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Chapter 1

1. Abstract

The future prospect of inorganic nanoparticles in the field of nanomedicine has augmented research in this area over the past decade. The present focus is on their utilization as multi-functional nanodevice that incorporates the therapeutic capability of these nanomaterials and their ability to specifically deliver drugs and nucleic acids to tissues, cells and organelles. These inorganic nanoparticulates can also operate as sensors and contrast agents leading to enhanced diagnostic potential of the diseased state. The intense research contributions in therapeutics, drug and gene delivery have centered on noble metal, magnetic, inorganic phosphate, silica and carbon nanomaterials. The hydrophobicity of carbon nanomaterials and toxicity issues are major deterrents related to their in vivo use. The enhanced surface plasmon resonance (SPR) of noble metal nanoparticles such as gold and silver, and the superparamagnetic iron oxide nanoparticles (SPIONS) have been extensively used for therapy and diagnosis. In addition, silver nanoparticles demonstrate significant microbicidal property applicable for control of superficial bacterial and fungal infections. The primary role of inorganic phosphate nanoparticles in targeted nucleic acid delivery has been amply demonstrated in recent studies. Surface functionalized calcium phosphate and magnesium phosphate nanoparticles can be targeted in vivo with significant expression of the entrapped gene. Studies related to the use of calcium phosphate nanoparticles as adjuvants for DNA vaccines have indicated promising application possibilities in this area. Silica nanoparticles, nanotubes, nanotesttubes and organically modified siloxane (ORMOSIL) derived nanoparticles have been successfully used for gene delivery, drug delivery and photodynamic therapy. Another important application of silica nanoparticles is in enzyme therapeutics which needs to be further explored in vivo. Investigations pertaining to inorganic nanomaterials have generated diverse hybrid nanostructures with interesting properties and multiple applications. It is postulated that as therapeutic nanodevice, these nanostructures have immense capability in revolutionizing the concept of disease diagnosis and cure.

Inorganic Nanoparticles for Therapeutics, Drug and Gene Delivery Susmita Mitraa and Amarnath Maitrab

a Amity Institute of Nanotechnology, Amity University, Noida, India. b Delhi University, Delhi, India

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2. Introduction

Intense research in nanobiotechnology and the associated innovative advancements in biomedical applications lay a strong basis for a customized and personalized medicine in the future. The important areas of investigation in nanomedicine encompasses targeted drug and gene delivery, nanoparticle cancer therapy, disease screening by diagnostic imaging, creation of nanoscaffolds for tissue engineering, development of implantable nanoporous devices, nano-robotics, as well as nanomaterial enhanced sensing of disease and drug effects.1-6 The tools and techniques in nanomedicine comprise of combinations of multifunctional nano-entities, which would further enhance our diagnostic and therapeutic capabilities, facilitate early detection and cure of disease, causing no side effects. The small size (<100nm) of nanoparticles (NPs) makes them desirable for many biological and biomedical applications because these nanoentities are easily endocytosed resulting excessive intracellular accumulation and consequent higher therapeutic effects. Demonstrating various physical properties such as mechanical, electrical, thermal, magnetic and optical properties, the inorganic NPs combined with biomolecules, drugs, and other reagents, can be used as nanoprobes. Gold NPs for example, exhibit specific optical absorption properties,7 while iron oxide NPs show superparamagnetic properties depending on their size.8 One can thus combine the immense surface to volume ratio of these nanostructures to deliver higher loads of compounds encapsulated or linked to their surface, while their presence can be measured due to their specific physical characteristics. Micron sized particles made from the same materials, however, do not exhibit such unique physical properties.9 The submicron size of NPs, with appropriate surface modification to enhance hydrophilicity and ensure steric stability, allows them to escape the reticuloendothelial system (RES) for in vivo applications. Further, conjugation with biomolecules is facilitated by the presence of appropriate surface functional groups. The 10nm-100nm size nanoparticles with appropriate surface modifications ensure long circulation and have potential accessibility to desired tissue and cellular domain when targeted. Thus, the nanoparticle (NP) becomes a versatile choice for site- specific drug / gene delivery, as well as imaging, and other diagnostic and therapeutic modalities.1 NPs can be targeted passively to solid tumors by the Enhanced Permeability Retention (EPR) effect.10 For active tumor targeting, NPs are formed by coating them with ligands such as monoclonal antibodies, aptamers, peptides, and various receptor-specific substrates that are recognized by tumor-cell-specific receptors.2 The nucleus is the desired target for certain cancer therapies that involve DNA–drug-binding interactions, gene therapy and antisense strategies that manipulate RNA splicing. Although viruses have been adapted to deliver genes to cell nuclei, the design of safer synthetic delivery systems remains a challenge. The functionally active peptide sequences of nuclear localization signals (NLSs) of many viral proteins are known. Such peptides could be synthesized with an appropriate terminal residue such as cysteine for gold NPs, and attached along with therapeutic agent.

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This chapter addresses the developments in therapeutic applications and carrier mediated drug and gene delivery. The focus is on inorganic NPs due to their size dependent properties and potential to act as carriers for therapeutics and prophylactics. Inorganic NPs entrapping biomolecules have demonstrated diverse applications in material science. Their excellent storage stability in comparison to organic NPs and the added advantage of protection from microbial attack has contributed to their importance for biomedical applications.3, 11 3.1 Noble metal nanoparticles The extinction of light (absorption plus scattering) by noble metal NPs is several orders of magnitude higher than that of any molecular chromophore. Both absorption and scattering properties of these NPs arises from resonant oscillation of their free electrons in the presence of light, and is known as localized surface plasmon resonance (LSPR). This can be considerably altered by surface modification or by electronic coupling between individual NPs.7 The LSPR can either radiate light (Mie scattering) which finds great utility in optical and imaging fields, or can be rapidly converted to heat if it is in the NIR region of the electro-magnetic spectrum. This has led to diverse applications of these nanostructured materials in several new areas of therapy. Nanostructured gold, silver-gold core-shell nanostructures and noble metal alloy nanomaterials have been investigated as photothermal scalpels for tumor cell ablation,12 drug and gene delivery.13, 14 Enzymes entrapped in sulphur containing cyclodextrin derivatives can be immobilized on metallic NPs (Au and Ag) by supramolecular interaction, for subsequent application in enzyme therapy.15 Silver NPs on the other hand demonstrate potent anti-microbial effects,16,17 paving a way to alternative antimicrobial therapy.18 The noble metal NPs are relatively easy to synthesize with a defined size, shape and surface chemistry. Surface modification with multiple polymers and ligands can also be achieved in a one-pot synthesis.19 Metal colloids of different sizes have been synthesized from metal salts, predominantly using either citrate or sodium borohydride as reducing agents.20 Surface functionalization of gold NPs utilizing thiol-containing carboxylic acids have produced NPs with surface –COOH groups that have been used as precursors for conjugating with target- specific biomolecules.20 Citrate molecules serve a dual role of reduction of the metal salt and capping of NPs for subsequent stabilization and biomolecule conjugation.20 The use of microemulsion, copolymer micelles, surfactants and other amphiphiles have also played a significant role in synthesis.21 Crystalline octahedral gold NPs can be formed following gold salt reduction with ascorbic acid and H2O2 as reaction promoter in the presence of cetyltrimethyl ammonium bromide.22 Gold nanoshells are synthesized by using aminated silica particles formed by Stober’s method,23 and spherical gold particles (1–3 nm diameter) as precursors.24 Gold nanowires,25 nanorods,7 nanorattles26 and hollow gold nanospheres or gold nanocages have also been reported.27,28 Gold nanocages of various size are synthesized using galvanic replacement reaction between nanocages of silver and Au3+ ions. They were recently synthesized in aqueous solution

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with HAuCl4, silver nanocubes and poly(vinyl pyrrolidone).28 A two-step procedure for generating cubic nanocages and nanoframes involves a first step synthesis of Au/Ag alloy nanoboxes by galvanic replacement reaction between Ag nanocubes and an aqueous HAuCl4 solution. Removal (dealloying) of Ag from alloy nanoboxes with aqueous etchant based on Fe (NO3)3 or NH4OH, is the second step involved. By increasing concentration of etchant, nanocubes could be converted to nanocages and subsequently to nanoframes with a shift of SPR band to 1200nm in the NIR region.28, 29 Silver NPs have the propensity to surface oxidation, thus have to be surface coated with robust and tailorable materials such as gold, silica or polymers.30 Polymer stabilized silver and gold nanostructures with predetermined size and shape were produced using organic compounds as well as biological molecules.31 Biological synthesis methods have been developed for both gold and silver NPs as environment friendly alternatives. Use of gelatin, sodium salt of carboxymethyl cellulose, Geranium leaf extract, Emblica officinalis fruit extract, soybean extract have demonstrated the formation of highly stable gold and silver NPs 32, 33 The antioxidant phytochemicals reduce metal salts and subsequently coat the NPs. The advantage of using natural polymers and plant extracts over the microbial methods of synthesis reported by them earlier 34 is the much faster formation of the nanoparticles.

3.1.1 Gold nanoparticles: In the context of biomedical applications, gold NPs amongst the noble metals appear to be the most promising, primarily due to their compatibility in the body fluid and easy surface modification with biomolecular functionalities. The ability to tune the surface of the particle provides access to cell-specific targeting and controlled drug release.35, 36 An importantcharacteristic of gold is its unique chemical property of persisting in the unoxidized state at the nano level, whereas the surface of less noble metal gets oxidized to a depth of several nanometers or more, often obliterating the nanoscale features. The SPR peaks of gold nanostructures can be tuned from the visible to the near infrared region by controlling the shape and structure in the form of nanospheres, nanorods, nanoshells, nanocages and nanoprisms.7 By increasing the size of gold nanospheres from 20 to 80nm, the magnitude of extinction and relative contribution of scattering to the extinction rapidly increases. Gold nanoshells have optical cross-sections comparable to and even higher than nanospheres. The resonance wavelength can be rapidly increased by either increasing the total nanoshell size or increasing the ratio of the core-to-shell radius. Nanorods with a higher aspect ratio along with a smaller effective radius are the best photoabsorbing NPs.7, 37 Gold nanorods show optical cross-sections comparable to nanospheres and nanoshells, however at much smaller effective size. Their optical resonance can be linearly tuned across the near-infrared (NIR) region by changing either the effective size or the aspect ratio.38 Core-shell nanoparticles having core of different dielectric material and gold shell have been the central to the development of photothermal cancer therapy and diagnostics for the past several years. Such core-shell nanoparticles having intense attenuation in the NIR (700–1300

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nm) are applicable to controllable photothermal therapy using near-infrared lasers which penetrate deep into tissues.39, 40 Blood provides strong optical transmission in this region, and this permits the photometric detection of nanoshells in whole blood.41 Nanoshell-based photothermal therapies in several animal models of human tumors have produced highly promising results. A novel ‘Trojan horse’ strategy overcomes the challenge of treating difficult to access hypoxic regions of the tumour.42 Mice bearing murine colon carcinomas,41 human breast carcinoma cells in culture,43 urological cancer44 and melanoma,45could be successfully treated with either PEG-SH modified or unmodified nanoshells following exposure to NIR light ( 820nm, 4 W/cm2). The plasmonic NPs can be used for photodynamic therapy (PDT) which employs chemical photosensitizers that generate single oxygen capable of tumor destruction.46, 47

PDT has emerged as an important paradigm in the management of cancer and other diseases.48 Highly efficient hydrophobic photosensitizer drugs, dyes as well as quantum dots, entrapped or conjugated to PEGylated AuNP “cage,” have been developed for passive and active targeting.48-

50 Gold is an excellent absorber of X-rays, leading to significant (>200%) dose enhancement following radiation therapy.51,52 Injecting cancer cells into mice followed by administration of AuNPs and irradiation with 250-kV X-rays, led to size reduction and eradication of tumors. The selective absorption of X-rays could be used as dual imaging and therapeutic probe.53, 54

Radioactive AuNPs present attractive prospects in cancer therapy. Both 198Au and 199Au have imagable gamma emissions that can be used for dosimetry and pharmacokinetic study.55, 56

Another approach involves encapsulation of radioisotopes within a nanocomposite device (NCD).Gold NCDs are synthesized as monodispersed hybrid NPs composed of radioactive guests immobilized in dendritic polymers, PAMAM and tectodendrimers as hosts. Tritium-labeled PAMAM –NCDs (5nm) were used in tumor models such as mouse B16 melanoma, human prostrate DU 145. The fast tumor uptake and long term retention could prove useful for therapy.55, 56 Non-invasive, deep tissue penetrating radiofrequency (RF) field induced hyperthermia have also been reported.57, 58 Specific targeting to cancer cells has the advantage of enhanced thermal ablation capability of the NPs. Colorectal metastases targeting of gold nanoshells by guanylyl cyclase C,59 EphrinA I- targeting to EphA2 receptor for pancreatic cancer cell (PC-3),60 anti-epidermal growth factor receptor (anti-EGFR) targeted to epidermal cancer cell,61 are some of the reported studies. Immuno-gold NPs were reported to show antiangiogenic properties through neutralizing angiogenic cytokines such as vascular endothelial growth factor.62 Au-Ag nanorods with molecular aptamers for target specificity have high absorption efficiency and thermal ablation capacity at low laser exposure.63 A SPR peak at around 800 nm is characteristic for 40 nm gold nanocages. Selective photothermal destruction of breast cancer cells can be achieved by immunotargeted nanocages.64 Gold nanoshells could be powerful drug carriers with near-infrared light assisted dual functions of light generation and controlled drug release. Optically active nanoshells (gold-gold sulfide) and composites of thermally sensitive hydrogels can photothermally modulate drug delivery.65 Incorporation of these nanoshells in liposome carriers

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can lead to enhanced therapy.66 Hollow nanoshells of gold entrapping enzyme Horseradish Peroxidase (HRP) was prepared in reverse micelles by leaching out silver chloride (AgCl) from HRP entrapped Au(shell)AgCl(core) NPs with dilute ammonia solution. Use of this soft chemical method allows the entrapped enzyme to remain active. Small substrate molecules such as o-dianisidine can easily enter through the pores of the nanoshell and can undergo enzymatic oxidation by H2O2.

27 These nanoshells could be used for enzymatic replacement therapy and optically tracked during in vivo delivery. Gold nanocages have hollow nanostructures with porous walls, thus can bind much more drug than any other filled nanoparticle. A new class of Au(x)Ag(1-x) nanostructures with dendrite morphology was used as photothermal agent destroying A594 lung cancer cells. The laser power required was also significantly reduced.67 Au NPs are reported to be good carriers of low molecular weight drugs. Thiol derivative of vanomycin conjugated to gold NPs, exhibit excellent antibiotic activity against vancomycin-resistant enterococci and E. coli within 1 min of near-infrared irradiation.68 Other examples include anticancer drug cisplatin, and insulin for treatment of diabetes mellitus.69

Au/Ni nanorods (200 nm length), were synthesized for gene transfer by electrochemical deposition of gold nanoparticles into porous channels of an Al2O3 membrane. The gold and nickel segments were selectively reacted with either thiol- or carboxyl-containing molecules for ligand (transferring) attachment. Gold nanorods can be excited by ultrafast laser-induced heating, and selectively release two different oligonucleotides from the nanorod surface by irradiation at the nanorods longitudinal surface plasmon resonance.71 Gold NPs intrinsically have DNA-delivering capacity, being electrostatically attached to the surface. Hetero-bifunctional molecules, such as 2-aminoethanethiol,72 N,N,N-trimethyl(11-mercaptoundecyl) ammonium,73 or thiol-modified polyethyleneimine,74 could be used to introduce amine groups onto their surface.The transfection efficiencies of the DNA-gold nanoparticle complexes were several fold higher than those of DNA-polyethyleneimine complexes used as standard transfection reagents.73, 74 PEG-SH stabilized DNA complexed particles suggested stable blood circulation and electric pulse controlled local gene delivery.72 Galactose-PEG-thiol nanoparticles led to hepatocyte-targeted DNA delivery in vivo.75 Au-NPs covalently bonded to low molecular weight (6kDa) chitosan can effectively transfer DNA vaccine.76 Intracellular delivery via the HIV-I tat peptide, a cell penetrating peptide,77 and the nuclear delivery of antisense oligonucleotides by gold NPs with targeting peptides and gene splicing oligonucleotides78 demonstrate effective gene therapy possibilities. Colloidal gold NPs act as efficient vectors for the intracellular delivery of DNAzymes that can cleave mRNA such as c-myc-mRNA,79 and for the safe and effective transfer of small interfering RNA (siRNA)80 for nucleic acid based therapeutics. 3.1.2 Silver nanoparticles: Silver products have been used as antimicrobials on wounds, burns and diabetic ulcers.81 Recent studies have revealed the reduction of chronic inflammatory response and reduction of bacterial levels with nanocrystalline silver dressings.16 In addition to the well established methods for

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syntheses of silver NPs, recent methods report capping agents such as poly (vinyl pyrrolidone) (PVP)82 for highly symmetrical Ag nanocubes and polyacrylic acid (PAA) for nanoparticles of < 50nm diameter.17 The low toxicity of Ag NPs to mammalian cells, and the sustained antimicrobial activity, has led to the development of sputtered nano-Ag wound dressings, antimicrobial tubing and related antimicrobial applications in hospitals. The lytic effect of nanocrystalline silver on the lipophilic dimorphic yeast, Malasezzia furfur, a major causative agent of dandruff, has led to the formulation of nano-Ag incorporated antifungal agent.17 This specific formulation is far less toxic than presently available antidandruff formulations. Yakaguchi et al., developed a novel adduct, GX-95, of Ag with nanometer-scale particles to peptidic hydrolysates from collagen. Strong and broad antifungal activity of the adduct against pathogenic yeasts and filamentous fungi (Cryptococcus neoformans, Candida albicans, Aspergillus fumigatus, Rrichophyton rubrum and Cladophialophora carionii) was noted.85 3.2 Magnetic nanoparticles

Nanoscale magnetic particles exhibit a variety of unique magnetic phenomena, and have emerged as important materials for medical and biotechnological applications. Besides magnetic separation, magnetic bio-sensing and magnetic resonance imaging (MRI), these nanomaterials have been applied for drug delivery, gene delivery and specifically hyperthermia therapeutics based on the heat they produce in an alternating magnetic field.86, 87 Hyperthermia, or increase in temperature between 42-48oC in the targeted tissue or cell, can modify or inhibit cell specific activities or release drugs in a precisely controlled manner due to thermo-labile covalent bonds.88

Hyperthermia based therapy was introduced from the mid 1970’s and a wide range of in vitro and in vivo applications are currently being developed.88 From the practical point of view magnetic nanoparticles (MNPs) are versatile tools requiring no invasive procedures for in vivo therapies. MNPs move in a preferred direction only if they experience a magnetic field gradient. The magnetic force acting on a particle is proportional to the magnetic flux density, to the volume of the particle, and to the field gradient. In vivo hydrodynamic forces due to blood flow counteract magnetic targeting and tissue retention. The forces due to linear blood flow are in the order of 4mm-5mm/s in capillaries, 10cm/s in the main arteries and 50cm/s in aorta.89 For magnetic iron oxide NPs, the flux densities required at targeted site is in the order of 0.1 to 1.0 T with field gradients ranging from 8T/m to over 100T/m for large arteries. Thus only a minor percentage of particles with around 50nm diameter can be trapped using rare-earth permanent magnets at the low flow rates of capillaries.90 The accuracy of magnetic targeting is also dependent on the depth of the target tissue in the body. The magnetic flux density and field gradients decrease rapidly with increasing distance from a magnetic pole. The lung and liver are harder to target than organs closer to the surface or extremities. Presence of large bones also

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hinders exposure to the appropriate magnetic gradient. Hence at present magnetic drug targeting is limited to superficial or surgically accessible areas of an organism.90 From the point of view of effective in vivo utilization of MNPs, particles exhibiting a stronger magnetic flux in external magnetic fields are desirable. The primary challenges are producing a focused field of sufficient magnitude and gradient, with the fabrication of NPs with sufficiently high moment. Magnetic metallic NPs of cobalt, iron and nickel achieve this aim, but further research has shown that these particles cannot be used directly since they are oxidized easily and release 2+ and 3+ charged metal ions that can exert toxic effects. Iron ions produce and catalyze oxygen radical formation, while cobalt and nickel induce adverse tissue reaction.91 In contrast to pure metallic magnetic particles, iron oxides and superparamagnetic iron oxide nanoparticles (SPION) coated and stabilized with hydrophilic polymers are stable under physiological conditions and are non-toxic. 91 Several alloy, core-shell and composite nanostructures of FeC, CoSiO2, FePt, CoPt, CoFe2O4, MnFe2O4 and FePt / Fe3O4 have been developed. Although FeC NPs have been used for in vivo applications, the biocompatible and biodegradable aspect of magnetite NPs has led to their extensive studies and clinical applications.86 The ferro-, ferri- or superparamagnetic coated iron oxide particles ensure colloidal stability, enhanced biocompatibility, phagocyte resistance, increased circulation time in body, functionalization ability and appropriate diagnostic properties. The equilibrium is influenced by van der Waals, electrostatic, steric and magnetic forces, as well as by Brownian motion. Thus the term ferrofluid can be used to describe a colloidal stable suspension of single domain NPs. Silica and gold coatings can enhance chemical stability and provide easy functionalization. Polysaccharide coatings such as dextran, starch and chitosan are biocompatible and offer a range of functionalization options.92, 93 The treatment of choice for superficial tumors and cancerous growths is hyperthermia based damage to the cells and tissues. MNPs can be guided by a strong magnetic field to the required site of therapy and exposed to an alternating magnetic field. Hyperthermia affects the activity of regulatory proteins, kinases and cyclins, which alters cell growth and differentiation, and can induce apoptosis.88 It can also enhance radiation and chemotherapy injury to tumor cells. In contrast, during thermal ablation as by noble metal nanoparticles, higher temperatures (>50oC) are generated. This can lead to necrosis and carbonization, thus need for radiation and chemotherapy can be eliminated. Thermal ablation is required for difficult to treat tumors and body areas far away from vital organs such as breast cancers; 88 for this purpose the superparamagnetic magnetite (Fe3O4) and maghemite ( -Fe2O3) have been extensively studied. The chemical synthesis method frequently used is Massart’s aqueous coprecipitation method, which leads to particles easily dispersible in water; 94 the size range can be tuned between 3-30 nm. Many methods for synthesis of magnetite and maghemite are available in literature, which have been extensively reviewed by Tartaj et al.95 The use of iron oxide hyperthermia was first proposed by Gilchrist et al., 96 who published a seminal paper in 1956 on the selective inductive heating of lymph nodes following introduction of maghemite particles sized between 20-100nm

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diameter. Once introduced into tissue the MNPs can be applied for multiple hyperthermia treatments. Subsequent studies involved embolization of tumor blood capillaries followed by hyperthermia.97 Investigations of magnetic micro- and nnanoparticles for targeted drug delivery began 30 years ago. Since then major progress has been made in particle design and synthesis techniques for in vivo targeting of therapeutic compounds. A variety of animal studies have demonstrated the efficacy of the technique, however only few phase I/II clinical trials have taken place. With recent advancements in the development of novel MNPs, there are ample possibilities of the technology progressing from the laboratory to the clinic.98, 99 The first generation MNPs used for drug and gene delivery clinical trials were nonspecific, wherein targeting was entirely based on external magnetic flux. The newer generation MNPs is being targeted to specific cell types and molecular targets via affinity ligands. These ligands are based on antibodies, aptamers, peptides from phage or small molecule screens.100, 101.Functional nano-magnetic particles (diam. 3nm) have been silanized with (3-aminoprpyl) triethoxysilane for subsequent covalent linking of amino groups with pharmaceuticals and biomolecules.102 Methotrexate immobilized on iron oxide NP surface via a poly (ethylene glycol) self assembled monolayer along with targeting ligand, chlorotoxin, could be successfully used for inducing cytotoxicity to 9L glioma cells.103, 104 Magnetofection, the magnetically enhanced delivery of nucleic acids associated with MNPs was first described in 2000. Following the initiation of this novel method of transfection, also known as MATra (magnetically assisted transfection), several studies have reported using either nucleic acid linked MNPs, or tagged NPs associated with cationic polymers or cationic lipid enhancers.105 NPs associated with siRNAs are suitable for siRNA delivery to cultured cells. Since intracellular delivery is still a limiting factor for transfection leading to temporary gene silencing based on inhibitory RNA/ siRNA or DNA based therapies, the present focus is on combining superparamagnetic NPs with magnetic forces to increase, direct and optimize intracellular delivery.106 The primary potential of this method lies in the extraordinarily rapid and efficient transfection at low vector doses, and the possibility of remotely controlled vector targeting in vivo.107 Airway epithelial cell transfection was carried out in vitro on permanent (16HBE 146) and primary airway epithelial cells, and ex vivo using native porcine airway epithelium.108 The very short incubation time in the ex vivo airway epithelium overcomes the fundamental limitations of therapy.108 A group of novel biodegradable polylactide MNP was synthesized by a modified emulsification-solvent evaporation methodology. MNP surface was modified using oleic acid and polyethyleneimine (PEI) oleate ion-pair for DNA binding. The in vitro expression in cultured arterial smooth muscle cells was established using Green fluorescent protein (GFP) reporter gene and cell growth inhibition after adiponectin plasmid transfection. Larger MNP (375nm) escaped lysosomal uptake and released DNA in perinuclear zone, leading to higher transfection.109 Surface coating with protamine sulfate can enhance association with plasmid DNA and cell surface, leading to high magnetic seeding percentage.110 The development of layered gold, semiconductor nanocrystals and MNP was carried out for use in multiple biological

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systems with focus on the eye. Biotin tagged oligonucleotide was conjugated to streptavidin coated NPs. The layered MNPs were tested on human hepatoma derived Huh-7 cells and retinal endothelial cells along with toxicity evaluation The study revealed easy uptake of MNPs by endothelial cells, thus neovasculature of proliferative retinopathies could be easily targeted. This technology has the potential for multiple uses in gene and drug delivery due to the presence of multilayer and the innocuous nature of the NPs.111 Magnetic nanocomposite particles were synthesized, such as amine-modified gold nanorod, decorated with multiple "pearls" of Fe3O4

NPs.112 This composite NP could be used for simultaneous targeting, dual-mode imaging, and photothermal ablation, or for targeting and controlled drug delivery,113 Nanosized magnetite was encapsulated in acrylate-based cationic co-polymer using the water replacement method. Negatively charged model drug aspirin loaded on the MNPs encapsulated cationic co-polymer displayed bi-phasic release (burst followed by prolonged slow release).113 Magnetic nanoparticle internalization by red blood cells (RBCs) was investigated as a possible application of nanomagnetism in diagnosis and therapy of RBC-related diseases. The internalization of surface-coated maghemite NPs was dependent on the hydrodynamic radii and particle concentration.114 Silica and iron oxide containing composite nanotubes synthesized by template synthesis method, could have important implications in biodistribution, subcellular trafficking and drug release.115

3.3 Inorganic Phosphate nanoparticles

Nanoparticles of the salts of divalent cations (Ca2+ and Mg2+) have diverse applications in medicine. These cations are involved in several cellular and sub-cellular functions, whereas the nanocrystals of carbonated hydroxyapatite (HaP) partake in the formation of the bony and cartilaginous tissues in the body. Diverse application possibilities have been demonstrated using calcium phosphate NPs (CaP NPs) and carbonated hydroxyapatite (HaP) following extensive investigations in the past eight years.3, 11 Gene delivery vectors, nanoscaffolds for tissue engineering and bone grafting have taken on the focus of the investigations, followed by drug delivery and therapeutic prospects. One of the early methods of gene transfer in cultured cells involved coprecipitating DNA with calcium phosphate, enabling effective in vitro transfection by way of endocytic pathway and Ca2+ion mediated endosomal release.11 This co-precipitation method has been used for over 30 years and has proven advantageous over other transfection species such as viruses and dendrimers in terms of superior biocompatibility and reduced immune response.116 But, due to large precipitate size and polydispersity and the extremely low transfection (10-15%) compared to viral vectors, in vivo application of these particles is not possible. Successful in vivo application is based on synthesizing virus-like particles (<100nm), with efficient DNA compaction and well dispersed uniform sized carriers.11 With the prospect of using CaP NPs as safe and cost effective non viral vectors, the aqueous core of water-in-oil microemulsions as

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nanoreactors for the formation of pDNA entrapped CaP NPs have been used117. Highly monodispersed NPs of narrow size distribution with upto 20% w/w pSV Gal could be obtained. These nanoparticles with polyacrylic acid or chitosan as capping agents can be stored easily as lyophilized powder and readily redispersed in aqueous buffer.117 Very high transfection efficiency has been demonstrated in HeLa cell line (more than 100% compared to polyfect) and DU145 cell line (1000 times higher compared to liposomal system).11 Using carbonated apatite nanocrystals upto 100-fold transgene expression in mammalian cells has been defined.118 By the incorporation of carbonate, fluoride or strontium the transfection activity could be dramatically controlled as it helps in generating nanosized particles as well as leads to endosome destabilization.118 Controlled stoichiometry (Ca/P ratio 100-300) and regulated mixing of the two precursors, makes it possible to obtain particles between 25-50nm with a two-fold increase of transfection efficiency in HeLa and MC3T3 –E1 cell lines.119 Effective delivery of CAP NPs with adsorbed pDNA was observed in an osteoblastic cell line and a fibroblastic cell line relative to commercial dendrimer vector.120. The transfection efficiency of multishell nanostructures incorporating EGFP-encoding DNA in T-HUVEC, HeLa, and LTK cell lines, was significantly higher than that of simple DNA-coated calcium phosphate NPs.121 DNA incorporation into the particle, with an outermost layer of DNA to give colloidal stability, enhanced the transfection. Despite the successful transfection noted in vitro, possibility of in vivo delivery is negated due to rapid degradation of surface adsorbed pDNA.122 Several in vivo studies related to targeted DNA delivery and applications have been reviewed by Maitra, using CaP NPs synthesized in microemulsion.11 Early studies had demonstrated the in vivo targeting of surface modified and ligand tagged CAP - NPs to liver parenchymal cells.122 Gene therapy using these non viral vectors has been successfully demonstrated in experimental autoimmune hepatitis. The plasmid expression vector pUMVC3-mIL2 encapsulated in the NPs effectively controlled the disease condition by way of reduction of antinuclear antibodies and reduced IgG level, to parenchymal cell surface antigen. Marked regression of inflammatory condition of mouse liver was also revealed by histopathological study.11 Recent investigations by Bhakta et al. have indicated the possible application of these NPs as adjuvant in DNA vaccination. A ten-fold increase in antibody (IgG 2a) along with lymphocyte proliferation and immunologic synapse formation was manifested in preliminary studies using pSV Gal plasmid expression vector.11 These authors have also demonstrated efficient in vitro gene delivery using magnesium phosphate and manganese phosphate NPs,123 as well as, in vivo and tissue targeted gene expression using magnesium phosphate NPs.124 Tissue regeneration and bone morphogenesis is enhanced using HaP NPs. These NPs have recently demonstrated the induction of bone morphogenetic protein (BmP2) in rat dental pulp stem cells and during odontogenic differentiation.125 pDNA-CaP co-precipitates (nanosized particles, 50-200nm) and porous collagen spheres were synthesized for possible application to regenerate bones and other tissues.126 Porous microspheres are also being considered as promising candidates for bone defect fillers, drug carriers and scaffolds for bone tissue

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regeneration. HaP microspheres (HA-1 and HA-2) were prepared using the chemical precipitation synthesis with H3PO4, Ca(OH)2 and a surfactant, SDS (sodium dodecyl sulfate). The HA powders were dispersed in a sodium alginate solution, and spherical particles were obtained by droplet extrusion coupled with ionotropic gelation in the presence of Ca2+. Subsequent sintering produced HA-1 and HA-2 microspheres with uniform size and interconnected microporosity. Osteoblastic-like MG-63 cells cultured on HA microspheres surfaces showed good adhesion and proliferation.127 Calcium-titanium-phosphate (CTP) and hydroxyapatite (HAP) powders have also been used for synthesis of the microspheres by sodium alginate method.128 A bio-functional hybrid gene-carrier with the advantage of promoting and facilitating development of stem cell-based therapy in regenerative medicine has been investigated very recently.129 Both naturally occurring and genetically engineered cell adhesive proteins, such as fibronectin and E-cadherin-Fc, were incorporated along with DNA into the growing nanocrystals of carbonated apatite. High affinity interactions with fibronectin-specific integrins and E-cadherin in embryonic stem cell surface accelerated transgene delivery for expression. Activation of protein kinase C (PKC) dramatically enhanced transgene expression probably by up-regulating both integrin and E-cadherin.129 Consideration of CaP particulates for radiotherapy and drug delivery has shown significant promise. -emitting radionuclides, 177Lu [T1/2 6.73d, E max 0.49 MeV, E 208 keV (11%)] could be envisaged as successful modalities for the treatment of primary and metastatic liver cancer. Hydroxyapatite (HA) particles of 20-60 micron size range having excellent biocompatibility and ease of labeling with lanthanides, could be retained in the liver ( 73%) after 14 days post arterial administration.130 Also (166)Ho-HA exhibited promising features as an agent for liver cancer therapy in preliminary studies that warrants further investigation.131 NanoCaP conjugated with cis-diamminedichloroplatinum (CDDP, cisplatin), showed a sustained release of CDDP from the nanoconjugates over time132 indicating an useful therapeutic formulation for solid tumors. A bone-specific drug delivery with bisphosphonates (BPs), specifically alendronate, chemically conjugated to hydroxyapatite could be an effective means to impart fine-tuned bioactivity to the pharmaceutical. Horse heart myoglobin (Mb), adsorbed onto biomimetic hydroxyapatite nanocrystals (nHA) and nHA/alendronate conjugate powdered samples, have potential use in bone implantation and as prospective drug-delivery device.133

3.4 Silica nanoparticles

Porous, hollow and solid silica particulates have carrier properties suitable for drug and gene delivery as well as enzyme therapeutics. The inert nature of the matrix, high biocompatibility and transparency, augments its use for in vivo applications.134 Entrapment of fluorescent dyes and photosensitive molecular entities, provides multifunctionality to the carrier.135 -137. The prospect of photodynamic therapy (PDT) along with fluorescent tracking in vivo is an exciting proposition

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for non invasive therapy.136 Inorganic silica nanomaterials have been synthesized using various biomimetic routes.133 Silica NPs can be synthesized by biomimetic methods,135 in microemulsion,132 and sol-gel processing.123 Highly ordered nanostructured surfaces were obtained with silica filled di-block polymeric micelles.138 Organically modified silica NPs (ORMOSIL), synthesized in surfactant micelles from organosilane precursors, were used for photodynamic therapy135 and gene delivery to the brain.139, 140 The NPs with appropriate surface functional groups can be used for delivery and targeting of surface attached drugs and genes.Silica NPs have been synthesized in the aqueous core of reverse microemulsion with entrapped enzymes such as Horse radish peroxidase (HRP), Asparaginase and Glucose oxidase.141, 142 These nanocarriers can be used for enzyme therapeutics and other biotechnological applications. The entrapped enzymes are not only protected from the environment, they can withstand relatively higher temperature and pH conditions and have also demonstrated enhanced activity.Inorganic hollow nanoparticles and nanotubes have attracted great interest in nanomedicine because of the generic transporting ability of porous material.115 In addition, inorganic porous nanomaterials are fundamentally advantageous for developing multifunctional nanomaterials due to their distinctive inner and outer surfaces.143 Hollow silica NPs have been synthesized in microemulsion by interfacial hydrolysis and polymerization of Tetra ethylorthosilicate (TEOS).144, 145 These particles can be used for enzyme or dye entrapment. Using alumina templates, both nanotubes(NT) and nanotestubes (NTT), open on only one end, have been synthesized from many different materials.146 These have great potential as drug delivery vehicles for biomedical applications. The tunable alumina template allows one to dictate both pore diameter and length. The nanotubes can be differentially functionalized on their inner and outer surfaces. “Corked nanotesttubes (NTT) can be designed by covalent capping to prevent premature payload leakage.146, 147 Breast cancer cell targeting was carried out using antibody functionalized on the outer surface of silica NTT. Fluorophore attached to the inner surface made possible the determination of the extent of NTT attachment to the cells; 148 this demonstrates targeted cell type specific drug delivery using silica NTT. Biomimetic synthesis of mesoporous silica matrices and nanospheres can permit the entrapment of organic molecules for protection in adverse environments such as the gastrointestinal tract.149, 150 Surface functionalized mesoporous silica NPs (MSN) can act as efficient drug delivery carriers for animal and plant cells, as they exhibit cell penetrating property. Thus, they can be envisaged as having a great potential for intracellular controlled release of drugs, genes and other therapeutic agents.151 Presently, the mesoporous silica NPs are being designed to act simultaneously as drug delivery, MRI, fluorescence imaging, magnetic manipulator and cell targeting agent.152, 153 These NPs serving multifarious roles may be considered as the new generation of nanodevices for advanced, integrated, therapeutic capabilities.

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4. Conclusion

The tremendous advancement of the application of inorganic nanoparticles as targeted drug delivery carriers has reached remarkable milestones parallel to liposomal and organic carriers in nanomedicine. Its success is due to low toxicity of many inorganic compounds, soft means of preparation, easy surface modification to render multifunctionality, dispersability in aqueous systems, long-term preservation without any microbial attack, inertness in in vivo systems, particularly particles made of noble metals, and many other useful physical and chemical properties such as surface plasmon resonance, superparamagnetism, fluorescence properties etc. Inorganic nanoparticles can be prepared ultra-small in size, down to less than 10nm diameter. The surface plasmon resonance properties of gold nanoparticles have been successfully used in the detection of diseased cells, treatment of tumors using NIR radiation, enzyme therapy by completely protecting the enzyme in hollow gold nanoparticles, preventing enzyme degradation and immunological reactions, and enhancing shape dependent biochemical reactions. Extensive use of silver nanoparticles against the microbes, fungus and even viruses has been reported. Superparamagnetic iron oxide nanoparticles (SPIONS) with multifunctional ability are now being used for magnetically directed drug and gene delivery. The use of calcium phosphate nanoparticles as gene delivery vector has become popular due to their ability to escape from endosome/lysosome, unusual stability of compacted calcium-DNA complex against DNAse degradation and assisting enhanced nuclear uptake of therapeutic DNA. The robustness and mesoporous character of silica nanoparticles including organically modified silica nanoparticles (ORMOSIL) have attracted the attention of many drug and gene delivery scientists. It appears that inorganic nanoparticles have tremendous potentiality in targeted delivery of drugs and genes.

5. References

[1] H.K. Sajja, M.P.East, H.Mao, Y.A.Wang, S.Nie, L.Yang, “Development of multi-functional nanoparticles for targeted drug delivery and noninvasive imaging of therapeutic effect,” Curr.Drug Discov.Technol., 6(1), 43-51 (2009).

[2] P. Debbage, “Targeted drugs and nanomedicine: present and future,” Curr. Pharm. Des., 15(2), 153-172 (2009).

[3] S. Bisht and A.N. Maitra, Inorganic nanoparticles as non-viral vectors for gene delivery, Handbook of Materials for Nanomedicine, Vladimir Torchilin and Mansoor M. Amiji, Pan Stanford Publishing Pte Ltd.:2009.

[4] M.S Muthu, S. Singh, “Targeted nanomedicines: effective treatment cancer, AIDS and brain disorder,” Nanomed., 4(1), 105-118 (2009).

15

[5] T.Murakami, K.Tsuchida, “Recent Advances in Inorganic Nanoparticle-Based Drug Delivery Systems,” Mini-Reviews in Medicinal Chemistry, 8, 175-183 (2008).

[6] X. Huang, P.K.Jain, I.H. El-Sayed, and M.A. El-Sayed, “Gold nanoparticles: interesting optical properties and recent applications in cancer diagnostics and therapy,” Nanomedicine, 2(5), 681-693, (2007).

[7] P.K.Jain, X.Huang, I.H.El-Sayed, M.A.El-Sayed, “Noble metals on the Nanoscale: Optical and Photothermal Properties and Some Applications in Imaging, Sensing, Biology and Medicine,”Acc. Chem. Res., 41 (12), 1578–1586 (2008).

[8] Y.W.Jun, J.W.Seo, J.Cheong, “Nanoscaling laws of magnetic nanoparticles and their applicabilities in biomedical sciences,” Acc Chem Res, 41(2) 179-189 (2008).

[9] A.Hernando, P.Crespo, and M.A.Garcia, “Metallic magnetic Nanoparticles,” Sci. World J., 5, 972–1001 (2005).

[10] H. Maeda, K.Greish, J.Fang, “The EPR Effect and Polymeric Drugs: A paradigm shift for cancer chemotherapy in the 21st Century,” Adv. Polym. Sci., 193, 103-121, (2006).

[11] A.N.Maitra “Calcium Phosphate Nanoparticles: second generation nonviral vectors in gene therapy, “Expert Rev. Mol. Diagn., 5(6), 893-905 (2005).

[12] M. Everts, “Thermal scalpel to target cancer,”Expert Rev.Med. Devices, 4(2), 131-136 (2007). [13] H. Liao, C.L. Nehl, J.H. Hafner, “Biomedical applications of plasmon resonant metal

nanoparticles,” Nanomed. 1(2), 201-208 (2006). [14] R.A. Sperling, P.Gil Rivera, F.Zhang, M.Zanella, W.J. Parak, “Biological applications of

gold nanoparticles,” Chem.Soc.Rev., 37(9), 1896-1908 (2008). [15] R. Cao, R.Villalonga, A. Fragoso, “Towards nanomedicine with a supramolecular

approach: a review,” IEE. Proc. Nanobiotechnol. 152(5), 159-164 (2005). [16] R.G. Sibbald, J. Contreras- Ruiz, P. Coutts, M. Fierheller, A. Rothman, K. Woo,

“Bacteriology, inflammation, and healing: a study of nanocrystalline silver dressings in chronic venous leg ulcers,” Adv. Skin Wound Care, 20(10),549-558 (2007).

[17] Md. Shamim, Chemical and biological studies with metal nanoparticles, PhD Thesis, Delhi University, India: May 2008.

[18] M.Rai, A.Yadav, A.Gade, “Silver nanoparticles as a new generation of antimicrobials,” Biotechnol. Adv., 27(1), 76-83 (2009).

[19] T. Jennings, G. Strouse, “Past, present and future of gold nanoparticles,” Adv. Exp. Med. Biol., 620, 34-47 (2007).

[20] E.S. Papazoglou and A. Parthasarathy, BioNanotechnology, Morgan and Claypool (Publisher), Connecticut: 2007.

[21] M-C. Daniel and D. Astruc, “Gold nanoparticles: Assembly, supramolecular chemistry, quantum-size-related properties, and applications towards biology, catalysis and nanotechnology,” Chem. Rev., 104, 293-346 (2004).

[22] C.Cao, S.Park, S.J. Sim, “.Seedless synthesis of octahedral gold nanoparticles in condensed surfactant phase,” J. Colloid Interface Sci., 322(1), 152-157(2008).

16

[23] W. Stöber, A. Fink, and E. Bohn, “Controlled growth of monodisperse silica spheres in the micron size range,” J. Coll. Interf. Sci., 26, 62–69 (1968). [24] T. Pham, J. B. Jackson, N. J. Halas, and T. R. Lee, “Preparation and characterization of

gold nanoshells coated with self-assembled monolayers,” Langmuir, 18(12), 4915–4920 (2002).

[25] L. Pei, K. Mori, M. Adachi, “Formation process of two-dimensional networked gold nanowires by citrate reduction of AuCl4 and shape stabilization,” Langmuir, 20(18), 7837–7843 (2004).

[26] Y Khalavka, J. Becker, C. Sönnichsen,“Synthesis of rod-shaped gold nanorattles with improved plasmon sensitivity and catalytic activity,” J Am.Chem.Soc., 131(5), 1871-1875 (2009).

[27] R.Kumar, A.N.Maitra, P.K. Patanjali, P.Sharma, “Hollow gold nanoparticles encapsulating horseradish peroxidase,” Biomaterials, 26(33), 6743-6753 (2005).

[28] J. Chen, F. Saeki, B. J. Wiley, H. Cang, M. J. Cobb, Z-Y. Li, L. Au, H. Zhang, M. B. Kimmey, X. Li, and Y. Xia, “Gold Nanocages: Bioconjugationand their potential use as optical imaging contrast agents,” Nano Lett., 5(3), 473–477 (2005).

[29] X.Lu, L.Au, J. McLellan, Z.Y.Li, M.Marquez, Y.Zia, “Fabrication of cubic nanocages and nanoframes by dealloying Au/Ag alloy nanoboxes with an aqueous etchant based on Fe(NO3) 3 or NH4OH,” Nano. Lett., 7(6), 1764-1769 (2007).

[30] S. Liu, Z. Zhang, M. Han, “Gram-scale synthesis and biofunctionalization of silica-coated silver nanoparticles for fast colorimetric DNA detection,” Anal. Chem., 77, 2595-2600 (2005).

[31] S.K. Bajpai, Y.M. Mohan, M. Bajpai, R. Tankhiwale, V. Thomas, “Synthesis of polymer stabilized silver and gold nanostructures,” J. Nanosci. Nanotechnol, 7(9), 2994-3010

(2007). [32] S.S. Shankar, A. Ahmad, M. Sastry, “ Geranium leaf assisted biosynthesis of silver nanoparticles,” Biotechnol. Prog., 19(6), 1627-1631(2003). [33] B.Ankamwar, C.Damle, A.Ahmed, M.Sastry, “Biosynthesis of gold and silver

nanoparticles using Emblica Officinalis fruit extract, their phase transfer and transmetallation in an organic solution,” J.Nanosci. Nanotechnol.,5(10),1665-1671 (2005).

[34] M. Sastry, A. Ahmad, M.I. Khan, R. Kumar, “Biosynthesis of metal nanoparticles using fungi and actinomycete,” Current Science, 85(2),162-170 (2003).

[35] G. Han, P. Ghosh, V.M. Rotello, “Multi-functional gold nanoparticles for drug delivery,” Adv. Exp. Med. Biol., 620, 48-56 (2007).

[36] P.Ghosh, G.Han, M.De, C.K.Kim, V.M.Rotello, “Gold nanoparticles in delivery applications,” Adv.Drug.Deliv.Rev., 60(11), 1307-1315 (2008).

17

[37] Z.Krpeti, P.Nativo, F. Porta, M. Brust, “A Multidentate Peptide for Stabilization and Facile Bioconjugation of Gold Nanoparticles,” Bioconjugate Chem., 20 (3), 619–624(2009).

[38] B. Pan, D.Cui, C.Ozkan, P. Xu, T.Huang, Q.Li, H.Chen, F.Liu, F.Gao, R.He, “DNA-Templated Ordered Array of Gold Nanorods in One and Two Dimensions,”J. Phys. Chem. C, 111 (34), 12572–12576 (2007).

[39] G.S. Terentyuk, G.N. Maslyakova, L.V. Suleymanova, N.G. Khlebtsov, B.N. Khlebtsov, G.G. Akchurin, I.L. Maksimova, V.V. Tuchin, “Laser-induced tissue hyperthermia mediated by gold nanoparticles: toward cancer phototherapy,” J. Biomed. Opt., 14(2), 021016 (2009).

[40] D. Lapotko, “Therapy with gold nanoparticles and lasers: what really kills the cells?” Nanomed., 4(3), 253-256 (2009).

[41] D. P. O'Neal, L. R. Hirsch, N. J. Halas, J. D. Payne, and J. L. West, “Photo-thermal tumor ablation in mice using near infrared-absorbing nanoparticles,” Cancer Lett., 209(2), 171 (2004).

[42] S. Lal, S.E. Clare, N.J.Halas,“Nanoshell-enabled photothermal cancer therapy: impending clinical impact,” Acc.Chem.Res.,41(12), 1842-1851(2008).

[43] L.R. Hirsch, R.J. Stafford, J.A. Bankson, S.R.Sershen, B.Rivera, R.E.Price, J.D. Hazle, N.J.Halas, J.L.West, “Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance,” Proc.Natl.Acad.Sci.USA, 1100(23), 13549-13554 (2003).

[44] J.M. Stern, J.A. Cadeddu, “Emerging use of nanoparticles for the therapeutic ablation of urologic malignancies,” Urol. Oncol., 26(1), 93-96 (2008).

[45] W. Lu, C. Xiong, G. Zhang, Q. Huang, R. Zhang, J.Z. Zhang, C. Li,“Targeted photothermal ablation of murine melanomas with melanocyte-stimulating hormone analog-conjugated hollow gold nanospheres,” Clin.Cancer Res.,15(3),876-886 (2009).

[46] X. Huang, P.K. Jain, I.H. El-Sayed, M.A. El-Sayed, “plasmonic photothermal therapy (PPTT) using gold nanoparticles,” Lasers med. Sci., 23(3), 217-228 (2008).

[47] Y. Zhang, K. Aslan, M.J. Previte, C.D. Geddes, “ Plasmonic engineering of singlet oxygen generation,” Proc. Natl. Acad. Sci USA., 105(6), 1798-1802 (2008). [48] D.K. Chatterjee, L.S. Fong, Y. Zhang, “Nanoparticles in photodynamic therapy: an

emerging paradigm,” Adv. Drug. Deliv. Rev., 60(15), 1627-1637 (2008). [49] Y. C. Cheng, A. Samia, J.D. Meyers, I. Panagopoulos, B.Fei, C. Burda, “Highly efficient

drug delivery with gold nanoparticle vectors for in vivo photodynamic therapy of cancer,” J. Am. Chen. Soc., 130(32), 10643-7 (2008).

[50] J.L. Li, L. Wang, X.Y.Liu, Z.P. Zhang, H.C.Guo, W.M. Liu, S.H. Tang,“In vitro cancer cell imaging and therapy using transferrin-conjugated gold nanoparticles,” Cancer Lett., 274(2), 319-326 (2009).

[51] J.F. Hainfeld, F.A. Dilmanian, D.N. Slatkin, H.M. Smilowitz, “Radiotherapy enhancement with gold nanoparticles,” J. Pharm. Pharmacol., 60(8), 977-985 (2008).

18

[52] W.N. Rahman, N. Bishara, T. Ackerly, C.F. He, P. Jackson, C. Wong, R. Davidson, M.Geso, “Enhancement of radiation effects by gold nanoparticles for superficial radiation therapy,” Nanomedicine, 5(2), 136-142 (2009).

[53] J.F. Hainfeld, D.Slatkin, and H.M. Smilowitz, “The use of gold nanoparticles to enhance radiotherapy in mice,” Phys. Med. Biol., 49(18), 309–315, (2004).

[54] T.Kong, J.Zeng, X.Wang, X.Yang, J.Yang, S.McQuarrie, A.McEwan, W.Roa, J. Chen, J.Z.Xing,“Enhancement of radiation cytotoxicity in breast-cancer cells by localized attachment of gold nanoparticles,” Small, 4(9):1537-1543 (2008).

[55] L.P. Balogh, S.S. Nigavekar, A.C. Cook, L. Minc, M.K.Khan, “Development of dendrimer–gold radioactive nanocomposites to treat cancer microvasculature,” Pharma.Chem., 2(4), 94–99, (2003).

[56] A. Bielinska, J.D.Eichman, I.Lee, J.R. Baker Jr., and L.Balogh, “Imaging {Au0-PAMAM}Gold dendrimer Nanocomposites in Cells,” J Nanoparticle Res., 4 (5), 395–403, (2002).

[57] J.Cardinal, J.R.Klune, E.Chory, G.Jeyabalan, J.S.Kanzius, M.Nalesnik, D.A.Geller, “Noninvasive radiofrequency ablation of cancer targeted by gold nanoparticles,” Surgery.144(2), 125-132 (2008).

[58] S.A. Curley, P. Cherukuri, K. Briggs, C.R. Patra, M. Upton, E. Dolson, P. Mukherjee, “Noninvasive radiofrequency field-induced hyperthermic cytotoxicity in human cancer cells using cetuximab-targeted gold nanoparticles,” J.Exp.Ther.Oncol., 7(4), 313-326 (2008).

[59] S.A.Waldman, P. Fortina, S. Surrey, T. Hyslop, L.J. Kricka, D.J.Graves, Opportunities for near-infrared thermal ablation of colorectal metastases by guanylyl cyclase C-targeted gold nanoshells,” Future Oncol.,2(6):705-716(2006). [60] A.M. Gobin, I.L.West, 2008, “EphrinA I-targeted nanoshells for photothermal ablation of

prostate cancer cells,” Int. J. Nanomedicine, 3(3), 351-358 (2008). [61] H. I. El-Sayed, X. Huang, and M. A. El-Sayed, “Selective laser photo-thermal therapy of

epithelial carcinoma using anti-EGFR antibody conjugated gold nanoparticles,” CancerLett., 239(1), 129-135 (2006).

[62] P. Mukherjee, R. Bhattacharya, P. Wang, L. Wang, S. Basu, J. A. Nagy, A. Atala, D. Mukhopadhyay, S. Soker, “Antiangiogenic properties of gold nanoparticles,” Clin. Cancer Res.,11, 3530-3534 (2005).

[63] Y.F. Huang, K. Sefah, S. Bamrungsap, H.T. Chang, W. Tan, “ Selective photothermal therapy for mixed cancer cells using aptamer-conjugated nanorods,”Langmuir, 24(20), 11860-11865 (2008).

[64] S.E.Skrabalak, L.Au, X.Lu, X.Li, Y.Xia, “Gold nanocages for cancer detection and treatment,”Nanomed., 2(5), 657-668 (2007).

[65] C.H. Loo, M-H. Lee, L.R. Hirsch, J.L.West, N.J. Halas, and R.A. Drezek, “Nanoshell bioconjugates for integrated imaging and therapy of cancer” In: Proc. SPIE—Int. Soc. Opt. Eng., Plasmonics in Biology and Medicine, 1–4, p.5327, 2004.

19

[66] P. Kasili, and T. Vo-Dinh, “Liposome-encapsulated gold nanoshells for nanophototherapy induced hyperthermia,” Int. J. Nanotechnol., 2(4), 397-410 (2005).

[67] K.W. Hu, C.C. Huang, J.R. Hwu, W.C. Su, D.B. Shieh, C.S. Yeh, “ A new photothermal therapeutic agent: core-fr ee nanostructured Au x Ag1-x dendrites,” Chemistry, 14(10), 2956-2964 (2008).

[68] H. Gu, P. L. Ho, E. Tong, L. Wang, B. Xu, , “Presenting vancomycin on nanoparticles to enhance antimicrobial activities,” Nano Lett., 3, 1261-1263 (2003).

[69] H.M. Joshi, D.R. Bhumkar, K. Joshi, V. Pokharkar, M. Sastry, “ Gold nanoparticles as carriers for efficient transmucosal insulin delivery,” Langmuir, 22(1), 300-305 (2006). [70] A. K. Salem, P. C. Searson, K. W. Leong, “Multifunctional nanorods for gene delivery,”

Nat. Mater., 2, 668-671 (2003). [71] A. Wijaya, S.B. Schaffer, I.G. Pallares, K. Hamad-Schifferli,“Selective release of multiple

DNA oligonucleotides from gold nanorods,” ACS Nano., 3(1), 80-86 (2009). [72] T. Kawano, M. Yamagata, H. Takahashi, Y. Niidome, S. Yamada, Y. Katayama, T.

Niidome, J. Control Rel., 111, 382 (2006). [73] K. K. Sandhu, C. M. McIntosh, J. M. Simard, S. W. Smith, V. M. Rotello, “Gold

nanoparticle-mediated transfection of mammalian cells,” Bioconjug. Chem.,13, 3-6 (2002).

[74] M. Thomas, A. M. Klibanov, “Conjugation to gold nanoparticles enhances polyethylenimine's delivery of plasmid DNA into mammalian cells,” Proc. Natl. Acad. Sci. USA,100, 9138-9143 (2003).

[75] J.M. Bergen, H.A.von Recum, T.T.Goodman, A.P.Massey, S.H.Pun,“Gold nanoparticles as a versatile platform for optimizing physicochemical parameters for targeted drug delivery,” Macromol. Biosci., 6(7), 506-516 (2006).

[76] X. Zhou, X. Zhang, X.Yu, X. Zha, Q. Fu, B.Liu, X. Wang, Y. Chen, Y, Chen, Y. Shan, Y. Jin, Y. Wu, J.Liu, W. Kong, J. Shen, “The effect of conjugation to gold nanoparticles on the ability of low molecular weight chitosan to transfer DNA vaccine,” Biomaterials, 29(1),111-117 (2008).

[77] C.C.Berry, “Intracellular delivery of nanoparticles via the HIV-1 tat peptide,” Nanomedicine, 3(3), 357-365 (2008).

[78] Y.Liu, S.Franzen, “Factors determining the efficacy of nuclear delivery of antisense oligonucleotides by gold nanoparticles,” Bioconjug. Chem., 19(5), 1009-1016 (2008).

[79] F. Tack, M. Noppe, A.Van Dijck, N. Dekeyzer, B.J. Van Der Leede, A. Bakker, W. Wouters, M. Janicot, M.E. Brewster, “Delivery of a DNAzyme targeting c-myc to HT29 colon carcinoma cells using a gold nanoparticulate approach,” Pharmazie, 63(3), 221-225 (2008).

[80] J.S. Lee, J.J.Green, K.T. Love, J. Sunshine, R. Langer, D.G. Anderson, “Gold, Poly(beta-amino ester) Nanoparticles for Small Interfering RNA Delivery,” Nano. Lett., 2009 May 7. [Epub ahead of print]

20

[81] S. Silver, T. le Phung, G. Silver, “ Silver as biocides in burn and wound dressings and bacterial resistance to silver compounds,” J.Ind.Microbiol. Biotechnol., 33(7), 627-634 (2006).

[82] Y. Sun, Y. Xia, “Shape-Controlled Synthesis of Gold and Silver Nanoparticles,” Science, 298 (5601), 2176- 2179 (2002).

[83] D. Roe, B.Karandikar, N. Bonn-Savage, B. Gibbins, J.B.Roullet, “Antimicrobial surface functionalization of plastic catheters by silver nanoparticles,” J.Antimicrob. Chemother., 61(4), 869-876 (2008).

[84] E. Weir, A. Lawlor, A. Whelan, F. Regan, “The use of nanoparticles in anti-microbial materials and their characterization,” Analyst, 133(7), 835-845 (2008).

[85] T.Yaguchi, K.Takizawa, H.Taguchi, R.Tanaka, T. Kubota, Y. Kubota, M. Kubota, K. Fukushima. “Antifungal activity of the novel adduct, GX-95, of silver with nanometer-scale particles to peptidic hydrolysates from collagen,” Nippon Ishinkin Gakkai Zasshi,. 48(2), 97-100 ( 2007).

[86] A.K.Gupta, R.R.Naregalkar, V.D. Vaidya, M. Gupta, “Recent advances on surface engineering of magnetic iron oxide nanoparticles and their biomedical applications,” Nanomed. 2(1), 23-39 (2007).

[87] Y.W.Jun, J.W.Seo, J.Cheon, “Nanoscaling laws of magnetic nanoparticles and their applicabilities in biomedical sciences,”Acc. Chem. Res., 41(2), 179-189 (2008).

[88] V. Labhasetwar and D.L. Leslie Pelecky, Biomedical Applications of Nanotechnology, John Wiley & Sons Inc(Publishers), New Jersey: 2007.

[89] U.O.Häfeli and M.Chastellain,“Magnetic Nanoparticles as Drug Carriers,” In:V.P.Torchilin (Editor). Nanoparticulates as drug carriers, Publisher, ICP, London, pp. 397-411 (2006).

[90] S.Nagel, “Theoretische und experimentelle Untersuchungen zum Magnetischen Drug Targeting,“ Greifswald: Ernst-Moritz-Arndt-Universit¨at Greifswald; 2004.

[91] D. Sellmyer and R. Skomski, Nanobiomagnets, Advanced magnetic Nanostructures, 2008. [92] J.Lee, Y.Lee, J.K.Youn, H.B. Na, T. Yu, H. Kim, S.M. Lee, Y.M.Koo, J.H.Kwak,

H.G.Park, H.N.Chang, M. Hwang, J.G. Park, J.Kim, T. Hyeon, “Simple synthesis of functionalized superparamagnetic magnetite/silica core/shell nanoparticles and their application as magnetically separable high-performance biocatalysts,” Small, 4(1),143-152 (2008).

[93] A.H. Lu, E.L.Salabas, F.Schüth, “Magnetic nanoparticles: synthesis, protection, functionalization, and application,” Angew.Chem.Int.Ed.Engl., 46(8),1222-1244 (2007).

[94] R. Massart, Magnetic fluids and process of obtaining them. US Patent No. 4,329,241, 1980. [95] P. Tartaj, M. del Puerto Morales, S. Veintenillas-Verdaguer, T. Gonzalez Carreno, C.J.

Serna, “The preparation of magnetic nanoparticles for applications in biomedicina,” Phys D Appl Phys, 36, 182-197 (2003).

[96] R. K. Gilchrist, R. Medal, W. D. Shorey, R. C. Hanselman, J. C. Parrott, and C. B.

21

Taylor, Ann. Surg. 146 (1957). [97] P.Moroz, S.K.Jones, B.N.Gray, Magnetically mediated hyperthermia: Current status and

future directions,” Int. J Hyperthermia, 18, 267-284 (2002). [98] J. Dobson, “Magnetic micro- and nano-particle-based targeting for drug and gene

delivery,”Nanomed, 1(1) 31-37 (2006). [99] S. C. McBain, H. H. Yiu, J. Dobson, “Magnetic nanoparticles for gene and drug

delivery,”Int. J Nanomedicine, 3(2) 169-180 (2008). [100] J.R.McCarthy, R.Weissleder, “Multifunctional magnetic nanoparticles for targeted

imaging and therapy,”Adv. Drug Deliv. Rev, 60 (11) 1241-1251 (2008). [101] X. H. Peng, X. Qian, H. Mao, A. Y. Wang, Z. G. Chen, S. Nie, D. M. Shin, “Targeted

magnetic iron oxide nanoparticles for tumor imaging and therapy,” Int. J Nanomedicine, 3(3) 311-321 (2008).

[102] S. Moritake, S.Taira, Y. Ichiyanagi, N. Morone, Y. S. Song, T. Hatanaka, S. Yuasa, M. Setou, “Functionalized nanomagnetic particles for an in vivo delivery system,” JNanosci. Nanotechnol., 7(3) 937-944 (2007).

[103] N. Kohler, C. Sun, A. Fichtenholtz, J. Gunn, C. Fang, M. Zhang, “Methotrexate-immobilized poly(ethylene glycol) magnetic nanoparticles for MR imaging and drug delivery,” Small, 2(6) 785-792 (2006).

[104] C. Sun, C. Fang, Z. Stephen, O Veiseh, S. Hansen, D. Lee, R. G. Ellenbogen, J. Olson, M. Zhang, Nanomed., 3(4) 495-505 (2008).

[105] O.Mykhaylyk, O. Zelphati, J. Rosenecker, C. Plank, “siRNA delivery by magnetofection,” Curr. Opin. Mol. Ther., 10(5) 493-505 (2008).

[106] J.Bertram, “MATra-Magnetic Assisted Transfection combining nanotechnology and magnetic forces to improve intracellular delivery of nucleic acids,” Curr. Pharm. Biotechnol., 7(4) 277-285 (2006).

[107] C. Plank, U. Schillinger, F. Scherer, C. Bergemann, J. S. Remy, F. Krötz, M. Anton, J. Lausier, J. Rosenecker, “The magnetofection method using magnetic force to enhance gene delivery,” Biol. Chem.,384(5) 737-747 (2003).

[108] S. W. Gersting, U. Schillinger, J. Lausier, P. Nicklaus, C. Rudolph, C. Plank, D.Reinhardt, J. Rosenecker, “Gene delivery to respiratory epithelial cells by magnetofection,” J Gene Med., 6(8) 913-922 (2004).

[109] M. Chorny, B. Polyak, I.S. Alferiev, K.Walsh, G. Friedman, R.J. Levy, “Magnetically driven plasmid DNA delivery with biodegradable polymeric nanoparticles,”FASEB J., 21(10) 2510-2519 (2007).

[110] S. Takeda, B. Terazono, F. Mishima, H. Nakagami, S. Nishijima, Y. Kaneda, “Novel drug delivery system by surface modified magnetic nanoparticles,” J Nanoscience Nanotechnol., 6(9-10) 3269-3276 (2006).

22

[111] T. Prow, J.N. Smith, R. Grebe, J.H. Salazar, N. Wang, N. Kotov, G. Lutty, J. Leary, “Construction, gene delivery, and expression of DNA tethered nanoparticles,” Mol.Vis.,12, 606-615 (2006).

[112] C.Wang, J.Chen, T. Talavage, J.Irudayaraj, “Gold nanorod/Fe3O4 nanoparticle ‘nano-pearl-necklaces’ for simultaneous targeting, dual-mode imaging, and photothermal ablation of cancer cells,” Angew. Chem.Int.Ed.Engl.,48(15), 2759-2763 (2009).

[113] P. Phanapavudhikul, S. Shen, W. K. Ng, R. B. Tan, “Formulation of Fe3O4/acrylate co-polymer nanocomposites as potential drug carriers,” Drug Deliv., 15(3) 177-183 (2008).

[114] M. A. Soler, S. N. Bao, G. B. Alcântara, V.H.Tibúrcio, G.R. Paludo, J.F. Santana, M.H. Guedes, E.C. Lima, Z. G. Lacava, P. C. Morais, “Interaction of erythrocytes with magnetic nanoparticles,” J Nanosci. Nanotechnol., 7(3) 1069-1071 (2007).

[115] S.J. Son, X. Bai, A. Nan, H. Ghandehari, S.B. Lee, “Template synthesis of multifunctional nanotubes for controlled release,” J Control. Release, 114(2) 143-152 (2006).

[116] C.E.Pedraza, D.C.Bassett, M.D.McKee, V.Nelea, U.Gbureck, J.E.Barralet, “The importance of particle size and DNA condensation salt for calcium phosphate nanoparticle transfection,” Biomaterials, 29(23), 3384-3392 (2008).

[117] S.Bisht, G.Bhakta, S.Mitra, A.Maitra, “pDNA loaded calcium phosphate nanoparticles: highly efficient non-viral vector for gene delivery,” Int J Pharm., 288, 157-168 (2005).

[118] E.H.Chowdhury, T.Akaike, “High performance DNA nano-carriers of carbonate apatite: multiple factors in regulation of particle synthesis and transfection efficiency,” Int. J. Nanomedicine, 2(1),101-106 (2007).

[119] D. Olton, J. Li, M. E. Wilson, T .Rogers, J. Close, L. Huang, P. N. Kumta, C. Sfeir, “Nanostructured calcium phosphates (NanoCaPs) for non-viral gene delivery: influence of the synthesis parameters on transfection efficiency,” Biomaterials, 28(6), 1267-1279 (2007).

[120] C.E. Pedraza, D.C. Bassett, M.D. McKee, V. Nelea, U. Gbureck, J. E. Barralet, “The importance of particle size and DNA condensation salt for calcium phosphate, nanoparticle transfection,” Biomaterials, 29(23), 3384-3392 (2008).

[121] V. V. Sokolova, I. Radtke, R . Heumann, M. Epple, Effective transfection of cells with multi-shell calcium phosphate-DNA nanoparticles,” Biomaterials, 27(16), 3147-3153 (2006).

[122] I. Roy, S. Mitra, A. N. Maitra, S. Mozumdar, “Calcium Phosphate Nanoparticles as novel non-viral vectors for targeted gene delivery,” Int. J.Pharm., 250, 25-33 (2003).

[123] G. Bhakta, S. Mitra, A.N. Maitra, “DNA Encapsulated Magnesium and Manganous Phosphate Nanoparticles: Potential Non-Viral Vectors for Gene Delivery,” Biomaterials, 26(14), 2157-2163 (2005).

[124] G. Bhakta, A. Srivastava, A. Maitra, “Magnesium phosphate nanoparticles can be efficiently used in vitro and in vivo as non-viral vectors for targeted gene delivery,”J.Biomed.Nanotech., 5, 106-114 (2009).

23

[125] X.Yang, X.F.Walboomers, J. Van den Dolder, F.Yang, Z.Bian, M.Fan, J.A. Jansen, “Non-viral bone morphogenetic protein-2 transfection of rat dental pulp stem cells using calcium phosphate nanoparticles as carriers,” Tissue Eng. Part A, 14(1), 71-81 ( 2008).

[126] H.Fu, Y.Hu, T. McNelis, J.O.Hollinger, “A calcium phosphate-based gene delivery system,” J.Biomed. Mater. Res. A, 74(1), 40-48 (2005).

[127] A.Y.Mateus, C.C.Barrias, C.Ribeiro, M.P.Ferraz, F.J.Monteiro, “Comparative study of nanohydroxyapatite microspheres for medical applications,” J Biomed. Mater. Res A.,86(2):483-493 (2008).

[128] C.C.Ribeiro, C.C.Barrias, M.A. Barbosa, “Preparation and characterisation of calcium-phosphate porous microspheres with a uniform size for biomedical applications,” J.Mater. Sci. Mater. Med., 17(5), 455-463 (2006).

[129] E.H.Chowdhury, “Self-assembly of DNA and cell-adhesive proteins onto pH-sensitive inorganic crystals for precise and efficient transgene delivery,” Curr. Pharm. Des., 14(22), 2212-2228 (2008).

[130] S.Chakraborty, T. Das, H.D.Sarma, M.Venkatesh, S.Banerjee, “Preparation and preliminary studies on 177Lu-labeled hydroxyapatite particles for possible use in the therapy of liver cancer,” Nucl. Med. Biol., 35(5), 589-597 (2008).

[131] T.Das, S.Chakraborty, H.D.Sarma, M.Venkatesh, S.Banerjee, “(166)Ho-Labeled Hydroxyapatite Particles: A Possible Agent for Liver Cancer Therapy,” Cancer Biother. Radiopharm., 2009 Feb 13. [Epub ahead of print]

[132] X.Cheng, L.Kuhn, “Chemotherapy drug delivery from calcium phosphate nanoparticles,” Int. J. Nanomedicine, 2(4), 667-674 (2007).

[133] M. Iafisco, B. Palazzo, G. Falini, M.D. Foggia, S. Bonora, S. Nicolis, L. Casella, N. Roveri, “Adsorption and conformational change of myoglobin on biomimetic hydroxyapatite nanocrystals functionalized with alendronate,” Langmuir, 24(9), 4924-4930 (2008).

[134] R.K.Sharma, S.Das, A.Maitra, “Surface modified ormosil nanoparticles,” Journal of colloid and interface science., 277(2), 342-346 (2004).

[135] I.Roy, T.Y.Ohulchanskyy, H.E.Pudavar, E.J.Bergey, A.R.Oseroff, J.Morgan, T. J.Dougherty, P.N.Prasad, “Ceramic-Based Nanoparticles Entrapping Water-Insoluble Photosensitizing Anticancer Drugs: A Novel Drug Carrier System for Photodynamic Therapy,”J. Am. Chem. Soc., 125 (26), 7860–7865 (2003).

[136] S.Kim, T.Y.Ohulchanskyy, H.E.Pudavar, R.K.Pandey, P.N.Prasad, “Organically Modified Silica Nanoparticles Co-Encapsulating Photosensitizing Drug and Aggregation-Enhanced Two-Photon Absorbing Fluorescent Dye Aggregates for Two-Photon Photodynamic Therapy,” J Am. Chem Soc.,129(9), 2669-2675 (2007).

[137] T.Y.Ohulchanskyy, I.Roy, L.N.Goswami, Y.Chen, E.J.Bergey, R.K.Pandey, A.R.Oseroff, P.N.Prasad, “Organically Modified Silica Nanoparticles with Covalently Incorporated

24

Photosensitizer for Photodynamic Therapy of Cancer,”Nano Lett., 7 (9), 2835–2842 (2007).

[138] A. Frömsdorf, A.Kornowski, S.Pütter, H.Stillrich, L.T.Lee, “Highly ordered nanostructured surfaces obtained with silica-filled diblock-copolymer micelles as templates,” Small, 3(5), 880-889 (2007).

[139] D.J.Bharali, I.Klejbor, E.K.Stachowiak, P.Dutta, I.Roy, N.Kaur, E.J.Berjey, P.N.Prasad, M.K.Stachowiak, “Organically modified silica nanoparticles: a nonviral vector for in vivo gene delivery and expression in the brain,” Proc. Natl. Acad. Sci. USA, 102, 11539-11544 (2005).

[140] I.Roy, M.K.Stachowiak, E.J.Bergey, “ Nonviral gene transfection nanoparticles: function and applications in the brain,” Nanomedicine, 4(2), 89-97 (2008).

[141] T.K.Jain, I.Roy, T.K.De, A.N.Maitra, “Nanometer Silica Particles Encapsulating Active Compounds: A Novel Ceramic Drug Carrier, ”J. Am. Chem. Soc., 120 (43), 11092–11095 (1998).

[142] R.K.Sharma, S.Das, A.Maitra, “Enzymes in the cavity of hollow silica nanoparticles,” Journal of colloid and interface science, 284(1), 358-361 (2005).

[143] S.J.Son, X.Bai, S.B.Lee, “Inorganic hollow nanoparticles and nanotubes in nanomedicine Part 2: Imaging, diagnostic, and therapeutic applications,” Drug Discov. Today, 12(15-16), 657-663 (2007).

[144] S.Das, T.K.Jain, A.N.Maitra, “Inorganic-organic hybrid nanoparticles from n-octyl triethoxy silane,” Journal of colloid and interface science., 252(1), 82-88 (2002).

[145] R.K.Sharma, S.Das, A.Maitra, “Surface modified ormosil nanoparticles,” Journal of colloid and interface science., 277(2), 342-346 (2004).

[146] H.Hillebrenner, F.Buyukserin, J.D.Stewart, C.R.Martin, “Biofunctionalization and capping of template synthesized nanotubes,” J Nanosci. Nanotechnol., 7(7), 2211-2221 (2007).

[147] H.Hillebrenner, F.Buyukserin, J.D.Stewart, C.R.Martin, “Template synthesized nanotubes for biomedical delivery application,” Nanomed., 1(1), 39-50 (2006).

[148] F. Buyukserin, C.D.Medley, M.O.Mota, K.Kececi, R.R.Rogers, W.Tan, C.R.Martin, “Antibody-functionalized nano test tubes target breast cancer cells,” Nanomed. 3(3), 283-292 (2008).

[149] A.Vinu, M.Miyahara, K.Ariga,. Assemblies of biomaterials in mesoporous media,” JNanosci. Nanotechnol., 6(6):1510-32 (2006).

[150] S.P. Rigby, M.Fairhead, C.F.van der Walle, “ Engineering silica particles as oral drug delivery vehicles,” Curr. Pharm. Des., 14(18), 1821-31(2008).

[151] I.L.Slowing, J.L.Vivero-Escoto, C.W.Wu, V.S.Lin, Mesoporous silica nanoparticles as controlled release drug delivery and gene transfection carriers. Adv. Drug Deliv. Rev., 60(11), 1278-88 (2008).

25

[152] M.Liong, J.Lu, M.Kovochich, T. Xia, S.G.Ruehm, A.E.Nel, F.Tamanoi, J.I.Zink, “Multifunctional inorganic nanoparticles for imaging, targeting, and drug delivery,” ACS Nano., 2(5), 889-896 (2008).

[153] J.M.Rosenholm, A.Meinander, E.Peuhu, R.Niemi, J.E.Eriksson, C.Sahlgren, M.Linden, “Targeting of porous hybrid silica nanoparticles to cancer cells,” ACS Nano., 3(1), 197-206 (2009).