Advanced Biomaterials and Biodevicess

86 Advanced Biomaterials and Biodevices tyrosine kinase inhibitor) [83]. The silk fibroin allows for the protection of the drug from metabolizing enzymes as well as its sustained release. Chitosan-coated liposomes have also been developed [84] and represent a novel approach for ocular drug delivery due to its muco-adhesivity and biocompatibility [85, 86]. Utilizing the layer-by-layer (L-b-L) approach, Fujimoto et al developed poly-lysine and poly-aspartic acid coated lipo- somes for the controlled release of macromolecular drugs [87]. Another example is the use of sugars like hydroxyl pyrene trisulfonic acid, alendro- nate and glucose to prepare L-b-L liposomes [88]. Interestingly, albumin- coated liposomes were shown to significantly reduce association of serum proteins, which could potentially allow for better circulation times [89]. Recently, it was shown that coating of liposome with a combination of PEG and polyvinyl alcohol provides for a better long-circulating function [90]. Similarly a combination of albumin and PEG was used to enhance the effi- cacy of liposomal doxorubicin in tumor-bearing rats [91]. 3.2.3 Stimuli-Sensitive and Triggered Release Liposomes Coating of liposomes with an appropriate polymer allows for an increase in circulation time. We already know that long-circulating liposomes passively accumulate at sites of leaky vasculature like solid tumors and inflamed tissues via the EPR effect. But the accumulation of the liposome at the target site alone may not be sufficient for good therapeutic outcomes. In addition, it has been shown that though coating of the liposome with PEG increases the circulation time, it can reduce the cellular association and subsequent internalization by virtue of its steric hindrance as well as prevent release of the cargo. However, if after target accumulation, this protective coating could be removed under the action of local stimuli (like lowered pH in tumors, upregulated enzymes) or external stimuli (like heat and ultrasound), then the facilitated release of the encapsulated contents should lead to improvements in therapeutic effects. 3.2.3.1 pH-Sensitive Liposomes Such liposomes are engineered mainly for the release of their con- tents in areas of lowered pH such as the tumor microenvironment, late endosomal vehicles etc..Bellavance et al. developed a novel cationic pH-sensitive liposome formulation for the cytosolic delivery of the encapsulated cargo [92]. Coating of liposomes with an acid-labile PEG polymer was recently suggested [93]. Similarly, the use of the acid-labile cholesterol-vinyl ether–PEG conjugate, which could detach from the

Recent Advances with Liposomes as Drug Carriers 87 liposome at lower pH conditions was demonstrated [94], and such sen- sitive polymers have been successfully used with liposomal prepara- tions for the delivery of DNA [95]. In another interesting study, Hiraka et al. developed pH-sensitive liposomes to deliver Fe-porphyrin for its anti-cancer effects [96]. The hypothesis was that Fe-porphyrin would cause cell death by the generation of reactive oxygen species. Non- PEG polymers like polyampholytes [97] and poly(hydroxypropyl) methacrylamides [98] have also been successfully used to take advantage of this pH-triggered effect. Although a majority of pH-sensitive release systems depend on an exogenous change in pH, there exist few examples of release by internal acidification. Recently Wehunt et al. designed a car- rier composed of pH-sensitive lipids, wherein the application of an HCL cotransporter allows for internal acidification and subsequent release of the encapsulated cargo [99]. In another approach, Yao et al. made use of a pH-sensitive peptide incorporated into the liposome with the idea of fus- ing with endosomal membranes at low pH and subsequently delivering the contents into the cell effectively [100]. 3.2.3.2 Enzyme-Sensitive Liposomes Another approach uses liposomal coatings degradable by extra-cellular enzymes [101]. Recently, there has been a lot of interest in the use of matrix metallo-proteinases (MMPs) to trigger the release of liposomal contents [102]. Various MMPs have been implicated in a variety of disease mod- els and thus serve as viable targets for assisted release of nanocarriers. Banerjee et al. demonstrated the use of an MMP-9 cleavable lipopeptide [103]. The cleavage of the lipopeptide compromised the structural integrity of the liposome thus releasing the encapsulated carboxyfluorescein dye. Phospholipase 2-sensitive liposomes were also developed for the delivery of siRNA [104]. Liposomes with a glutathione-reducible PEG coating were also used to effectively deliver DNA to the cells [105]. 3.2.3.3 Ultrasound-Triggered Liposomes For the development of ultrasound-sensitive liposomes, it is imperative that the liposome contains substantial air pockets in addition to its drug cargo, i.e. could be considered liposomal microbubbles [106]. It was hypothesized that the application of external ultrasound at the target site would allow the liposomes accumulated there to break open and release their cargo [107]. Studies have also shown that the liposome-membrane composition greatly affects the sensitivity of liposomes to ultrasound [108]. Echogenic lipo- somes have thus been successfully developed for the delivery of drugs like

88 Advanced Biomaterials and Biodevices doxorubicin [109–111], methylprednisolone succinate [110] and cisplatin [112]. In the field of gene delivery, they have been used for the delivery of DNA [113], RNA [114] and other oligonucleotides [115]. 3.2.3.4 Hyperthermia-Triggered Liposomes Hyperthermia has been used widely in nanocarrier research to increase vascular permeability of the target tissue [116] and to trigger the drug release from thermo-sensitive nanocarriers [117]. This combination strategy allows for the efficient delivery of drugs [118] as well as to help in image-guided therapies [119, 120]. The use of magnetic liposomes to induce cell death by magnetic fluid hyperthermia [121, 122] and combine this with the delivery of cytotoxic molecules was demonstrated recently by Clares et al. [123]. 3.2.3.5 Other Triggered Release Liposomes In another interesting approach, liposomes, which release their cargo in a glucose-sensitive manner were prepared by incorporating a hydrophobi- cally modified glucose oxidase into the membrane [124]. Another recent advance in the field of triggered release is the use of photo-sensitive lipo- somes that can degrade on irradiation with UV light [125] or where the PEG coating can be removed by photoactivation thus allowing for the improved uptake of the liposome [126]. Similarly, in another approach, the permeability of the liposomal membrane was increased by irradiating the membrane-bound photosensitizer with red light [127]. Wu et al. have also demonstrated the use of near infrared-triggerable systems for the con- trolled release of gold nanoparticles [128]. 3.3 Actively Targeted Liposomes Actively targeted liposomes reach the target site the same way as passively targeted ones, i.e. by the EPR effect. The role of the ligands comes into play mainly when liposomes arrive at the target tissue, where they are able to bind and subsequently internalize into the cell (See Figure 3.1 and Figure 3.2). Therefore, it is imperative that they have longer blood circulation times as well as enhanced binding efficiencies. Most of the liposomal examples discussed in this section include a combination of targeting and longev- ity functionalities. Though traditionally these have been thought of as multi-functional, a study of the recent literature suggests that targeting

Recent Advances with Liposomes as Drug Carriers 89 Figure 3.1 Extravasation of Liposome and subsequent binding to cellular receptors. The liposomes extravasate out of systemic circulation and accumulate at the pathological site by the EPR effect. Here the dual-ligand targeted liposome is able to bind to the complimentary receptors on the cell surface. Figure 3.2 Receptor mediated endocytosis of dual-targeted liposome. (1)Liposome specifically binds to membrane-bound receptors (2) Receptor binding initiates the formation of clathrin-coated pits (3) Clathrin-coated pits pinch off of the membrane (4) Liposome is internalized via formation of an endosome and trafficked into the cell.

90 Advanced Biomaterials and Biodevices and longevity are presumed to go hand in hand. A number of approaches have already been established for the incorporation of targeting ligands into liposomes so as to maintain this dual functionality [129, 130]. Initially, ligands were attached directly to the surface of the liposome along with the PEG lipids. But the steric hindrance of PEG seemed to partially block the action of the targeting moiety. To overcome this barrier, a majority of the small molecule ligands are now attached to the distal end of the PEG chain. 3.3.1 Antibody-Targeted Liposomes It has been repeatedly shown that incorporation of antibodies onto the surface of liposomes increases their potency by allowing the multivalent binding to target receptors [131]. Liposomal trastuzumab and rituximab have been proven to have potent activities both in vitro and in vivo [132]. Milatuzumab, a CD74 antagonistic monoclonal antibody (mAb), when incorporated into a liposomal carrier, was shown to be significantly more toxic to chronic lymphocytic leukemia cells [133]. Antibody-targeted liposomes are frequently used for the site-specific delivery of hydropho- bic drugs for the treatment of cancer [134–136]. One of the many advan- tages of using mAbs is the potential for an additive or synergistic effect between the signaling antibody and the encapsulated drug. Another added advantage is that specific antigens expressed in one tumor cell line but not in others can be targeted [137]. Our group has successfully used cancer- specific anti-nucleosome monoclonal antibody 2C5 for tumor delivery of liposomal doxorubicin [138–140] as well as diagnostic and imaging agents [141]. More recently, immunoliposomes targeting specific receptors like the transferrin receptor [142] and growth factor receptors EGFR [143] and Her-2 [144] have been developed. This not only helps the selective deliv- ery of therapeutic cargo but also serves to competitively block off recep- tor access to its natural ligands thereby preventing further tumor growth. Similar preparations have also been successfully used to deliver siRNA to extra-hepatic targets [145]. Immunoliposomes have also found applications for the treatment of dis- eases other than cancer. Recently, anti-P-selectin immunoliposomes were shown to selectively target areas affected by myocardial infarction [146]. Although antibody-mediated targeting of nanocarriers is generally char- acterized by high affinity for the target tissue or organ, continued adminis- tration can lead to adverse reactions in the body. With this in mind, Cheng et al. have used the single chain fragments of the variable region (ScFv) of the anti-CD19 monoclonal antibody to target liposomal doxorubicin towards CD19-expressing cells [147]. Though liposomes with the chain

Recent Advances with Liposomes as Drug Carriers 91 fragment had increased accumulation at the target site when compared to the non-targeted liposomes, it was still 3-fold lesser than the liposomes with the whole antibody. Epithelial cell adhesion molecule-targeted doxo- rubicin immunoliposomes also showed potent anti-tumor activity in a murine xenograft model [148]. Similarly, topotecan immunoliposomes with anti-CD166 ScFv were shown to induce prostate cancer-specific cyto- toxicity [149]. 3.3.2 Single Ligand-Targeted Liposomes 3.3.2.1 Targeting the Folate Receptor Over the past decade, the folate receptor (FR) has been established as a promising target for the delivery of biological actives since it is upregulated in a variety of malignant tumors [150, 151]. Compared to other targets, FR targeting has been shown to reduce drug toxicity due to its location on the apical side of the epithelium rather than the luminal side. Folate-targeted liposomes represent a very popular approach for the delivery of hydropho- bic [152–154] as well as hydrophilic drugs [155, 156]. A similar approach was applied for the delivery of photosensitizers for the photodynamic ther- apy of cancer [157]. In another recent study, FR targeted liposomal doxo- rubicin was combined with FR upregulation using all-trans retinoic acid and the role of the targeting ligand on the circulation time was evaluated [158]. For important reviews see references [159, 160]. 3.3.2.2 Targeting the Transferrin Receptor Another popular approach is the targeting of the transferrin (Tf) recep- tor [161]. Tf receptors present on the surfaces of cells allow for the uptake of iron and have been found to be overexpressed in a broad spectrum of tumors. Targeting these receptors not only allows for the delivery of drug cargo into the cell, but also blocks the natural function of the receptor thus allowing for a dual effect. Tf is also involved in the transport of iron to the brain and has been used as an effective targeting strategy for the penetration of the blood brain barrier [162, 163]. A popular approach for incorpora- tion of the Tf ligand has been to conjugate it to a functionalized PEG chain and subsequently post-insert it into the liposome. An added advantage of using ligands like Tf are that the nanoparticles are endocytosed into the cell and thus by-pass drug efflux pumps like P-gp [164]. In a recent study, Tf-liposomes loaded with C6-ceramide were successfully used to target the lysosome [165]. Tf-liposomes thus represent an effective vehicle for the delivery of anticancer drugs [166], DNA [167–169]and siRNA [170].

92 Advanced Biomaterials and Biodevices 3.3.2.3 Targeting with Sugar Moieties An interesting concept in the targeting of liposomes has been the use of sugar moieties [171]. Recently, the targeting potential of mannose-coated liposomes was demonstrated [172] and subsequently mannose- and lac- tose-coated liposomes have been successfully used for the cellular delivery of genes [173] and antigens [174]. Sialylneolacto-N-tetraose c (LSTc)- bearing liposomes were used to bind to influenza A virus thereby inhibit- ing infection [175]. Fucosylated liposomes were shown to effectively target Kupffer cells to deliver NF-kB decoys [176]. Another interesting approach has been the use of lectins like Sialyl Lewis X as targeting moieties [177]. These lectins target E-selectins, which are generally overexpressed during inflammation[178]. 3.3.2.4 Peptides as Targeting Moieties Peptide-targeted liposomes have many advantages and have been widely used for the delivery of chemo-therapeutic drugs [179], DNA[180] and siRNA [181, 182]. They are smaller and more labile than antibodies and can be easily chemically synthesized depending on the target-selectivity. In recent years, advances in the field have led to a development of phage- coat peptide libraries for the efficient targeting of nanocarriers [183]. These can be specifically engineered [184] to bind to specific cell surface recep- tors [185] and proteins [186] with high affinity. A few studies have been carried out in order to optimize the development of these phage-directed nanocarriers [187]. Use of these peptide ampiphiles allows for a more sta- ble liposomal structure [188], which results in prolonged in vivo stability. Peptide-targeted liposomes have been proposed as delivery vehicles for boron neutron capture therapy [189] as well as for the targeting of brain endothelium [190] and lung tumor models in vivo [191]. Recently, the targeting of angiogenic chaperone proteins like BiP/GRP78 using specific peptides have been shown to be an effective strategy to suppress tumor growth [192]. In an interesting study by Rivera-Fillat et al., the antian- giogenic effects of the liposomal doxorubicin targeted by thrombospon- din peptidomimetics was demonstrated [193]. The targeted formulation reduced tumor growth by almost 60% in a murine HT29 tumor model. Another interesting strategy has been to target growth factor receptors on tumors with peptides [194]. Similarly, truncated human basic fibro- blast growth factor peptide was used to target liposomal doxorubicin and paclitaxel to prostate and melanoma tumors, respectively, in mice [195]. Falciani et al. have very elegantly demonstrated the use of a branched

Recent Advances with Liposomes as Drug Carriers 93 neurotensin peptide for the delivery of liposomal doxorubicin [196]. The branched peptide had higher targeting efficiency to cancer cells than its linear form on account of its ability to bind multivalently to receptor clus- ters. It is important to keep in mind that in general, the downside of an increased targeting ability is faster uptake by the MPS. Another interest- ing concept is the development of poly(amino acid) peptide sequences, like oligo-arginine for the efficient cell-penetration of liposomes [197] and the subsequent delivery of macromolecular drug [198] and genetic cargo [199]. Such sequences have also been successfully used for the targeting of intracellular organelles [200, 201]. In the past decade, the RGD peptide and its analogs has been widely used in the targeting of cell-surface integrins [202]. The potential of RGD peptides in the targeting of integrins of tumour vasculature with liposomes containing a vascular disrupting agent ZD6126 [203] as well as siRNA has been well demonstrated [204]. Another popular cell-penetrating peptide is the transactivating transduction peptide (TATp). Recently, it was shown that TATp anchored to cholesterol enhanced liposomal delivery to the brain [205, 206]. 3.3.2.5 Targeting with Other Ligands Apart from the targeting moieties discussed above, various other ligands including small molecules have also been used for the liposomal deliv- ery of biological actives [207]. Hydroxy-apatite targeting liposomes were used to deliver hydrophobic drugs specifically to bone tissue [208]. The potential of hyaluronic acid-modified liposomes as vehicles for gene delivery into the liver endothelial cells [209] as well as the delivery of anti-cancer drugs like gemcitabine to pancreatic cancer cells have been demonstrated [210]. Similarly, interleukin 13-liposomes containing doxorubicin were shown to be a viable option for the treatment of glio- blastoma multiforme [211]. Liposomal doxorubicin targeted with oval- bumin was also evaluated as a potential immune suppressant [212]. The potential of the wheat germ agglutinin in the pulmonary delivery of lipo- somal calcitonin, a model peptide drug, to alveolar epithelial cells was demonstrated [213]. Recently, triphenylphosphonium, a lipophilic cation, was used to deliver liposomal paclitaxel [214] and sclareol [215] specifically to mito- chondria. Similarly, octadecyl-rhodamine B was used for the intracellular targeting of lysosomes as a model for the treatment of lysosome-stor- age diseases [216]. Anginex-coated fluorescently labeled paramagnetic

94 Advanced Biomaterials and Biodevices liposomes were developed as an angiogenesis targeting agent for imaging applications [217]. Another interesting approach was the use of an adenovirus as a target- ing moiety to deliver genetic cargo to cancer cells [218]. 3.3.3 Dual-Targeted Liposomes To take the targeting strategies of nanoparticulates one step further, a new approach to targeted drug delivery has emerged in the last five years. Dual-targeting employs the use of two different targeting ligands attached to the surface of the same carrier. Many target cell types like tumor cells express many different types of receptors and/or antigens on their surface. The attachment of more than one ligand onto the lipo- some allows for a higher target binding efficiency thus increasing the accumulation of liposomal cargo [219]. One of the main focuses of this dual targeting strategy has been in anticancer therapy [220–223]. Chen et al. have used liposomes targeted with RGD and octeotride to selec- tively target cancers of the GI tract with greater efficiency than mono- ligand-modified liposomes [224]. In another study, liposomes with hepatocyte targeting and nuclear targeting capabilities were used for the efficient delivery of DNA [225]. In a recent study by Gao et al., doxoru- bicin liposomes targeted with Tf and folic acid were developed with the assumption that Tf would help facilitate penetration across the BBB and the folate would allow for effective internalization into the tumor vascu- lature [226]. Similarly, daunorubicin-loaded liposomes targeted with Tf and p-aminophenyl-α-D-manno-pyranoside were also developed [227]. In vivo, the dual targeted liposomes were able to increase the survival time and reduce tumor volume when compared to the single ligand formulations. The benefits of dual targeting were also demonstrated by Jiang et al. who developed paclitaxel-loaded liposomes targeted with hyaluronic acid (HA) and an arginine-rich cell-penetrating peptide in the hope that in the tumor microenvironment, when the HA is hydro- lyzed by hyaluronidase, the underlying CPP would become exposed allowing for the further penetration of the liposome [228]. A number of groups have also successfully demonstrated synergistic effects of using two different ligands on cell uptake [229, 230] while maintaining the specificity of the formulation [231]. This synergism usually results from the multivalent binding of both the targeting ligands. That aside, it is important to keep in mind that employing multiple ligands can result

Recent Advances with Liposomes as Drug Carriers 95 in a shortened circulation time due to the induction of a heightened immune response. Therefore, synergistic ligand combinations should be to able to use lower ligand concentrations and yet achieve sufficient targeting. Antibodies have also been employed in dual targeting strategies. Ko et al. have combined the use of an anti-myosin mAb for specificity and a TATp for effective internalization of the liposome into the cell’s nuclear compartment for gene delivery [232]. Liposomes targeted with anti- CD137 and an engineered interleukin-2 protein were shown to enhance tumor antigen-specific T cells [233]. The use of liposomes with two differ- ent antibodies in order to improve their therapeutic effects has also been demonstrated [234, 235]. Using multiple antibodies however increases the associated costs significantly and can also decrease the shelf-life of the formulation. 3.4 Multifunctional Liposomes So far we have reviewed long-circulating, ligand and/or antibody-tar- geted, stimuli-sensitive and triggered-release liposomes. The more we look into meeting the demands of modern therapeutics, the more obvi- ous is the need for the development of multi-functional drug delivery systems, which combine the various properties of the aforementioned systems thus allowing for the simultaneous execution of multiple func- tions [236]. In this section, we seek to review these liposomal systems, which have made use of a combination of functions like co-delivering more than one drug type and/or combining targeted and stimuli-sensi- tive release systems for the delivery of drugs. Combining targeting and triggered-release strategies, Hansen et al. demonstrated the use of an UV-activatable cell-penetrating peptide [237]. Stimuli-sensitive PEG coats have also been successfully used, which shields the targeting moi- eties under ‘normal’ conditions but detach in the presence of external stimuli, such as changed pH thus exposing the targeting moiety [238]. Kale et al. used hydrazone-functionalized PEG coats which detached at lower pH exposing the underlying cell-penetrating peptide and allow- ing for efficient internalization of the liposome and subsequent deliv- ery of its genetic cargo [239]. Another useful strategy for the efficient delivery of drug cargo has been to combine a fusogenic polymer with a targeted liposome so that once the targeted liposome reaches the tar- get cell and has been efficiently endocytosed, the polymer fuses with

96 Advanced Biomaterials and Biodevices the endosome at a lower pH releasing the encapsulated contents in the cytoplasm in the vicinity of the nucleus [240]. Recently, targeted liposo- mal formulations were complexed with polyethylenimine (PEI) for the efficient delivery of DNA [241, 242] and siRNA [243]. Similarly, lipo- somes containing dendrimer-DNA complexes have also been developed [244]. Octaarginine-targeted fusogenic liposomes loaded with bleomy- cin demonstrated strong anti-tumor activity in a murine 4T1 tumor model [245]. Similarly, liposomes targeted with peptides that released their contents in a pH –sensitive [246] as well as temperature-sensitve manner were developed [247]. Antibodies have also successfully been used with triggered release systems [248]. pH-sensitive doxorubicin liposomes with anti-HER2 antibodies [249] as well as anti-EGFR gem- citabine liposomes [250] have been developed. Taking multi-functional approaches a step further, dual ligand-targeted liposomes with stimuli- sensitive functions have been suggested. Recently, Koren et al. showed the efficiency of doxorubicin liposomes targeted with 2C5 antibody and TATp (see Figure 3.3) [251]. The targeted liposome was coated with PEG-hydrazone-PE which dissociates at a lower pH, typical of tumor Fibroblasts MCF-7 B16-F10 (a) (b) (c) (d) (e) Figure 3.3 Fluorescence microscopy showing the internalization of rhodamine-PE- labeled liposomes. (a) Plain liposomes (b) TATp-modified liposomes (c)2C5-modified liposomes (d)TATp-2C5-hydrazone-modified liposomes pre-incubated at pH 7.4 and (e) TATp-2C5-hydrazone-modified liposomes pre-incubated at pH 5 are shown. (Red-rhodamine; Blue-hoechst nuclei staining) From [251] Reproduced with the permission of Elsevier.

Recent Advances with Liposomes as Drug Carriers 97 microenvironments thus exposing the targeting ligands. Similarly, Zhu et al. developed a liposomal formulation targeted with 2C5 and TATp and coated with a MMP-2 sensitive PEG-based polymer [252]. In recent years, there have been a number of studies involving the delivery of a combination of drugs encapsulated in the same liposome with the hope of eliciting a synergistic effect [253, 254]. Drug resistance represents a major obstacle in the treatment of many cancers with con- ventional liposomal systems encapsulating just one drug. Liposomes for the combined delivery of transforming growth factor inhibitor as well as interleukin-2 for increased tumor immunotherapy were developed [255]. Belogurov et al. demonstrated the use of mannosylated liposomes encap- sulating three myelin basic protein peptides for the effective treatment of experimental autoimmune encephalomyelitis in rats [256]. Liposomes co-loaded with irinotecan and phytic acid were also developed and their cytotoxic effects on colon tumors were evaluated in vitro and in vivo in a murine xenograft model [257]. Inhalable liposomes co-loaded with paclitaxel and a vitamin E analog significantly inhibited murine mam- mary tumors when compared to single treatments [258]. Liposomes con- taining a combination of specific P-gp inhibitors and anti-cancer drugs also represent a novel approach to overcoming multi-drug resistance in tumors [259]. Liposomes co-loaded with vincristine and quinacrine have also proved to be efficient in overcoming resistant human leukemia in a murine xenograft model [260]. In a recent study, Raju et al developed vita- min E – conjugated Trastuzumab liposome containing docetaxel [261]. To try to overcome multidrug resistance at a gene level, many groups have employed the use of liposomal systems co-loaded with either siRNA [262] or DNA [263, 264] and a cytotoxic drug. Similarly, anti-ganglioside liposomes co-loaded with c-myc antisense oligoneucleotide and doxo- rubicin showed significant tumor growth inhibition [265]. The hypoth- esis is that the oligonucleotide sensitizes the cell to doxorubicin. Another approach has been the use of apoptosis-inducing ligands like TRAIL to sensitize the cell to conventional anti-cancer drugs to improve therapeu- tic effects [266]. Similarly, peptide-targeted liposomes containing pacli- taxel and the gene encoding for TRAIL showed increased cytotoxicity in a murine glioma model [267]. Recently Sawant et al. demonstrated the use of palmitoyl ascorbate (PA)-modified liposomes loaded with pacli- taxel as a novel combination for the treatment of cancer [268, 269]. It was shown that PA causes cytotoxicity by increasing the formation of reactive oxygen species.

98 Advanced Biomaterials and Biodevices 3.5 Conclusions and Future Directions In summary, significant progress has already been made in the area of liposomal therapeutics, and liposomes have come a long way as the car- rier of choice for the delivery of pharmaceutical drugs. As this chapter has reviewed current research on liposomes within a six year window, certain trends have been identified. There has not been too much focus on exploratory research with ‘classical’ and long-circulation liposomes as these have already been well established over the past two decades. Current research in the use of passively targeted liposomes has laid a lot of onus on the use of stimuli-sensitive and triggered release systems enabling us with more control to prevent off-target toxicities [270, 271]. In the case of targeted liposomes, there has been much more focus on the use of engineered peptides from phage libraries for the selec- tive delivery of drugs to the target site [272]. There is also a lot of evi- dence to suggest that targeted liposomes even when PEGylated are still taken up by the MPS or have been seen to cause non-specific interac- tions in healthy tissues, which express the target receptor. The use of dual-ligand combinations seeks to address both these issues. Using two different ligands not only allows for more selectivity, but also allows us to go to a much lower overall ligand concentration if a synergistic relationship is established between the ligands used. However, the use of high-affinity ligands, especially in tumor therapies, may also result in the ‘barrier effect’ [273]. The liposomes bind to the first cell in the tumor with so much affinity that they are unable to penetrate into the tumor tissue effectively. This accumulation at the first line of cells blocks off access to the other formulations preventing deeper penetration. It is becoming more evident that diseases like cancer are evolving to become able to overcome traditional chemotherapeutics by developing drug resistance etc. To combat this, many researchers have been develop- ing strategies like the use of combination therapeutics, such as loading different drugs into the same liposome while simultaneously according it stimulus-sensitive and targeting functions in the hope of developing the ideal ‘smart’ carrier. Another contemporary approach has been the use of various combination therapies such as treatment with radiation or a chemotherapeutic agent to sensitize the cells to the primary treatment modality. These have been summarized in Table 3.2. The results from all these approaches certainly seem encouraging and represent a very prom- ising path for liposomal drug delivery systems.

Table 3.2 Examples of Liposomes In Combination Therapies (Pre-clinical and Clinical). Treatment 1 Treatment 2 Malignancy Reference [274] Oral metronomic S-1 dosing PEGylated Oxaliplatin Liposomes Colorectal Cancer [275] [276] PEGylated Doxorubicin Liposomes Carboplatin Advanced Ovarian Cancer [277] Recent Advances with Liposomes as Drug Carriers 99 Liposomal doxorubicin targeted with Liposomal Vincristine with CD13- HER-2 Positive Breast [278] anti-HER2 Fab’ fragments targeted NGR peptides Cancer [279] Liposomal Honokiol Radiation Therapy Lewis Lung Carcinoma [280] [281] Temperature-sensitive liposomal Radiation Therapy Human Colorectal and hypoxanthine followed by regional Lung Cancer [282] hyperthermia [283] Bevacizumab PEGylated liposomal doxorubicin Recurrent Ovarian Cancer [284] (Continued) Liposomal Curcumin Oxaliplatin Colorectal Cancer Mitochondria targeted ionidamine Mitochondria targeted epirubicin Drug Resistant Lung liposomes liposomes Cancer Aminopeptidase A-targeted doxorubicin Aminopeptidase N-targeted doxorubicin Human Neuroblastoma liposomes liposomes Radiation Peptide-targeted liposomal doxorubicin Lewis Lung Carcinoma selective for irradiated tumors IL-8 silencing liposomal SiRNA Docetaxel Ovarian Cancer

Table 3.2 (Cont.) 100 Advanced Biomaterials and Biodevices Treatment 1 Treatment 2 Malignancy Reference Trabectedin Liposomal doxorubicin Recurrent Ovarian Cancer [285] Squamous Cell Carcinoma [286] Liposomal hemoglobin Radiation therapy Prostrate Cancer [287] Malignant Brain Tumor [288] Liposomal curcumin Liposomal reveratrol Glioblastoma Multiforme [289, 290] Liposomal topotecan PEGylated liposomal doxorubicin Mammary [291] Atherosclerotic plaque-specific liposomal High intensity focused ultrasound Adenocarcinoma doxorubicin [292] Non-Small Cell Lung Radiofrequency ablation Liposomal quercetin followed by liposo- Carcinoma [293] mal doxorubicin Head and Neck Squamous Liposomal nitic oxide synthase Cisplatin Cell Carcinoma Ultrasound-guided collagenase-2 Technetium99-liposomal doxorubicin

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Recent Advances with Liposomes as Drug Carriers 117 258. Latimer, P., et al., Aerosol delivery of liposomal formulated paclitaxel and vitamin E analog reduces murine mammary tumor burden and metastases. Exp Biol Med (Maywood), 2009. 234(10): p. 1244–52. 259. Patel, N.R., et al., Reversal of multidrug resistance by co-delivery of tariqui- dar (XR9576) and paclitaxel using long-circulating liposomes. Int J Pharm, 2011. 416(1): p. 296–9. 260. Liang, G.W., et al., Enhanced therapeutic effects on the multi-drug resistant human leukemia cells in vitro and xenograft in mice using the stealthy lipo- somal vincristine plus quinacrine. Fundam Clin Pharmacol, 2008. 22(4): p. 429–37. 261. Raju, A., M.S. Muthu, and S.S. Feng, Trastuzumab-conjugated vitamin E TPGS liposomes for sustained and targeted delivery of docetaxel. Expert Opin Drug Deliv, 2013. 10(6): p. 747–60. 262. Chen, Y., et al., Multifunctional nanoparticles delivering small interfering RNA and doxorubicin overcome drug resistance in cancer. J Biol Chem, 2010. 285(29): p. 22639–50. 263. Wang, H., et al., PLGA/polymeric liposome for targeted drug and gene co- delivery. Biomaterials, 2010. 31(33): p. 8741–8. 264. Xiao, W., et al., Co-delivery of doxorubicin and plasmid by a novel FGFR- mediated cationic liposome. Int J Pharm, 2010. 393(1–2): p. 119–26. 265. Pastorino, F., et al., Increase of therapeutic effects by treating melanoma with targeted combinations of c-myc antisense and doxorubicin. J Control Release, 2008. 126(1): p. 85–94. 266. Guo, L., et al., TRAIL and doxorubicin combination enhances anti-glioblas- toma effect based on passive tumor targeting of liposomes. J Control Release, 2011. 154(1): p. 93–102. 267. Sun, X., et al., Co-delivery of pEGFP-hTRAIL and paclitaxel to brain glioma mediated by an angiopep-conjugated liposome. Biomaterials, 2012. 33(3): p. 916–24. 268. Sawant, R.R., et al., Palmitoyl ascorbate-modified liposomes as nanoparticle platform for ascorbate-mediated cytotoxicity and paclitaxel co-delivery. Eur J Pharm Biopharm, 2010. 75(3): p. 321–6. 269. Sawant, R.R., et al., Palmitoyl ascorbate liposomes and free ascorbic acid: comparison of anticancer therapeutic effects upon parenteral administra- tion. Pharm Res, 2012. 29(2): p. 375–83. 270. Zhu, L. and V.P. Torchilin, Stimulus-responsive nanopreparations for tumor targeting. Integrative Biology, 2013. 5(1): p. 96–107. 271. Preiss, M.R. and G.D. Bothun, Stimuli-responsive liposome-nanoparticle assemblies. Expert Opin Drug Deliv, 2011. 8(8): p. 1025–1040. 272. Zhang, X.-X., H.S. Eden, and X. Chen, Peptides in cancer Nanomedicine: drug carriers, targeting ligands and protease substrates. Journal of Controlled Release, 2012. 159(1): p. 2–13. 273. Kaasgaard, T. and T.L. Andresen, Liposomal cancer therapy: exploiting tumor characteristics. Expert Opin Drug Deliv, 2010. 7(2): p. 225–243.

118 Advanced Biomaterials and Biodevices 274. Doi, Y., et al., Combination therapy of metronomic S-1 dosing with oxali- platin-containing polyethylene glycol-coated liposome improves antitumor activity in a murine colorectal tumor model. Cancer Sci, 2010. 101(11): p. 2470–5. 275. Ferrero, J.M., et al., Second-line chemotherapy with pegylated liposomal doxorubicin and carboplatin is highly effective in patients with advanced ovarian cancer in late relapse: a GINECO phase II trial. Ann Oncol, 2007. 18(2): p. 263–8. 276. Hare, J.I., E.H. Moase, and T.M. Allen, Targeting combinations of liposomal drugs to both tumor vasculature cells and tumor cells for the treatment of HER2-positive breast cancer. J Drug Target, 2013. 21(1): p. 87–96. 277. Hu, J., et al., Liposomal honokiol, a potent anti-angiogenesis agent, in combi- nation with radiotherapy produces a synergistic antitumor efficacy without increasing toxicity. Exp Mol Med, 2008. 40(6): p. 617–28. 278. Jeong, S.Y., et al., Enhancement of radiotherapeutic effectiveness by temperature- sensitive liposomal 1-methylxanthine. Int J Pharm, 2009. 372(1–2): p. 132–9. 279. Kudoh, K., et al., Effects of bevacizumab and pegylated liposomal doxoru- bicin for the patients with recurrent or refractory ovarian cancers. Gynecol Oncol, 2011. 122(2): p. 233–7. 280. Li, L., et al., Liposomal curcumin with and without oxaliplatin: effects on cell growth, apoptosis, and angiogenesis in colorectal cancer. Mol Cancer Ther, 2007. 6(4): p. 1276–82. 281. Li, N., et al., Development of targeting lonidamine liposomes that circum- vent drug-resistant cancer by acting on mitochondrial signaling pathways. Biomaterials, 2013. 34(13): p. 3366–80. 282. Loi, M., et al., Combined targeting of perivascular and endothelial tumor cells enhances anti-tumor efficacy of liposomal chemotherapy in neuroblas- toma. J Control Release, 2010. 145(1): p. 66–73. 283. Lowery, A., et al., Tumor-targeted delivery of liposome-encapsulated doxo- rubicin by use of a peptide that selectively binds to irradiated tumors. J Control Release, 2011. 150(1): p. 117–24. 284. Merritt, W.M., et al., Effect of interleukin-8 gene silencing with liposome- encapsulated small interfering RNA on ovarian cancer cell growth. J Natl Cancer Inst, 2008. 100(5): p. 359–72. 285. Monk, B.J., et al., Trabectedin plus pegylated liposomal Doxorubicin in recurrent ovarian cancer. J Clin Oncol, 2010. 28(19): p. 3107–14. 286. Murayama, C., et al., Liposome-encapsulated hemoglobin ameliorates tumor hypoxia and enhances radiation therapy to suppress tumor growth in mice. Artif Organs, 2012. 36(2): p. 170–7. 287. Narayanan, N.K., et al., Liposome encapsulation of curcumin and resveratrol in combination reduces prostate cancer incidence in PTEN knockout mice. Int J Cancer, 2009. 125(1): p. 1–8. 288. Yamashita, Y., et al., Convection-enhanced delivery of a topoisomerase I inhibitor (nanoliposomal topotecan) and a topoisomerase II inhibitor

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4 Fabrication, Properties of Nanoshells with Controllable Surface Charge and its Applications Parul Khurana, Sheenam Thatai* and Dinesh Kumar Department of Chemistry, Banasthali Vidyapith, Rajasthan, India Abstract Nanomaterials plasmonics is an emerging research field that deals with the fab- rication and optical characterization of noble metal nanoparticles of various size, shape, structure and plasmon resonances over VIS-NIR spectral band. The recent advances in synthesis, characterization, electromagnetic simulation, and surface functionalization of plasmonic nanoparticles by porous silica-coated nanostruc- tures have led to a publication storm in discoveries and potential applications of plasmon-resonant nanomaterials. The advanced and high-quality synthesis of porous silica-coated nanostructures is increasing research interests for its impor- tant properties and diverse applications, as for catalytic, detection of heavy metal ions in water, colorimetric diagnostics, photothermal therapy, surface enhanced Raman scattering (SERS) detection, and so forth. Here we will focus on up-to- date synthesis strategies, improved properties and emerging applications of silica- coated metal nanoparticles. We have synthesized core-shell particles with metal (Ag, Au) shell and SiO2 as core. The particles will be investigated using UV-Vis- NIR spectroscopy, FTIR, Raman spectroscopy, X-ray diffraction, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Further interac- tion of these core-shell materials with carbon nanotubes (CNT) has been investi- gated using optical spectroscopy and Raman spectroscopy and will be discussed. Nanoshell materials with controllable surface charge (zeta potential) has allowed optimization using the adsorption of CNT for ultrasensitive analysis using SERS. Keywords: Core-shell nanomaterials, carbon nanotubes, surface plasmon resonance (SPR), surface enhanced Raman spectroscopy (SERS), raman enhancement *Corresponding author: [email protected] Ashutosh Tiwari and Anis N. Nordin (eds.) Advanced Biomaterials and Biodevices, (121–146) 2014 © Scrivener Publishing LLC 121

122 Advanced Biomaterials and Biodevices 4.1 What is Nanotechnology? Nanotechnology, is one of the new technologies which refers to the devel- opment of devices, structures as well as systems whose size varies from 1 to 100 nanometers (nm). The last decade had seen advancement in every side of nanotechnology such as nanoparticles and powders; nanolayers and coats; electrical, optic and mechanical nanodevices; and nanostructured biological materials [1, 2]. Presently, nanotechnology is estimated to be influential in all fields of science and technology. It deals with the extreme miniaturization and creation of materials with a very fine structure. Furthermore at the extreme end, nanotechnology is defined as involving the manipulation of tiny objects at the dimension of molecules and atoms and self-assembly of these objects. Materials would then be controlled at the nanoscale level. Nanotechnology relates to the design, creation, and utilization of mate- rials whose constituent structures exist at the nanoscale. These constituent structures can, by convention, be up to 100 nm in size. Nanotechnology is a growing field that explores electrical, optical, and magnetic activity as well as structural behaviour at the molecular and sub molecular level. Nanoscale structures permit the control of fundamental properties of materials without changing the materials chemical status. Designing at the nanoscale is work- ing in a world where physics, chemistry, electrical engineering, mechanical engineering and even biology become unified into an integrated field [3]. “Building blocks” for nanomaterials include carbon-based components and organics, semiconductors, metals, and metal oxides. Nanomaterials are the infrastructure/building blocks for nanotechnology. Nanotechnology is playing an increasingly important role in the develop- ment of biosensors. The sensitivity and performance of biosensors is being improved by using nanomaterials for their construction. The use of these nanomaterials has allowed the introduction of many new signal transduc- tion technologies in biosensors. Nanotechnology plays a major role in the development of new products to substitute existing chemicals and materials with improved performance and potential cost savings. Reduced consump- tion of materials also benefits the environment [4]. Moreover, nanotechnol- ogy gives possibilities to organise and develop production processes. 4.2 Nanomaterials and Their Uses Nanomaterials form a special class of materials that lie in the domain that is in between two extremes i.e. bulk solid and molecules. Nanomaterials

Fabrication, Properties of Nanoshells 123 exhibit properties distinctively different from their bulk and molecular counterparts. The properties of these materials are characterized by their nanometer dimensions. They are considered to be the building blocks for nanotechnology and are referred to particles with at least dimension of 100 nm. A nanometer is approximately 100,000 times smaller than the diameter of a human hair. Some nanomaterials occur naturally but some of these materials are designed and already being used in many commer- cial products such as sunscreens, cosmetics, sporting goods, stain-resistant clothing, tires, electronics as well as many other everyday items and are used in medicine for purposes of diagnosis, imaging and drug delivery [5]. If the physical size of the material is reduced below this nanometer scale, its properties change and become sensitive to its size and shape [6]. Surfaces and interfaces are important in explaining nanomaterials behav- ior [7]. The reason for variation of properties with size of the materials is due to extremely high surface to volume ratio. In bulk materials, only a relatively small percentage of atoms will be at or near a surface or inter- face but in nanomaterials, the small feature size ensures that many atoms will be near interfaces. Number of surface atoms increases with decreas- ing particle size. For example, in a cube of edge size 1 cm, the percentage of surface atoms would be 10–5 % of the bulk atoms, for a cube of edge size 10 nm, percentage of surface atoms would be 10 % of the bulk atoms whereas for a cube of edge 1 nm every atom can be a surface atom. Surface properties such as energy levels, electronic structure and reactiv- ity can be quite different from interior states and give rise to different mate- rial properties. Reducing the size of nanoparticles has a profound effect on the energy level spacing as the system becomes more confined. Particles in these size ranges have been used by several industries and humankind for thousands of years; however, there has been a recent resurgence because of the ability to synthesize and manipulate such materials. Nanomaterials find use in a variety of different areas, such as electronic, magnetic and optoelectronic, biomedical, pharmaceutical, cosmetic, energy, environmental, catalytic and materials applications. There are novel UV-blocking coatings on glass bottles which protect beverages from dam- age by sunlight and longer-lasting tennis balls using butyl rubber/nano- clay composites. Many finds application in cosmetics, sun-block creams, self-cleaning windows and nanoscale silica is being used as filler in a range of products, including cosmetics and dental fillings. Major emphasis is also being put on ensuring broader social improvements and sustainable devel- opment [8]. Nanomaterials are not simply another step in miniaturization, but a different area entirely. The nanoworld lies midway between the scale of atomic and quantum phenomena, and the scale of bulk materials.

124 Advanced Biomaterials and Biodevices 4.3 Classification of Nanomaterials Nanomaterials can be nanoscale in one dimension (e.g. surface films), two dimensions (e.g. strands or fibres), or three dimensions (e.g. particles). They can exist in single, fused, aggregated or agglomerated forms with spherical, tubular, and irregular shapes. Common type of nanomaterials includes nanotubes, dendrimers, quantum dots and fullerenes. They have applications in the field of nanotechnology, and displays different physical chemical characteristics from normal chemicals (i.e., silver nano, carbon nanotubes, fullerene, photocatalyst, silica). 0D Classification 1D 3D of nanomaterials 2D Nanostructured materials are classified as zero dimensional, one dimen- sional, two dimensional and three dimensional nanostructures. It is clearly shown in Figure 4.1: Figure 4.1 Classification of nanomaterials (a) 0 D spheres and clusters (b) 1 D nanofibers, wires and rods (c) 2 D films, plates and networks (d) 3 D nanoparticles.

Fabrication, Properties of Nanoshells 125 0 D nanomaterials • Nanoparticles, clusters and quantum dots. • Amorphous or Crystalline. • Single crystalline or Polycrystalline. • Composed of single or multi-chemical elements. • Exhibit various shapes and forms. • Exist individually or incorporated in a matrix. • Metallic, ceramic or polymeric. 1 D nanomaterials • Nanotubes, nanorods and nanowires. • Amorphous or Crystalline. • Single crystalline or Polycrystalline. • Chemically pure or impure. • Stand-alone materials or embedded in within another medium. • Metallic, ceramic or polymeric. 2 D nanomaterials • Super-thin films, multilayered films, nanolayers and nanocoatings. • Amorphous or Crystalline. • Made up of various chemical compositions. • Used as a single layer or as multilayer structures. • Deposited on a substrate. • Integrated in a surrounding matrix material. • Metallic, ceramic or polymeric. 3 D nanomaterials • Dispersion of nanoparticles, bundles of nanowires and nanotubes. • Involve the presence of features at the nanoscale. • Made up of various chemical compositions. • Metallic, ceramic or polymeric. It is of great interest to synthesize 0 D to 3 D nanomaterials with a con- trolled structure and morphology.

126 Advanced Biomaterials and Biodevices 4.4 Nanoparticles Nanoparticles are a class of materials with properties distinctively different from their bulk and molecular counterparts. Colloidal dispersions of metal nanoparticles having size much smaller than wavelength of visible radia- tion exhibit intense colours. Art of making coloured glass is known over thousand years. Small amount of metal nanoparticles such as gold, silver, copper etc. doped in glass give rise to beautiful colours [9]. Windows of churches, palaces etc. are often decorated with such glasses. One such win- dow of Milan cathedral, having small gold particles as colorant. Although technique of making coloured glass had been known since long time, it was not known that it is because of the presence of the metal nanoparticles in glass, until Michael Faraday in 1857 synthesized gold nanoparticles. He got ruby red and pink colour dispersion of gold particles. Nanoparticles can be synthesized by growing, shaping or assembling the materials by: Physical methods Hybrid Synthesis of Chemical methods nanoparticles methods Biological methods Broadly speaking synthesis of nanomaterials can be divided in to two cat- egories viz. top down and bottom up approach [11]. In “top-down” approach, macroscopic particles can be reduced to the nanosize by removal of material. This approach involves milling, machining and lithography techniques. On the other hand “bottom up” approach involves aggregation of atoms to form particles of definite size, shape or structure. Bottom up approach consists of physical as well chemical methods. The advantages of chemical methods over physical methods are that they are relatively simple, inexpensive, low temperature techniques and do not require sophisticated equipment. The materials can be synthesized in various shapes, sizes and structures [12].

Fabrication, Properties of Nanoshells 127 Figure 4.2 Stained-glass window in Milan Cathedral, Italy, made by Niccolo da Varall between 1480 and 1486, showing the birth of St. Eligius, patron saint of goldsmiths. The red colours are due to colloidal gold [9, 10]. Nanoparticles are designed at the nanometer level has advantage of their small size as they possess novel properties that are typically not observed in their conventional or bulk counterparts. Nanoparticles have a much larger surface area to volume ratio which is the basis of their novel physical-chemical properties [13, 14]. The emergence of these novel prop- erties on the nanoscale is attributable to the lack of symmetry at the inter- face or to confinement of electrons that does not scale linearly with size. In nanoparticles, various atoms perhaps half or more in some cases, are near interfaces. Surface properties such as energy levels, electronic struc- ture and reactivity can differ markedly from those in interior states. Since properties depend in this way on size, rather than on the material, reliable and continuous change can be achieved using a single material. In 1908, observation of brilliant colors of colloidal metal nanoparticles could be explained using classical Mie theory [15]. It is the most simple, exact and only solution to Maxwell’s equations which is relevant to the particles. The physical origin of the strong light absorption by noble metal nanoparticles is the coherent oscillation of the conduction band elec- trons induced by interaction with an electromagnetic field. The resonance between these oscillations and incident radiation, gives rise to an intense peak in the visible range known as surface plasmon resonance (SPR). This

128 Advanced Biomaterials and Biodevices resonance exhibited by nanoparticles is a small size effect but not a quan- tum size effect. It is absent in the individual atoms as well as bulk solid. Now with advances of science and technology, the morphology of metal- lic nanoparticles has been understood. Thus, nanoparticles have potential applications in electronics and photonics, catalysis, information storage, chemical sensing and imaging, environmental remediation, drug delivery and biological labelling [12]. 4.5 Nanocomposites Material Core-shell nanoparticles constitute a special class of nanocomposite mate- rials. They consist of concentric particles, in which particles of one mate- rial are coated with thin layer of another material by special procedures [16–18]. Since the time the nanoparticles have been developed, there is a constant attempt to protect their surfaces which are unstable due to sur- face strains, dangling bonds, susceptible to oxidation, coalescence etc. Therefore the coating of other material is often done on such particles to passivate their surface. Recent euphoria among the scientists to make dif- ferent core shell assemblies is a result of immense applications emerging out for such assemblies. They are highly functional materials with tailored properties, which are quite different than either of the core or shell mate- rial. Indeed they show modified and improved properties than their single component counterparts or nanoparticles of same size. Therefore they are preferred over nanoparticles. Their properties can be modified by chang- ing either the constituting materials or core to shell ratio [19]. Properties of shell materials (can be metal or semiconductor) having thickness in nanometers, become important when they are coated on dielectric cores to achieve higher surface area. So they are referred to as core@shell particles. Synthesis of core-shell can be useful for creating novel materials with different morphologies as it is not possible to synthesize all the materials in desired morphologies. Core particles of different morphologies such as rods, wires, tubes, rings, cubes etc. can be coated with thin shell to get desired morphology in core-shell structures [20]. These materials can be of economic interest also as precious materials can be deposited on inex- pensive cores. The core-shell nanostructures can be fabricated by using various meth- ods including chemical deposition [22], seed induced deposition [23], layer- by-layer synthesis [24], inverse micelle [25–26], sol-gel condensation [27], sonochemical approach [28], electroless plating [29] and others. Fabrication of nanoshells is often a multi-step process, which requires a strict control of

Fabrication, Properties of Nanoshells 129 Figure 4.3 Schematic representation of some possible mixing patterns: (a) core-shell (b) subcluster segregated (c) mixed [21]. the each synthesis step in order to obtain structures with desired properties. The control of each step of the synthesis allows obtaining nanostructures with a narrow dispersion of particle sizes and desired degree of coverage of the core surface by metal nanoparticles, and simultaneously prevents an excessive precipitation and coagulation of these nanoparticles. Some of the core-shell nanocomposites prepared by researchers are like Au@Ag, Cu@Ag, Ag@SiO2, Au@CuO2, Fe3O4@Au, Au@Pd etc. Here we will report technique to prepare controlled core-shell nanoparticles (SiO2@M). In this technique, the shell (M= Ag, Au etc.) is formed when the core (SiO2) reacts with metal ions and the size of the core and thickness of the shell will be controlled by the synthetic route and controlled condi- tions such as stirring speed, temperature, pH and concentration of solu- tion. Core-shell nanoparticles with a core of dielectric material silica (SiO2) of suitable diameter (400–450 nm) will be prepared using Stober’s method. Both silver and gold nanoshells will be grown on silica microspheres with diameters ranging from 50 to 60 nm by the reduction of silver nitrate or gold hydroxide onto silica microspheres resulted in increasing coverage of silver or gold on the core. The core-shell nanoparticles produced by this technique can be used as surface enhanced Raman scattering probes. 4.6 Spherical Silica Particles Silica is stable in water and at elevated temperatures and also good insula- tor. One of the advantages of SiO2, especially for optical applications, is its transparency to electromagnetic radiation in wavelength range from 300 to 800 nm. Silica nanoparticles in the form of precipitated amorphous silica, sols, colloids, and pyrogenic silica, are used as additives to polymers and rubbers in order to improve their mechanical properties, additives to

130 Advanced Biomaterials and Biodevices Metal NPs Interfacing Small molecules Interface Silicates/silanes Citrate salts Silica-coating Surfactants Synthetic polymers PVP Polyelectrolytes Bio-polymers Enzyme Gelatin Silica-coated metal NPs Figure 4.4 Silica-coating strategies [31]. liquid phases for stabilization of suspensions [30], and also as fillers in wide range of polymer products, including dental materials [31]. Silica nanopar- ticles are also used for the synthesis of various nanoshells are depicted in Figure 4.4, consisting of a solid siliceous core and a nanoshell or vice versa Silica particles having sizes ranging from 40 nm to several micrometers by controlled hydrolysis of tetraethylortho-silicate (TEOS) in ethanol, fol- lowed by condensation (polymerization) of the dispersed phase material. The reactions involved is (hydrolysis) Si(OR) 4+ 4H2O Si(OH) 4+ 4ROH (condensation) Si(OH) 4 SiO2 + 4H2O It was found that there are five primary parameters affect on the size of silica particles. These parameters were (1) concentration of TEOS (2) con- centration of ammonia (3) concentration of water (4) alcohol used as the solvent (5) reaction temperature. Silica particles were synthesized by authors using Stöber procedure [33, 34]. Hydrolysis and successive condensation of silica precursor TEOS was carried out in alcoholic medium in a base catalyzed reaction using ammonium hydroxide. In a typical preparation method mixture of ethanol (15 mL), distilled water (3 mL) and ammonium hydroxide (0.75 mL) was stirred for 30 mins to form a homogeneous solution. TEOS (1.2 mL) was

Fabrication, Properties of Nanoshells 131 Figure 4.5 Schematic diagram of experimental apparatus. (1) Water bath, (2) microfeed pump, (3) stirrer, (4) reactor, (5) N2 gas, (6) EtOH/water/NH3 solution, (7) EtOH/TEDS solution [32]. NH4OH Ethanol Milli Q water After addition of 1.2 mL TEOS & stirring white coloured ppts of silica is formed Figure 4.6 Graphical setup for the fabrication of silica particles. added to this solution and total solution was stirred for three hours to get white color precipitate. This precipitate was washed with water 3–4 times to remove traces of NH4OH and dried to collect in powder form. This pro- cedure yields silica particles which are highly monodispersed. This route is highly reproducible, produces monodispersed particles and gives high yield. As characterization of nanoparticles is done using spectroscopic and microscopic techniques. Therefore here silica particles are characterized using SEM and TEM. These particles prepared acts as core for preparation of core-shell nano- composites like SiO2@Ag, SiO2@Au etc.

132 Advanced Biomaterials and Biodevices Figure 4.7 SEM images of monodispersed SiO2 particles. 4.7 Silver Nanoparticles Silver has been used as an antibacterial agent since ancient times. Romans stored wine in silver urns to prevent spoilage. Chinese emperors ate with silver chopsticks for better health whereas Americans used to put silver coins in milk and water to avoid spoilage in old times. Indian tradition of using silver utensils and feeding babies with silver spoon is due to the same reason. Till date silver is being used as antibacterial agent and many commercial products are introduced which exploit antibacterial prop- erty of silver. Silver inhibits the growths of bacteria by deactivating the bacteria’s oxygen metabolism enzyme. In turn this destroys bacteria’s cell membranes, stopping the replication of bacteria’s DNA. Recent research shows that antibacterial properties of silver are retained or even enhanced in nano form similar to its bulk form. Although the literature describes a broad range of superb and innovative methods for obtaining nanosilver, only a few of them are concerned with the main stream production. Laser ablation Photo Different Chemical reduction method of reduction preparation of Ag NPs Microwave irradiation

Fabrication, Properties of Nanoshells 133 Chemical reduction is the most common of all methods to obtain silver nanoparticles. The most important advantages it offers include high yield of non-aggregated nanoparticles, low price and ease of performance. This method is based on the reduction of silver nitrate (AgNO3) with a reducing agent in the presence of a suitable amount of stabilizer, which controls the growth of silver nanoparticles undisturbed by aggregation. The reducing agents used are sodium borohydride (NaBH4), tri-sodium citrate, hydra- zine (N2H4) and glucose [35]. Polyoxyethylene sorbitan monolaurate, a sta- bilizer commercially known as TWEEN [36] was used to obtain colloidal silver. The most important parameters of this method are: starting concen- tration of AgNO3, ratio of molal concentrations of reducing agent to silver nitrate, and the concentration of the stabilizer [37]. The most popular and best known method to obtain nano silver is the reduction of silver nitrate with a reducing agent e.g. sodium borohydride or tri- sodium citrate. Photoreduction with UV light is also one of the leading methods. In addi- tion, regardless of the vast number of publications on “green” technologies with which to obtain nanosilver, research into developing a fully environmentally- friendly production method for Ag NPs is still in progress [39, 40]. Laser ablation, a new method to obtain nanosilver which has been frequently analysed in the last decade, involves the superficial reaction between liquid and a solid body suspended in it. In this process a pulsat- ing laser beam directed at the surface of a solid body causes a “discharge” of material from the surface of the solid body, which then migrates to the surrounding liquid (reducing liquid medium) in a bubble form. Vacuum ion sputtering is another method used to synthesize nanosilver. Microwave irradiation is another method used to synthesize nanoparticles, and it was successfully used to obtain nanosilver. When Figure 4.8 TEM images of silver nanoparticles at various citrate concentrations (a) low citrate concentration (0.5×10-4 M) (b) intermediate citrate concentration (1.5×10-4 M) while, MT represents multiple twinned particles [38].

134 Advanced Biomaterials and Biodevices a microwave beam goes through the dielectric coating of the material, radiation energy transforms into thermal energy and the material’s temperature increases [41]. Here, silver colloid was prepared by using chemical reduction method [42, 43]. All solutions of reacting materials were prepared in Milli Q water. In typical experiment 50 mL of 2 mM AgNO3 was heated to boiling. To this solution 50 mL of 2◊10–4 trisodium citrate was added drop by drop. The solution was refluxed at 80°C for 30 min. After 15 min, clear solution will change to pale yellow. Then it was removed from the heating element and cooled to room temperature. Tri-sodium citrate being a weak reducing agent cannot perform reduction at room temperature and merely serves as a capping agent. Silver nanoparticles were isolated from the colloidal solu- tion by centrifugation at 5000 rpm for 20 min and then washed 3–4 times with water. These nanoparticles were used as shell in preparation of core-shell nanocomposites. 4.8 Gold Nanoparticles Gold nanoparticles (GNPs) have attracted a wide range of interest because of increasing applications in sensors, biosensors and many emerging areas of nanotechnology. SPR bands of gold nanoparticles originate because of the coherent excitation of free conduction electrons on nanoparticle sur- faces as electromagnetic waves interact with nanoparticle surfaces and the wavelengths of these bands depend on size, shape, and interparticle dipole interactions [44]. For example, the study of the optical properties of Au nanoparticles prepared by reverse micelles showed a red shift with the particle size, which is expected from Mie theory. The nanoparticles are either inside the reverse micelles (for the smaller particles) or capped with a monolayer of surfactants. The comparison of the optical proper- ties of nanoparticles with the predictions from Mie theory showed that the resonance band of gold nanoshells on polystyrene cores could be tuned across the visible and near-infrared range of the electromagnetic spectrum by varying the amount of gold in the shells [45]. Au NPs have been studied for their tunable optical properties in a wide spectral range as shown in Figure 4.11. The particle shows SPR band in the visible range of electro- magnetic spectrum, position of which is dependent on various factors such as size, shape and dielectric constant of the medium in which they are dis- persed [44]. Previous studies suggest that SPR band of metal nanoparticles could only be slightly varied by changing their sizes i.e in the range of 1 to

Fabrication, Properties of Nanoshells 135 1.2 30nm 40nm 1.0 50nm 60nm Normalized Abs. 70nm 0.8 80nm 90nm 100nm 0.6 0.4 0.2 0.0 400 500 600 700 800 λmax (nm) Figure 4.9 UV-Vis spectra for Au nanoparticles with different particle sizes in aqueous solution [45]. 400 nm Light color 750 nm Silver spheres Silver rods Gold spheres Gold rods Gold/silver alloyed spheres Gold shells with hollow interiors Silver plates Silver cubes Figure 4.10 Position of SPR band can be tuned using gold and silver nanoparticles of various structures and morphologies [44]. 100 nm. In contrast SPR band can be tuned in the region from visible to near-infrared by varying the shape and size of the nanoparticles [45, 46]. It has been shown that when a transition in shape from sphere to rods is made, a single broad plasmon absorption band splits into two plasmon bands. Peak at shorter wavelength due to transverse plasmon resonance while peak at higher wavelength due to longitudinal plasmon resonance. The wavelength of longitudinal plasmon band is tunable from visible to near-infrared by changing the aspect ratio [47] There are many different ways to synthesize gold NPs: • Citrate Reduction Method [45, 46] is first reported by Turkevich and is popularly used to generate spherical gold

136 Advanced Biomaterials and Biodevices nanoparticles. Simply put, gold salt, reducing agent and citrate are stirred in water and metal nanospheres are reduced. 2HAuCl4 + 3C6H8O7 (citric acid) 2Au + 3C5H6O5 (3-ketoglutaric acid) + 8HCl + 3CO2 During the process, the temperature, the ratio of gold to citrate, and the order of addition of the reagents control the size distribution of gold nano- spheres. The most popular one for a long time has been that using sodium citrate reduction of HAuCl4 in water. Citrate reduction method Two phase Synthesis Nanosphere reactions of gold lithography nanoparticles Biological method Nanosphere lithography [50] method is an inexpensive synthetic proce- dure to generate arrays of metal nanoparticles. Polystyrene nanospheres are drop-coated onto piranha-cleaned and base-treated glass substrates and are allowed to dry, forming a hexagonal closed-packed monolayer or spheres. Such monolayer can act as a template or deposition mask for metal deposition. Metal is then deposited onto and in between the spheres by thermal evapo- ration, creating particles in the voids of the polystyrene spheres. Following metal deposition, the samples are sonicated in ethanol to remove the polysty- rene nanosphere mask, leaving an array of triangular shaped metal nanopar- ticles on the substrate. This generates monodisperse, uncapped nanoparticles in geometric arrays over a large surface area of the substrate [51]. Two phase reactions [49, 52] method has been widely used to produce small nanoparticles (1–5 nm) with narrow dispersity. A gold-thiol bond is used to stabilize these particles. Samples generated with this method are