Engineering of Nanobiomaterials: Applications of Nanobiomaterials

By Alexandru Grumezescu

Engineering of Nanobiomaterials presents the most recent information regarding the specific modifications of nanomaterials and of their synthesis methods, in order to obtain particular structures for different biomedical purposes. This book enables the results of current research to reach those who wish to use this knowledge in an applied setting.

Engineered nanobiomaterials, designed from organic or inorganic raw materials, offer promising alternatives in many biomedical applications. In this book, eminent researchers from around the world discuss the various applications, including antibacterial therapy, biosensors, cancer therapy, stimuli-responsive drug release, drug delivery, gene therapy and visual prostheses. In each case, advantages, drawbacks and future potential are outlined.

This book will be of interest to students, postdoctoral researchers and professors engaged in the fields of materials science, biotechnology and applied chemistry. It will also be highly valuable to those working in industry, including pharmaceutics and biotechnology companies, medical researchers, biomedical engineers and advanced clinicians.

• An up-to-date and highly structured reference source for students, researchers and practitioners working in biomedical, biotechnological and engineering fields
• A valuable guide to recent scientific progress, covering major and emerging applications of nanomaterials in the biomedical field
• Proposes novel opportunities and ideas for developing or improving engineering technologies in nanomedicine/nanobiology

• Language: English
• Publisher: Elsevier Science
• Release date: Jan 14, 2016
• ISBN: 9780323417341

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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 nanostructure 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

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

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

http://grumezescu.com/

About the Series (Volumes I–XI)

The increased fabrication 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, named nanobiotechnology. This complex research and applicative field brings together significant knowledge from physical, chemical, biological, and technological sciences.

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 application of these materials of the future medicine, starting with their fabrication, specific engineering and characterization and ending with the most investigated applications such as tissue engineering, antimicrobial, and cancer therapies, and the development of different medical and cosmetic use products. These books brings 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 entitled Applications of Nanobiomaterials contains 165 chapters, organized into 11 volumes which are ready to present a novel and up-to-date approach related to this intriguing domain developed in the last years. Each chapter was carefully composed and illustrated to highlight the relevance of nanobiomaterials on most biomedical fields, revealing the most recent applications on a 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 biotechnological settings.

These 11 volumes cover almost all aspects related to the applications of nanobiomaterials and are named as 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: NanobBiomaterials in Drug Delivery

Volume X: Nanobiomaterials in Galenic Formulations and Cosmetics

Volume XI: Nanobiomaterials in Dentistry

About this Book

Volume II, Engineering of Nanobiomaterials, provides an up-to-date approach about the engineering and distinct applications of the most utilized nanobiomaterials designed from organic or inorganic raw materials.

Engineered nanobiomaterials are currently offering promising alternatives in many biomedical applications, such as antibacterial therapy, biosensors, cancer therapy, stimuli-responsive drug release, drug delivery, gene therapy, and visual prostheses.

The great number of nanotechnology applications on the biomedical field is the motivation behind this volume, to summarize current research findings concerning the engineering of nanobiomaterials. Volume II contains 15 chapters, prepared by outstanding international researchers from Canada, the United States of America, Germany, Poland, Ukraine, Israel, India, and China.

In Chapter 1, entitled Engineering of stimuli-sensitive nanopreparations to overcome physiological barriers and cancer multidrug resistance, Ağardan et al., present the advantages of nanopreparations (liposomes, micelles, and polymeric nanoparticles) with functional moieties to facilitate prolonged circulation, targetability, enhanced intracellular penetration, and stimuli-sensitivity. The microenvironment of the tumors (reduced pH, increased local temperature, and altered redox status) is a guideline for the design of stimuli-sensitive drug-delivery systems. The authors also discuss the most important causes of ineffective chemotheraphy and multidrug resistance.

Sokovnin et al., in Chapter 2, entitled Production of complex metal oxide nanopowders using pulsed electron beam in low-pressure gas for biomaterials application, give an overview about nanopowders of a pure and metal doped oxide produced by a pulsed electron beam evaporation method in low-pressure gas for biomaterials application.

Chapter 3, Bioabsorbable engineered nanobiomaterials for antibacterial therapy, presents the current knowledge of nanofiber fabrication techniques for the development of technical and medical textiles which exhibit antimicrobial properties and are tailored for stimuli-responsive drug release.

In the Chapter 4, Organic electronic materials for gene delivery, the authors discuss the potential of organic electronic materials (i.e., fullerenes, graphenes, and conjugated polymers) to be utilized as nonviral gene delivery vectors and as novel theranostic devices. These represent an exciting new class of nonviral vectors that are at the frontier of novel approaches towards gene therapy.

Safarik et al., in Chapter 5, Magnetic modification of cells, focus their attention on the presentation of available procedures used to prepare magnetically responsive cells from originally diamagnetic precursors. Immunomagnetic techniques have become widely used tools in cell biology, medicine, and microbiology. The unique properties of magnetically responsive cells enable their selective magnetic separation, MRI imaging, cancer treatment, etc. Cell labeling methods using magnetic particles have been widely developed, usually showing no adverse effect on cell proliferation and functionalities while conferring magnetic properties to various cell types.

In Chapter 6, Aptamers as functional bionanomaterials for sensor applications, Tom et al., present an up-to-date review focused on the development of aptamers as recognition elements. The authors also describe their incorporation into materials and sensor devices, with the ultimate goal of detecting various chemical and biological targets with high sensitivity and selectivity.

Cardoso et al., in Chapter 7, entitled Gene delivery mediated by gemini surfactants: structure–activity relationships, describe the structural characteristics of gemini surfactants that are correlated with their biological impact on cells, in terms of adverse effects produced and with capacity to deliver and promote the expression of nucleic acids. Examples from the literature illustrate the ability of these complexes to circumvent biological barriers associated with the different steps leading to gene expression, with emphasis on membrane translocation and endosomal escape.

Chapter 8, Nanobiomaterials for bionic eye: vision of the future, discusses the current role of nanobiomaterials in visual prosthesis progress and touches on the future possibilities of this technology.

Perchyonok et al., in Chapter 9, Bioactive-functionalized interpenetrating network hydrogel (BIOF-IPN), reveal the advances in basic material science, biotechnology, and designer functional materials. It has the specific aim of exploiting molecular mechanistics, understanding of conventional free radicals towards finding, and harnessing a broad range of biologically relevant processes such as bio-adhesion, biocompatibility, biorepair, and topical drug delivery. The advantage of the novel intelligent designed functional material—BIOF-INP—is its amalgamation of molecular design, mechanism, bioanalytical advancement, and molecular biology. Furthermore, it presents the ability to easily refocus the aim of the problem, making its applications only limited by the imagination.

Singh et al., in Chapter 10, Engineered nanomaterials for biomedicine: advancements and hazards, describe the ways in which the interaction of nanomaterials with biomolecules can be controlled for beneficial biomedical applications. This chapter highlights different types of engineered nanomaterials currently used in biomedical applications and provides a critical overview of the recent advancements and their potential hazards and drawbacks.

Han et al., in Chapter 11, Mechanism of nanomachining semiconductor and ceramic blades for surgical applications, investigate the mechanisms of ductile machining ceramic/silicon using DEM/MD method.

Chapter 12, Design and implementation of an electrospinning system, aims to provide a brief introduction to the electrospinning process for obtaining nanofibers by using personalized, up-to-date novel modeling techniques. Moreover, the feasibility of suppressing the bending instability due to the complicated oscillations of a polymer jet was investigated with experimental observations and mathematical models are also developed.

Sharma et al., in Chapter 13, Engineered nanoparticles as a precise delivery system in cancer therapeutics, summarize the recent advancement of nanoparticle engineering for tumor-targeted therapies, including theranostic applications, clinical trials, and recent patents.

Oza et al., in Chapter 14, Inorganic nanoflotillas as engineered particles for drug and gene delivery, highlight the techniques used for nanocrystalline synthesis, advanced processing by polymers, such as antibodies, attachment of drug/genes, and delivery of such engineered nanoparticles at the desired site. They possess capabilities of integrating different functionalities like provision of effective contrast for imaging modalities, synaptic delivery, and thermal therapies. This chapter is an endeavor towards comprehension of complex nanostructures which are emerging as a future avenue for nanomedicine.

Gopi et al., in Chapter 15, Chemical and green routes for the synthesis of multifunctional pure and substituted nanohydroxyapatite for biomedical applications, discuss the synthesis methods of pure and substituted nano-hydroxyapatite through various chemical and green routes for multidirectional biomedical applications.

Chapter 1

Engineering of stimuli-sensitive nanopreparations to overcome physiological barriers and cancer multidrug resistance

N. Başaran Mutlu Ağardan¹,² and Vladimir P. Torchilin¹,³,    ¹Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, MA, USA,    ²Department of Pharmaceutical Technology, Gazi University Faculty of Pharmacy, Ankara, Turkey,    ³Department of Biochemistry, King Abdulaziz University, Jeddah, Saudi Arabia

Abstract

Cancer nanotechnology is an interdisciplinary area of research, whose main objective is delivery of therapeutic agents to tumors, that destroy cancer cells with minimum damage to normal cells. The advantages of nanopreparations such as liposomes, micelles, and polymeric nanoparticles over conventional therapies are well-established. Currently, research has been focused on further engineering of nanopreparations with functional moieties to facilitate prolonged circulation, targetability, enhanced intracellular penetration, and stimuli-sensitivity. The particular characteristics of the tumor microenviroment, such as reduced pH, increased local temperature, and altered redox status, provide guidelines for design of stimuli-sensitive drug delivery systems that respond to these specific differences. Another important goal for stimuli-sensitive drug delivery systems is avoidance of multidrug resistance, which is one of the most important causes of ineffective chemotherapy.

Keywords

Stimuli-sensitive nanopreparations; drug delivery; biological barriers; cellular barriers; physiological barriers; tumor microenvironment; multidrug resistance; cancer

1.1 Introduction

Cancer is a major health problem worldwide in terms of mortality and morbidity. The main therapeutic approach in clinics for the treatment of both localized and metastatic tumors is chemotherapy. Although chemotherapy is the most common approach, the crucial problem is that anticancer drugs often lack specificity and selectivity for cancer cells. Since the cancer therapeutics are highly hydrophobic and cytotoxic, they fail in the treatment because of a lack of effectiveness and side effects. In addition to the toxicity that is generally observed in rapidly dividing cells in the body (such as hair, spleen, liver), many widely used chemotherapeutics include solvents (such as castor oil and polysorbate 80) to provide drug solubility which is a key factor for systemic/intravenous administration. These solvents are known to be directly involved with adverse effects. Currently, conventional chemotherapeutics are substantially limited by (i) systemic cytotoxicity, (ii) insufficient ability to bypass biological barriers, (iii) poor biodistribution and nonspecific delivery, and (iv) development of drug resistance.

Nanotechnology, although not a new concept, has gained significant momentum in the past few decades, especially in the area of cancer research. Briefly, the expected features of an effective nanoparticulate system are selective accumulation in the target tissue/organ and an ability to penetrate target cells with loaded drug. Although nanoparticulate drug delivery systems have several advantages over conventional therapeutics such as increasing drug stability in vivo and blood circulation time, it has been established that only a small fraction of an administered dose reaches the tumor site for its intended effect. The primary reasons for an inability to reach the target site selectively are physiological and cellular barriers. They are the main protective mechanisms which limit access to organs/tissues. Additionally, the failure of conventional chemotheraphy is often directly associated with multidrug resistance (MDR), which is most frequently caused by insufficient drug concentration at the tumor site. When tumors are exposed to nonoptimal amounts of drug, they often develop resistance. Higher doses of drug are then needed to elicit a therapeutic response.

Tumor cells have some unique properties and a microenviroment which is perhaps more heterogeneous than the other cells of the body. Stimuli-sensitive drug delivery systems can be engineered based on the specific properties of the tumor microenvironment, including reduced pH, increased local temperature, altered redox status, and enzyme activities. This chapter focuses on the development of stimuli-sensitive nanopreparations with the ability to overcome physiological barriers and cancer MDR.

1.2 Barriers in Drug Delivery

The key factor for a successful drug delivery system is reaching the target area selectively with the loaded drug. Therefore, the drug delivery system has to be stable in blood and be transported across the barriers to reach target cells. The body has developed many offensive and defensive mechanisms to remove foreign materials such as bacteria, viruses, medical devices, and drugs. Hence, comprehension of the barriers of the body is crucial for engineering of nanoparticulate drug delivery systems. The barriers can be classified into two main parts as: physiological and cellular barriers.

1.2.1 Physiological Barriers

1.2.1.1 The mononuclear phagocyte system—opsonization

Stability of the drug delivery system in the systemic circulation is the first necessity. Blood is a dynamic fluid which contains many proteins (albumin, fibrinogen, complement system proteins, etc.), enzymes, and immune cells. When nanoparticles enter the systemic circulation, the plasma proteins can bind unselectively and reversibly on their surface. This adsorption process, opsonization, determines the fate of the drug carriers’ pharmocokinetics and pharmacological effects. The opsonization process permits macrophages of the mononuclear phagocytic system (MPS) (also known as the reticuloendothelial sytem or RES) to readily recognize and eliminate the drug carrier before it reaches its target site (Owens and Peppas, 2006; Serpe, 2006). Once nanoparticles are coated with plasma proteins (opsonized), the macrophages of the liver (Kupffer cells) and spleen rapidly take up these particles.

The physiochemical characteristics of nanoparticles such as shape, size, charge, and surface chemistry are determinative for opsonization and clearence (Kiwada, 1997; Dobrovolskaia et al., 2008; Albanese et al., 2012). To be effective, the nanoparticulate drug delivery systems must be camouflaged so as to be unrecognized by the MPS and to avoid phagocytosis by macrophages.

The most common approach to avoid MPS effects and obtain prolonged circulation times by minimizing clearence is by coating nanoparticulate systems with hydrophilic polymers such as poly(ethylene glycol) (Gref et al., 1995; Storm et al., 1995). This type of polymer forms dense conformational clouds which deny surface interaction of drug delivery systems (i.e., liposomes, nanoparticles, polymeric micelles, dendrimers, quantum dots) with macromolecules (Torchilin and Trubetskoy, 1995), although, it has been noted that repetitive administration of pegylated nanoparticles may cause immunogenic responses where the production of antibodies can promote the blood clearance of nanoparticles. This is known as the accelerated blood clearance (ABC) phenomenon and is demonstrated for clinically used pegylated liposomes (Ishida et al., 2006; Abu Lila et al., 2013).

1.2.1.2 Tumor vasculature and nanoparticle extravasation

There are three main subcompartments for healthy and neoplastic tissues: vascular, interstitial, and cellular (Jain, 1987). After escaping from the MPS, a nanoparticulate system has to pass through the vascular endothelial layer to reach the interstitial compartment. The most important factors that affect the flow of particles through the vasculature are the rate of blood flow and vascular morphology. In normal tissues, there is a balanced array of proangiogenic and antiangiogenic molecules, which provides for an efficient and regular network of blood vessels to supply the metabolic demands of the tissue. It is known that, in comparision to normal tissues, blood vessel systems of tumor tissues are highly different and irregular (Cairns et al., 2006; Baxter and Jain, 1990). Until they reach a diameter of 1–2 mm, tumors can use the vasculature of their host tissues to provide their nutrient and oxygen supplies by passive diffusion (Nishida et al., 2006). In time, cancerous tissues grow along with their metabolic demands. At this point, tumors orchestrate the building of their own vasculature system by recruiting surrounding mature blood vessels which sprout new blood vessel capillaries for themselves. This angiogenic process is a very important factor in the progress of cancer and is determinative for metastasis (Kerbel, 2000). Tumors express high levels of endogenous angiogenic factors including cytokines, vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)-α, which activate surrounding cells for initiation and maintenance of enhanced angiogenesis (Weis and Cheresh, 2011).

The main structural abnormalities of the tumor vasculature, referred to as vascular chaos, can be divided into three main types. First, they have an abnormal vessel wall characterized by incomplete or absent endothelial lining, discontinous basement membrane, a deficiency of pericytes, contractile wall components, and cell receptors. Secondly, they have an abnormal vascular architecture with the main properties including contour irregularites, tortuosity, elongation of vessels, existence of arteriovenous shunts, and a loss of the normal hierarchical arrangement of arterioles–capillaries–venules. Lastly, they have an abnormal vascular density which causes a heterogeneous distribution of vascularization and dilation of intercapillary space. This property results in an increased diffusion distance. The heterogeneous blood vessel distribution of tumors is the one of the factors that complicates drug delivery to tumors; it causes heterogeneous tumor perfusion and extravasation, thus promoting drug resistance and tumor progression (Vaupel et al., 1989; Jain, 2013). In addition to an irregular vasculature, tumor vasculature has wide interendothelial junctions and large pore diameters up to 100 nm, which lends a relatively leaky property to the vessels. Roberts and Palade demonstrated the strong opening effect of VEGF on endothelial intercellular junctions that increased capillary and venular leakage, within 10 min from topical application to rat muscle and mouse skin (Roberts and Palade, 1995).

Destruction of the tumor vasculature by blockage of VEGF signaling, to deprive the tumor of oxygen and nutrients (antiangiogenic therapy), has been a widely investigated issue. Antiangiogenic factors are best used in combination with targeted systems. Otherwise, this treatment could be harmful to healthy tissues and may cause hypoxia and poor drug delivery in tumors (Jain, 2005). Besides an altered blood vessel structure, tumors have an inefficient lymphatic drainage. Nanoparticles readily extravasate into the tumor space through its enlarged pores and stay there as a result of poor lymphatic drainage. This is the basis for passive targeting to solid tumors first explained by Matsumura-Maeda and later named the enhanced permeability and retention effect (EPR) (Matsumura and Maeda, 1986; Maeda and Matsumura, 1989; Iyer et al., 2006; Sriraman et al., 2014). Due to this unique characteristic EPR effect associated with solid tumors, a somewhat selective delivery of macromolecular anticancer drugs and drug delivery systems to the tumor site is achievable. A significant EPR effect-based targeting and a prolonged time for accumulation in the target by a drug delivery system are provided by long circulation and the optimized particle size (200–800 nm) to allow passage through the permeabilized vasculature (Hobbs et al., 1998; Torchilin, 2011). In a study by Pandey and coworkers, an inhibitor of platelet aggregation, clopidogrel, was used to increase the delivery of a cisplatin nano-formulation in a murine syngeneic 4T1 breast cancer model. Compared to self-assembling cisplatin nanoparticles (SACNs) alone, combination therapy with clopidogrel and SACNs was associated with a fourfold greater delivery of cisplatin to tumor tissue, a greater reduction in tumor growth as well as a higher survival rate (Pandey et al., 2014).

1.2.1.3 Tumor microenvironment

1.2.1.3.1 Extracellular matrix

This section will focus on the barrier function of the tumor microenvironment. Specific changes in tumor tissues and attempts at engineering drug delivery systems which respond to these micro changes will be discussed in stimuli sensitive drug delivery systems part (Section 1.3).

After extravasation, the drug delivery system faces another barrier, the tumor interstitium. The interstitium of a tissue is the space between the cells and the vascular compartment. The tumor microenvironment is entirely different from normal tissues (Figure 1.1). It has been defined as the total functional and structural combination of cancerous and noncancerous cells (endothelial cells, fibroblasts, and immune cells), in addition to the dynamic microenvironment in which they live. It is created by the tumor and controlled by tumor-induced interactions. The major component of the tumor interstitium is an extracellular matrix (ECM), which is a complex assembly of collagens, elastins, and proteoglycans. In addition, it includes soluble components such as cytokines, chemokines, and polypeptide growth factors (van Kempen et al., 2003; Whiteside, 2008; Morin, 2003). The ECM of the tumor microenvironment is denser than a normal tissue ECM, which causes poor drug penetration. Grantab and coworkers developed multicellular layers (MCLs) by growing tumor cells on a semipermiable Teflon support membrane. They aimed to quantify the penetration of anticancer drugs commonly used in chemotherapy (doxorubicin, 5-fluorouracil, paclitaxel methotrexate) as a function of cellular adhesion and packing density. The properties of MCLs grown from two epithelioid and round subclones of a colon carcinoma cell line were compared. One pair of epithelioid and round sublines differed in expression of α-E-catenin, and both pairs generated MCLs with different packing densities. The penetration of drugs through MCLs derived from these cell lines was significantly greater through the round (loosely packed) (HCT-8Ra, HCT-81R1) than the epithelioid (tightly packed) (HCT-8Ea, HCT-8E11) sublines. The tumor packing density constituted a barrier to effective drug penetration, even for small drug molecules (Kwon et al., 2012; Grantab et al., 2006).

Figure 1.1 A schematic view of the tumor microenvironment compared with normal tissue.

As a distinctive feature, tumor cells may alter their microenvironment in the presence of chemotherapeutics to promote their survival. Sherman-Baust et al. demonstrated a collagen VI-promoted resistance in vitro by cultivation of cisplatin-sensitive ovarian cancer spheroids in the presence of cisplatin and confirmed an increased collagen VI expression in vivo in ovarian tumors (Sherman-Baust et al., 2003). In a comprehensive study by Netti and coworkers, the relationship between interstitial penetration differences of a macromolecule (IgG) and the collagen network stiffness was intestigated using four different cell lines: human colon adenocarcinoma (LS174T), human glioblastoma (U87), human soft tissue sarcoma (HSTS 26T), and murine mammary carcinoma. U87 and HSTS 26T showed more resistance to IgG diffusion that was directly proportional to the complexity of their collagen networks. Following this result, tumors were treated with collagenase to decrease collagen levels. By this method, an increase in the diffusion coefficients of IgG was achieved (Netti et al., 2000).

It is well known that factors such as size, shape, charge, and hydrophobicity of macromolecules and nanoparticles influence their transport in the ECM. It was shown that small particle size dominated the effect of molecular convection while the movement of larger molecules and nanoparticles was mechanically hindered by the interactions with the interstitial matrix. Also, branched proteins convected more slowly compared to linear molecules of equal molecular mass. In addition, negative surface charge is known to increase convection through matrix repulsion (Reddy et al., 2006). Another important component of the ECM, glycosaminoglycan, affects interstitial transport by increasing the viscosity of the interstitial fluid. In addition it carries a highly negative charge which can decrease the diffusion of molecules/particles by forming aggregates. The structure of the interstitial matrix is highly heterogeneous and this can cause an irregular diffusion of particles. High-collagen-fiber-containing areas impede particle diffusion rates by creating steric hindrance and also by their high viscosity. However, low-collagen-fiber-containing areas have low viscosity and the diffusion of particles is similar to that of water (Afratis et al., 2012; Alexandrakis et al., 2004; Jain and Stylianopoulos, 2010).

1.2.1.3.2 Increased interstitial fluid pressure

The growing and differentiating tumor produces new, abnormally leaky microvessels (neoangiogenesis), although it is incapable of generating its own functional lymphatic system (Vaupel, 2004). The leaky blood vessels, poor lymphatic drainage of the tumor’s center and low perfusion results from the increased interstitial fluid pressure (IFP) in most solid tumors. The elevated IFP in tumors limits the entrance of therapeutics by decreasing the driving forces for extravasation of fluids and macromolecules and by creating a convective flux of fluids towards the periphery of tumors (Boucher et al., 1990). The IFP in normal tissues is approximately 0 mmHg (balanced by lymphatic drainage) while it is 10–30 mmHg on average and even as high as 60 mmHg for some tumors. The IFP does not represent a homogeneous distribution, it is high in the inner parts/center of the tumor while it decreases abruptly in the tumor margins and surrounding tissues (Fukumura and Jain, 2007).

In a study by Rofstad and coworkers, it was observed that tumors which metastasized to lymph nodes showed higher IFP than those that did not metastasize. In addition, tumors with high IFP had large hypoxic fractions when compared with tumors with low IFP (Heldin et al., 2004; Rofstad et al., 2014). Feretti and her group demonstrated that treatment with cytotoxic or cytostatic agents significantly decreased the IFP in ectopic and orthotopic tumor models (Ferretti et al., 2009). In a study by Fan and coworkers, a molecular targeting drug, imatinib, was loaded in sterically stabilized liposomes (SSL-IMA) to reduce the tumor IFP and increase the delivery of liposomal doxorubicin (SSL-DOX) into tumors. A significant reduction of tumor IFP occurred with a single intravenous injection of 20 mg/kg SSL-IMA in a mouse B16 melanoma model which lasted for 50 h. In addition they observed inhibition of tumor growth and induced apoptosis with the combination of SSL-IMA and SSL-DOX liposomes (Fan et al., 2013). In a new approach, vascular endothelial growth factor C (VEGF-C) was used to enhance tumor-associated lymphangiogenesis and reduce the IFP by increasing lymphatic drainage of the tumor tissue. They used a xenograft mouse model created by inoculating A431 epidermoid vulva carcinoma cells to NMRI nu/nu mice. It is noted that tumor growth and IFP were lowered significantly in 16 days following peritumoral injection of VEGF-C in comparison to control or VEGF-A-treated animals (Hofmann et al., 2013).

1.2.1.3.3 Hypoxia

Another important result of impaired blood flow in tumor tissue is reduced and heterogeneous oxygen delivery (hypoxia). Most human tumors contain hypoxic regions with an approximate ratio of 50–60%. There are two main types of hypoxia which exist in human tumors: chronic and acute. The presence of chronic hypoxia, also known as diffusion-limited hypoxia, in human tumors was first postulated by Thomlinson and Gray in 1955, based on their studies of the distribution of necrosis relative to blood vessels (Thomlinson and Gray, 1955). Acute, perfusion-limited or cycling hypoxia was demonstrated to be caused mainly by temporary, local perfusion defects or bouncing variations in red blood cell (RBC) fluxes resulting in fluctuations of the microvascular oxygen supply. Currently, there is no definite conclusion about the prominence of acute or chronic hypoxia in solid tumors (Bayer and Vaupel, 2012; Brown and Wilson, 2004).

Oxygen concentrations in tumors are highly out-of-balance, both spatially and temporally, and often at very low levels in many regions. Normal tissue pO2 (partial pressure of oxygen) values vary between 10 mmHg and 80 mmHg depending on the tissue type, whereas tumors contain regions where the pO2 is even lower than 5 mmHg (Brown and Wilson, 2004). It has been reported that oxygen can diffuse readily to within 100–200 µm from the nearest blood vessel and since tumor cells are typically farther from blood vessels, they are not oxygenated sufficiently (Koch, 2002). Hypoxia affects many metabolic pathways in tumors: tyrosine kinase-mediated signaling, epithelial-to-mesenchymal transition, metastasis, and suppression of immune reactivity (Wilson and Hay, 2011).

The main problem caused by hypoxic tumor cells is their resistance to radiotherapy and chemotherapy. There are several possible reasons for the resistance of hypoxic cells to chemotherapy. First of all, hypoxic regions are known to be abnormally distant from blood vessels, and so they are not sufficiently exposed to chemotherapeutics. Cell proliferation is also affected by the distance from blood vessels. In addition to distance from blood vessels, there is a lack of sensitivity of hypoxic cells to p53-mediated apoptosis. This may also decrease the effectiveness of some chemotherapeutics which show their effect using this pathway. It has been demostrated that disruption of the p53 gene results in alteration of the responses to the treatment with 5-fluorouracyl and adriamycin in isogenic human cancer cell lines (Bunz et al., 1999). In addition, there is a consideration about hypoxia’s effect on upregulatation of genes involved in drug resistance, including P-glycoprotein (P-gp) encoding genes, and also hypoxia-inducible factor 1a (HIF-1a). Doublier and coworkers showed the induction of doxorubicin resistance by HIF-1 activation in MCF 7 3-D spheroids, via P-glycoprotein expression (Doublier et al., 2012).

Hypoxia is generally accompanied by acidosis and causes a very well-known feature of tumors, reduced pH. Engineering of pH-sensitive drug-sensitive systems is a very promising strategy and will be discussed in detail in "stimuli sensitive drug delivery systems (Section 1.3)."

1.2.2 Cellular Barriers

The cell membrane is a dynamic structure which consists of lipid bilayers and proteins held together by noncovalent interactions. While the lipid bilayer in its structure provides fluidity, it also serves as a relatively impermeable barrier to the passage of most water-soluble molecules (Alberts et al., 2002). Under normal conditions, there is an ion gradient in the membrane structure which results in a −10 to −100 mV across the membrane with a net negative charge on the cytosolic face of the membrane (Shin et al., 2013). Most of the pharmaceutical agents and genes exert their therapeutic effects in specific organelles such as the nucleus, mitochondrion, or lysosome of the cell. Cytosolic drug delivery is especially useful for drugs which undergo extensive exportation from the cell via efflux transporter systems such as P-gp and MDR proteins (Hillaireau and Couvreur, 2009). After reaching the tumor interstitium, drug-loaded carriers need to cross the barrier of cell membrane to reach the cytoplasm and deliver their payloads to different subcellular compartments (Torchilin, 2009). While low-molecular-weight, lipophilic compounds are known to be taken up through the cell membrane via passive diffusion processes, it is not that easy for larger molecular structures as well as nanoparticles (Plank et al., 1998). The majority of nanoparticles are taken up by cells via specific endocytosis (clathrin-mediated, caveolae-mediated, and clathrin- and caveolae-independent endocytosis, or macropinocytosis) and release their payloads intracellularly (Hillaireau and Couvreur, 2009; Wang et al., 2012).

To provide an efficient intercellular drug delivery, the physicochemical properties of a nanocarrier such as particle size, shape, surface charge and properties, and lipophilicity have to be optimized. Higher positive-charged nanoparticles exhibit a stronger affinity for the negatively charged cell membrane while repulsive forces are promoted by negatively charged particles. He and coworkers prepared a series of polymeric nanoparticles with different particle size (150–500 nm) and zeta potentials (−40 mV to +35 mV). They observed that nanoparticles with a particle size around 150 nm and a zeta potential below +15 mV, escaped from phagocytic uptake. In addition, nonphagocytic cellular uptake of polymeric nanoparticles was found to be cell-line-dependent and affected not only by particle size and surface charge, but also by nanoparticle composition (He et al., 2010). For cellular uptake, there is another issue that should be considered. PEGylation of the nanoparticulate drug delivery system may cause a low interaction with the cancer cell membrane by steric hindrance and reduce endocytosis (Sui et al., 2011). Compared to normal cells, cancer cells overexpress many cell-surface receptors like transferrin, folic acid, integrins, and glucose transporters. This feature of cancer cell membranes has been widely used to enhance targeting by receptor-mediated cellular internalization of nanocarriers (Koshkaryev et al., 2012). Cell-penetrating peptides (e.g., Tat-peptide, penetratin, octa-arginin), are another promising approach for intracellular delivery of plasmid DNA, oligonucleotides, small interfering RNAs (siRNAs), proteins and peptides, contrast agents, and drugs, as well as various nanocarriers (Koren and Torchilin, 2012).

Subsequent to cellular internalization, the nanocarrier may need to reach an intracellular target to release its drug content. The function of endosomal vesicles is to transport internalized particles or macromolecules to different intracellular organelles (golgi, mithocondria, lysosome, nucleus, etc.). Inside the cell, the nanocarrier faces another barrier, lysosomal degradation. However, although internalized endocytic vesicles are transported mainly to a lysosomal compartment for degradation, a second possibility is recycling of the carrier back to the cell surface. Strategies must be considered to avoid possible degradation and to faciliate endosomal escape. The most common strategy is the use of cationic lipids or polymers with the ability to fuse with the endosomal membrane, destabilize the drug carrier, and provide release of its payload to the cytoplasm. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) has been used extensively as a fusogenic lipid, typically for liposomal and micellar carrier formulations. DOPE exists in two conformations, lamellar and hexagonal. The transformation of a lamellar to a hexagonal conformation, occurs at the low pH of the endosomal compartment and provides rapid fusion with endosomal membranes, resulting in the release of drug payloads into the cytoplasm (Koltover et al., 1998; Dominska and Dykxhoorn, 2010; Biswas and Torchilin, 2014). This strategy have been used extensively for cellular delivery of DNA and siRNA (Zhang et al., 2010; Kapoor et al., 2012). Conjugation of DOPE to low-molecular-weight polyethyleneimine (PEI) has also given promising results by enhanced endosomal escape and overcoming doxorubicin resistance in breast cancer cells (Navarro et al., 2012).

Even after passing all these barriers, only a small portion of the administered dose manages to enter cells and show its intended cytotoxic effect. In addition to these barriers, it is well known that most tumors have overexpressed efflux pumps with the ability to export a large variety of chemicals/drugs. The overexpression of these efflux pumps occurs when cells receive levels of drugs that do not kill them, and hence provide cells the oppurtunity to adapt and protect themselves against the chemotherapeutic agent. This mechanism is indicated to be directly related to MDR in cancer. The most typical efflux pump in the cell membrane is a member of the ATP-binding cassette family (ABC) of transporters, protein 1 (MDR1) or P-glycoprotein (P-gp) with a molecular weight of 170 kDa. In some tumors, although the P-gp expression was low before chemotheraphy, it increased during chemotheraphy. To reduce drug efflux and improve the treatment efficacy, formulation of nanocarriers including anticancer agents with P-gp inhibitiors (especially third-generation ones such as tariquidar) is the most common approach (Thomas and Coley, 2003; Modok et al., 2006; Fletcher et al., 2010; Ozben, 2006). The increased accumulation of paclitaxel in cancer cells was reported to be achieved by long-circulating paclitaxel and tariquidar coloaded liposomes (Patel et al., 2011). Similarly, coloading of paclitaxel and tariquidar to poly(d,l-lactide-co-glycolide) nanoparticles enhanced therapeutic efficacy of dual-agent nanoparticles and was correlated with increased accumulation of paclitaxel in otherwise drug-resistant tumor cells. This strategy is a promising approach to overcome tumor drug resistance (Patil et al., 2009).

1.3 Stimuli-Sensitive Nanopreparations

The special changes in the tumor microenvironment and their important barrier functions have been covered in details in Section 1.2.1.3. Nanopreparations that have been engineered to incorporate stimulus-responsive components to take advantage of the changes in tumor microenvironment have distinct properties which allow them to bypass those barriers and achieve targeted intracellular drug delivery (Figure 1.2). This section will focus on the major changes in use for stimuli-sensitive nanoprepations.

Figure 1.2 Stimuli-responsive drug-targeting strategies via nanopreparations.

1.3.1 pH-Sensitive Nanopreparations

Low pH is a well-established property of tumors that has been worked into the designs of stimuli-sensitive drug delivery systems. In 1930, Warburg and co-workers showed that tumor cells preferentially convert glucose and other substrates to lactic acid, even under aerobic conditions. Lactic acid has a pK of 3.7. Hence it is apparent that the intracellular fluid becomes acidic. It was thought that lactic acid was the main source of tumor acidity for years. However, it is now known that the decreased pH is also related to the high levels of CO2 and the increased expression and activity of vacuolar-type proton pumps (Griffiths, 1991; Helmlinger et al., 2002; Fais et al., 2007). Deficiencies in tumor perfusion, hypoxia, and metabolic abnormalities associated with transformation and uncontrolled cell growth also contribute to the low pH of tumors.

An ideal pH-sensitive drug delivery system has to protect its payload in blood at a pH of 7.3–7.4 and release drug when the preparation reaches the interstitial space of tumors (pH 6.8–7.2) or the intracellular endosomal compartments such as endosomes (pH 5–6) and lysosomes (pH 4.5–5.5) (Gerweck and Setharaman, 1996; Shi et al., 2014; Boeckle et al., 2006). If high concentrations of cytotoxic drug have been delivered intracellularly, it is possible to exceed the efflux capacity of drug transporters (e.g., P-gp) and kill the tumor cells efficiently. Drug release can occur in the presence of the slightly acidic extracellular fluids of tumor tissue after tissue accumulation via the EPR effect. Likewise, pH-triggered drug release can occur in endosomes or lysosomes, which have an even lower pH value than extracellular fluids, when assisted by hydrolysis (with enzymes such like catepsin B) or by dissociation after uptake by cells via the endocytic pathway (Schmaljohann, 2006; Liu et al., 2013).

For pH-triggered drug release, one of the most common approaches is the use of protonizable components (amino, imidazolyl, and carboxyl groups) that can induce nanocarrier destabilization and drug release following protonization at acidic pH. For example, adriamycin-loaded poly(L-histidine)-b-PEG and PLLA-b-PEG mixed micelles were evaluated for extracellular tumor targeting. While the system was nonionized (nonprotonated) and stable at pH 7.4, a sharp transition to the ionized form occurred by protonization of the histidine residues of the poly(L-histidine) at pH 6.6. This transition destabilized the micelle structure with a significant increase in the critical micelle concentration and led to rapid release of a loaded anticancer drug (Lee et al., 2003). There has been much effort on discovery of pH-sensitive polymers using both acidic (e.g., carboxylic and sulfonic acids) and basic (e.g., ammonium salts) groups, to accept or release protons in response to the changes in local pH. These polymers undergo pH-sensitive conformational changes by dissociation, destabilization (by collapsing or swelling), or via the changes of the partition coefficient between the drug and vehicle (Liu et al., 2014). Nanocarriers with acid-labile chemical bonds may also be useful for pH-triggered drug release. There are specific chemical bonds like the ortho ester bond, the acetal/ketal bond, and the hydrazone bond which can breakdown under acidic conditions. The drug can be bound to the drug delivery system via any of these bonds and release drug content at acidic pH (Prabaharan et al., 2009; Liu et al., 2014). The folate-conjugated amphiphilic hyperbranched block copolymer, H40-P(LA-DOX)-b-PEG-OH/FA, was synthesized as a targeted anticancer drug delivery carrier by Prabaharan and coworkers. Doxorubicin was conjugated onto hydrophobic segments of the block copolymer via an acid-labile hydrazone linkage. The results indicated that doxorubicin was released rapidly at acidic pHs typical of the extracellular tumor microenvironment and the endocytic vesicles of tumor cells due to the hydrolysis of the hydrazone linkage (Prabaharan et al., 2009). Relatively new CO2-generating pH-responsive systems are another promising strategy that enables intracellular drug release in acidic lysosomes and may be effective in overcoming MDR. In a study by Liu and coworkers, the bicarbonate ion was firstly encapsulated in the liposomes which were composed of HSPC, Chol, and mPEG2000–DSPE. Liposomes in an acidic medium (pH 5.0), released CO2 gas following formation of carbonic acid and H+ and led to effective drug release from the liposomes. As a result, the drug-loaded liposomes circumvented the breast cancer cells’ resistance (MCF-7R) to doxorubicin due to enhanced cellular uptake and endosomal escape (Liu et al., 2012b).

1.3.2 Redox Potential Sensitive Nanopreparations

Glutathione (GSH) is a crucial tripeptide (γ-glutamyl cysteinyl glycine) generated in the cell cytoplasm that acts as the main reducing agent in cells and is considered the major thiol-disulfide redox buffer of the cells (Schafer and Buettner, 2001; Li et al., 2011). Its intracellular concentration (2–10 mM) is especially prominent in certain regions, such as the cytosol, mitochondrion, and nucleus (reducing). This is just 0.1–1% of the concentration (about 2–20 µM) in blood and the ECM (mildly oxidizing). Blood is generally an environment in which disulfide exchange reactions are minimal, because the redox balance of the blood is regulated and maintained continuously by the surrounding cells (RBCs, endothelial cells) and organs (liver) (Moriarty-Craige and Jones, 2004; Giustarini et al., 2008). By contrast, the glutathione concentration in a tumor mass is 100-fold higher than the extracellular level of glutathione in normal tissues. This extreme concentration difference makes GSH a candidate stimulus for drug delivery.

Due to this potential gradient between the extra- and intracellular redox environments, disulfide bonds are increasingly being examined as stimulus-responsive linkers for drug delivery systems. Intracellular reduction of disulfide bonds is most likely mediated by thiol–disulfide exchange reactions of redox molecules, mainly GSH and additionally thioredoxin, acting alone or with the help of redox enzymes. The reduction of the disulfide bonds results in a rapid disruption of the nanocarrier as well as increased cytotoxic activity of cancer therapeutics (Li et al., 2011; Li et al., 2012; Songa et al., 2011; Yin et al., 2013; Brülisauer et al., 2014; Caia et al., 2014). Redox- sensitive systems are usually stable in the tumor ECM. However, cell-secreted thiols and cell-surface thiols can cause disulfide cleavage in the extracellular space of tumor cells. The degree of this cleavage depends on the characteristics of the nanocarrier. For example, if the nanocarrier is PEGylated, the nanocarrier is protected by steric hindrance. Poly(L-lysine)-based DNA nanoparticles were developed by Sun and Davis. Poly(L-lysine) was conjugated through an N-terminal cysteine to a PEG chain by either of two different bonds: a disulfide bond (SS) (an unhindered disulfide) or a thioether bond (CS) (a sterically hindered disulfide) to obtain PEG–peptide conjugates. The nanoparticles containing the hindered disulfides avoided cleavage in the extracellular space of the two different cancer cell lines (HeLa and HuH7), while the nanoparticles with the unhindered bonds underwent cleavage by an extracellular mechanism which was not clear. The rate of dePEGylation was shown qualitatively to be cell-line-dependent, suggesting different amounts of thiols had been secreted from different types of cells (Sun and Davis, 2010).

Subsequent to cellular uptake of a redox-sensitive nanopreparation, it was proposed that high acidity of the endosomes slowed the thiol–disulfide exchange due to protonation of the reactive thiolate (Jensen et al., 2009). Although redox enzymes present on the outer cell membrane (i.e., PDI—protein disulfide isomerase) are transferred to the inner membrane of the endosome during the invagination process, it is a fact that the catalytic activity of PDI is pH-dependent (it shows maximum activity at neutral pH) (Saito et al., 2003). In a study by Liu and coworkers, redox-responsive polyphosphate nanosized assemblies based on amphiphilic hyperbranched multiarm copolyphosphates (HPHSEP-star-PEPx) with redox-responsive backbone are prepared. The glutathione-mediated intracellular drug delivery was investigated in the HeLa (human cervical carcinoma) cell line. The results suggested that DOX-loaded HPHSEP-star-PEPx micelles show higher cell proliferation inhibition against glutathione monoester pretreated HeLa cells compared to the nonpretreated ones. A redox-responsive micellar nanopreparation assembled from the single disulfide bond-bridged block polymer of poly(ε-caprolactone) and poly(ethyl ethylene phosphate) (PCL-SS-PEEP) achieved more drug accumulation and retention in MCF-7/ADR breast cancer cells. The system rapidly released its DOX payload in response to the intracellular reductive environment and also significantly enhanced the cytotoxicity of doxorubicin to MCF-7/ADR cancer cells. The nanoparticulate drug carrier with either a PEG or a poly(ethyl ethylene phosphate) (PEEP) shell increased the DOX influx but decreased the efflux of DOX by the multidrug-resistant mechanisms, in comparison with the direct incubation of MCF-7/ADR cells with DOX, which led to high cellular retention of doxorubicin (Liu et al., 2011; Wang et al., 2011). These systems may be effective for overcoming MDR in