Ashwani Kumar Sharma

Multifunctional self-assembled cationic peptide nanostructures efficiently carry plasmid DNA in vitro and exhibit antimicrobial activity with minimal toxicity

In this study, a modified dehydropeptide, Boc-FΔF-εAhx-OH, was conjugated with an aminoglycoside antibiotic, neomycin, to construct a multifunctional conjugate, Pep-Neo. The amphiphilic conjugate (Pep-Neo) was able to self-assemble into cationic nanostructures in an aqueous solution at low concentrations. Nanostructure formation was evidenced by TEM and dynamic light scattering analyses. The average hydrodynamic diameter of the self-assembled Pep-Neo nanostructures was found to be ∼279 nm with a zeta potential of +28 mV. The formation of nanostructures with a hydrophobic core and cationic hydrophilic shell resulted in an increased local concentration of cationic charge (ca. in 50% aqueous methanol, i.e. disassembled structure, zeta potential decreased to +17.6 mV), leading to efficient interactions with negatively charged plasmid DNA (pDNA). The size and zeta potential of the resulting Pep-Neo/pDNA complex were found to be ∼154 nm and +19.4 mV, respectively. Having been characterized by physicochemical techniques, the complex was evaluated for its toxicity and ability to deliver nucleic acid therapeutics. The flow cytometry results on MCF-7 cells revealed that Pep-Neo/pDNA complex transfected ∼27% cells at a w/w ratio of 66.6 while the standard transfection reagent, Lipofectamine, could transfect only ∼15% cells. MTT and hemolysis assays showed the non-toxic nature of the projected conjugate at various concentrations. Further, these nanostructures were shown to encapsulate hydrophobic drugs in the core. Finally, Pep-Neo nanostructures showed efficient antibacterial activity against different strains of Gram-positive and -negative bacteria. Interestingly, unlike neomycin, which is highly effective against Gram-negative bacteria, these nanostructures showed considerably high efficiency against Gram-positive strains, highlighting the promising potential of these nanostructures for various biomedical applications.

Synthesis and antimicrobial activity of aminoglycoside-conjugated silica nanoparticles against clinical and resistant bacteria

Functionalization of silica nanoparticles with different cationic moieties makes them suitable for being an effective antimicrobial agent against various clinical pathogenic microbes. Here, we report the synthesis, characterization and evaluation of aminoglycoside-conjugated silica nanoparticles [S–X NPs, where X = gentamicin (G), neomycin (N) or kanamycin (K)] for their antimicrobial activity against clinical pathogens and kanamycin-resistant E. coli. These functionalized silica nanoparticles exhibited enhanced broad-spectrum antimicrobial activity against clinical Gram-positive and Gram-negative bacteria as well as kanamycin-resistant E. coli strain with minimal cytotoxicity. The results show the potential of these conjugates to combat drug resistance.

Azobenzene-aminoglycoside: Self-assembled smart amphiphilic nanostructures for drug delivery.

Here, we have designed and synthesized a novel cationic amphiphilic stimuli-responsive azobenzene-aminoglycoside (a small molecule) conjugate, Azo-AG 5, and characterized it by UV and FTIR. Light responsive nature of Azo-AG 5 was assessed under UV-vis light. Self- assembly of Azo-AG 5 in aqueous solutions into nanostructures and their ability to act as drug carrier were also investigated. The nanostructures of Azo-AG 5 showed average hydrodynamic diameter of ∼ 255 nm with aminoglycoside moiety (neomycin) and 4-dimethylaminoazobenzene forming hydrophilic shell and hydrophobic core, respectively. In the hydrophobic core, eosin and aspirin were successfully encapsulated. Dynamic light scattering (DLS) measurements demonstrated that the nanoassemblies showed expansion and contraction on successive UV and visible light irradiations exhibiting reversible on-off switch for controlling the drug release behavior. Similar behavior was observed when these nanostructures were subjected to pH-change. In vitro drug release studies showed a difference in UV and visible light-mediated release pattern. It was observed that the release rate under UV irradiation was comparatively higher than that observed under visible light. Further, azoreductase-mediated cleavage of the azo moiety in Azo-AG 5 nanoassemblies resulted in the dismantling of the structures into aggregated microstructures. Azo-AG 5 nanostructures having positive surface charge (+9.74 mV) successfully interacted with pDNA and retarded its mobility on agarose gel. Stimuli responsiveness of nanostructures and their on-off switch like behavior ensure the great potential as controlled drug delivery systems and in other biomedical applications such as colon-specific delivery and gene delivery.

Enzyme sensitive smart inulin-dehydropeptide conjugate self-assembles into nanostructures useful for targeted delivery of ornidazole

Molecular self-assembly of biodegradable amphiphilic polymers allows rational design of biocompatible nanomaterials for drug delivery. Use of substituted polysaccharides for such applications offers the ease of design and synthesis, and provides higher biofunctionality and biocompatibility to nanomaterials. The present work focuses on the synthesis, characterization and potential biomedical applications of self-assembled polysaccharide-based materials. We demonstrated that the synthesized amphiphilic inulin self-assembled in aqueous medium into nanostructures with average size in the range of 146-486nm and encapsulated hydrophobic therapeutic molecule, ornidazole. Hydrophophic dehydropeptide was conjugated with inulin via a biocompatible ester linkage. Dehydrophenylalanine, an unusual amino acid, was incorporated in the peptide to make it stable at a broader range of pH as well as against proteases. The resulting core-shell type of nanostructures could encapsulate ornidazole in the hydrophobic core and released it in a controlled fashion. By taking the advantage of inulin, which gets degraded in the colon by colonic bacteria, the effect of enzyme, inulinase, present in the microflora of the large intestine, on inulin-peptide degradation followed by drug release has been studied. Altogether, small peptide conjugated to inulin offers novel scaffold for the future design of nanostructures with potential applications in the field of targeted drug delivery.

Self-assembled amphiphilic mixed α/β-tetrapeptoid nanostructures as promising drug delivery vehicles

We are currently investigating self-assembled nanostructures from short oligomers of β-amino acids and their subsequent use as drug delivery systems. Successful demonstration by our group of self-assembled nanovesicular structures, generated from a β-alanine homotetramer, as carriers for the hydrophobic drug L-Dopa has prompted us to design even more efficient drug delivery systems from the β-amino acid containing peptoid. In the present study, we report a systematic study on the self-assembly of an amphiphilic mixed α/β-tetrapeptoid, H-βAla-βAla-Lys-βAla-NH2. The secondary structure of the tetrapeptoid in a self-assembled state is studied using Fourier Transform Infrared (FTIR) spectroscopy, and Circular Dichroism (CD). These studies are strongly indicative of the presence of the β-sheet conformation, a requisite for self-assembly. Dynamic Light Scattering (DLS) measurements reveal that the tetrapeptoid self-assembles to stable nanovesicular structures having an average hydrodynamic size of ∼343 nm and a zeta potential of ∼21 mV. The morphology of the self-assembled cationic structures in aqueous solutions was studied by Transmission Electron Microscopy (TEM). Supportive evidence regarding the conformation of the peptoid is provided by a conformational search using computational methods. The theoretically predicted minimum energy structure depicts a curved strand type of conformation. Four of these curved strands are then subjected to a geometry optimization that resulted in the formation of an assembly with a hydrophobic core and a hydrophilic exterior. Successful entrapping of hydrophobic molecules of drugs, aspirin and L-Dopa, into the generated nanostructures is revealed by DLS measurements and TEM micrographs. Absorption spectroscopic studies were conducted to study the interaction between the generated nanostructures and each of the hydrophobic drugs, aspirin, and L-Dopa with significant association constant values of the order of 106 to 105, respectively. These described nanostructures from the tetrapeptoid may be developed into efficient drug delivery systems.

Amphiphilic azobenzene-neomycin conjugate self-assembles into nanostructures and transports plasmid DNA efficiently into the mammalian cells

The present study demonstrates the use of self-assembled nanostructures of cationic amphiphilic azobenzene-neomycin (a small molecule) conjugate, Azo-Neo, as delivery vector for plasmid DNA. These nanostructures efficiently condensed nucleic acid and formed more compact nanoassemblies. DLS analysis showed size and zeta potential of the resulting Azo-Neo/pDNA nanoassemblies ∼153.7nm and +7.26mV, respectively. The nanoassemblies were characterized by physicochemical techniques and evaluated for its toxicity and ability to deliver nucleic acid therapeutics. The flow cytometry results on MCF-7 and HEK293T cells revealed that Azo-Neo/pDNA nanoassemblies transfected ∼31% and 23% cells, respectively, at a w/w ratio of 250, while the standard transfection reagent, bPEI/pDNA complex, could transfect only ∼21% and 29% cells, respectively, at its best w:w ratio of 2.3. MTT and hemolysis assays showed the non-toxic nature of the projected nanoassemblies and nanostructures, respectively, at various concentrations. Further, Azo-Neo nanostructures showed efficient antibacterial activity against different strains, laboratory strain of Staphylococcus aureus (MTCC 740) as well as MRSA strains (Staphylococcus aureus ATCC 33591, ATCC 43300 and ATCC 700699). These results ensure the great potential of these nanostructures in gene delivery and antimicrobial applications.

Enhanced antibacterial activity of tetramethylguanidinium-conjugated linear polyethylenimine polymers

ABSTRACT In the present study, varying amounts of tetramethylguanidinium moiety have been conjugated to linear polyethylenimine to obtain linear polyethylenimine-tmg (LPTG) polymers. Incorporation of hydrophobic and highly basic moiety in the polymeric backbone resulted in the significant improvement in the antibacterial activity which was confirmed by zone of inhibition and MIC assays. Further, the results of transmission electron microscopy and confocal studies revealed that the projected LPTG polymers possessed higher antibacterial activity than the native polymer. In addition, these modified polyethylenimine (PEI) polymers were capable of reducing auric chloride into stable gold nanoparticles. These polyamine-stabilized gold nanoparticles can be used in various biomedical applications. GRAPHICAL ABSTRACT

Multifunctional biosynthesized silver nanoparticles exhibiting excellent antimicrobial potential against multi-drug resistant microbes along with remarkable anticancerous properties

This study demonstrates the therapeutic potential of silver nanoparticles (AgNPs), which were biosynthesized using the extracts of Citrus maxima plant. Characterization through UV-Vis spectrophotometry, Dynamic Light Scattering (DLS), Fourier Transform Infrared spectroscopy (FTIR), X-ray Diffraction (XRD) and Transmission Electron Microscopy (TEM) confirmed the formation of AgNps in nano-size range. These nanoparticles exhibited enhanced antioxidative activity and showed commendable antimicrobial activity against wide range of microbes including multi-drug resistant bacteria that were later confirmed by TEM. These particles exhibited minimal toxicity when cytotoxicity study was performed on normal human lung fibroblast cell line as well as human red blood cells. It was quite noteworthy that these particles showed remarkable cytotoxicity on human fibrosarcoma and mouse melanoma cell line (B16-F10). Additionally, the apoptotic topographies of B16-F10 cells treated with AgNps were confirmed by using acridine orange and ethidium bromide dual dye staining, caspase-3 assay, DNA fragmentation assay followed by cell cycle analysis using fluorescence-activated cell sorting. Taken together, these results advocate promising potential of the biosynthesized AgNps for their use in therapeutic applications.

Fabrication of cationic nanostructures from short self-assembling amphiphilic mixed α/β-pentapeptide: Potential candidates for drug delivery, gene delivery, and antimicrobial applications

The present article describes designing and fabrication of nanostructures from a mixed α/β-pentapeptide, Lys-βAla-βAla-Lys-βAla, which majorly contains non-natural β-alanine residues in the backbone with two α-lysine residues at 1- and 4-positions. The amphiphilic pentapeptide showed the ability to self-assemble into cationic nanovesicles in an aqueous solution. The average size of peptide nanostructures was found to be ~270 nm with a very high cationic charge of ~+40 mV. TEM micrographs revealed the average size of the same nanostructures ~80 nm bearing vesicular morphology. CD and FTIR spectroscopic studies on self-assembled pentapeptide hinted at random coil conformation which was also correlated with conformational search program using Hyper Chem 8.0. The pentapeptide nanostructures were then tested for encapsulation of hydrophobic model drug moieties, L-Dopa, and curcumin. Transfection efficiency of the generated cationic nanostructures was evaluated on HEK293 cells and compared the results with those obtained in the presence of chloroquine. The cytotoxicity assay performed using MTT depicted ~75-80% cell viability. The obtained nanostructures also gave positive results against both Gram-negative and Gram-positive bacterial strains. Altogether the results advocate the promising potential of the pentapeptide foldamer, H-Lys-βAla-βAla-Lys-βAla-OEt, for drug and gene delivery applications along with the antimicrobial activity.