Kishore Paknikar

Extracellular synthesis of silver nanoparticles by a silver-tolerant yeast strain MKY3

Silver nanoparticles in the size range of 2-5 nm were synthesized extracellularly by a silver-tolerant yeast strain MKY3, when challenged with 1 mM soluble silver in the log phase of growth. The nanoparticles were separated from dilute suspension by devising a new method based on differential thawing of the sample. Optical absorption, transmission electron microscopy, x-ray diffraction and x-ray photoelectron spectroscopy investigations confirmed that metallic (elemental) silver nanoparticles were formed. Extracellular synthesis of nanoparticles could be highly advantageous from the point of view of synthesis in large quantities and easy downstream processing.

Cellular responses induced by silver nanoparticles: In vitro studies.

A systematic study on the in vitro interactions of 7-20 nm spherical silver nanoparticles (SNP) with HT-1080 and A431 cells was undertaken as a part of an on-going program in our laboratory to develop a topical antimicrobial agent for the treatment of burn wound infections. Upon exposure to SNP (up to 6.25 microg/mL), morphology of both the cell types remained unaltered. However, at higher concentrations (6.25-50 microg/mL) cells became less polyhedral, more fusiform, shrunken and rounded. IC(50) values for HT-1080 and A431 as revealed by XTT assay were 10.6 and 11.6 microg/mL, respectively. When the cells were challenged with approximately 1/2 IC(50) concentration of SNP (6.25 microg/mL), clear signs of oxidative stress, i.e. decreased GSH ( approximately 2.5-folds in HT-1080, approximately 2-folds in A431) and SOD ( approximately 1.6-folds in HT-1080, 3-folds in A431) as well as increased lipid peroxidation ( approximately 2.5-folds in HT-1080, approximately 2-folds in A431) were seen. Changes in the levels of catalase and GPx in A431 cells were statistically insignificant in both cell types. DNA fragmentation in SNP-exposed cells suggested apoptosis. When the apoptotic thresholds of SNP were monitored with caspase-3 assay the concentrations required for the onset of apoptosis were found to be much lower (0.78 microg/mL in HT-1080, 1.56 microg/mL in A431) than the necrotic concentration (12.5 microg/mL in both cell types). These results can be used to define a safe range of SNP for the intended application as a topical antimicrobial agent after appropriate in vivo studies.

Silver nanoparticles in therapeutics: development of an antimicrobial gel formulation for topical use.

Silver is an effective antimicrobial agent with low toxicity, which is important especially in the treatment of burn wounds where transient bacteremia is prevalent and its fast control is essential. Drugs releasing silver in ionic forms are known to get neutralized in biological fluids and upon long-term use may cause cosmetic abnormality, e.g., argyria and delayed wound healing. Given its broad spectrum activity, efficacy and lower costs, the search for newer and superior silver based antimicrobial agents is necessary. Among the various options available, silver nanoparticles have been the focus of increasing interest and are being heralded as an excellent candidate for therapeutic purposes. This report gives an account of our work on development of an antimicrobial gel formulation containing silver nanoparticles (SNP) in the size range of 7-20 nm synthesized by a proprietary biostabilization process. The typical minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against standard reference cultures as well as multidrug-resistant organisms were 0.78-6.25 microg/mL and 12.5 microg/mL, respectively. Gram-negative bacteria were killed more effectively (3 log(10) decrease in 5-9 h) than Gram-positive bacteria (3 log(10) decrease in 12 h). SNP also exhibited good antifungal activity (50% inhibition at 75 microg/mL with antifungal index 55.5% against Aspergillus niger and MIC of 25 microg/mL against Candida albicans). When the interaction of SNP with commonly used antibiotics was investigated, the observed effects were synergistic (ceftazidime), additive (streptomycin, kanamycin, ampiclox, polymyxin B) and antagonistic (chloramphenicol). Interestingly, SNP exhibited good anti-inflammatory properties as indicated by concentration-dependent inhibition of marker enzymes (matrix metalloproteinase 2 and 9). The post agent effect (a parameter measuring the length of time for which bacterial growth remains suppressed following brief exposure to the antimicrobial agent) varied with the type of organism (e.g., 10.5 h for P. aeruginosa, 1.3 h for Staphylococcus sp. and 1.6 h for Candida albicans) indicating that dose regimen of the SNP formulation should ensure sustained release of the drug. To meet this requirement, a gel formulation containing SNP (S-gel) was prepared. The antibacterial spectrum of S-gel was found to be comparable to that of a commercial formulation of silver sulfadiazine, albeit at a 30-fold less silver concentration. As part of toxicity studies, localization of SNP in Hep G2 cell line, cell viability, biochemical effects and apoptotic/necrotic potential were assessed. It was found that SNP get localized in the mitochondria and have an IC(50) value of 251 microg/mL. Even though they elicit an oxidative stress, cellular antioxidant systems (reduced glutathione content, superoxide dismutase, catalase) get triggered and prevent oxidative damage. Further, SNP induce apoptosis at concentrations up to 250 microg/mL, which could favor scarless wound healing. Acute dermal toxicity studies on SNP gel formulation (S-gel) in Sprague-Dawley rats showed complete safety for topical application. These results clearly indicate that silver nanoparticles could provide a safer alternative to conventional antimicrobial agents in the form of a topical antimicrobial formulation.

Nanotoxicology and in vitro studies: The need of the hour

Nanotechnology is considered as one of the key technologies of the 21st century and promises revolution in our world. Objects at nano scale, take on novel properties and functions that differ markedly from those seen in the corresponding bulk counterpart primarily because of their small size and large surface area. Studies have revealed that the same properties that make nanoparticles so unique could also be responsible for their potential toxicity. Nanotechnology is rapidly advancing, with more than 1000 nanoproducts already on the market. Considering the fact that intended as well as unintended exposure to nanomaterials is increasing and presently no clear regulatory guideline(s) on the testing/evaluation of nanoparticulate materials are available, the in vitro toxicological studies become extremely relevant and important. This review presents a summary of nanotoxicology and a concise account of the in vitro toxicity data on nanomaterials. For nanomaterials to move into the applications arena, it is important that nanotoxicology research uncovers and understands how these multiple factors influence their toxicity so that the ensuing undesirable effects can be avoided.

Interactions of silver nanoparticles with primary mouse fibroblasts and liver cells

Primary cells are ideal for in vitro toxicity studies since they closely resemble tissue environment. Here, we report a detailed study on the in vitro interactions of 7-20 nm spherical silver nanoparticles (SNP) with primary fibroblasts and primary liver cells isolated from Swiss albino mice. The intended use of silver nanoparticles is in the form of a topical antimicrobial gel formulation for the treatment of burns and wounds. Upon exposure to SNP for 24 h, morphology of primary fibroblasts and primary liver cells remained unaltered up to 25 microg/mL and 100 microg/mL SNP, respectively, although with minor decrease in confluence. IC(50) values for primary fibroblasts and primary liver cells as revealed by XTT assay were 61 microg/mL and 449 microg/mL, respectively. Ultra-thin sections of primary cells exposed to 1/2 IC(50) SNP for 24 h, visualized under Transmission electron microscope showed the presence of dark, electron dense, spherical aggregates inside the mitochondria, and cytoplasm, probably representing the intracellular SNP. When the cells were challenged with approximately 1/2 IC(50) concentration of SNP (i.e. 30 microg/mL and 225 microg/mL for primary fibroblasts and primary liver cells, respectively), enhancement of GSH (approximately 1.2 fold) and depletion of lipid peroxidation (approximately 1.4 fold) were seen in primary fibroblasts which probably protect the cells from functional damage. In case of primary liver cells; increased levels of SOD ( approximately 1.4 fold) and GSH ( approximately 1.1 fold) as compared to unexposed cells were observed. Caspase-3 activity assay indicated that the SNP concentrations required for the onset of apoptosis were found to be much lower (3.12 microg/mL in primary fibroblasts, 12.5 microg/mL in primary liver cells) than the necrotic concentration (100 microg/mL in primary fibroblasts, 500 microg/mL in primary liver cells). These observations were confirmed by CLSM studies by exposure of cells to 1/2 IC(50) SNP (resulting in apoptosis) and 2 x IC(50)) cells (resulting in necrosis). These results clearly suggest that although silver nanoparticles seem to enter the eukaryotic cells, cellular antioxidant mechanisms protect the cells from possible oxidative damage. This property, in conjunction with the finding that primary cells possess much higher SNP tolerance than the concentration in the gel (approximately 20 microg/g), indicates preliminary safety of the formulation and warrants further study for possible human application.

Perspectives for nano-biotechnology enabled protection and nutrition of plants.

Indiscriminate use of pesticides and fertilizers causes environmental pollution, emergence of agricultural pests and pathogens, and loss of biodiversity. Nanotechnology, by virtue of nanomaterial related properties, has potential agro-biotechnological applications for alleviation of these problems. The literature pertaining to the role of nanotechnology in plant and soil systems demonstrates that nanomaterials may assist in a) the controlled release of agrochemicals for nutrition and protection against pests and pathogens, b) delivery of genetic material, c) sensitive detection of plant disease and pollutants and d) protection and formation of soil structure. For instance, porous silica (15nm) and biodegradable, polymeric chitosan (78nm) nanoparticles displayed slow release of encapsulated pesticide and fertilizer, respectively. Further, nanosized gold (5-25nm) delivered DNA to plant cells while iron oxide (30nm) based nanosensors detected pesticides at minute levels. These functions assist the development of precision farming by minimizing pollution and maximizing the value of farming practice.

Biosorption of lead and zinc from solutions using Streptoverticillium cinnamoneum waste biomass

Mycelial wastes of microbial origin from fermentation industries have been recognized as potential biosorbents for decontamination of waste waters containing heavy metals. Dried, nonliving, granulated biomass of Streptoverticillium cinnamoneum was used for the recovery of lead and zinc from solutions. It was found that pretreatment of the biomass with boiling water for 15 min increased the biosorption of lead and zinc by 52 and 41%, respectively. The optimum pH range for lead uptake was 3.5-4.5 while for zinc it was 5.0-6.0. The lead and zinc adsorption data when applied to Freundlich and Langmuir isotherm equations showed good correlation (r2 = 0.97) and hence equal conformity to both models. The Scatchard plots indicated clearly that more than one type of binding sites were involved in the adsorption of lead and zinc by the biomass. The maximum loading capacity of S. cinnamoneum biomass was found to be 57.7 mg/g for lead and 21.3 mg/g for zinc with boiling water pretreatment. The loaded metals could be desorbed effectively with dilute hydrochloric acid, nitric acid and 0.1 M EDTA. Treatment with 0.1 M sodium carbonate permitted reuse of the desorbed biomass although the metal loading capacity in the subsequent cycles decreased by 14-37%. The metal biosorbent granules prepared are a value-added product that has the potential for removal/recovery of lead and zinc from dilute solutions on a commercial scale.

Microbial Synthesis of Semiconductor PbS Nanocrystallites

The use of microbes as producers of semiconductor nanocrystals is demonstrated. When torulopsis yeast is challenged with lead, it builds intracellular spherical crystallites of PbS, 2-5 nm in diameter (see Figure for an HR-TEM image) and pure by X-ray diffraction. The crystals, which can be isolated by freeze-thawing, show a sharp absorption maximum at 330 nm, corresponding to a bandgap of 3.75 eV.

Recovery of gold from solutions using Cladosporium cladosporioides biomass beads

A fungal isolate, Cladosporium cladosporioides was used for biosorption of gold from solutions. The fungal biomass was granulated by mixing it with a matrix derived from keratinous material of natural origin. The resulting biosorbent beads adsorbed 100 mg gold per gram from a solution of gold. Maximum biosorption of gold (80%) occurred under acidic pH conditions (pH 1–5). The contact time required for 80% biosorption of gold could be reduced to 20 min by pre-soaking the beads in deionized distilled water. Gold uptake by the beads was found to increase linearly as a function of metal concentration. The data could be fitted into Freundlich model of adsorption isotherms. A column packed with 3 g biosorbent beads was used for continuous adsorption of gold. The gold loading capacity obtained in the system was to the tune of 110 mg g−1. Gold was removed from an electroplating unit effluent with 55% efficiency in batch experiment and the loading capacity was 36 mg g−1. It was found that gold could be removed from solutions in the presence of carbonate and complexing agents like citrate, sulfite and thiosulfate albeit with less efficiency. The beads were found to biodegrade in soil in about 140 days. The process, thus, has the prospect of becoming an efficient and environmental friendly method to recover gold from aqueous solutions.

Applications of bacterial cellulose and its composites in biomedicine

Bacterial cellulose produced by few but specific microbial genera is an extremely pure natural exopolysaccharide. Besides providing adhesive properties and a competitive advantage to the cellulose over-producer, bacterial cellulose confers UV protection, ensures maintenance of an aerobic environment, retains moisture, protects against heavy metal stress, etc. This unique nanostructured matrix is being widely explored for various medical and nonmedical applications. It can be produced in various shapes and forms because of which it finds varied uses in biomedicine. The attributes of bacterial cellulose such as biocompatibility, haemocompatibility, mechanical strength, microporosity and biodegradability with its unique surface chemistry make it ideally suited for a plethora of biomedical applications. This review highlights these qualities of bacterial cellulose in detail with emphasis on reports that prove its utility in biomedicine. It also gives an in-depth account of various biomedical applications ranging from implants and scaffolds for tissue engineering, carriers for drug delivery, wound-dressing materials, etc. that are reported until date. Besides, perspectives on limitations of commercialisation of bacterial cellulose have been presented. This review is also an update on the variety of low-cost substrates used for production of bacterial cellulose and its nonmedical applications and includes patents and commercial products based on bacterial cellulose.