Diksha Jha

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.

Enhanced Antimicrobial Activity of Amine-Phosphonium (N-P) Hybrid Polymers Against Gram-Negative and Gram-Positive Bacteria

The authors report synthesis, characterization and evaluation of a series of linear polyethylenimine (lPEI)-grafted butyltriphenylphosphonium bromide (LBTP) polymers (N-P hybrid polymers) for their antimicrobial activity on various Gram-positive and Gram-negative bacteria. Polymers with ∼5.8–13.8% substitution of butyltriphenylphosphonium bromide (BTP) on the backbone of lPEI showed enhanced charge density as compared to native lPEI confirming the conjugation of BTP onto lPEI. These modified polymers displayed low hemolytic activity and excellent antimicrobial activity against these two types of bacteria with one of the modified polymers, LBTP-40, was found to exhibit high antimicrobial activity in all the strains. GRAPHICAL ABSTRACT

Zinc Oxide Nanoparticles Dispersed in Ionic Liquids Show High Antimicrobial Efficacy to Skin-Specific Bacteria

Zinc oxide (ZnO) nanoparticles have been shown in the literature to have antibacterial properties and have been widely used in antibacterial formulations. However, one of the problems with ZnO nanoparticles is their tendency to aggregate, thereby causing damage to normal cells and lowering their antibacterial efficacy during application. In this work, we have attempted to avoid this by using a combination of ZnO nanoparticles and ionic liquids, a class of low melting salts containing organic cations and organic/inorganic anions that show antibacterial property as well, and tested the antibacterial activity of this dispersion. ZnO nanoparticles of 60 nm were dispersed in two different ionic liquids-choline acetate (IL1) and 1-butyl-3-methylimidazolium chloride (IL2)-to achieve high dispersibility, whereas ZnO dispersed in phosphate-buffered saline was taken as a control. These dispersions were tested on four strains- Escherichia coli, Bacillus subtilis, Klebsiella pneumoniae, and Staphylococcus epidermidis. Maximum efficiency was obtained for ZnO nanoparticles dispersed in imidazolium-based ionic liquids against skin-specific S. epidermidis. Skin infections induced by S. epidermidis are prevalent in hospital-acquired diseases. In most cases, traditional antibiotic-based therapies fail to combat such infections. Our strategy of developing a dispersion of ZnO nanoparticles in ionic liquids shows superior antibacterial efficacy in comparison to that shown individually by ZnO nanoparticles or ionic liquids. We have also established that the mechanism of killing this skin-specific bacterium is possibly through the production of reactive oxygen species leading to bacterial cell lysis. Further, we showed that this formulation is biocompatible and nontoxic to normal keratinocyte cells even under coculture conditions.

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.

Evaluation of antimicrobial activity and cytotoxicity of pegylated aminoglycosides

Here, in this study, we have investigated the antibacterial activity and cytotoxicity of pegylated aminoglycosides (gentamicin, kanamycin, and neomycin). The antibacterial activity of pegylated aminoglycosides, prepared in two different ratios (1:1 and 1:2, aminoglycoside:polyethylene glycol), was determined against some common pathogens, namely, Gram-positive (Bacillus cereus, Staphylococcus aureus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa), by zone of inhibition and minimum inhibitory concentration assays. The activity of pegylated aminoglycosides was found to be decreased as compared to their respective native aminoglycosides. Moreover, aminoglycoside:polyethylene glycol (1:1) derivatives showed much higher activity as compared to aminoglycoside:polyethylene glycol (1:2) derivatives, that is, an increase in the content of polyethylene glycol decreased the activity considerably. This decrease in antibacterial activity was found to be the most prominent in Gram-positive bacteria, S. aureus. On the other hand, pegylation significantly decreased the cytotoxicity as determined by hemolysis and MTT assays. These results advocate exploration of different types of crosslinkers consisting of a small degree of amphiphilicity, which could facilitate better interactions with the bacterial cell membranes and inhibit induction of bacterial resistance.

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.

Bifunctionally engineered polyethylenimines as efficient DNA carriers and antibacterials against resistant pathogens

In this study, we have designed and developed two series of bifunctional conjugates by tethering polyethylenimine with streptomycin. By varying the amount of streptomycin, conjugates, polyethylenimine-streptomycin, have been synthesized and characterized spectroscopically. Gel electrophoresis assay revealed a slight decrease in the cationic charge density on the conjugates as these retarded the mobility of pDNA at higher w/w ratios. Further, transfection studies showed that both the series of conjugates transfected the mammalian cells efficiently with low-molecular weight polyethylenimine-streptomycin conjugates were more competent (∼9-fold enhancement with respect to native bPEI) exhibiting high cell viability too. Besides, both the series of conjugates displayed excellent antibacterial activity on pathogenic bacteria, even better than native streptomycin on resistant strains. Altogether, these results ensure the promising potential of the projected bifunctional conjugates as safe and efficient gene delivery vectors as well as antibacterials for future biomedical applications.

Regulation of Gene Expression by Quantitative Real Time PCR in Low DoseIsotretinoin Treated Acne Patients

Acne vulgaris is a common human skin disorder, hunting the younger age groups of people ranging from 10-35 years. It is not a deadly disease, but it is a major fact, that most of the suicides in adolescents are due to acne vulgaris. Accumulation of Propionibacterium acnes, sebum production, follicular hyper keratinisation and inflammation are some of the significant causes of pathogenesis of acne. Many medications including antibiotics like tetracycline, minocycline, erythromycin, clindamycin are given to the patients, but due to improper medication habit of the patient and certain resistance mechanisms of smart pathogens leads to the resistance of such bacteria against these antibiotics. Till date, isotretinoin and retinoic acid, are the best treatment for Acne vulgaris. This study was undertaken to analyze the regulation of genes expression after 1 and 8 weeks of isotretinoin (dose: 0.5 mg/kg/ day) treatment given toacne patients. Upregulation in the expression of some prime genes like LCN2, KRT23, SERPINA3 accounts for the initiation of the immune response against the pathogens causing acne. Down regulation of genes like PDE6A, COL1A1, ALOX15B, MMP-2, INSIG1 etc. again demonstrates that the gene products which can convert sebum, fats and cholesterol to triglycerides would no further be beneficial for P. acnes inhabitation. The aim of this study was to understand the action of isotretinoinin the regulation of gene expression in acne patient.