Pallab Sanpui

The antibacterial properties of a novel chitosan–Ag-nanoparticle composite

Escherichia coli expressing recombinant green fluorescent protein was used to test the bactericidal efficacy of a newly synthesized chitosan-Ag-nanoparticle composite. The composite was found to have significantly higher antimicrobial activity than its components at their respective concentrations. The one-pot synthesis method led to the formation of small Ag nanoparticles attached to the polymer which can be dispersed in media of pH< or =6.3. The presence of a small percentage (2.15%, w/w) of metal nanoparticles in the composite was enough to significantly enhance inactivation of E. coli as compared with unaltered chitosan. Fluorescence spectroscopy indicated that bacterial growth stopped immediately after exposure of E. coli to the composite, with release of cellular green fluorescent protein into the medium at a faster rate than with chitosan. Fluorescence confocal laser scanning and scanning electron microscopy showed attachment of the bacteria to the composite and their subsequent fragmentation. Native protein gel electrophoresis experiments indicated no effect of the composite on bacterial proteins.

SIGNALING GENE CASCADE IN SILVER NANOPARTICLE INDUCED APOPTOSIS

Nanoscale materials are presently gaining much importance for biological applications especially in the field of medicine. The large numbers of nanomaterial based products that are currently being developed - with projected applications in medicine - have inspired a growing interest in exploring their impact on cellular gene expression. The present study examines the effects of silver nanoparticles (NPs) on genes expression in an endeavor to assess the fundamental mechanisms that contribute to silver NP induced programmed cell death. Here, we have used RT-PCR to study the gene expression, flow cytometry analyses to probe the extent of apoptosis (FACS) and atomic force microscopy (AFM) to follow the cell membrane topology change induced by Ag NPs. The gene expression study revealed that Ag NP induced p53-mediated apoptotic pathway through which most of the chemotherapeutic drugs trigger apoptosis (programmed cell death). The results also suggest that Ag NPs could be attributed as therapeutic agent for biomedical and pharmaceutical applications.

Fabrication of antibacterial silver nanoparticle—sodium alginate–chitosan composite films

A new and simple ecofriendly method for the synthesis of silver nanoparticles (Ag NPs) using a natural biopolymer, sodium alginate as both reducing and stabilizing agent is reported. The synthesized NPs were characterized using UV-Vis spectroscopy, transmission electron microscopy (TEM) and selected area electron diffraction pattern (SAED). The alginate capped NPs (Alg–Ag NPs) were found to be antibacterial. The Alg–Ag NPs were blended with varying amounts of chitosan to form polyelectrolyte complex that was cast into stable films. The films were characterized by field emission scanning electron microscopy (FESEM), optical microscopy, Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). The water uptake and mechanical properties of the films were also studied. The blended film demonstrated excellent antibacterial activity against both Gram negative and Gram positive bacteria with more activity against Gram positive bacteria. Thus, the developed films have a potential to be used for various antibacterial applications in biotechnology and biomedical fields.

Silver nanoparticles impregnated alginate-chitosan-blended nanocarrier induces apoptosis in human glioblastoma cells.

Herein, a green method for the development of a novel biodegradable silver nanoparticles (NPs) impregnated alginate-chitosan-blended nanocarrier (Ag NPs-Alg-Chi NC) is reported. The synthesis of Ag NPs-Alg-Chi NC is based on the polyelectrolyte complex formation between alginate and chitosan. The composite NC is characterized by ultraviolet-visible spectroscopy, transmission electron microscopy, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, and X-ray diffraction. The Ag NPs in the NC are found to elicit anticell proliferative effect on refractory U87MG (human glioblastoma) cells at IC50 of 2.4 μg mL(-1) for Ag NPs. The cell cycle analysis shows extensive DNA damage. Elevation in reactive oxygen species level indicates induction of oxidative stress in treated cells. Mitochondrial dysfunction in cell death is evident from the depolarization of mitochondrial membrane potential (ΔΨm ). Fluorescence and SEM images of the treated cells reveal nuclear and morphological changes characteristic of apoptosis, which is further confirmed by TUNEL assay. The induction of apoptosis at low concentration of Ag NPs present in Ag NPs-Alg-Chi NC in comparison with free Ag NPs makes it a promising tool for cancer therapy.

Green fluorescent protein for in situ synthesis of highly uniform Au nanoparticles and monitoring protein denaturation.

Purified recombinant green fluorescent protein (GFP) expressed in E. coli was used for single-step synthesis of gold nanoparticles (Au NPs) with extraordinary size specificity in aqueous medium. The fluorescence of GFP offered a probe for concomitant changes in the protein during the course of synthesis, in addition to the monitoring of the time-dependent formation of Au NPs by the surface plasmon resonance. Reaction of AuCl(4)(-) with the protein produced spherical Au NPs having diameters ranging from 5-70 nm. Remarkably, addition of 1.0x10(-5) M AgNO(3) in the medium produced uniform spherical Au NPs with particle diameter of 2.2+/-0.5 nm. Fluorescence spectroscopic measurements suggest that during synthesis of Au NPs in absence of AgNO(3), partial denaturation of the protein occurred resulting in the lowering of fluorescence intensity. On the other hand, when the NPs were synthesized in the presence of AgNO(3) complete denaturation of the protein with complete loss of fluorescence could be observed, which was further confirmed by native and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). However, use of AgNO(3) only resulted neither in the formation of NPs nor had any significant effect on the fluorescence of GFP.

Single-walled carbon nanotubes increase pandemic influenza A H1N1 virus infectivity of lung epithelial cells.

BackgroundAirborne exposure to nanomaterials from unintended occupational or environmental exposures or as a consequence of product use may lead to adverse health effects. Numerous studies have focused on single-walled carbon nanotubes (SWCNTs) and their ability to cause pulmonary injury related to fibrosis, and cancer; however few studies have addressed their impact on infectious agents, particularly viruses that are known for causing severe disease. Here we have demonstrated the ability of pristine SWCNTs of diverse electronic structure to increase the susceptibility of small airway epithelial cells (SAEC) to pandemic influenza A H1N1 infection and discerned potential mechanisms of action driving this response.MethodsSmall airway epithelial cells (SAEC) were exposed to three types of SWCNTs with varying electronic structure (SG65, SG76, CG200) followed by infection with A/Mexico/4108/2009 (pH1N1). Cells were then assayed for viral infectivity by immunofluorescence and viral titers. We quantified mRNA and protein levels of targets involved in inflammation and anti-viral activity (INFβ1, IL-8, RANTES/CCL5, IFIT2, IFIT3, ST3GAL4, ST6GAL1, IL-10), localized sialic acid receptors, and assessed mitochondrial function. Hyperspectral imaging analysis was performed to map the SWCNTs and virus particles in fixed SAEC preparations. We additionally performed characterization analysis to monitor SWCNT aggregate size and structure under biological conditions using dynamic light scattering (DLS), static light scattering (SLS).ResultsBased on data from viral titer and immunofluorescence assays, we report that pre-treatment of SAEC with SWCNTs significantly enhances viral infectivity that is not dependent on SWCNT electronic structure and aggregate size within the range of 106 nm – 243 nm. We further provide evidence to support that this noted effect on infectivity is not likely due to direct interaction of the virus and nanoparticles, but rather a combination of suppression of pro-inflammatory (RANTES) and anti-viral (IFIT2, IFIT3) gene/protein expression, impaired mitochondrial function and modulation of viral receptors by SWCNTs.ConclusionsResults of this work reveal the potential for SWCNTs to increase susceptibility to viral infections as a mechanism of adverse effect. These data highlight the importance of investigating the ability of carbon-nanomaterials to modulate the immune system, including impacts on anti-viral mechanisms in lung cells, thereby increasing susceptibility to infectious agents.

Synthesis, characterization and enhanced bactericidal action of a chitosan supported core–shell copper–silver nanoparticle composite

A simple two-step seed-mediated method has been developed to synthesize chitosan (CS)-supported core–shell metal nanoparticles (NPs) in aqueous solution. CS-supported copper NPs (CS–Cu NPs), when treated with silver nitrate (AgNO3) solution, resulted in Ag shells on Cu NP cores in the composite. The Cu@Ag NPs in the CS composite were characterized and found to be spherical in shape with an average particle size of 14.0 ± 3.4 nm. The antibacterial efficacy of the CS–Cu@Ag NP composite was evaluated on Gram-negative Escherichia coli and Gram-positive Bacillus cereus bacteria using turbidity measurement as well as flow-cytometry. Results demonstrated enhanced bactericidal activity of the composite. Analytical techniques, used to elucidate the mode of bactericidal action, revealed that the superior antibacterial activity of the CS–Cu@Ag NP composite was due to synergistic effects of CS and Cu@Ag NPs. The proposed mode of antibacterial action involves the electrostatic binding of CS to the bacterial cell wall and simultaneous interaction of Cu@Ag NPs with membrane proteins leading to perforation of the cell membrane and release of intracellular material.

Protein-based multifunctional nanocarriers for imaging, photothermal therapy, and anticancer drug delivery

We report a simple approach for fabricating plasmonic and magneto-luminescent multifunctional nanocarriers (MFNCs) by assembling gold nanorods, iron oxide nanoparticles, and gold nanoclusters within BSA nanoparticles. The MFNCs showed self-tracking capability through single- and two-photon imaging, and the potential for magnetic targeting in vitro. Appreciable T2-relaxivity exhibited by the MFNCs indicated favorable conditions for magnetic resonance imaging. In addition to successful plasmonic-photothermal therapy of cancer cells (HeLa) in vitro, the MFNCs demonstrated efficient loading and delivery of doxorubicin to HeLa cells leading to significant cell death. The present MFNCs with their multimodal imaging and therapeutic capabilities could be eminent candidates for cancer theranostics.

Keratin mediated attachment of stem cells to augment cardiomyogenic lineage commitment

The objective of this work was to develop a simple surface modification technique using keratin derived from human hair for efficient cardiomyogenic lineage commitment of human mesenchymal stem cells (hMSCs). Keratin was extracted from discarded human hair containing both the acidic and basic components along with the heterodimers. The extracted keratin was adsorbed to conventional tissue culture polystyrene surfaces at different concentration. Keratin solution of 500μg/ml yielded a well coated layer of 12±1nm thickness with minimal agglomeration. The keratin coated surfaces promoted cell attachment and proliferation. Large increases in the mRNA expression of known cardiomyocyte genes such as cardiac actinin, cardiac troponin and β-myosin heavy chain were observed. Immunostaining revealed increased expression of sarcomeric α-actinin and tropomyosin whereas Western blots confirmed higher expression of tropomyosin and myocyte enhancer factor 2C in cells on the keratin coated surface than on the non-coated surface. Keratin promoted DNA demethylation of the Atp2a2 and Nkx2.5 genes thereby elucidating the importance of epigenetic changes as a possible molecular mechanism underlying the increased differentiation. A global gene expression analysis revealed a significant alteration in the expression of genes involved in pathways associated in cardiomyogenic commitment including cytokine and chemokine signaling, cell-cell and cell-matrix interactions, Wnt signaling, MAPK signaling, TGF-β signaling and FGF signaling pathways among others. Thus, adsorption of keratin offers a facile and affordable yet potent route for inducing cardiomyogenic lineage commitment of stem cells with important implications in developing xeno-free strategies in cardiovascular regenerative medicine.

Heterologous expression and functional characterization of phytaspase, a caspase-like plant protease

Following the cloning and expression of tobacco (Nicotiana tabacum) phytaspase gene in Escherichia coli BL21, the recombinant protease was purified by affinity chromatography for further characterization. Circular dichroism (CD) spectroscopy and in silico analysis revealed structural similarities of recombinant phytaspase with other plant serine-proteases. Molecular docking studies showed favourable binding of synthetic peptide substrate for caspase 8 (Ac-VETD-AMC) to the reactive pocket of recombinant phytaspase indicating its potential in assessing functional activity of recombinant phytaspase. In silico findings were supported by caspase 8-like activity of purified phytaspase demonstrated in vitro. The Michaelis constant (KM) and specificity constant (kcat/KM) of phytaspase for hydrolyzing Ac-VETD-AMC were found to be 1.587μM and 4.67×103M-1min-1, respectively. Transient expression of phytaspase in lung epithelial adenocarcinoma cells (A549) resulted in reduced IC50 value of doxorubicin. This is the first report of functional expression of mature phytaspase in bacterial system as well as its transfection to sensitize A549 cells at lower doxorubicin concentration.