Sukdeb Pal

Synthesis of highly antibacterial nanocrystalline trivalent silver polydiguanide.

Highly monodispersed nanoparticles of a trivalent silver polydiguanide complex are synthesized by oxidation of the monovalent silver, followed by stabilization of the oxidized higher-valent metal through complexation with a polydiguanide ligand in a reverse microemulsion at room temperature. The synthesized nanoparticles have excellent photostability and displayed superior antibacterial activity toward Gram-positive and Gram-negative prokaryotes of clinical interest in vitro compared to silver sulfadiazine. These nanoparticles may serve as a new generation antibacterial metallopharmaceutical in wound care.

Shape-Dependent Skin Penetration of Silver Nanoparticles: Does It Really Matter?

Advancements in nano-structured materials have facilitated several applications of nanoparticles (NPs). Skin penetration of NPs is a crucial factor for designing suitable topical antibacterial agents with low systemic toxicity. Available reports focus on size-dependent skin penetration of NPs, mainly through follicular pathways. Herein, for the first time, we demonstrate a proof-of-concept study that entails variations in skin permeability and diffusion coefficients, penetration rates and depth-of-penetration of differently shaped silver NPs (AgNPs) via intercellular pathways using both in vitro and in vivo models. The antimicrobial activity of AgNPs is known. Different shapes of AgNPs may exhibit diverse antimicrobial activities and skin penetration capabilities depending upon their active metallic facets. Consideration of the shape dependency of AgNPs in antimicrobial formulations could help developing an ideal topical agent with the highest efficacy and low systemic toxicity.

Metallopharmaceuticals based on silver(I) and silver(II) polydiguanide complexes: activity against burn wound pathogens

OBJECTIVES The in vitro pharmacodynamics of silver(I) and silver(II) complexes of a polydiguanide ligand, chlorhexidine, were assayed to examine the value of the bactericidal endpoint as an alternative means of evaluating their antibacterial activities against burn wound pathogens. METHODS Synthesis of silver(I) chlorhexidine [Ag(I)CHX] was accomplished by in situ precipitation of the complex from a feebly acidic or neutral aqueous solution of AgNO(3) and chlorhexidine, whereas silver(II) chlorhexidine [Ag(II)CHX] was synthesized by oxidation of Ag(I), followed by complexation of the oxidized metal with chlorhexidine. Their antibacterial potencies were assessed in vitro by determining the MICs and MBCs for four Gram-positive and four Gram-negative bacteria. Time-kill assays using three different concentrations of these agents were also performed. RESULTS The MICs of Ag(I)CHX and Ag(II)CHX were much lower than those of chlorhexidine, AgNO(3) and silver sulfadiazine. The time-kill study provided quantitative information on actual times required to reach the bactericidal endpoint using a particular concentration of the active agent. The lethality rates of Ag(I)CHX and Ag(II)CHX against the tested bacteria were 2× to 8× faster than those of chlorhexidine or AgNO(3) at a concentration equal to or 4× MIC. CONCLUSIONS Ag(I)CHX and Ag(II)CHX showed superior antibacterial activity and faster killing kinetics compared with chlorhexidine and AgNO(3). These complexes may serve as new-generation antibacterial agents in wound care.

Quantitation of surface coverage of oligonucleotides bound to chip surfaces: a fluorescence-based approach using alkaline phosphatase digestion

Silanized chip surfaces provide a reliable substrate for immobilization of oligonucleotides. The ability for rapid and sensitive detection of oligonucleotide surface coverage on these chips is crucial for their wide and effective applications in biotechnology. In this paper, two different silanization procedures were used to covalently bind fluorescent-labeled single-stranded DNA onto silicon dioxide or nitride chip surfaces. Effects of surface functionalization techniques for different surfaces, and immobilization conditions, including buffers and solution ionic strength, on surface probe coverage were investigated, quantifying the endpoint probe density by fluorescent measurement upon digestion with alkaline phosphatase (ALP). Digestion of surface-immobilized oligonuleotides with ALP released the fluorophore-tagged probe fragments back into the solution. The detection of DNA was accomplished by laser-induced fluorescence detection of the solution containing those cleaved fragments. The probe surface density on gold thin film, determined by ALP-digestion, was found to coincide well with that measured using the conventional alkanethiol-based fluorescence-displacement technique for the same system. The developed method has important implications for evaluating the performance of different oligonucleotide immobilization strategies. Also, it has the potential to serve as a sample-thrifty, time saving, and therefore routine tool to realize more realistic, practical quantification of the surface coverage of oligonucleotides immobilized on any solid surfaces.

A multifunctional composite of an antibacterial higher-valent silver metallopharmaceutical and a potent wound healing polypeptide: a combined killing and healing approach to wound care

The present study relates to a combined killing and healing approach for the treatment of infected wounds. Herein we report a multifunctional, including antimicrobial and wound healing, composite containing a conjugate of a bi-valent silver polydiguanide that demonstrated high antibacterial activity in vitro and a potent wound healing polypeptide, histatin-1, for the treatment of infected wounds. The synthesis of silver(II) chlorhexidine [Ag(II)CHX] was accomplished by the oxidation of Ag(I), followed by the complexation of the oxidized metal with chlorhexidine (CHX), whereas the metal complex conjugate of the solid phase-synthesized histatin polypeptide (Hst-1), Hst-1-[Ag(II)CHX], was realized by mixing the starting materials in aqueous solution. The change in the Hst-1 structure upon binding with the silver complex was examined by circular dichroism spectroscopy. The wound healing applicability of the histatin polypeptide and its metal complex conjugate was tested using the synthesized Hst-1 and Hst-1-[Ag(II)CHX] complex on 3T3-L1 preadipocytes in a cell-spreading assay. The antibacterial activity of the silver metal complex and its Hst-1 conjugate was tested against several gram positive and gram negative bacteria, including Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-resistant coagulase negative staphylococcus (MRCNS) by a broth microdilution method. The results of these experiments revealed that the polypeptide and silver(II) polydiguanide complex retained their individual wound healing and antimicrobial activity even in their conjugate. The conjugate of an antibacterial higher-valent silver polydiguanide complex with a potent wound healing polypeptide (Hst-1) showed promise as a new multifunctional therapeutic wherein the killing and healing functions of the constituent materials are preserved together for the development of new-generation wound-care agents.

Monitoring the (photo)genotoxicity of photosensitizer drugs: direct quantitation of single-strand breaks in deoxyribonucleic acid using an oligonucleotide chip.

Oligonucleotide chip-based assays can be a sample-thrifty, time-saving, routine tool for evaluation of chemical-induced DNA strand breaks. This article describes a novel approach using an oligonucleotide chip to determine photosensitizer-induced DNA single-strand breaks. Surface coverage of fluorophore-labeled oligonucleotides on silicon dioxide chip surfaces was determined on alkaline phosphatase digestion. Fluorescence maxima (at 520 nm) of the solutions were converted to molar concentrations of the fluorescein-modified oligonucleotide by interpolation from a predetermined standard linear calibration curve. The photosensitizing activity of chlorpromazine and triflupromazine toward DNA single-strand breaks was then studied at different drug doses and also as a function of photoirradiation time. Photoinduced single-strand breaks calculated using the method described here agreed with values predicted by theoretical extrapolation of the single-strand breaks obtained for plasmid DNAs from agarose gel electrophoresis, and thereby indirectly validated the chip-based assays. Under UV irradiation (>or=93.6 kJ/m2) chlorpromazine (>or=0.08 mM) was found to have significant photogenotoxicity. However, triflupromazine did not exhibit any (photo)genotoxicity over the concentration range studied (0.04-0.20mM). The method developed will be useful for quantitative screening of drug genotoxicity in terms of induction of breaks in DNA.

Quantitative Classification of DNA Damages Induced by Submicromolar Cadmium Using Oligonucleotide Chip Coupled with Lesion-Specific Endonuclease Digestion

Implementation of proper analytical tool for systematic investigation and quantitative determination of different classes of cadmium ion-induced DNA damages, especially at low metal ion concentrations, is still lacking. Using lesion-specific enzymes that cleave DNA at specific classes of damage and a fluorometric approach developed for quantifying fluorophore-labeled oligonucleotides bound to chip surfaces, we determined the frequencies of different lesions (strand breaks, oxidized purines, oxidized pyrimidines, or abasic sites) induced by submicromolar Cd(2+). Cd(2+)-treated oligonucleotide chips were digested with various endonucleases (Fpg protein, endonuclease III, endonuclease IV), producing a de novo single strand break (SSB) at their substrate modifications. The frequency of SSB and double strand break (DSB) was computed from the difference of pre- and post-Cd(2+)-treatment oligonucleotide coverage on the chip. While the frequency of SSBs and oxidized bases were successfully quantified even at 0.5 μM of Cd(2+), DSB frequency could be easily quantitated at 8.7 μM [Cd(2+)]. The numbers of abasic sites were below the oligonucleotide detection limit (2.4 amole; equivalent to 0.24 fM for a reaction volume of 100 μL). SSBs were found to constitute about 85-90% of single strand damages, while oxidized bases comprise only 4-7% of the total at 0.9 to 8.7 μM [Cd(2+)].

Oligonucleotide chip assay for quantification of gamma ray-induced single strand breaks.

An oligonucleotide chip assay was designed for direct quantification of single strand breaks (SSBs) induced by gamma-ray irradiation. The oligonucleotides used were 20-mers, which were short enough to produce only a single strand break within a single oligonucleotide. The two ends of the oligonucleotides were labeled with fluorescein and biotin, respectively. The biotinylated ends of the oligonucleotides were immobilized on a silicon wafer chip treated with (3-aminopropyl)triethoxysilane (APTES), glutaraldehyde, and avidin. The DNA fragments cleaved by gamma-ray irradiation were detected by a laser-induced fluorescence (LIF) detection system. The gamma-ray-induced SSBs were quantified using a calibration curve (fluorescence intensity versus gamma-ray dose) without the need for complicated mathematical calculation based on gel-based separation. The experimentally determined gamma-ray-induced SSBs yield was almost equal to the theoretical value derived from gel electrophoresis of plasmid DNAs and DNA surface coverage.

Quantitation of ultraviolet-induced single-strand breaks using oligonucleotide chip

A simple, accurate and robust methodology was established for the direct quantification of ultraviolet (UV)-induced single-strand break (SSB) using oligonucleotide chip. Oligonucleotide chips were fabricated by covalently anchoring the fluorescent-labeled ssDNAs onto silicon dioxide chip surfaces. Assuming that the possibility of more than one UV-induced SSB to be generated in a small oligonucleotide is extremely low, SSB formation was investigated quantifying the endpoint probe density by fluorescence measurement upon UV irradiation. The SSB yields obtained based on the highly sensitive laser-induced fluorometric determination of fluorophore-labeled oligonucleotides were found to coincide well with that predicted from a theoretical extrapolation of the results obtained for plasmid DNAs using conventional agarose gel electrophoresis. The developed method has the potential to serve as a high throughput, sample-thrifty, and time saving tool to realize more realistic, and direct quantification of radiation and chemical-induced strand breaks. It will be especially useful for determining the frequency of SSBs or lesions convertible to SSBs by specific cleaving reagents or enzymes.

Feasibility of bioengineered two-stages sequential batch reactor and filtration-adsorption process for complex agrochemical effluent.

In the present study, the feasibility of a bioengineered two-stages sequential batch reactor (BTSSBR) followed by filtration-adsorption process was investigated to treat the agrochemical effluent by overcoming factor affecting process stability such as microbial imbalance and substrate sensitivity. An air stripper stripped 90% of toxic ammonia, and combined with other streams for bio-oxidation and filtration-adsorption. The BTSSBR system achieved bio-oxidation at 6 days hydraulic retention time by fending off microbial imbalance and substrate sensitivity. The maximum reduction in COD and BOD by heterotrophic bacteria in the first reactor was 87% and 90%, respectively. Removal of toxic ammoniacal-nitrogen by autotrophic bacteria in a post-second stage bio-oxidation was 97%. The optimum filtration and adsorption of pollutants were achieved at a filtration rate of 10 and 9 m(3)m(-2)h(-1), respectively. The treatment scheme comprising air stripper, BTSSBR and filtration-adsorption process showed a great promise for treating the agrochemical effluent.