Surface Chemistry of Nanobiomaterials: Applications of Nanobiomaterials

China

Preface of the series

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

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

In this context, the scientific series entitled Applications of Nanobiomaterials was motivated by the desire of the Editor, Alexandru Mihai Grumezescu, and others to bring together comprehensive, up-to-date and relevant findings on the field of biological applications of 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

About the Series Set (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, called nanobiotechnology. This complex research brings together significant knowledge from physical, chemical, biological, and technological sciences in an applicative field.

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

The multivolume set Applications of Nanobiomaterials contains 165 chapters, organized into 11 volumes which are ready to present a novel and up-to-date approach related to this intriguing domain. 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 biotechnologies.

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: Nanobiomaterials in Drug Delivery

Volume X: Nanobiomaterials in Galenic Formulations and Cosmetics

Volume XI: Nanobiomaterials in Dentistry

About Volume III

Volume III, Surface Chemistry of Nanobiomaterials, is an up-to-date book that highlights the recent progress made in the field of surface chemistry of relevant nanobiomaterials. This is one of the most important fields developed during the last decade, able to predict the applicability domain in nanomedicine, depending on the functional groups presented on the surface. Different types of nanobiomaterials such as quantum dots (QDs), carbon nanotubes, silver nanoparticles, copper oxide, zinc oxide, magnesium oxide, magnetite, hydroxyapatite, graphene, etc., and their related functionalization strategies are presented and discussed.

Volume III contains 15 chapters, prepared by outstanding international researchers from Canada, Argentina, Spain, France, Romania, Ukraine, Turkey, Saudi Arabia, Israel, India, Malaysia, and Japan.

In Chapter 1, Surface functionalized hybrid nanomaterials: implications in biosensing and therapeutics, Srabanti Ghosh et al. review recent innovative strategies to produce surface functionalized hybrid nanostructures. Their research show that the successful assembly of dendrimers offers various advantages including simple processability, excellent luminescence, and biomemetic properties, and is useful in developing diagnostic tools and biosensing systems for molecules of biological relevance.

Ahmad Amiri et al., in Chapter 2, Microbial toxicity of different functional groups treated carbon nanotubes, give an overview of the functionalization of carbon nanotubes and their promising applications in nanobiotechnology. Their studies demonstrate that carbon nanotubes (CNTs) hold significant antimicrobial activities against different pathogens, such as Escherichia coli, Staphylococcus aureus, and Salmonella enterica. Recent results have shown that CNTs represent promising alternatives to antibiotic treatment for the annihilation of multidrug-resistant bacterial strains.

Chapter 3, Making the hospital a safer place by the sonochemical coating of textiles by antibacterials nanoparticles, reviews the research that has been done for the functionalization of textiles with inorganic nanoparticles (Ag, CuO, ZnO, MgO) by sonochemical methods. The nanoparticles have been deposited on the surfaces of various fabrics (cotton, nylon, polyester) using ultrasound irradiation. The coating can be performed by an in situ process. One of the key aspects of their research is that some coated fabrics showed excellent antibacterial properties after they were washed for up to 65 cycles in hospital washing machines.

In Chapter 4, Nano-microporous structured surfaces prepared by the breath figures approach and their biorelated applications, Juan Rodríguez-Hernández et al. present the most recent advances on the formation of multiscale structured porous interfaces using the breath figures methodology. This approach takes advantage of water condensation during the solvent evaporation of a polymer solution to create porous interfaces. The authors summarize the applications of such materials in biorelated applications, which include, among others, control of cell adhesion and growth or the selective immobilization of bacterial cells.

Andrea Mathilde Mebert et al., in Chapter 5, Surface chemistry of nanobiomaterials with antimicrobial activity, report an up-to-date overview about biofilms and antimicrobial coatings. Two approaches are described in this study: (i) using a controlled-release nanostructured coating, in which the antibiotic is released from the biomedical device and intercepts bacteria in the vicinity; (ii) application of a molecular surface layer of covalently immobilized (grafted) molecules or on the modification of surface nanotopography, which can prevent bacterial attachment to materials surfaces by either killing bacteria or changing physicochemical characteristics of the surfaces (hydrophobicity/hydrophilicity, charge, surface free energy, nanoroughness).

In Chapter 6, Nanotechnology from particle size reduction to enhancing aqueous solubility, Doaa Hasan Alshora et al. highlight the correlation between a drug’s aqueous solubility and its particle size. The production of drug particles in nanoscale for enhancing drug aqueous solubility, dissolution rate, as well as bioavailability, is also discussed. This research also reveals the application of nanotechnology as a solubility-enhancing technique in the development of pharmaceutical formulations. Moreover, the stability aspects of nanotechnology and relationship to solubility are illustrated.

Ie.V. Pylypchuk et al., in Chapter 7, Formation of biomimetic hydroxyapatite coatings on the surface of titanium and Ti-containing alloys: Ti-6Al-4V and Ti-Zr-Nb, present the surface modification by creation of special functional groups on the titanium surface followed by formation of a self-assembled hydroxyapatite layer. According to this study, the titanium surface was modified by –OH, –Si-OH, and –COOH functional groups. By comparative investigation it was demonstrated that the closest to natural stoichiometric hydroxyapatite Ca/P ratio was obtained on the samples modified by carboxyl groups.

In Chapter 8, Interaction between nanoparticles and cell membrane, Hideki Nabika et al. give an overview of the interactions of artificial nanoscale materials, including metal, semiconductor, polymeric, and metal oxide nanoparticles and clusters with lipid membranes, in order to evaluate the parameters that control the material interactions with the membrane surface. An understanding of these interactions is important for the application of nanoscale materials as therapeutic drugs, and also to design safe nanoscale materials for other products.

Chapter 9, Antimicrobial studies of metal and metal oxide nanoparticles, aims to bring the substantial antimicrobial effect of nanomaterials and size- and morphology-dependent studies. The chapter particularly focuses on silver (Ag)-, copper (Cu)-, gold (Au)-, and zinc (Zn)-based nanomaterials.

Chapter 10, by Alina Maria Holban et al., Inorganic nanoarchitectonics designed for drug delivery and anti-infective surfaces, reviews the actual and most promising approaches developed on the field of inorganic nanostructured assemblies for the efficient delivery of antimicrobial drugs and for the development of anti-infectious surfaces for medical use. The technological progress made in this engineering field offers promising alternatives for the emergence of novel personalized antimicrobial strategies, based on versatile bioactive nanostructured systems optimized for the prevention and treatment to severe infections. Recent progress is starting to be efficiently integrated in the development of customized surfaces with specific properties, able to inhibit microbial attachment and biofilm formation in order to limit microbial colonization of medical surfaces and indwelling devices and to reduce mortality and morbidity associated with such severe infections.

Jobin John Jacob et al., in Chapter 11, The chemistry of magnetosomes, highlights the world of magnetosomes and their surface chemistry. This research reveals the unique properties of magnetosome magnetite crystals over synthetic nanoparticles, such as narrow size distribution, superior shape control, high purity with limited defects, better T2 reducing effect, and chain-like alignment.

Chapter 12, Nanomaterials and natural products for UV-photoprotection, by Maira Gaspar Tosato, discusses recent studies on natural sunscreens introduced or associated with nanoscale systems, such as liposomes, micelles, and lipid nanoparticles. The evaluated natural products include UV filters and antioxidants from the families of polyphenols, steroids, xanthines, carotenoids, tocopherols, and mycosporine-like amino acids. Emphasis is given in the analysis of the nanomaterial designs in relation to the effects on the photochemical and photophysical behavior and bioavailability of natural sunscreen agents.

Xueru Zhang et al., in Chapter 13, Progress in graphene-based optical and electrochemical aptasensors, report the recent progress about the mechanisms and paradigms of FRET (Forster resonance energy transfer), impedimetric, amperometric, electro-chemiluminescence, and FET (field effect transistors) graphene aptasensors. Future prospects in the development of graphene aptasensors are also highlighted.

Udita Agrawal et al., in Chapter 14, Improved oral bioavailability of bioactives through lipid-based nanoarchitectures, describe cutting-edge technologies with a specific focus on the delivery of the lipid carriers containing bioactive agents within the gastrointestinal (GI) system, and also the transport mechanism of the GI tract, barriers to drug absorption, current status, and limitations of this nanotechnology.

Chapter 15, Scientometric overview regarding the surface chemistry of nanobiomaterials, prepared by Ozcan Konur, presents an up-to-date overview of the research on the surface chemistry of nanobiomaterials; however, there has not been any scientometric study on the surface chemistry of nanobiomaterials. Following a scientometric overview of the research in surface chemistry of materials and nanomaterials, as well as the surface engineering of nanobiomaterials, brief information on a selected set of 25 citation classics in the field of the surface chemistry of nanobiomaterials is presented to inform major stakeholders about the influential papers in this dynamic research field as the first-ever study of its kind. It was found that the major research areas in surface chemistry of nanobiomaterials are surface plasmons and nanobiomaterials with emphasis on the localized surface plasmon resonance and the surface chemistry of nanobiomaterials.

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Chapter 1

Surface functionalized hybrid nanomaterials

Implications in biosensing and therapeutics

Srabanti Ghosh,    Department of Chemical, Biological and Macromolecular Sciences, S. N. Bose National Centre for Basic Sciences, Kolkata, West Bengal, India

Abstract

In the arena of nanoscience and nanotechnology, hybrid materials have shown great promise for various biomedical applications including cell type recognition, disease diagnosis, targeted therapeutics, intracellular imaging, and drug/gene delivery as well as optoelectronic device fabrication. In particular, functionalized semiconductor nanoparticles (NPs) or quantum dots (QDs) are among the most exciting nano platforms, due to their unique combination of physicochemical and optical properties. A pre-requisite for moving toward such applications is fabrication of NPs with well-controlled size and shape distributions, well-defined surface chemistry, biocompatibility, and a unique optical and electronic property is the basis of all such biological applications. Central to tackling these issues are surface functionalization of nanomaterials and elucidating the interfaces and interface between nanomaterials and biomolecules. Importantly, this interface demonstrates focusing effects on the in vitro and in vivo applications of nanomaterials, such as multiplexed detections, biosensing, multimodal bioimaging, drug delivery, and phototherapy.

Keywords

Nanomaterials; hybrid materials; quantum dots; polymers; bioconjugation; dendrimer

1.1 Introduction

In the arena of nanoscience and nanotechnology, hybrid materials have shown great promise for various biomedical applications including cell type recognition, disease diagnosis, targeted therapeutics, intracellular imaging, and drug/gene delivery as well as optoelectronic device fabrication (Vivero-Escoto and Huang, 2011; Ghosh et al., 2012; Biju, 2014). In particular, functionalized semiconductor nanoparticles (NPs) or quantum dots (QDs) are among the most exciting nano platforms, due to their unique combination of physicochemical and optical properties (Alivisatos, 1996). A pre-requisite for moving toward such applications is fabrication of nanoparticles with well-controlled size and shape distributions, well-defined surface chemistry, biocompatibility, and a unique optical and electronic property is the basis of all such biological applications (Palui et al., 2014). Central to tackling these issues are surface functionalization of nanomaterials and elucidating the interfaces and interface between nanomaterials and biomolecules. Importantly, this interface demonstrates focusing effects on the in vitro and in vivo applications of nanomaterials, such as multiplexed detections, biosensing, multimodal bioimaging, drug delivery, and phototherapy (Figure 1.1).

Figure 1.1 Examples of nanomaterials and their functional groups for biological applications.

To achieve binding specificity and targeting ability, QDs can be linked to monoclonal antibodies, peptides, oligonucleotides, a small inhibitor, or a hydrophilic segment (Kim et al., 2013). The integration of these QDs with biological macromolecules greatly expands the impact of optical imaging, sensing, and also of therapeutic strategies. Synthetic organic polymers are found to be excellent for this purpose as they meet all the criteria expected for an ideal medium. The selection of the polymeric matrix is crucial for the optimization of the properties of the nanocomposite derived. In this chapter, an overview of the recent developments using amphiphilic polymers to interface inorganic nanocrystals with biological media is given. The chapter starts with a brief description of the most common strategies to functionalized QDs as well as routes for growing a few representative high-quality hybrid polymer/semiconductor nanocomposites (i.e., QDs), and provides a few representative examples of where these hydrophilic platforms have been used as sensors and therapeutic applications.

1.2 Surface Functionalization

The unique physical properties of QDs, when utilized in conjunction with their remarkable biomolecular recognition capabilities, could lead to biological electronics and optical devices, including probes and sensors. Currently, in the field of biomedical diagnosis using nanoparticles, one of the key challenges is to design and develop a surface structure with multiple functionalities that can function to track and exert specific biological actions in cells. The surface functionalization of organometallically synthesized QDs to interface with biomolecules remains a major technical hurdle. In addition, due to complexity in the biological system, the nanostructures are often required to have several key features which are as follows:

• The surface of the nanoparticles must be treated with specific functional groups for the attachment of biological molecules.

• For diagnosis, strong luminescence from the QDs is expected in the desired visible range for in vivo imaging.

• Certain geometrical dimensions of the nanoparticles would be preferred for the storage and release of the treating drugs.

The surface-capped nanocrystals possess excellent size-dependent luminescence properties, which can be harnessed for various biomedical applications. If the capping ligand on the surface has a free bioactive end group, the QDs are consequently biofunctionalized using bioactive thiols, such as cysteine, glutathione, thioglycolic acid, etc., as capping agents () or amino (NH2), groups facing the solvent, ensuring its solubilization in water. A disadvantage of this process is the formation of a thick silica-like layer around the QDs which enlarges the particles significantly (Michalet et al., 2001). This might hamper the targeting of nanocrystals into the nano-sized cellular compartments. The surface of nanoparticles can be further modified with proteins and nucleic acids as well. So far, few attempts have been made to render the QDs more biocompatible and the surface of the QDs is modified with a suitable molecule to make it amenable to several biological manipulations (Alivisatos, 2004). The results suggested that the surface coating must be sufficiently stable and it is highly desirable to use nontoxic and biocompatible substrates. For example, ligand exchange using bifunctional molecules with thiol terminating ends (–SH) (Wu et al. 2003; Wang et al., 2007a,b), and the modifications of QDs surface chemistry using oligomers have been previously studied (Ma et al., 2008). Alternatively, the native hydrophobic ligands can be retained on the QD surface, and rendered water-soluble through the adsorption of amphiphilic polymer that contains both a hydrophobic segment (mostly hydrocarbon) and a hydrophilic segment (such as polyethylene glycol (PEG) or several carbohydrate groups). Several polymers have been reported including octylamine-modified polyacrylic acid, PEG-derivatized phospholipids, block copolymer, amphiphilic polyanhydride, calixarenes, or cyclodextrines (Carion et al., 2007; Pellegrino et al., 2004; Feng et al., 2005). The bioactive end-groups of the surface cap can be further conjugated to biological macromolecules yielding hybrid materials with high specificity and sensitivity, which could be of immense utility for in vivo imaging of cancerous cells and drug delivery (Riegler and Nann 2004; Lai et al., 2003). Folic acid has been shown to be one of the most promising ligands for targeting a range of human carcinomas, including breast, ovary, kidney, lung, neck, and brain, and myeloid cancers that are known to express an FA receptor (Ross et al., 1994). However, these processes involve multiple reactions with generally low yield. A new direction of biological surface modification of QDs with proteins, antibodies, and peptides received a great deal of attention, and can be used in life sciences for luminescence tagging, drug delivery, and implantable microdevices, as well as molecular electronics (Wolcott et al., 2006; Medintz et al., 2005, 2007; Smith et al., 2008). Bioconjugation of QDs, that is, the attachment of biospecific ligands to them, represents the convolution of nanotechnology and biotechnology and yields hybrid materials by incorporating unique optical and electrical properties of QDs and highly selective binding of oligonucleotides and proteins. The following simple bioconjugation techniques can do this kind of grafting of biological recognition moiety. For example, if the surface cap of a nanocrystal has COOH or NH2 end groups, it can be covalently coupled to a protein or enzyme using linker molecules (glutaraldehyde, carbodiimide) conventionally used for protein crosslinking (Wong, 1991). Another approach uses electrostatic interactions between QDs and charged adapter molecules or proteins modified in order to incorporate charged domains (Goldman et al., 2002). As, for example, streptavidin-coated QDs can be used in combination with biotinylated proteins or antibodies. By extension, a generic approach using (i) an antibody against a specific target, (ii) a biotinylated secondary antibody against the first, and (iii) a streptavidin-coated QD which allows QD labeling of most types of target. Such can be of immense utility in enhancing the efficiency of diagnostic and therapeutic techniques (Wolcott et al., 2006; Medintz et al., 2005; Smith et al., 2008). Ligand exchange and bioconjugate reactions of gold nanoparticles and quantum clusters are summarized in Figure 1.2.

Figure 1.2 Ligand exchange and bioconjugation reactions of gold nanoparticles and quantum clusters. Reproduced from Biju (2014) with permission from The Royal Society of Chemistry.

Conventionally, the luminescent imaging of diseased cells is carried out by using organic fluorophores such as rhodamine G, RITC, etc. Similarly, quantification of proteins and nucleic acids is based on their fluorescent enhancement effect on organic dyes such as diaminophenylindole (DAPI), bisimidazole, and ethidium bromide (EB). However, these organic fluorophores suffer from inherent functional limitations such as narrow excitation bands and broad red-tailing luminescence spectra, low resistance to photodegradation, photobleaching, and random on/off light emission (blinking) (Weng and Ren, 2006; Parak et al., 2003; Alivisatos et al., 2005). The luminescent QDs promise to be an attractive alternative to biolabeling and biosensing applications as they overcome most of their functional limitations. They have a high photobleaching threshold and emit bright and steady fluorescence. However, with the multilayer coating, the final size of the QD biolabels would typically be 12–20 nm, which might be too bulky to gain access to cells for in vitro and in vivo imaging. In addition, the large dimensions would dramatically lower the labeling efficiency on specific target sites within the cells. Therefore, to address these concerns, improvements in the surface-coating techniques are required to efficiently functionalize QDs for biomedical applications. Thus, poly(amidoamine) PAMAM dendrimers are being extensively studied for their potentially important applications in biotechnology and medicine (Svenson, 2009; Kannan, 2006; Thomas et al., 2005). PAMAM dendrimers are synthetic spherical macromolecules with a well-defined surface, comprising a core, branching sites, and a large number of terminal groups as shown in Figure 1.3. The presence of multiple surface functional groups and cavities allows encapsulation of various targeting ligands, imaging dyes, and therapeutic drug molecules, providing a unique platform for cancer targeting, imaging, and therapeutic (Majoros et al., 2005, 2006; Latallo et al., 2005). The biomimetic properties and low cytotoxicity of dendrimer molecules make them potentially useful for many biological applications such as gene transfection, diagnostics, and drug delivery, as well as nanoscale building blocks (Christine et al., 2005; Gillies and Réchet, 2005). Additionally, small molecules can pass into the cell membrane and be used to deliver material such as drug or genetic materials into the cells (Shukla et al., 2005). Decorating the surface of dendrimers with fluorescent tags yields potential signal amplification systems for use in clinical diagnostics (Singh, 2001). Further, nucleic acid (DNA) binds to dendrimers much like histones, providing complexes that are capable of carrying genetic material into cells (Wang et al., 2003). Dendrimers with gadolinium are being tested for their use as magnetic resonance imaging contrast agents (Worden et al., 2006).

Figure 1.3 Schematic representation of PAMAM dendrimer structure with (a) amine (NH2), (b) hydroxyl (OH), and (c) carboxyl (COOH) terminal groups. The blue, red, purple, green, and cyano balls represent core amine (N), secondary amine of repetitive unit (NH), terminal NH2, terminal OH, and terminal COOH groups of dendrimer, respectively. Reproduced from Ghosh et al. (2012) with permission from the American Chemical Society.

The self-assembly of dendrimer on nanoparticles could provide a means to modifying the nanoparticles for various biomedical applications. Worden et al. (2006) demonstrated the preparation of gold nanoparticle–dendrimer conjugates by simple chemical reaction using an active agent, di-iso-propylcarbodiimide (DIPCDI), but theses conjugates are soluble in organic medium. Furthermore, Shi et al. have synthesized the self-assembly of modified dendrimers with carboxyl termini onto iron oxide nanoparticle surfaces, which were able to be taken up by cells regardless of the repelling force between the negatively charged cell surfaces and the negatively charged particles (Frankamp et al., 2005; Shi et al., 2007; Landmark et al., 2008). This implies that the surface charge of dendrimer-stabilized magnetic iron oxide nanoparticles in biological medium is an important factor influencing their biological performance. Shi et al. (2009) demonstrated a facile approach to modifying carbon nanotubes with multifunctional poly(amidoamine) (PAMAM) dendrimers for cancer cell targeting and imaging, thereby providing many possibilities for various applications in biomedical sensing, diagnosis, and therapeutics. Hence, QD–dendrimer conjugates can be used as novel luminescent multifunctional nanostructured materials. In general, formation of nanoparticle assemblies depends on noncovalent interactions, however, the inherent instability due to various experimental parameters, such as low pH, higher temperature, and ionic strength, etc., create difficulty during application. Hence, an alternative useful approach to prepare QD assemblies is interfacing through covalent binding (Mamedova et al., 2001). Highly luminescent, water-soluble, semiconductor nanoparticles capped with thiol have been synthesized in which the carboxyl group, as well as the NH2 group, of the surface ligands remains free. In order to obtain multifunctional, highly luminescent nanomaterial, we need to carry out the conjugation procedure. The two functional groups, carboxyl (COOH) and amine (NH2), can be crosslinked by two well-known techniques.

1. Schiff’s base linkage using glutaraldehyde (G) as linker.

2. Peptide bond linkage using NHS (N-hydroxysulfo-succinimide) and EDC (1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride).

To synthesize a stable highly luminescent covalently linked CdTe–dendrimer conjugate in aqueous medium, a linker molecule, glutaric dialdehyde (G), has been used as shown in Figure 1.4 (Ghosh et al., 2009a,b,c). In this strategy, after formation of the conjugation with dendrimer, the photoluminescence efficiency of CdTe QDs having narrow emission bandwidths remained unaffected (Ghosh and Saha, 2009).

Figure 1.4 Schematic representation of the synthetic route used to prepare the QD–dendrimer conjugate.

The successful assembly of QDs and dendrimers with desired functionality has significant implications in material research and demands most extensive inquiries into the luminescent electronic and chemical properties of these unique building blocks as they are incorporated into new and functional nanostructured materials. Another example, water-soluble polymer-conjugated drug molecules are superlative for drug delivery and using biocompatible as well as biodegradable polymers such as chitosan shows the efficacy in selectivity of drug molecule delivery (Haldar et al., 2006; Kenawy et al., 2007). Chitosan is known as a common biopolymer having excellent biocompatibility and wound-healing activities (Illum, 1998). Methylglyoxal (MG, a bioactive normal metabolite) has significant antimicrobial and anticancer activity but due to the limited stability in air and strong enzyme-induced degradation there is difficulty during its application (Együd and Szent-Györgyi, 1968; Talukdar et al., 2008; Pal et al., 2009; Bock et al., 1957). Alternatively, a green approach has been used by using multivalent chitosan polymers for the synthesis of nanoformulated drug (Figure 1.5) (Ghosh et al., 2014a,b).

Figure 1.5 Schematic representations of polymer-based nanoformulations of methylglyoxal (MG). The structure of chitosan-based nanoformulated methylglyoxal (NMG) showing methylglyoxal entrapped within the chitosan shell. Reproduced from Ghosh et al. (2014a,b) with permission from The Royal Society of Chemistry.

However, due to the limited solubility of chitosan, another mutlifunctional PAMAM dendrimer has been utilized for encapsulation of methylglyoxal for biomedical applications. Recently, Yang et al. (2010) reported folic-acid-modified chitosan nanoparticles demonstrated enhanced drug release in the cellular lysosome which can be used for detection of oral cancer. Furthermore, Yang et al. (2013) suggested folic-acid-conjugated chitosan nanoparticles improved protoporphyrin IX accumulation in colorectal cancer cells which can be used as an ideal vector for fluorescent endoscopic detection. Mathew et al. (2010) conjugated folic acid with carboxymethyl chitosan to produce nanoparticles capable of targeting and controlling the release of 5-fluorouracil, an anticancer drug used in chemotherapy. Park et al. (2000) suggested grafting dextran to galactosylated chitosan for targeting genes to hepatocytes. Another simple way to functionalize or to fabricate hybrid nanomaterials is to use functionalized organic molecules or polymers and in situ synthesis of nanomaterials directly as a template. For example, dendrimers are spherical macromolecules containing connectors and symmetric branching units built around a linear polymer core. The controlled synthesis of dendrimer generates well-defined and symmetrical polymer with narrow molecular weight. Dendrimers contain a huge number of regularly spaced external and internal functionalities which make them an attractive candidate for biomedical applications such as gene transfection, diagnostics, enzyme mimics, and as antiprion agents. On the other hand, dendrimers can be considered as spherical artificial proteins that may be utilized as building blocks (Yellepeddi et al., 2009; Tomalia, 1993). As the dendrimers are aliphatic molecules, it is difficult to observe them directly in cells. In this aspect, the attachment of fluorescent markers, radioactive substituent, or surface complexes of metal ions can be used to overcome this issue. However, the conjugation of marker molecules to the surface of dendrimers may dramatically change the solubility as well as alter other surface-related properties. This problem can easily be solved by incorporating fluorescent QDs in the dendrimer matrix, that is, forming dendrimer hybrid nanocomposites (DNCs) as shown in Figure 1.6 (Ghosh et al., 2008).

Figure 1.6 Preparation of semiconductor/dendrimer nanocomposites by reactive encapsulation. Reproduced from Ghosh et al. (2012) with permission from the American Chemical Society.

Dendrimer-based nanocomposites are novel organic–inorganic hybrid materials where very small and uniformly dispersed inorganic guest domains are physically trapped within nanoscopic-sized organic hosts (Zhao et al., 1998; Balogh et al., 1999). The PAMAM dendrimer features (i) interior amine and amide groups that can interact with ionic metal precursors through coordination chemistry or ligand-exchange reactions; (ii) interior void space and structural flexibility that can accommodate supermolecular guest species; and (iii) exterior chemical groups (amine, hydroxyl, carboxyl, etc.) that permit further functionalization, tethering to surfaces, and assembly into higher-order structures. The first two of these features make PAMAM useful as a template and stabilizer for synthesizing nanoparticles with controlled size, shape, and composition (Esumi, 2003; Crooks et al., 2002). The third feature provides the means to stabilize the nanoparticles in various phases and/or deliver them to desired locations on solid surfaces (Wu et al., 2004). Therefore, it is possible to synthesize revolutionally new chemical entities which were not available before, employing a molecular-size scaffold with well-defined and exactly known structure as a nanoreactor. In DNCs, most of the interactions between guest atoms and their microenvironment (metal–metal and metal–solvent interactions) are substituted with the metal–dendrimer and dendrimer–solvent interactions. For this reason, nanocomposite solutions may be stable for a very long time in appropriately selected solvent systems. Pioneering research on the synthesis of metal nanoclusters was reported at the same time by several groups by using dendrimers as both nanoreactors and stabilizers (Zhao et al., 1998; Balogh et al., 1999). DNCs units are being used as building blocks for highly ordered nanostructures including self-assembled ultrathin multilayer and smart nano-devices (Lebedeva et al., 2007; Balogh et al., 2007; Khan et al., 2008). Although PAMAM dendrimers have been used for preparation of metal nanoparticles for the past decade, the reports regarding semiconductor/dendrimer nanocomposites are limited. Semiconductor nanoparticles having controlled sizes, and well-defined luminescence properties, have already been prepared in the dendrimer matrix (Sooklal et al., 1998; Lakowicz, 1999; Lakowicz et al., 1999). Such materials might be useful as biological labels, because QDs have highly desirable characteristics compared to organic dyes and because the luminescent properties of QDs are not affected by the surrounding dendrimers to an appreciable extent. A study conducted by Lakowicz et al. revealed that CdS NPs fabricated in the presence of starburst dendrimer exhibited polarized emission with an anisotropy value of 0.3. This is an important property discovered for use as a biophysical probe. The high and nonzero anisotropy observed in CdS/dendrimer nanocomposites suggests that the excited state dipole is oriented in a fixed direction within the nanoparticles (Lakowicz et al., 1999). Hence, these dendrimer nanocomposites can be expected to be useful analogs of proteins or other macromolecules and as internal cellular markers, which could report the rate of rotational diffusion (Lakowicz et al., 1999). However, these nanocomposite particles were unstable and underwent further aggregation to form micrometer-scale flocs, in contrast to a colloidal chemical method, an alternative simple and a single-step approach for synthesizing highly monodispersed, water-soluble, fluorescent CdS/dendrimer nanocomposites by adopting steady-state gamma radiation route. A systematic investigation has provided an effective control over particle size distribution by optimizing the key parameters, dose rate, dendrimer core, terminal group, and generation, etc. The ambient conditions make this methodology amenable for the synthesis of other semiconductor/dendrimer nanocomposites which can be used for tailor-made applications from biomedical to device making (Ghosh et al., 2009a,b,c). In the quantum confinement regime, CdS emits in the near-UV to green region, ZnS emits in the UV region, while CdTe and ZnTe nanoparticles show robust luminescence through the entire visible spectrum, a property valuable for luminescent imaging and biosensor applications. Furthermore, the synthesis of CdTe and ZnTe/dendrimer nanocomposites has been reported and a good control over size distribution can be achieved by manipulating the supersaturation as well as synthesis temperature (Ghosh et al., 2008, 2009a,b,c). Interestingly, this method provides a simple route to narrow down the size distribution without using any postpreparative treatment (Priyam et al., 2009). Dendrimer nanocomposites containing nanoparticles are known to exhibit unusual properties like enhanced fluorescence, anisotropy, antioxidant action, and tunable zeta potential, which make them suitable for a wide range of applications. In this regard, the amalgamation of biomimetic properties of dendrimer macromolecules with excellent luminescence properties of semiconductors, such as CdS, CdTe, etc., that could yield novel hybrid materials ideally suited for various biomedical applications.

1.3 Applications in the Biomedical Field

The era of nanotechnology has been accompanied with the development of a new generation of therapeutic approaches for in vitro diagnostics as well as in vivo imaging. A series of functional nanomaterials, for example core/shell nanoparticles, graphene nanosheets, carbon-nanotube, silica nanoparticles, gold nanoparticles, iron oxide nanoparticles, QD, and polymeric nanoparticles, etc., have been developed for therapeutic and biosensor applications. Fluorescent QDs are being extensively used for myriads of bioapplications such as biosensing, biolabeling, in vivo imaging, and photodynamic therapy (Figure 1.7) (Ghasemi et al., 2009; Jaiswal et al., 2004; Azzazy et al., 2007).

Figure 1.7 Biomedical applications of QDs. (a) Schematic representation of the four-color multiplex assay of QDs with antibodies against the four different toxins immobilized by capture antibodies on the surface. (b) Mechanistic representation for induction of photodynamic processes by QDs. (c) Simultaneous five-color imaging in fixed human epithelial cells using QD conjugates. The colors allowed localization of cellular proteins and substructures, such as nuclei (cyano), cell proliferation protein (magenta), mitochondria (orange), microtubules (green), and actin filaments (red). Courtesy of Quantum Dot Corp.

Alivisatos and co-workers first demonstrated the use of QDs for bioimaging by successfully modifying the QDs surface with conjugated biomolecules such as oligonucleotides and antibodies (Bruchez et al., 1998). In fact, QDs have been used for studying early embryonic development in vivo, tracking tumor cell metastasis (Gao et al., 2004). These promising breakthroughs demonstrate the feasibility of utilizing QDs to study biological systems and detecting terminal illnesses. Indeed, the beauty of the myriad of colors emitted by QDs permits long-term multicolor imaging of multiple cells for investigating a range of phenomena in cell and developmental biology demonstrated by Simon and co-workers (Jaiswal et al., 2003). Recently, we reported the modulation of a glycolytic enzyme critically involved in the glycolysis of cancer cells, glyceraldehyde-3-phosphate dehydrogenase activity by surface functionalized QDs (Ghosh et al., 2014a,b). In vitro activities of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme critically involved in the glycolysis of cancer cells, are selectively inactivated in a controlled way by the QDs at a significantly low concentration (nM). Maximum inactivation (97–100%) of GAPDH activity from EAC cell was observed with IC50 values of 3.2 and 6.3 nM for 3MC-GAPDH (Figure 1.8a). In contrast, CdSMPA QDs inactivate N-GAPDH and R-GAPDH at higher concentrations of QDs (IC50 values of ~11 nM for N-GAPDH and 15 nM for R-GAPDH). Since the active site of GAPDH is surrounded by positively charged amino acid residues, negatively surface functionalized QDs can effectively block the active site. Subsequently, the accessibility of negatively charged substrates (such as GAP) to the catalytic center is reduced, resulting in the inhibition of GAPDH activity depending on the concentration of MPA-coated QDs. Cumulative kinetic studies suggest that due to the site-specific interactions of QDs with enzyme molecules, the QDs undergo both reversible and irreversible inhibition mechanisms. When the surface charge and structure of the inhibitor mimics the substrate molecule, inhibitors may bind to enzymes via noncovalent interaction following competitive or noncompetitive inhibition. The Lineweaver–Burk (L-B) plots for R-GAPDH (Figure 1.8b) show a noncompetitive type of inhibition suggesting that CdSMPA may not affect substrate binding to the active site of enzyme directly. This process blocks enzyme-binding possibilities to substrates, reducing enzyme activity and rate of chemical reaction of enzyme and substrate. We have calculated the equilibrium constant (Ki) from the Dixon plot (Figure 1.8c) (Dixon, 1972).

Figure 1.8 (a) Effect of CdSMPA on R-GAPDH (black asterisk and line), N-GAPDH (blue square and line), 3MC-GAPDH (red dot and line), and EAC-GAPDH (magenta star and line). (b) The L-B double reciprocal plot of R-GAPDH in absence of CdSMPA (black square and line) and presence of 9.8 nM (red dot and line), and 19 nM (blue star and line) CdSMPA with various concentrations of GAP. A decrease in Vmax values suggests a noncompetitive inhibition mechanism. (c) Dixon plot for R-GAPDH in which measurement of the hydrolysis of the substrate at four different GAP concentrations, 0.04 (black square and line), 0.06 (blue star and line), 0.1 (green dot and line), and 0.2 mM (red diamond and line) with CdSMPA concentrations. (d) L-B double reciprocal plot for N-GAPDH in presence of 9.8 nM CdSMPA (red dot and line) and absence of CdSMPA (black square and line) with various concentrations of GAP. Reproduced from Ghosh et al. (2014a,b) with permission from the PCCP Owner Societies.

The Ki value of 21±0.3 μM for R-GAPDH is much lower than the effective concentration of GAP (2.5–25 mM) suggesting a stronger affinity of CdSMPA toward GAPDH in comparison to the substrate. A competitive inhibition with Ki~39±0.2 µM is observed for N-GAPDH (Figure 1.8d). In contrast, GAPDH from tumor muscles shows irreversible inhibition of enzyme activity by CdSMPA. The QDs may react with an amino acid side chain of enzyme molecule to form adducts, specifically altering the active site of enzymes without affecting protein structure (Lundblad, 2004).

The surface functional groups of QDs have strong influence using NAC (CdSNAC) and cysteine (CdSCys)-coated CdS QDs to compare with CdSMPA (Figure 1.9a–c). CdSMPA showed the strongest inhibition up to ~90% on R-GAPDH, whereas CdSNAC and CdSCys show 80% and 70% inhibition, respectively (Figure 1.9c). For CdXMPA, MPA form a highly negative-charged surface by exposing –COOH groups which can interact more strongly with enzyme further supported with high negative zeta potential value (Figure 1.9d).

Figure 1.9 Schematic surface structures of CdX QDs synthesized using (a) 3-mercapto propionic acid, MPA and (b) N-acetyl L-cysteine NAC, (c) cysteine hydrochloride Cys as capping agent. (d) Effect of CdS QDs with different thiol groups; 3-mercaptopropionic acid (red bar), N-acetyl L-cysteine (green bar), and cysteine hydrochloride (blue bar) on the activity of GAPDH. Rabbit muscle GAPDH in the absence of CdS QDs as control (black bar) is shown. The inhibition of GAPDH at maximum concentration of 60 nM follows the trend as CdSCysGhosh et al. (2014a,b) with permission from the PCCP Owner Societies.

Due to the presence of different amino acid residues at the active site of the enzyme molecules, differential interaction may be achieved between QDs and enzyme molecule. The sarcoma tissue was developed by intramuscular injection of 3-methyl-cholanthrene in the left hindleg of mice and confirmed by histological examination as highly differentiated sarcoma tissue (Figure 1.10).

Figure 1.10 Images represent (a) normal mice and (b) tumor generated (in left hindleg) mice, (c) histological examination of mouse skeletal muscle with 3MC induced sarcoma tissue at different stages; initial stage, intermediate stages with progression of malignancy and full-grown tumor. The stain used was eosin and hematoxylin.

GAPDH was purified by gel filtration and ion exchange chromatography from normal mouse muscle or sarcoma tissue. GAPDH from normal tissues is a homotetramer having four identical 35 kDa subunits. In contrast to normal cells, the GAPDH purified from both EAC cells as well as sarcoma mouse muscle is a heterodimer of 55 and 33 kDa along with the presence of a critical lysine residue in the active site (Smith and Macquarrie, 1979). This indicates that due to structural and active site modification associated with malignancy, CdSMPA inhibits GAPDH activity via differential mechanistic pathways. Furthermore, a similar behavior was observed in human cervical cancer cells, as well as malignant EAC cells and sarcoma-180 cells, where CdSMPA can selectively inhibit cancer cells but normal cells are viable more than 90% at a concentration upto 50 μg/ml. No distinct morphological changes have been observed for normal myoblast cells treated with 50 μg/ml CdSMPA indicating myoblast cells remain healthy after treatment with CdSMPA (Figure