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Samrat Roy Choudhury, Biological Sciences Division, Indian Statistical Institute, 203, B. T Road, Kolkata-700108, India. E-mail: email@example.com
Antimicrobial resistance continues to be an inexorable threat for the biomedical and biochemical researchers. Despite the novel discoveries in drug designing and delivery, high-throughput screening and surveillance data render the prospects for new antimicrobial agents as bleak as ever. The advent of nanotechnology, however, strengthens pharmacology by offering effective therapeutics to treat this aforementioned problem. Several nanoparticles of the known elements have already been reported for their antimicrobial efficacy. Nanosized fabrication of elemental sulphur with suitable surface modifications offers to retrieve the use of sulphur (man's oldest known ecofriendly microbicide) as a potential antimicrobial agent. Sulphur nanoparticles (SNPs) are effective against both conventionally sulphur-resistant and sulphur-susceptible microbes (fungi and bacteria). Moreover, biocompatible polymers present on the surface of SNPs minimize toxicity during application. Here, we focus on various aspects of physicochemical features of SNPs and their biochemical interactions with microbes. The present review also illustrates the effects of SNPs on plants and animals in terms of cytotoxicity and biocompatibility.
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Microbial pathogenesis is a persistent problem for the agro-medical industries. Despite the administration of several synthetic antimicrobial agents (AAs), effective control against pests and pathogens has remained unsatisfactory (Piemental et al. 1992; Allsop 2002). Conventional pesticides are applied to crop fields via periodic broadcasting and spraying, while antimicrobial drugs are administered through oral, mucosal and transdermal routes. Very high and possibly highly toxic concentrations are applied initially, and these often decrease rapidly below the ‘minimum effective concentration’ (MEC) level (Jutsum et al. 1998). As a result, repeated applications of AAs have become mandatory to obtain effective control over the target pathogens. Nonetheless, the toxic effects of AAs are being magnified in the food chain and result in chronic and acute health disorders (Metcalf et al. 1971; Abel 2006; Eskenazi et al. 2008). AAs thus must be designed to meet the demands of efficacy and suitability to the mode of application and minimizing the damage to the environment.
Nanotechnology offers effective new tools and therapeutics to support health issues and agro-medical productivity by mitigating pathogenic invasions. Surface-modified sulphur nanoparticles (SNPs) with potential antimicrobial efficacy can be claimed as one of the important discoveries of nanotechnology. Here, we summarize the physicochemical and antimicrobial properties of these novel microbicides with their probable biocompatible or bioadverse attributes. The study also discusses the need of this group of drugs for the developing countries from the commercial point of view.
Nanodrugs and Pesticides – A Novel Therapeutics for Agro-Medical Sectors
The word ‘nano’ (derived from the Greek word nanos) means dwarf, and in the measurement scale, it is 10−9 of a metre. In particular, nanoparticles that are essentially below 100 nm in size are found to have enhanced surface reactivity (owing to high surface/volume ratio) and translocation potential (Halperin 1986; Nanda et al. 2003). At nanoscale, intermolecular interactions are predominated by Van der Waals forces, stronger polar and electrostatic or covalent interactions that influence their physical properties such as boiling point or surface plasmon resonance. The potential benefits of nanotechnology have been recognized by many industries, and a few commercial products have already being manufactured. Achievements and discoveries in nanotechnology are beginning to influence the agro-medical and allied industries. This includes important aspects such as safety in the molecular synthesis of new drugs and food products (Weiss et al. 2006; Shi et al. 2010) and enhances the crop productivity by minimizing pathogenic invasions in agricultural and horticultural fields. Application of nanotechnology in food, agro-medical and allied sectors can be summarized as follows:
Nanoparticles of medical importance are usually embedded or encapsulated in polymeric coatings. Polymers are chosen, based on their efficacy to circulate in bloodstream for a longer period of time (Tan et al. 1993; Torchilin 1998).
Specific functional groups (proteins, peptides or DNA) on the surface of nanoparticles ensure targeted delivery in the host cell (Cui et al. 2003). Moreover, choice of matrix substance/polymeric coatings (surfactants) of the nanoparticles enables their controlled release and degradation (Zalipsky 1995).
Nanosensors have been developed to detect the presence of any number of bacterial, fungal or viral pathogens rapidly and accurately in packed and imported foodstuffs. Given their small size, nanosensors can gain access into the tiny crevices where pathogens often hide (Cui et al. 2001; Sanguansri and Augustin 2006).
Nanoencapsulated drugs and pesticides can be used effectively over a given period of time interval, and their design enables (Tiju and Morrison 2006; Ximena et al. 2010) them to resist severe environmental fluctuations (i.e. leaching, evaporation and photolytic, hydrolytic or microbial degradation) that act to eliminate conventionally applied pesticides.
Nanoparticles have higher in vivo and in vitro translocation potential owing to their size in comparison with the micron-sized particles (Zhu et al. 2008). Moreover, biocompatible surfactants and diminutive size make nanoparticles easily biodegradable (Anton et al. 2008).
Nanodrugs and pesticides are needed in very small amounts to achieve the desired effectiveness. Hence, bulk volume requirement of conventional drugs and pesticides during application and the cost to afford that volume can be cut down sufficiently.
Elemental Sulphur: The Oldest Known Antimicrobial Agent
Pungent sulphurous smell has always tended to fascinate humans and has led to the elevation of this element to an empyrean status. As per the Biblical Pentateuch, destruction of the twin city Sodom and Gomorrah was purported to be mediated by brimstone (sulphur) and fire. However, sulphur – multivalent inorganic nonmetal – is not only associated with death and destruction, but also has been in use since time immemorial for its purifying and beneficial properties. The Egyptians were familiar with sulphurous antiseptic cream for treating bacterial infections, while the Chinese used to inject colloidal sulphur to treat rheumatoid arthritis (Mitcell 1996). So, from the divine fearsome substance, across the mystic alchemists' world to the group 16 (VIB) of the modern periodic table, the origin and broad-spectrum implications of sulphur have been profusely explored.
Elemental sulphur (ES) and sulphur-rich compounds bear an interesting interaction with biological systems. Diverse floral and microbial communities assimilate the reduced form of ES through sulphhydrylation pathway (Foglino et al. 1995; Vermeij and Kertesz 1999), convert them into less toxic sulphides and finally reduce into thiol (sulphur compound with lowest state of oxidation). Formation and incorporation of thiol into methionine is quintessential for the initiation of protein synthesis machinery. Moreover, diverse plant families are found to accumulate ES or sulphur-rich organic compounds as phytoalexin in order to combat pathogenic invasions (Resende et al. 1996; Williams et al. 2002; Cooper and Williams 2004; Williams and Cooper 2004). Hence, it is interesting to note that while sulphur plays a crucial role in protein synthesis at a basal quantity, a high concentration of sulphur is, however, considered to be toxic against micro-organisms. ES upon incorporation or adsorption by microbial cells generates a number of volatile compounds like hydrogen sulphide or pentathionic acid (Owens 1963). These compounds in turn impair a cluster of enzymes (especially – SH containing) involved in microbial respiration or denature certain proteins and lipids (McCallan 1949; Libenson et al. 1953; Brock and Madigan 1991). This nonsystemic and contact AA was widely used in agro-medical sectors in its wettable form, dust form, colloidal form or in combination with other synthetic and organic pesticides (Baldwin 1950; Rose et al. 1999). Various forms of ES have been used to control powdery mildews contamination in gooseberries, grapes, strawberries, etc., scab in apples and certain smut and rust diseases of hops and ornamental plants (Baldwin 1950). Earlier studies have also reported the antimicrobial efficacy of ES against a number of Gram-positive bacterial spp. of agricultural and medical importance (Massey and Snider 1936; Libenson et al. 1953). Antimicrobial efficacy of ES, hence, has been exploited for decades for treating bacterial and fungal infections. However, it was noticed that ES and sulphonamides (sulphur-containing antibiotics) were barely microbicidal and typically fungistatic or bacteriostatic, respectively, in nature (Huovinen et al. 1995). Moreover, genetic modifications and concomitant acquired resistance in target pathogens depreciate the microbicidal efficacy of ES. At the same time, owing to the large volume requirement and huge cost to afford, sulphur has lost its worldwide popularity among farmers and agro-medical industries. Nanotechnology offers endless opportunities to re-explore the biological properties of ES by manipulating the size and surface to enhance their antimicrobial efficacy.
Engineering of the Surface-Modified Sulphur Nanoparticles
Preparation of SNPs
Synthesis of surface-functionalized SNPs reported so far was mainly via two different wet chemical (liquid-phase precipitation and water-in-oil microemulsion) methods (Guo et al. 2005, 2006; Deshpande et al. 2008). In liquid-phase precipitation method, micron-sized sulphur particles were brought down into nanosize by precipitating with weak acids (formic/acetic acid, etc.), being encapsulated in polymeric coatings (Torchilin 1998). Guo et al. (2006) and Deshpande et al. (2008) later on reported the synthesis of SNPs via water-in-oil microemulsion technique using different starting materials, surfactants, co-surfactants, aqueous and oil phases (Guo et al. 2006; Deshpande et al. 2008). Furthermore, Xie et al. 2009 reported the preparation of cystine-stabilized SNPs via liquid synthesis method. Our research group has prepared SNPs with suitable modifications to the standard procedures reported elsewhere (Guo et al. 2005, 2006). With suitable alterations in surfactants, oil phases and physical factors (temperature, pH, etc.), we have succeeded in synthesizing and stabilizing orthorhombic and monoclinic nanoallotropes of sulphur (Fig. 1) and estimated their concentration with suitable analytical tools (Kumar et al. 2011). The stock concentration of α-SNPs and β&!szlig;-SNPs was estimated to be 5194·3 μg ml−1 and 18 000 μg ml−1, respectively. Syntheses of these batches of SNPs were carried out at room temperature and with minimal equipment facility.
Physicochemical characterizations of SNPs
Nanoparticles are generally characterized for their size with transmission electron microscopy (TEM), shape with scanning electron microscopy (SEM), surface topology with atomic force microscopy (AFM), surface modification with Fourier transform infra-red (FT-IR) spectroscopy, allotropic composition with X-ray diffraction (XRD) pattern, thermal stability with thermogravimetric analysis (TGA) and purity with energy-dispersive X-ray spectroscopy. SNPs prepared by Xie et al. (2009) were ‘insect-like’ in shape and were around 50–100 nm in size, while SNPs prepared by Guo et al. (2006) and Deshpande et al. (2008) were spherical in shape and were around 20 nm and 5–15 nm, respectively, in size. The SEM and AFM micrographs revealed that the synthesized orthorhombic and monoclinic SNPs by our research group were ‘spheroid’ (Fig. 2a,b) and ‘tetrapod-like’ (Fig. 2c,d) in shape with 10 and 50 nm (as evident from TEM micrographs) in size, respectively. TEM micrographs were used to confirm both the size and shape of the SNPs (Fig. 3). Noteworthy, under TEM, monoclinic SNPs were identified as nanorods. These nanorods presumably conglomerate to form the bigger ‘tetrapod’-like structures as observed in the TEM micrographs (inset of Fig. 3b).
Orthorhombic SNPs (α-SNPs) were prepared, using three different polysulphide agents (calcium polysulphide, sodium polysulphide and ammonium polysulphide), of which SNPs prepared from sodium polysulphide were smallest in size (10 nm; Fig. 3a) and largest from ammonium polysulphide (50 nm). On the other hand, monoclinic SNPs (α-SNPs) of 50 nm (Fig. 3b) in size were prepared from the combination of sodium polysulphide as the inorganic sulphur reactant, conjugate of Span-80 and Tween-80 as surfactant, 1-butanol as co-surfactant and cyclohexane as oil phase. Allotropic natures of the prepared SNPs were determined with X-ray diffraction pattern after comparison with the standard peaks of α- and α&!szlig;-sulphur using Joint Commission on Powder Diffraction Standards (JCPDS) files. Another crucial part of the aforementioned syntheses was the stabilization of SNPs with biocompatible polymers, in absence of which nanoparticles tend to agglomerate and fail to retain their nanosized configuration. The α-SNPs were coated with polyethylene glycol-400 (PEG-400), while α-SNPs were encapsulated with a conjugate of Span 80–Tween 80. The surfactant agents chosen to encapsulate the SNPs were inert and biocompatible in nature. FT-IR spectroscopy was used to determine the surface modification of ES, α-SNPs and α-SNPs (Roy Choudhury et al. 2011). TGA revealed that SNP allotropes were stable even beyond 300°C, which makes them eminently suitable to apply in tropical crop fields where the temperature often reaches up to approx. 50–60°C (Roy Choudhury et al. 2011). Colloidal stability and coagulation kinetics of the prepared SNPs were also studied along a gradient of temperature, time-drive and dilution using dynamic light scattering. Both the SNPs revealed adequate stability under the stated physical conditions (Roy Choudhury et al. 2012a).
Hydrophilization of the prepared SNPs
Elemental sulphur is a known hydrophobic substance, is insoluble in most of the hydrophilic media and hence is a nonspecific AA for a number of reasons. Firstly, being insoluble in water, the targeted delivery of ES to the living cells is extremely difficult. Secondly, living cells are equipped with distinct efflux systems to mitigate the load of ES toxicity (Sato et al. 2011). Finally, insolubility in hydrophilic media also results in the formation of uneven sulphate crystals and hinders their uniform assimilation (Hawkesford 1999).
Surfactants are generally chosen to provide optimum stability, bioavailability and biodegradation of the nanoparticles. The α-SNPs were encapsulated with PEG-400. PEGylation is considered as the simplest and cheapest method (Zalipsky 1995; Otsuka et al. 2003) to enhance the translocation potential of different colloidal drugs in the systemic circulation. PEG-400 coating on the SNPs' surface not only hydrophilizes the ES core but also makes their distribution even and controlled in any polar solvents like water, agar and broth media. On the other hand, conjugate of Span 80 and Tween 80 was used to stabilize monoclinic sulphur in their nanoform, which is otherwise highly metastable in nature. Both Span-80 and Tween-80 are neutral lipids, while ES is lipophilic in nature. The selected surfactant agents, hence, are believed to facilitate the sustained release of α-SNPs in the colloidal media and ensure their prolonged activity as an antimicrobial agent.
Antimicrobial Efficacy of SNPs
Our research group is presumably the first to report the antimicrobial efficacy of SNPs against both the conventionally sulphur-resistant and sulphur-susceptible microbial pathogens (fungi and bacteria). Modified agar dilution (poisoned food technique; Roy Choudhury et al. 2011) and broth dilution methods were used to determine the microbial growth, minimum effective concentrations (MEC) and minimum inhibitory concentrations (MICs) of SNPs. MECs were defined as the lowest concentration at which SNPs impart their first line of inhibition against the microbes. MICs were defined as the lowest concentration of SNPs, at which there was no visible growth in the test media. In addition, spore germination slide bioassay was used to enumerate the number of spores for the treated fungal isolates. The antifungal efficacy testing of SNPs against Erysiphe cichoracearum (powdery mildew) was performed using ‘pot culture’ technique and SNP solutions were sprayed on the live fungal cultures. All the antimicrobial assays were performed in comparison with ES-treated and untreated replica. SNP-imposed effects on the microbial surface were also visualized at the ultrastructural level using field emission scanning electron microscopy (FE-SEM).
The antifungal efficacy of SNPs
As mentioned earlier, the rationale of our study was to evaluate the broad-spectrum antimicrobial efficacy of SNPs against both the sulphur-susceptible and sulphur-resistant fungal pathogens. For the same purpose, synthesized SNPs were tested against two known sulphur-susceptible fungi, namely E. cichoracearum (powdery mildew) and Fusarium oxysporum, and one known sulphur-resistant fungus Aspergillus niger. A. niger is an ubiquitous fungal pathogen and is a mycotoxigenic food and feed contaminant and is the causative agent of invasive aspergillosis in immunocompromised patients (Pfohl-Leszkowicz and Manderville 2007), while F. oxysporum and E. cichoracearum are responsible for the wide range of vascular wilts (Baldwin 1950) and powdery mildews (Maraite and Meyer 1971), respectively, in plants. Earlier findings (Roy Choudhury et al. 2011) have revealed that MEC of the α-SNPs was as low as 50 ppm for F. oxysporum, while the MEC of α-SNPs was 400 ppm (data not shown). MEC and MIC of α-SNPs were 125 ppm and 8000 ppm, respectively, for A. niger. In contrast, MIC of α-SNPs against A. niger was 32 000 ppm (Fig. 4). At MEC, SNPs not only retarded the fungal growth but also inhibited any visible spore formation. Subsequent biochemical studies revealed that SNPs were capable of altering the lipid fatty acid contents and composition in fungi (Roy Choudhury et al. 2012b).
Fungal infestations of E. cichoracearum were also effectively controlled with SNP treatment, as revealed from the pot culture assay. At a concentration of 1000 ppm, α-SNPs sufficiently reduced mildew contaminations from the infected leaves (data not shown). Light microscope images revealed that at 1000 ppm, cleistothecia of E. cichoracearum were atrophied remarkably (Fig. 5a,b). The antifungal effect of α-SNPs on E. cichoracearum is yet to be seen. Various degrees of surface deformities were also observed for A. niger (data not shown) and F. oxysporum (Fig. 5c,d) under FE-SEM after treatment with SNPs.
Agar dilution method suggested that inhibition in radial growth of the fungi was directly proportional to the increase in concentration and reduction in particle size of SNPs. Spore germination slide bioassay for both A. niger and F. oxysporum revealed that at the subinhibitory concentrations, SNPs inhibited the sporulation completely (Roy Choudhury et al. 2011). Inhibition in spore formation can be correlated with the retardation in fungal growth and alteration in the lipid constituents. In each of the aforementioned bioassays, ES failed to control fungal growth and sporulation at the equivalent concentrations of SNPs. Moreover, micron-sized ES failed to impart any fungicidal activity even against the sulphur-susceptible fungi and proved to be barely fungistatic.
The antibacterial efficacy of SNPs
Recent work by our research group has showed the α-SNPs were highly effective against multidrug-resistant, Gram-negative bacilli (GNB; Roy Choudhury et al. 2012c). All the GNB isolates were found to possess a family of ‘carbapenamase’ enzyme called the New Delhi metallo-α-lactamase-1 (NDM-1). The GNB isolates harbouring NDM-1 are considered as the recent most threat for the medical science and research, because they are resistant to almost all the commercially viable α-lactamase inhibitors. SNPs inhibited the growth of these bacterial isolates at a concentration as low as ≤18·82 mg l−1 (equivalent to 4000 ppm). The antimicrobial efficacy of α-SNPs was also tested against the Gram-positive bacterial isolates. The growth of Gram-positive organisms was inhibited at a concentration as low as 2·36 mg l−1 (equivalent to 500 ppm). The α-SNPs were also capable of controlling the bacterial growth (not reported yet), however, at a concentration higher than α-SNPs, while ES failed to impart any antibacterial effect against the tested isolates.
Exact mode of action of these novel AAs is yet to be examined in comparison with ES. Further biochemical and biophysical studies are urgently required to determine the route and site of action for these novel microbicides.
The acaricidal efficacy of SNPs
Earlier studies have reported that ES has an acaricidal (efficacy to kill mites) efficacy against the black currant gall mites (Eriophyes ribis) and red spider mites (Tetranychus urticae; Goodwin and Martin 1928; Auger et al. 2003). SNPs owing to their supramolecular reactivity are expected to control mite infections in agricultural and horticultural fields. Researchers at the entomology section of the Indian Agricultural Research Institute (IARI), India, have already started working on this field.
Biocompatibility Studies with SNPs
With a small size and a large surface area, nanoparticles are able to generate unwarranted reactive oxygen species in the environment (Nel et al. 2006). Inhaled or instilled, these ultrafine particles (below 100 nm) can induce severe pulmonary infections, oxidative stress and injuries, inflammation, genotoxicity (mutation of DNA and proteins) or cytotoxicity. Several metallic (silver, copper, etc.), nonmetallic (carbon, silica, etc.) and oxide (titanium dioxide, zinc oxide, etc.) nanoparticles have already been reported for their biological efficacy. However, upon infusion through biomembrane barriers (blood–brain barrier), these nanoparticles induce severe toxic effects on different cell structures and mutate different macromolecules (Hussain et al. 2005). Bioadverse attributes of these nanoparticles, hence, demand their restriction in use as pesticide and drug formulations. Moreover, with decreasing size, nanoparticles might induce several health disorders, even if the same material is relatively inert or biosafe in bulk form (e.g. carbon black or TiO2). Hence, the toxic effects (if any) of SNPs on the plant and mammalian cells were also needed to be determined, although ES is known to be a nontoxic substance (only a respiratory irritant) even at 50 000 ppm (Krieger 2010).
Effects of SNPs on phytophysiology
Phytotoxic and agro-beneficial properties of SNPs were evaluated on mung (Vigna radiata) plants (P. Patra, S. Roy Choudhury, S. Mandal, A. Basu, A. Goswami, R. Gogoi, C. Srivastava, K. Kumar and M. Gopal, 2012, accepted for publication in Springer Proceedings). Mung seeds were germinated with an increasing concentration gradient of SNPs. The extent of phytotoxicity (if any) was assessed on physical features (relative root and shoot length, dry weight and area of leaves), photosynthetic pigments (chlorophyll, carotene and xanthophyll contents) and mitochondrial stress indicator level (thiol, and glutathione reductase). A simultaneous study was undertaken to understand the effect of SNPs on overall plant growth and nutrition. The nutritive values of SNPs were determined in terms of total lipid, carbohydrate and protein contents. The α-SNPs were found to increase root/shoot length and area of the second leaf of the treated plants. In contrast, α-SNPs were failed to impart any marked effects on the aforementioned features of plants. Moreover, it is worth mentioning that, at the highest applied concentrations, both the SNPs significantly induced the level of intracellular thiol content (data not shown). Enhancement in thiol content insinuates that SNPs might work in an identical route of ES.
Cytotoxicity of SNPs against the human derived lung-fibroblast cell line
Cytotoxic effects of α-SNPs on the human derived hepatoma (HepG2) cell line (ATCC HB8065) have been reported recently using WST-1 (water-soluble tetrazolium) dye–based assay (Roy Choudhury et al. 2012c). Initial findings revealed that even at the highest applied concentration (94·08 mg l−1), α-SNPs failed to impart any marked toxicity to the HepG2 cells. In contrast, the cell viability of HepG2 cells was gradually decreased from 100·04 to 58·2% upon serial exposure to α-SNPs (S. Roy Choudhury et al., unpublished data). Preferential toxicity to the microbial cells and insignificant cytotoxicity to the mammalian cells insinuate the emergence of SNPs as the future putative antimicrobial drug. However, in vivo cytotoxicity (both acute and chronic toxicity) studies with SNPs on both the animal and plant models should have been performed prior to their commercialization as a viable drug or pesticide.
Cost-effectiveness of SNPs
In comparison with the conventional AAs, nanodrug and pesticides would be required in very small amounts; hence, the cost of manufacturing and disseminating these nanoproducts is expected to be much cheaper. Cost-effectiveness of these nanoproducts is also expected to make them more accessible and affordable especially for the developing countries. The technology for the preparation and evaluation of antimicrobial efficacy of SNPs by our research group has already been patented with Institute of Technology Management Unit of the IARI, India (Gopal et al. 2011). Investment for innovative drugs and pesticides is a significant component of government expenditure and is becoming an integral part of the technology regulatory approval process. This may be achieved either by the active cooperation of industries or by strict evaluation of expert government advisory committees. It is expected that the concerned regulatory boards will not overlook the importance of safety and cost-effectiveness, even though the product is agro-medically important.
The physicochemical studies have been attempted to revive the antimicrobial efficacy of sulphur in its nanoform employing simple methods and minimal equipment facilities for their preparation. At nanoscale, sulphur particles were proved to be better AAs in comparison with ES. SNPs are able to inhibit the growth of both the sulphur-susceptible and sulphur-resistant microbial pathogens effectively at their MECs. Moreover, biocompatible polymers used as the surface-stabilizing agents reduced the toxicity of SNPs during application and ensure their targeted delivery into the microbial cytosol. Initial results of the biosafety assays revealed no significant cytotoxicity of SNPs against the mung plants and human derived lung-fibroblast cell line. Further biochemical and biophysical studies are urgently warranted to explore the site and mode of antimicrobial activity of SNPs in comparison with the ES.
Samrat Roy Choudhury is a recipient of Senior Research Fellowship [s/93 (0143)/12 EMR-I] from the Council of Scientific and Industrial Research (CSIR), India (2012–2013). The research was funded by NAIP-ICAR-World Bank (Comp-4/C3004/2008-09), ICAR-National Fund (NFBSFARA/GB-2019/2011-12) and Department of Biotechnology (DBT), Govt. of India (BT/BIPP0439/11/10, BT/PR15217/NNT/28/506/2011, BT/PR9050/NNT/28/21/2007 and BT/PR8931/NNT/28/07/2007) and ISI plan project for 2008–2011. The authors would like to thank Mrs Indrani Roy for critical reading of the manuscript.