Manab Deb Adhikari

NIR- and FRET-based sensing of Cu2+ and S2- in physiological conditions and in live cells.

We have synthesized a new indole functionalized rhodamine derivative L(1) which specifically binds to Cu(2+) in the presence of large excess of other competing ions with visually observable changes in their electronic and fluorescence spectral behavior. These spectral changes are significant enough in the NIR and visible region of the spectrum and thus enable naked eye detection. The receptor, L(1), could be employed as a resonance energy transfer (RET) based sensor for detection of Cu(2+) based on the process involving the donor indole and the acceptor Cu(2+) bound xanthene fragment. Studies reveal that L(1)-Cu complex is selectively and fully reversible in presence of sulfide anions. Further, fluorescence microscopic studies confirmed that the reagent L(1) could also be used as an imaging probe for detection of uptake of these ions in HeLa cells.

Selective sensing and efficient separation of Hg2+ from aqueous medium with a pyrene based amphiphilic ligand

We have synthesized a new fluorogenic compound L, which can selectively bind and sense Hg2+ in aqueous medium over a broad pH range. It exhibits excellent selectivity for Hg2+ over a large number of competitive cations (Fe3+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Ag+, Cd2+, Pb2+, Ca2+, Mg2+, Na+, K+). The sensing ability of the ligand is studied by fluorescence and UV-vis spectroscopy. Hg2+ present even in a nanomolar range can be detected. The effectiveness of L for detecting Hg2+ inside live human cancer cells (HeLa) is also examined. The hydrophobic part of L is efficiently employed for the quantitative extraction of Hg2+ from an aqueous medium into the organic layer. The extraction ability of L is also estimated by NMR, fluorescence and atomic absorption spectroscopy showing that approximately 99% of the Hg2+ ions are extracted. These results imply that the compound has potential application for sensing and removal of Hg2+ ions from waste water in a large cross-section of mercury threatened zones around the world.

Membrane-Directed High Bactericidal Activity of (Gold Nanoparticle)–Polythiophene Composite for Niche Applications Against Pathogenic Bacteria

The use of nanoscale materials as bactericidal agents represents a novel paradigm in the development of therapeutics against drug-resistant pathogenic bacteria. In this paper the antimicrobial activity of a water soluble (gold nanoparticle)-polythiophene (AuNP-PTh) composite against common bacterial pathogens is reported. The nanocomposite is broad-spectrum in its bactericidal activity and exhibits a membrane-directed mode of action on target pathogens. The therapeutic potency of AuNP-PTh is demonstrated by experiments which reveal that the nanocomposite can breach the outer membrane defense barrier of Gram-negative pathogens for subsequent killing by a hydrophobic antibiotic, inhibit the growth of model gastrointestinal pathogens in simulated gastric fluid, and significantly eradicate bacterial biofilms. The high bacterial selectivity and lack of cytotoxicity on human cells augers well for future therapeutic application of the nanocomposite against clinically relevant pathogenic bacteria.

Tuning the bactericidal repertoire and potency of quinoline-based amphiphiles for enhanced killing of pathogenic bacteria

The overwhelming challenge posed by drug-resistant pathogenic bacteria underscores the need for potent bactericidal agents, which exhibit broad-spectrum activity and a mode of action that does not favor development of resistance. In the present study we report the synthesis and bactericidal activity of structurally diverse quinoline-based amphiphiles, having a fluorescent head group and varying hydrophobic chain length. A structure-guided bactericidal efficacy and broad-spectrum activity of the amphiphiles was apparent in screening experiments against a panel of common pathogenic bacteria. Structure–function studies by fluorescence-based assays revealed that the charge and hydrophobic chain length of amphiphiles were key structural determinants that radically boosted the bactericidal activity. The most potent amphiphile N-methyl 8-dodecoxy quinolinium iodide (compound 6) exhibited a dose-dependent bactericidal activity on target pathogens and could even inhibit the growth of a presumptive methicillin-resistant S. aureus (MRSA) strain. Fluorescence-based mechanistic studies and transmission electron microscope (TEM) analysis indicated that the initial binding of compound 6 to bacteria probably involved electrostatic interaction, whereas the hydrophobic chain of the amphiphile promoted membrane insertion, which culminated in large scale membrane disruption and loss in cell viability. Although the bactericidal activity of compound 6 was independent of bacterial transmembrane potential, interaction of the amphiphile with pathogenic bacteria resulted in rapid dissipation of membrane potential. Interestingly, compound 6 displayed high antimicrobial selectivity and did not affect the viability of human HT-29 cells. It is envisaged that the therapeutic regime of the bactericidal scaffold of compound 6 can be further expanded by rational structural design for generating potent bactericidal agents.

Synthetic amphiphiles as therapeutic antibacterials: lessons on bactericidal efficacy and cytotoxicity and potential application as an adjuvant in antimicrobial chemotherapy

In this paper, we present a critical assessment of the therapeutic potential of low molecular weight pyridine-based synthetic amphiphiles based on structure-guided bactericidal activity and a rational evaluation of their cytotoxic potential. Fluorescence-based structure-function studies revealed that the amphiphiles were membrane-acting and displayed a hierarchical pattern of bactericidal activity, which could be correlated with their charge density and hydrophobicity. The membrane-targeting activity of the most potent cationic amphiphile (compound 6) was vindicated as it induced extensive membrane-disruption and dissipation of the transmembrane potential (ΔΨ) in pathogenic bacteria. At concentrations equivalent to the minimum inhibitory concentration (MIC) against the Gram-positive pathogen S. aureus MTCC 96, none of the amphiphiles exerted any cytotoxic effect on model human cell lines (HeLa, MCF-7 and HT-29). However, at elevated concentrations, a distinct gradation in the cytotoxic effect was manifested, which is probably accounted by the charge density and conformational flexibility of the amphiphiles. A viable therapeutic application of compound 6 is demonstrated in combinatorial assays, wherein the proclivity of the amphiphile to disrupt bacterial membranes at very low concentration is exploited to enhance the uptake and bactericidal efficacy of erythromycin against Gram-negative pathogenic bacteria.

A facile method for estimating viable bacterial cells in solution based on “subtractive-aggregation” of gold nanoparticles

In this study we report a new method for sensitive estimation of bacterial cells in solution based on a systematic decrease in the degree of poly-L-lysine (PLL)-mediated aggregation of gold nanoparticles (Au NPs). The quantification of cells was done by interacting bacterial cell suspensions with PLL and recovering the unbound PLL fraction which was subsequently used to initiate aggregation of Au NPs. A progressive decline in nanoparticle aggregation evidenced by a reduced broadening of the surface plasmon resonance (SPR) peak of Au NPs revealed a strong correlation with increasing bacterial cell numbers. Using this method we could readily estimate both Gram-negative strains of Escherichia coliMTCC 433, Enterobacter aerogenesMTCC 2822 and Pseudomonas aeruginosaMTCC 2488 as well as Gram-positive bacteria such as Bacillus cereusMTCC 1305, Staphylococcus aureusMTCC 740 and Listeria monocytogenes Scott A. The limit of detection was as low as 10 colony forming unit (CFU) of bacterial cells. The method was specific for viable cells as heat-killed bacterial cells failed to reveal the characteristic decline in the broadening of the SPR peak of Au NPs. We also demonstrate that a centrifugal filter device could be used for rapid estimation of bacterial cells in solution based on the same principle used herein. The application potential of the method was further validated in an antimicrobial susceptibility test, wherein estimation of viable cell numbers of target bacteria Leuconostoc mesenteroidesNRRL B640 following treatment with nisin, a membrane permeabilizing cationic peptide, was on par with conventional plating. The present method of bacterial cell estimation offers a distinct advantage over other conventional methods in terms of ease of operation, rapidity, high sensitivity and quantitative detection of viable cells.

Amphiphile-mediated enhanced antibiotic efficacy and development of a payload nanocarrier for effective killing of pathogenic bacteria

Synthetic amphiphiles have emerged as potent bactericidal scaffolds owing to their high propensity to interact with bacterial cells and a membrane-directed mode of action, which is likely to overcome resistance development in pathogenic bacteria. In this study, we highlighted a membrane-acting quinolinium-based cationic amphiphile (compound 1) as an adjuvant for antibiotic-mediated eradication of pathogenic bacteria and demonstrated the generation of an amphiphile-loaded nanocarrier for potential antibacterial therapy. Treatment of Gram-negative pathogenic bacteria E. coli MTCC 433 and P. aeruginosa MTCC 2488 with 1 resulted in significant augmentation of the activity of erythromycin and a decrease in the minimum inhibitory concentration of the antibiotic. Interestingly, 1 promoted large-scale eradication of P. aeruginosa MTCC 2488 biofilm and could also enhance the anti-biofilm activity of tobramycin in combination. For potential therapeutic applications, a 1-loaded bovine serum albumin-based nanocarrier was developed, which exhibited sustained release of 1 both in physiological and acidic pH and the released amphiphile displayed antibacterial as well as anti-biofilm activities. Interestingly, the nanocarrier also displayed the signature membrane-directed activity of 1 against tested pathogenic bacteria. The high bactericidal and anti-biofilm activities in conjunction with a lack of cytotoxic effect on HT-29 human cell lines enhance the merit of the amphiphile-loaded nanocarrier as a potentially therapeutic antibacterial against clinically relevant drug-resistant pathogenic bacteria.

Magnetic nanoparticles for selective capture and purification of an antimicrobial peptide secreted by food-grade lactic acid bacteria

Biocompatible iron oxide nanoparticles (IONPs) were utilized for charge-based selective capture of the cationic bacteriocin pediocin secreted by food-grade lactic acid bacteria (LAB), resulting in the generation of a nanocomposite, which could be readily separated from other secreted metabolites. Interestingly, pediocin activity was conserved and a membrane-directed antibacterial activity typically associated with pediocin was manifested in the nanocomposite. Efficient sequestration of pediocin was also achieved through facile magnetic separation of IONP-pediocin composites and following desorption at pH 2.0, the recovered IONPs could be recycled for a subsequent round of pediocin adsorption. The steady state adsorption isotherm of pediocin with IONPs followed a Langmuir isotherm model. Following the reversible adsorption-desorption process with IONPs, 16-fold purification of pediocin could be achieved. The HPLC profile of the desorbed pediocin revealed a similar retention time to pediocin purified by an established cell-adsorption method and the HPLC eluted fraction also displayed the signature membrane-directed pediocin activity. The facile capture of pediocin, magnetic separation and the possibility of salvaging IONPs for reuse, accompanied by high retention of pediocin activity during the purification process, enhance the merit of IONPs as robust and effective purification tools for a potentially therapeutic antimicrobial peptide.

Quantitative Appraisal of the Probiotic Attributes and In Vitro Adhesion Potential of Anti-listerial Bacteriocin-producing Lactic Acid Bacteria

Estimation of bile tolerance, endurance to gastric and intestinal environment and adhesion potential to intestinal cells are significant selection criteria for probiotic lactic acid bacteria (LAB). In this paper, the probiotic potential of native bacteriocin-producing LAB isolated previously from indigenous source has been determined through quantitative approaches. Among fifteen anti-listerial bacteriocin-producing native LAB, ten strains were found to be bile tolerant. The presence of bile salt hydrolase (bsh) gene in native Lactobacillus plantarum strains was detected by PCR and confirmed by nucleic acid sequencing of a representative amplicon. Interestingly, three native LAB strains exhibited significant viability in simulated gastric fluid, analogous to the standard LAB Lactobacillus rhamnosus GG, while an overwhelming majority of the native LAB strains demonstrated the ability to survive and remain viable in simulated intestinal fluid. Quantitative adhesion assays based on conventional plating method and a fluorescence-based method revealed that the LAB isolates obtained from dried fish displayed significant in vitro adhesion potential to human adenocarcinoma HT-29 cells, and the adhesion level was comparable to some of the standard probiotic LAB strains. The present study unravels putative probiotic attributes in certain bacteriocin-producing LAB strains of non-human origin, which on further in vivo characterization could find specific applications in probiotic food formulations targeted for health benefits.

2-Alkylmalonic Acid: Amphiphilic Chelator and a Potent Inhibitor of Metalloenzyme

The present investigation demonstrates the potential of 2-alkylmalonic acid amphiphile as inhibitor of metalloenzymes like Taq DNA polymerase and alpha-amylase. A dose-dependent inhibition of Taq DNA polymerase was observed when a polymerase chain reaction (PCR) was performed in the presence of amphiphiles while in the case of alpha-amylase the inhibition was found to be independent of the inhibitor concentration. Control experiments revealed that both the chelating as well as the amphiphilic nature of the inhibitor was essential for enzyme inhibition. The fluorescence intensity and lifetime of alpha-amylase were also found to decrease in the presence of the amphiphiles. Steady-state fluorescence quenching studies suggested that removal of the metal ion from the enzyme leads to a decrease in the solvent accessibility of tryptophans, indicating change in the tertiary structure of the protein. It is proposed that removal of metal ion from the active sites of the enzyme by the amphiphilic compound possibly leads to disruption of the native conformation of the enzyme which is responsible for loss of its activity.