibility of shorter PEG chains. Moreover, as molecular weight is increased above 2000, it is believed that the blood circulation half-life of the PEGylated particles is also increased because of increased chain flexibility of higher molecular weight (MW) PEG polymers (Gref et al., 2000).

1.1.2.3.2 Active targeting: nanoparticles with surface (corona) tagged proteins

The ubiquitous distribution of cytotoxic drugs in the body is responsible for the side effects that often lead to termination of treatment. Multifunctional polymeric nanoparticles (with core-corona architecture) targeted specifically to receptors expressed on cancer cells, and not on healthy cells in the body will obviate this problem. Some proteins (especially antibody) can be used as active ligands or targeting moieties to help nanoparticles loaded with chemotherapeutic or other drugs in the core to reach particular sites in the body (site-specific or targeted delivery). Multifunctional polymeric nanoparticles, with drug-loaded core and ligand-tagged corona, are capable of specific delivery of large amounts of therapeutic agents per targeting biorecognition event, compared to simple immunotargeted drugs (i.e., antibody drug conjugate) (Shenoy and Amiji, 2005). Consequently, efficacy will be maximized and development of resistance will be difficult. Proteins such as albumin, antibody, growth factors, transferrin, cytokines, and low-density lipoprotein have been investigated for use as active ligands (Mo and Lim, 2005; Acharya et al., 2009; Akala, 2010). Fig. 1.1 gives a schematic targeting of nanoparticles to HER-2 receptors using a monoclonal antibody (E.O. Akala, unpublished document, 2016a). Abraxane—albumin-bound nanoparticle of paclitaxel—is an example of US Food and Drug Administration (FDA)-approved, protein-based, active ligand for the treatment of metastatic breast cancer (Miele et al., 2009).

Figure 1.1 Multifunctional nanoparticles interact with HER-2 receptor on breast cancer cell.

Monoclonal antibodies (mAb) have been widely investigated as bioprobes in diagnostics as well as the delivery of drugs to specific tumors (Huh, 2005; DeNardo et al., 2005). OX26 mAb can help nanoparticles to cross the blood–brain barrier and diffuse in the brain tissue, in order to transport drugs for the treatment of neurological and psychiatric disorders (Aktas et al., 2005). Nanoparticles can also be coated with mAb for cell surface antigen and used as a bait for the detection or isolation of various kinds of cells including lymphocyte and tumor cells (Zhao et al., 2004a; Natarajan et al., 2008).

1.1.2.3.3 Active targeting: nanoparticles with surface (corona) tagged peptides

Recent developments have been made in the nanoparticle targeting field, with emphasis on peptides that home to vascular zip codes in target tissues and provide a tissue- and cell-penetrating function (Ruoslahti, 2012). It is believed that the vasculature of each tissue is unique, in terms of its protein expression, and these molecular differences are referred to as vascular zip codes (Ruoslahti, 2002). These selectively expressed proteins provide targets for the specific delivery of diagnostic and therapeutic compounds to the vasculature of desired tissues. Currently, a variety of tumor-targeting peptides are in preclinical and clinical development. Recently, a strategy has been developed to enhance tumor penetration of chemotherapeutic drugs through use of iRGD peptide (CRGDK/RGPDC). This peptide comprises two sequence motifs: RGD, which binds to αvβ3/5 integrins on tumor endothelia and tumor cells, and a cryptic CendR motif (R/KXXR/K-OH). Once integrin binding has brought iRGD to the tumor, the peptide is proteolytically cleaved to expose the cryptic CendR motif. The truncated peptide loses affinity for its primary receptor and binds to neuropilin-1, activating a tissue penetration pathway that delivers the peptide along with attached or co-administered payload into the tumor mass (Alberici et al., 2013).

1.1.2.3.4 Polymeric nanoparticles for targeted delivery of bioactive agents for HIV/aids treatment

Highly active antiretroviral (ARV) therapy (HAART), for chronic suppression of HIV replication, has revolutionized the treatment of HIV/AIDS. Many patients are now in their second decade of treatment, with levels of plasma HIV RNA below the limits of detection of clinical assays (Ogunwuyi et al., 2016). However, HAART is not a panacea. Current treatments must be maintained for life. Although great progress has been made in antiretroviral therapy, HIV continues to replicate in anatomical and intracellular sites where ARV drugs have restricted access (Benson et al., 2014; Richman et al., 2009; Edagwa et al., 2014; Ogunwuyi et al., 2016). Given the restricted access of HAART to anatomical reservoirs, nanotechnology has been considered a platform that can be brought to bear in order to circumvent some of the challenges in HIV/AIDS treatment. Nanotechnological platforms that deliver antiretroviral agents to HIV reservoirs (drug polymer conjugates, dendrimers, micelles, liposomes, solid nanoparticles, nanosuspensions, polymeric nanoparticles and cell-mediated nanoparticles) have been discussed recently (Edagwa et al., 2014). Polymeric nanoparticles have higher drug loading capacity and the potential to target HIV reservoirs by the manipulation of their surface characteristics (Amiji et al., 2006; Mamo et al., 2010; Khali et al., 2011). Polymeric nanoparticles are preferentially taken up by the M cells of Peyer’s Patches of the GALT 1. Core-corona (shell) poly-(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) containing ritonavir (RTV), lopinavir (LPV), and efavirenz (EFV) have been fabricated using multiple emulsion-solvent evaporation procedures (Shibata et al., 2013). Recently, polymeric nanoparticles were fabricated, loaded with four different ARV drugs of different physicochemical properties: zidovudine (nucleoside reverse transcriptase inhibitor), lamivudine (nucleoside reverse transcriptase inhibitor), nevirapine (non-nucleoside reverse transcriptase inhibitor), and raltegravir (HIV integrase strand transfer inhibitor). These nanoparticles efficiently inhibited HIV-1 infection in CD4+ T (CEM-T) cells and peripheral blood mononuclear cells (PBMCs); they hold promise for the treatment of HIV/AIDS. The ARV-loaded nanoparticles with PEG on the corona may facilitate the tethering of ligands for targeting specific receptor(s) expressed on cells of HIV reservoirs (Ogunwuyi et al., 2016). It is known that the cure (sterilizing or functional HIV/AIDS cure) of HIV-1 infection is impeded by the existence of latent HIV-1 reservoirs in which the integrated HIV-1 provirus is transcriptionally inactive. Activation of HIV-1 transcription requires the viral Tat protein and host cell factors, including protein phosphatase-1 (PP1). A library of small compounds that targeted PP1 was recently developed and a compound, SMAPP1, was identified which induced HIV-1 transcription. However, this compound has a limited bioavailability in vivo and may not be able to reach HIV-1-infected cells and induce HIV-1 transcription in patients. SMAPP1 has been packaged in polymeric nanoparticles core with the surface (corona) tagged with polyethylene glycol. The in vitro availability of SMAPP1 was analyzed, as well as the effect on HIV-1 transcription in a cell culture. SMAPP1 was efficiently packaged in the nanoparticles, resulting in improved stability, and was released during a 120-h period (Smith et al., 2015).

1.1.2.3.5 Nanotechnology approaches for the delivery of exogenous siRNA for HIV therapy

Combination therapy, also known as HAART, is the current mainstay of HIV-1 therapy (Mamo et al., 2010; Ogunwuyi et al., 2016; Adesina and Akala, 2015). Nucleic acid-based therapeutics that involve the use of antisense oligonucleotides, aptamers, ribozymes, miRNAs, and RNA interference (RNAi) among others, for therapeutic purposes have emerged as an alternative to HAART. The use of these various agents, either as single agents or in combination with other nucleic acids or chemical antiviral agents, holds the potential to overcome some of the challenges associated with HAART and show greater efficacy in blocking viral replication and decreasing the incidence of resistant strains. As one of the most potent nucleic acid-based approaches, RNAi has become popular due to its potential to mediate sequence-specific posttranscriptional gene silencing and represents a viable option to currently available anti-HIV therapy (Zhou et al., 2013). It is triggered by oligonucleotides that are about 21–23 nucleotides long and are capable of inducing the destruction of complementary mRNA. The RNAi technique has been successfully utilized to target HIV replication; however, the main limitations to the successful utilization of this technique in vivo are the inability of naked siRNA to cross the cell membrane by diffusion, due to its strong anionic charge and large molecular weight, and stability in the blood en route to the biophase (Adesina and Akala, 2015).

Limitations to the successful utilization of RNAi in vivo can generally be divided into two categories: the inability of unprotected siRNA to cross the cell membrane by passive diffusion, due to its strong anionic charge and large molecular weight; and the lack of methods and delivery systems to safely and efficiently deliver siRNA molecules into target cells (biophase) and induce the RNAi response (Weber et al., 2008; Kim and Kim, 2009; Mahajan et al., 2011; Adesina and Akala, 2015). Other problems include: rapid degradation of siRNA by endogenous nucleases, nonspecific distribution, low endosomal escape efficiency, removal by glomerular filtration, and development of RNAi viral mutants due to the high mutation rate of HIV-1 (Naito et al. 2007; Wang et al., 2010; Cun et al., 2011). To facilitate the use of therapeutic siRNAs in vivo, various attempts have been made to deliver siRNAs using two different categories of vectors, namely, viral and nonviral vectors (Liu et al., 2014). Furthermore, the delivery of genes and siRNA has been accomplished using recombinant proteins and physical methods such as electroporation, microinjection, and biolistic particle delivery (gene gun) (Sokolova and Epple, 2008). Delivery methods using nonviral carriers employ synthetic and/or natural compounds to deliver nucleic acids. Nonviral carriers exhibit considerably reduced transfection efficiencies and they are generally considered to be less effective than viral methods. Unlike viral methods, however, the materials used in the fabrication of these carriers are less toxic and less immunogenic compared to viral vectors. Other advantages of nonviral carriers include biocompatibility, the potential for targeting or site-specific drug delivery, the ease of production of these carriers—especially in laboratory-scale formulations, and the potential for repeat administration without stimulating the immune system. Nanotechnological (nonviral) approaches for exogenous siRNA or DNA delivery for treatment of HIV/AIDS include the use of liposomes, polymeric nanoparticles, dendrimers, quantum rods, carbon nanotubes, and inorganic nanoparticles (Adesina and Akala, 2015).

Core-shell nanoparticles have been fabricated from a PEG–PLGA copolymer and loaded with polyethylenimine (PEI)–siRNA complexes in the core (Gu et al., 2015). The nanoparticles were targeted to HLA-DR+ cells by conjugating the nanoparticle surface to an anti-HLA-DR antibody for site-specific delivery. The targeted nanoparticles were transported across the vaginal epithelial layer and internalized by mKG-1 cells at significantly higher concentrations compared to untargeted nanoparticles, leading to knockdown of SNAP-23 gene/protein expression. Thus, the nanoparticle system has the potential for use as vaginal pre-exposure prophylaxis for the prevention of HIV infection. In another report, a novel siRNA delivery technology was described: using a single siRNA nanocapsule delivery technology achieved by forming a degradable polymer shell around a single siRNA molecule, Yan et al. (2012) were able to deliver siRNA to target cells and demonstrate the release of the siRNA.

1.1.2.3.6 Core-shell nanoparticles for imaging, diagnosis and treatment

The most commonly used imaging techniques are positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), and various optical imaging techniques (bioluminescence and fluorescence) that have high sensitivity. Among these techniques, MRI is the most commonly studied technique and a considerable amount of research has been devoted to the use of magnetic particles as contrast agents. Particles of gadolinium, iron oxides, gold, silver and other metals are currently being investigated (Lammers et al., 2010; Ahmed et al., 2012). Though MRI is a noninvasive technique routinely used clinically for diagnostic imaging, it is believed that magnetic resonance sensitivity is significantly low in comparison with optical and nuclear imaging (Artemov et al., 2003). To improve MRI to the level where detection of molecular markers becomes possible, special contrast agents which significantly amplify the MR signals are often used. Advances in the application of MR methods for breast cancer research have become possible from the development of contrast agents that generate receptor-target or molecular-target contrast. The noninvasive MRI has a big role in functional imaging: cancer diagnosis and staging, in which the contrast agents in nanoparticle core have found applications in defining tumor margins, characterizing tumor perfusion and identifying tumor-bearing lymph nodes (Bogin et al., 2002; Glunde et al., 2009). Dynamic contrast-enhanced MRI has been used to characterize breast lesions based on contrast enhancement and enhancement patterns of the lesions over time (Heywang et al., 1986). Molecular imaging is a powerful tool that can aid the oncologist in noninvasively assessing the phenotypic signature of a tumor in an individual patient and thus help in making therapeutic decisions, and also in the detection of tumor response. The detection of tumor response requires probing of the downstream effects of the drug on tumor cell viability or on other processes, for example, angiogenesis, hypoxia, apoptosis, tumor metabolism, or gene expression (McLarty and Reilly, 2007). The available modalities are many, but functional MRI is one of the most important.

The targeted contrast agents can be directed to cell surface receptors using antibodies or smart agents activated by specific enzymes, or by the expression of MR-detectable reporters (Glunde et al., 2009). The core-corona (shell) nanoparticles for imaging and diagnosis comprise a metallic core and a polymer shell. Polymer-coated magnetic nanoparticles are used for magnetic resonance contrast agents. In the development of multifunctional nanoparticles, ligand-targeted supramagnetic iron oxide nanoparticles (SPIONs) provide enhancement of HER2-(or other receptors) positive breast, prostate and ovarian cancers on magnetic resonance (MR) images. The use of vascular imaging to detect the effects of anti-angiogenic and anti-vascular drugs exemplifies the incorporation of MRI into breast cancer treatment (Bhujwalla, 2001). Another example is the image-guided placement of nano-devices and micro-devices in tumors for slow release of therapeutic agents and gene delivery, which holds promise for the treatment of breast cancer (Gimi et al., 2005). There has been considerable research focusing on the development of magnetically guided drug delivery systems, because an externally applied magnetic field allows the drug-loaded SPION to be concentrated at a target region (Jeon et al., 2016).

1.2 Materials for the Fabrication of Polymeric Core-Shell Nanostructures

Polymers (nature-versatile devices) are used extensively in nearly all areas of human endeavors: from aircrafts to medical devices. Polymers that have been investigated for the fabrication of polymeric nanoparticles include natural macromolecules (biopolymers) and synthetic polymers (Ogunwuyi et al., 2015). The major technique in the design of polymeric nano-devices is based on physical or chemical combination of drug(s) with polymers. Given the complexities of natural polymers, research efforts are shifting towards synthetic polymers, because they can be synthesized reproducibly and predictably, thereby permitting the selection of materials of formulation with uniform and controlled composition.

Over the years, certain requirements are expected of polymers for the fabrication of drug delivery devices in general, including polymeric core-shell nanostructures (Paul, 1976; Malinova and Wolfang, 2010): (1) The drug should show good diffusion and solubility characteristics in the polymer to provide the desired release control. (2) The polymer must be compatible with the host environment (e.g., not toxic or antagonistic in medical applications). (3) The polymer must be stable (should not degrade or change undesirably). (4) The polymer must be compatible with the bioactive agent (no undesirable reactions or physical interactions). (5) The polymer must exhibit optimum mechanical properties. (6) The polymer must be easily manufactured, fabricated into desired shape, easily sterilized and inexpensive. (7) The polymeric material biocompatibility has to be defined only in the precise context of material use: a polymer may be biocompatible in one application but not biocompatible in another (should be compatible with blood if contact with blood is desirable; should be compatible with the tissue in question if not blood). (8) Polymeric nanoparticles are of the same size as biological entities; consequently, they can readily interact with biomolecules on both the cell surface and within the cell, and show considerable hazard for human body. (9) Polymer molecular weight distribution, charge, hydrophobicity, etc., have a profound effect on the polymer biocompatibility. The molecular weight of non-biodegradable polymers should be <40 kDa to ensure renal elimination. (10). It is believed that polycations are significantly more toxic than water soluble natural polymers and polyanions; however, a few polycation-based systems (chitosan for example) have been developed and tested in clinical applications.

1.2.1 Natural Polymers

Among the natural macromolecules available for the manufacture of polymeric nanoparticles are proteins such as collagen, albumin, gelatin, legumin, or vicilin, as well as polysaccharides like alginate, agarose, starch, dextran, hyaluronic acid, alginate and chitosan. They are mainly used in polymeric nanoparticle fabrication techniques that involve dispersion of preformed polymers, except if they are chemically modified. The advantage of using polysaccharides or proteins in the fabrication of drug delivery systems is their biodegradability in vivo. For example, chitosan and its derivatives have shown excellent biocompatibility, biodegradability, low immunogenicity, and biological activities (Malinova and Wolfang, 2010). As indicated earlier, the main disadvantage of natural polymers is their structural complexity which makes modification and purification very difficult and challenging.

1.2.2 Synthetic Polymers

The most commonly used synthetic polymers for the fabrication of nanoparticles are PLA, PLGA, poly(glycolic acid) (PGA), poly(ε-caprolactone) (PCL) and poly(β-hydroxybutyrate) (PHB). Belonging to the family of polyesters, these polymers are known to exhibit adequate biodegradability and biocompatibilty. Under physiological conditions, polyesters are generally degraded by hydrolysis into products which are well tolerated by various tissues. For example, the degradation products from PLA, PGA, and PLGA, namely glycolic acid and lactic acid, are physiological substances easily eliminated through the Krebs cycle (Akala, 2010). The successful use of polymers of lactic acid and glycolic acid (PLA, PGA, and PLGA) as biodegradable drug delivery systems and as biodegradable sutures led naturally to an evaluation of other aliphatic polyesters, and to the discovery of the degradability of PCL in vivo (Schindler et al., 1977; Ogunwuyi et al., 2015). Several studies using copolymers of poly-caprolactone have shown that it is biocompatible. Poly-caprolactone undergoes hydrolytic degradation to give an intermediate which produces 6-hydroxycaproic acid. 6-Hydroxycaproic acid is broken down to acetyl-CoA units via β-oxidation (fatty acid metabolism) for further degradation via the Krebs cycle. Thus PCL is degradable to products that are physiologically metabolized by the body. Among United States Food and Drug Administration-approved polyesters such as PLA, PGA and PLGA, PCL possesses unique properties, such as higher hydrophobicity and neutral biodegradation end products, which do not disturb the pH balance of the degradation medium (Pitt et al., 1981; Woodward et al., 1985). Over the years, many types of drug delivery systems, including nanoparticles, have been developed using PCL as polymeric material (Ogunwuyi et al., 2015). Aside from poly (esters), other classes of biodegradable synthetic polymers that have been used in the fabrication of nanoparticles are as follows: polyorthoesters, polyanhydrides, polycarbonates, polyphosphazenes, polyphosphoesters, and polyamides. Some of the polymers/copolymers that have been used in the fabrication of nanoparticles are shown in Table 1.1.

Table 1.1

Commonly Used Polymers for Drug Delivery Applications

1.2.3 Materials for the Metallic Core of Core-Shell Nanoparticles

Earlier in this chapter, the fourth generation nanoparticles have been dubbed theranostics: multifunctional nanoparticles which allow for a combination of diagnostic agent with a therapeutic agent and a reporter of therapeutic efficacy in the same nanodevice package (Kelkar and Reineke, 2011). In addition to nanoparticles that are designed for therapeutic or diagnostic purposes, diagnostic (contrast agent) and therapeutic (anti-cancer drug) agents can be combined within a single, multifunctional nanoparticle, known as theranostic nanomedicine or nanotheranostics (Schleich et al., 2015). The advantageous properties of an ideal theranostic nanoparticle are as follows: (1) the ability for selective tumor accumulation, (2) the capacity to deliver therapeutic doses of anti-cancer drugs and to detect tumors at their earliest stage, and (3) the nanovector must be biocompatible and biodegradable (Lammers et al., 2011). Furthermore, nanotheranostics can be used for various applications: (1) noninvasive assessment of biodistribution and target site accumulation, (2) drug release monitoring, (3) the enhancement of therapeutic efficacy via triggered drug release, and (4) prediction of therapeutic response (Lammers et al., 2010; Schleich et al., 2015). Moreover, nanotheranostics can be used to preselect patients, leading to their significant potential for personalized nanomedicine (chemo-therapeutic interventions). Theranostic strategies include situations where patients are pre-selected based on data from initial target site accumulation studies.

The diagnostic aspect of theranostics primarily revolves around imaging mechanisms. Among these imaging techniques, MRI is the most commonly studied technique and a considerable amount of research has been devoted to the use of magnetic particles as contrast agents. Particles of gadolinium, iron oxides (superparamagnetic iron oxide nanoparticles (SPIONs)), gold, silver and other metals are currently being investigated. With the formation of metallic core-polymeric shell nanoparticles, a magnetic resonance contrast agent with enhanced stability and biocompatibility under physiological conditions are achievable (Han et al., 2007). The most popular among the magnetic nanoparticles are SPIONs. To avoid aggregation and prevent surface oxidation, which are crucial for their clinical use, it is necessary to coat the synthetized SPIONs. SPIONs have been coated with a wide variety of materials, such as biocompatible polymers (dextran and its derivatives, siloxane, poly(ethylene glycol), poly(lactic acid), poly(ε-caprolactone), and chitosan); inorganic materials (gold and silica) and monomeric stabilizers (carboxylates, phosphates) (Laurent and Mahmoudi, 2011; Santhosh and Ulrih, 2013; Laurent et al., 2014). However, it was difficult to achieve a narrow particle size distribution using dextran, starch, albumin, silicones or PEG as the corona, or shell, of iron oxides (superparamagnetic iron oxide nanoparticles (SPIONs)), gold, silver and other metals as core materials. This challenge has been obviated by using PVP. PVP-coated iron oxide nanoparticles with a narrow particle size distribution have been prepared (Lee et al., 2006).

1.3 Methods for the Fabrication of Polymeric Core-Shell Nanostructures

There are two main methods for the fabrication of polymeric core-shell nanoparticles: dispersion of preformed polymers and polymerization of monomers (i.e., in-situ polymerization) (Soppimantha, 2001; Vauthier and Bouchemal, 2009; Rao and Geckeler, 2011; Sanna et al., 2011).

1.3.1 Methods for Preparation of Nanoparticles From Preformed Polymers

1.3.1.1 Emulsification solvent evaporation

The polymer and the drug are first dissolved in a water-immiscible volatile solvent, such as dichloromethane or chloroform, which is then emulsified in an aqueous solution containing a surfactant. The emulsification is brought about by subsequent exposure to a high-energy shearing source, such as an ultrasonic device or homogenizer. The organic phase is evaporated, resulting in a fine aqueous dispersion of nanoparticles. The nanoparticles are collected by centrifugation and washed with distilled water to remove surfactant residues or any free drug, followed by lyophilization and storage. Although the solvent evaporation method can be a simple method for the preparation of polymeric nanoparticles, it is possible for the coalescence of the nanodroplets to occur during the evaporation process which may affect the final particle size and morphology (Rao and Geckeler, 2011; Dinarvand et al., 2011; Nagacarma et al., 2012).

1.3.1.2 Emulsification solvent diffusion

An oil/water (o/w) emulsion is prepared with oil phase, containing polymer (and drug) and an organic solvent. It is emulsified with the aqueous phase, containing an emulsifier in a high shear mixer, which is followed by addition of water to induce the diffusion of organic solvent, resulting in formation of nanoparticles. The selected organic solvent must be partially soluble in water (for the diffusion step) and must have the capacity to dissolve both the polymer and the stabilizing emulsifier. Suitable solvents include benzyl alcohol, propylene carbonate, ethyl acetate, isopropyl acetate, methyl acetate, methylethyl ketone, benzyl alcohol, butyl lactate, and isovaleric acid (Moinard-Checot et al., 2008; Dinarvand et al., 2011; Mishra and Patel, 2010).

1.3.1.3 Double emulsion method

This generally involves the formation of a water/oil (w/o) emulsion between an organic solution of the polymer (which may or may not contain the drug) and an aqueous solution by sonication. After the primary emulsion formation, a surfactant-containing aqueous solution is then added to the primary emulsion and sonicated to obtain the double emulsion (w/o/w). The organic solvent is then evaporated, either by gentle magnetic stirring at room temperature or under reduced pressure. Nanoparticles are then recovered by centrifugation or dialysis (Zambaux et al., 1998; Gryparis et al., 2007). The method is popular with the fabrication of nanoparticles containing water soluble drugs, like proteins.

1.3.1.4 Nanoprecipitation

Nanoprecipitation is performed using systems containing three basic ingredients: the polymer, the polymer solvent, and the nonsolvent for the polymer. The polymer solvent should be organic, miscible with water, and easily removed by evaporation. For this reason, acetone is the most frequently used solvent with this method. Sometimes it exists as a binary blend of solvents (as a blend of ethanol and acetone). Polymer, drug, and lipophilic surfactant (e.g., phospholipids) are dissolved in a semipolar, water-miscible solvent, such as acetone or ethanol. The solution is then poured or injected into an aqueous solution containing a surfactant under magnetic stirring. Nanoparticles are formed immediately by rapid solvent diffusion. The solvent is then removed from the suspension under reduced pressure (Vauthier and Bouchemal, 2009; Dinarvand et al., 2011; Nagacarma et al., 2012).

1.3.1.5 Microphase-inversion

This technique of nanoparticles preparation involves adding, dropwise, a dilute solution of polymer in an organic solvent such as acetone, into an aqueous solution of surfactant. The surfactant concentration used is usually higher than its critical micelle concentration (CMC). As the polymer solution is added dropwise to the aqueous phase, the organic solvent quickly mixes with water and the hydrophobic polymer chains undergo intrachain contraction and association to form small particles, which are stabilized by the adsorption of surfactant on the particle surface. The organic solvent is then removed under reduced pressure and the resulting nanoparticles are recovered by an appropriate method (Zhao et al., 2004b).

1.3.1.6 Salting out

The method does not demand thermal treatment at any stage of sample processing and therefore, may be especially useful for the incorporation of thermolabile drugs. Salting out is based on the separation of a water-miscible solvent from aqueous solution via a salting out effect. Polymer and drug are initially dissolved in a solvent such as acetone, which is subsequently emulsified into an aqueous gel, containing the salting out agent (electrolytes, such as magnesium chloride, calcium chloride, and magnesium acetate, or non-electrolytes such as sucrose) and a colloidal stabilizer such as polyvinylpyrrolidone or hydroxyethylcellulose. This o/w emulsion is diluted with a sufficient volume of water or aqueous solution to enhance the diffusion of acetone into the aqueous phase, thus inducing the formation of nanoparticles. Both the solvent and the salting out agent are then eliminated by cross-flow filtration. The greatest disadvantages are exclusive application to lipophilic drugs and the extensive nanoparticle washing steps (Rao and Geckeler, 2011; Mishra and Patel, 2010; Nagacarma et al., 2012).

1.3.1.7 Dialysis

Polymer is dissolved in an organic solvent and placed inside a dialysis tube with a known molecular weight cut off. The displacement of the solvent inside the membrane is followed by the progressive aggregation of polymer, due to a loss of solubility and the formation of homogeneous suspensions of nanoparticles (Rao and Geckeler, 2011).

1.3.1.8 Supercritical fluid technology

The need to develop environmentally safer methods for the production of polymeric nanoparticles has motivated research on the utility of supercritical fluids as more environmentally friendly solvents, with the potential to produce nanoparticles with high purity and without any trace of organic solvent. Two principles have been developed for the production of nanoparticles using supercritical fluids.

1.3.1.8.1 Rapid expansion of supercritical solution (RESS)

In the traditional RESS, the solute is dissolved in a supercritical fluid to form a solution, followed by the rapid expansion of the solution through an orifice or a capillary nozzle into ambient air. The high degree of supersaturation, accompanied by the rapid pressure reduction in the expansion, results in homogenous nucleation and thereby, the formation of well-dispersed particles (Rao and Geckeler, 2011; Mishra and Patel, 2010; Nagacarma et al., 2012).

1.3.1.8.2 Rapid expansion of supercritical solution into liquid solvent (RESOLV)

A simple, but significant modification to RESS involves expansion of the supercritical solution into a liquid solvent instead of ambient air (dubbed RESOLV). Although in RESS technique no organic solvents are used for the formation of polymeric nanoparticles, the prime products obtained using this technique are microscaled rather than nanoscaled materials, which is the main drawback of RESS. In order to overcome this drawback, a new supercritical fluid technology known as RESOLV has been developed. In RESOLV the liquid solvent apparently suppresses the particle growth in the expansion jet, thus making it possible to obtain primarily nanosized particles (Rao and Geckeler, 2011; Nagacarma et al., 2012).

1.3.2 In-Situ Polymerization

In-situ polymerization of monomers/macromonomers—including crosslinkers—is another method for the fabrication of nanoparticles. The method allows for one-pot synthesis of nanoparticles and it offers advantages, such as the following: Easy functionalization of the nanoparticles’ surface, which is needed either to modify the biodistribution of the nanoparticles for long blood circulation by avoiding capture by the reticuloendothelial system (passive targeting). Site-specific uptake in cells (active targeting) by tethering a ligand to nanoparticle surface that can achieve biorecognition by virtue of the receptors expressed on the surface of cells (e.g., cancer cells) (Landfester and Mailander, 2013; Adesina et al., 2014a; Ogunwuyi et al., 2015). Nanoparticles that are pH-sensitive can be designed by incorporating appropriate monomers or crosslinkers, which can respond to pH changes in the body (Gillies and Fréchet, 2004). It is possible to control or sustain the release of drugs and other substances incorporated in the nanoparticles from the interior by using degradable or hydrolyzable crosslinker (Akala and Okunola, 2013; Adesina et al., 2014b; Adesina et al., 2014a), or enzymes can be used for specific cleavage: preparation of enzyme responsive nanocapsules with payload-release properties (Landfester et al., 2010; Landfester and Mailander, 2013). Other stimuli include light, temperature, and reduction of disulfide crosslinker in a reductive environment in the cell (Oliveira et al., 2011). Theranostic nanoparticles can be easily developed by in-situ polymerization. A case in point is the molecular and functional imaging of breast cancer receptors using a noninvasive technique. The superparamagnetic iron oxide nanoparticles (SPIONs) can be used as a contrast agent in MRI for identifying micrometastases and for breast cancer diagnosis. The targeted contrast agents can be directed to cell surface receptors using affibody molecules or antibody. Furthermore, antibody or affibody-decorated nanoparticles containing SPIONs will be suitable for MRI monitoring of treatment progression for both big tumors and metastases. Image-guided placement or accumulation of the drug- and contrast agent-loaded nanoparticles on the surface of breast tumors, followed by internalization, is possible (Kramer-Marek et al., 2009; Reilly, 2009).

All these advantages of in-situ polymerization derive from the possibility of simultaneous encapsulation of relevant hydrophobic/hydrophilic drugs, contrast agent, nucleic acids, fluorochromes and, by copolymerization, adding surface functionalities in one batch process, without further modifications as the case is with nanoparticle fabrication by dispersion of preformed polymers. Any attempt to modify the surface of nanoparticles fabricated by dispersion of preformed polymers often results in a substantial loss of encapsulated drugs or other materials.

Among the techniques available for in-situ polymerization for the fabrication of nanoparticles are emulsion polymerization, microemulsion polymerization, miniemulsion polymerization, dispersion polymerization, and suspension polymerization (Arshady, 1992; Yin et al., 2002; Hong et al., 2007; Landfester et al., 2010; Adesina et al., 2014b; Akala and Okunola, 2013; Hong, Landfester and Mailander, 2013; Adesina et al., 2014a).

1.3.2.1 Suspension polymerization

In this method, the initiator is soluble in the monomer and this monomer solution is insoluble in the polymerization medium. The monomer phase is then suspended in the medium in the form of small droplets, usually by stirring and the incorporation of a suitable droplet stabilizer as suspending agent such as polyvinylpyrrolidone (PVP). Polymerization is then initiated and the monomer droplets are converted to the corresponding polymer particles of approximately the same size.

1.3.2.2 Interfacial polymerization

This method has been used for the preparation of poly(alkylcyanoacrylate) nanoparticles. One of the advantages of these polymers is their very rapid polymerization–occurring during seconds–initiated by ions present in the medium (Couvreur et al., 2002). Cyanoacrylate monomer and drug were dissolved in a mixture of an oil and absolute ethanol. This mixture was then slowly extruded through a needle into a well-stirred aqueous solution, with or without some ethanol or acetone containing a surfactant. Nanocapsules were formed spontaneously by polymerization of cyanoacrylate after contact with initiating ions present in the water. The resulting colloidal suspension can be concentrated by evaporation under vacuum. Poly(isobutylcyanoacrylate) (Lambert et al., 2000) and poly(isohexylcyanoacrylate) (Lenaerts et al., 1995) have been used in nanoparticle production by this process. Examples of drugs encapsulated are insulin (Damge et al., 1988) and photoactivatable cytotoxic compounds used in photodynamic tumor therapy, such as phthalocyanines in an injectable vehicle (Lenaerts et al., 1995).

1.3.2.3 Interfacial polycondensation

Polymeric nanoparticles are also prepared by the interfacial polycondensation of the lipophilic monomer, such as phtaloyldichloride and the hydrophilic monomer, diethylenetriamine, with or without a surfactant (Montasser et al., 2002). A modified interfacial polycondensation method was also developed. In this case, polyurethane polymer and poly (ether urethane) copolymers were chosen and successfully applied as drug carriers for alpha-tocopherol. Polyurethane and poly (ether urethane)-based nanocapsules were synthesized by interfacial reaction between two monomers (Bouchemal et al., 2004).

1.3.2.4 Emulsion polymerization

Emulsion polymerization is an important method for the production of colloidal polymer dispersions, with a wide diversity of industrial applications such as paints, coatings, adhesives and polishes, or medical and biochemical applications like drug delivery systems and immunoassay (Wutzel and Samha, 2006). Two types of emulsion polymerization are discernible, based on the nature of the continuous phase of the emulsion: conventional emulsification polymerization (o/w emulsion) in which the continuous phase is aqueous and inverse emulsification polymerization (w/o emulsion) in which the continuous phase is organic.

1.3.2.4.1 Conventional emulsion polymerization

The belief is that the swollen micelles are the sites of conventional emulsification polymerization. The monomer is emulsified in the non-solvent phase with surfactant, resulting in the formation of monomer swollen micelles and stabilized monomer droplets. The mechanism of polymerization is called micellar polymerization reaction (nucleation and propagation) which takes place in the presence of a chemical or physical initiator. Once generated in the continuous phase, free reactive monomers would initiate the reaction within the micelles. Furthermore, because the monomer is slightly soluble in the surrounding phase, the monomer molecules would reach the micelles by diffusion from the monomer droplets through the continuous phase, so polymerization would take place in the micelles. The second mechanism of emulsion polymerization in conventional emulsification polymerization is called homogenous nucleation and propagation process. Thus, if the monomer is sufficiently soluble in the continuous phase, nucleation and propagation stages will occur in this phase, resulting in the formation of oligomers (primary polymer chains). Thus throughout the polymer chain growth, both micelles and monomer droplets play the role of monomer reservoir. Regardless of the mechanism involved, the reaction stops when full consumption of monomer or initiator is achieved. The drug to be incorporated into the nanoparticles may be present during polymerization or can be subsequently added to the preformed nanospehres so it can be either incorporated into the matrix or simply adsorbed at the surface of the nanoparticles (Vauthier-Holtzscherer et al., 1991; De Jaeghere et al., 1999).

1.3.2.4.2 Inverse emulsification polymerization

This technique is emulsification polymerization in an organic continuous phase. The belief is that, in inverse emulsification polymerization, the very water soluble monomers used in the system cannot diffuse from the micelles through the organic phase. Consequently, particle formation would only occur from the fusion of small nucleated inverse monomer micelles of constant size (Adamsky and Beckman, 1994).

Studies have been carried out on the factors that affect nanoparticles produced from emulsion polymerization techniques. The final number of particles was found to increase to the 0.56 power of the surfactant concentration. Thus, a higher amount of surfactant generates a higher number of micelles with a higher surface area for radical entry and, therefore, a higher possibility of initiation for the production of particles can be expected. It was also observed that the number of particles increased with the initiator concentration (N α [I]⁰.³⁷). An increase in the reaction temperature also yielded a higher number of particles due to the higher decomposition rate of the thermal initiator in the system, which therefore induced higher values of N with smaller particle diameters (Wutzel and Samha, 2006).

1.3.2.5 Mini-emulsion polymerization

The term miniemulsion generally implies a method that allows one to create small, stable droplets in a continuous phase by applying high shear stress. Under high shear—ultrasonication, for example—the broadly distributed macrodroplets from a usual (macro) emulsion are broken into narrowly distributed and defined small nanodroplets in a size between 50 and 500 nm. The size of the droplets mainly depends on the type and the amount of the emulsifier used in the particular system. In addition to the emulsifier, a co-stabilizer is required, which acts as an osmotic pressure agent within the droplets. The co-stabilizer has a good solubility in the dispersed phase and also possess a lower solubility in the continuous phase than the dispersed phase itself. In the case of a direct (oil-in-water) miniemulsion, this agent is a (ultra)hydrophobe, and in the case of an inverse (water-in-oil) miniemulsion, it represents an (ultra)lipophobe (Landfester et al., 2010; Rao and Geckeler, 2011; Nagacarma et al., 2012). The process allows the encapsulation of hydrophilic and hydrophobic liquids and solids, molecularly dissolved dyes or other components in polymeric shells. In combination with a specific functionalization of the nanoparticles’ or nanocapsules’ surfaces and the possibility to release substances in a defined way