Abhayraj Shrikrishna Joshi

Objective: The nanoparticles prepared from lignin polymer and its methacrylated derivative have gained significant attention in the biomedical field owing to their superior biosafety profile. Here, this study aims towards the synthesis of the methacrylated lignin nanoparticles (MLNPs) using the nanoprecipitation method and understanding their biocompatibility in A549 cells using the resazurin assay. Results: In our study, the nanoprecipitation method yielded a stable suspension of MLNPs having sub-200 nm size. When incubated with A549 cells, initially, these nanoparticles showed cytotoxicity and reduced biocompatibility from the well-known resazurin assay. However, careful microscopic examination of the cells showed healthy and well-adhered A549 cells with normal cobblestone-like epithelial morphology. Hence, we performed a detailed analysis of MLNPs to understand their interaction with resazurin with respect to concentration and time while keeping pristine lignin nanoparticles (LNPs) as a control. We found that LNPs and MLNPs interfered with resazurin assay, leading to false-positive results. Our study highlights a significant drawback in the use of resazurin assay for these nanoparticles and emphasizes the critical need for alternative approaches to evaluate their biocompatibility. In an attempt to find alternative, conventional MTT assay offered highly reproducible biocompatibility results without any interference during evaluating the cytotoxicity of these nanoparticles. Graphical

A single cell or cell population exhibits the fundamental phenomenon of cell migration during developmental processes or disease progression. Vast literature suggests that, in vitro 2-dimensional or 3-dimensional cell migration assay is one of the most commonly used assays in cancer, wound healing research, and developmental biology research. The data obtained from this assay are often analyzed using various proprietary or open-source programs. Proprietary software are costly and not always accessible to everyone. Whereas the open-source programs are free, easy to access, and user friendly. However, not all researchers are aware of these open-source programs. Despite the increasing availability of these programs, many researchers still rely on proprietary software, due to a lack of comparative analyses and practical guidance on their implementation. Hence, this review aims to provide insights into these open-source tools and serves as a practical guide to both biologists and computational researchers for their specific analytical needs.

Nisin is a naturally occurring bioactive small peptide produced by Lactococcus lactis subsp. lactis and belongs to the Type A (I) lantibiotics. Due to its potent antimicrobial activity, it has been broadly employed to preserve various food materials as well as to combat a variety of microbial pathogens. The present review discusses the antimicrobial properties of nisin and different types of their derivatives employed to treat microbial pathogens with a detailed underlying mechanism of action. Several alternative strategies such as combination, conjugation, and nanoformulations have been discussed in order to address several issues such as rapid degradation, instability, and reduced activity due to the various environmental factors that arise in the applications of nisin. Furthermore, the evolutionary relationship of many nisin genes from different nisin-producing bacterial species has been investigated. A detailed description of the natural and bioengineered nisin variants, as well as the underlying action mechanisms, has also been provided. The chemistry used to apply nisin in conjugation with natural or synthetic compounds as a synergetic mode of antimicrobial action has also been thoroughly discussed. The current review will be useful in learning about recent and past research that has been performed on nisin and its derivatives as antimicrobial agents.

Many bacteria have the capability to form a three-dimensional, strongly adherent network called ‘biofilm’. Biofilms provide adherence, resourcing nutrients and offer protection to bacterial cells. They are involved in pathogenesis, disease progression and resistance to almost all classical antibiotics. The need for new antimicrobial therapies has led to exploring applications of gold and silver nanoparticles against bacterial biofilms. These nanoparticles and their respective ions exert antimicrobial action by damaging the biofilm structure, biofilm components and hampering bacterial metabolism via various mechanisms. While exerting the antimicrobial activity, these nanoparticles approach the biofilm, penetrate it, migrate internally and interact with key components of biofilm such as polysaccharides, proteins, nucleic acids and lipids via electrostatic, hydrophobic, hydrogen-bonding, Van der Waals and ionic interactions. Few bacterial biofilms also show resistance to these nanoparticles through similar interactions. The nature of these interactions and overall antimicrobial effect depend on the physicochemical properties of biofilm and nanoparticles. Hence, study of these interactions and participating molecular players is of prime importance, with which one can modulate properties of nanoparticles to get maximal antibacterial effects against a wide spectrum of bacterial pathogens. This article provides a comprehensive review of research specifically directed to understand the molecular interactions of gold and silver nanoparticles with various bacterial biofilms.

Method validation for calculating the amount of polysorbate 80 adsorbed on PLGA nanoparticles for regulatory compliance.

Huntington disease (HD) is a genetic disorder caused by CAG expansion mutation in Huntingtin gene leading to polyglutamine (PolyQ) expansion in the N-terminus part of Huntingtin (Httex1). Expanded PolyQ, through complex aggregation pathway forms aggregate in neurons and present a potential therapeutic target. Here we show Httex1 aggregation suppression by arginine and arginine ethyl ester (AEE) in vitro, as well as in yeast and mammalian cell models of HD, bearing expanded polyQ. These molecules also rescue locomotion dysfunction in HD Drosophila model. Both molecules alter the hydrogen bonding network of polyQ to enhance its aqueous solubility and delay aggregation. AEE shows direct binding with NT₁₇ part of Httex1 to induce structural changes to impart an enhanced inhibitory effect. This study provides a platform for the development of better arginine based therapeutic molecule against polyQ-rich Httex1 aggregation.

Detailed study of the molecular mechanism behind the pathogenesis of Huntington’s disease (HD) suggests that the polyglutamine aggregation is one of the fundamental reasons. Despite of discovery of many potential molecules, HD therapy is yet limited to symptomatic relief. Among these molecules, few mechanism based peptide inhibitors of polyglutamine aggregation (QBP1, NT17 and PGQ9P2) have shown promising activity; however, poor blood-brain barrier (BBB) penetration, low bioavailability and low half-life may hinder their therapeutic potential. Hence, to deliver them to brain for assessing their efficacy, we have designed and synthesized peptide loaded poly-D, L-lactide-co-glycolide (PLGA) nanoparticles of size less than 200 nm by carbodiimide chemistry and nanoprecipitation protocols. For brain delivery, PLGA nanoparticles were coated with polysorbate 80 which aids receptor mediated internalization. Using in vitro BBB model of Madin-Darby Canine Kidney cells and healthy mice, the translocation of polysorbate 80 coated fluorescent nanoparticles was confirmed. Moreover, QBP1, NT17 and PGQ9P2 loaded PLGA nanoparticles showed dose dependent inhibition of polyglutamine aggregation in cell models of HD (Neuro 2A and PC12 cells) and improved motor performance in Drosophila model of HD. Additionally, no toxicity in cells and animals confirmed biocompatibility of the nanoparticulate formulations. Based on this work, future studies can be designed in higher animal models to test peptide loaded nanoparticles in HD and other polyglutamine expansion related diseases.

Huntington’s disease (HD) is a genetic disorder caused by a CAG expansion mutation in Huntingtin gene leading to polyglutamine (polyQ) expansion in the N-terminus side of Huntingtin (Httex1) protein. Neurodegeneration in HD is linked to aggregates formed by Httex1 bearing expanded polyQ. Initiation and elongation steps of Httex1 aggregation are potential target steps for the discovery of therapeutic molecules, for yet untreatable and cruel, HD. Here we report Httex1 aggregation inhibition by calmidazolium chloride (CLC) by acting on the initial aggregation event. Being hydrophobic, CLC was adsorbed to the vial surface and could not sustain inhibition effect for longer duration. Usage of bovine serum albumin (BSA) prevented its adsorption by forming BSA-CLC complex. This complex showed improved Httex1 aggregation inhibition by interacting with the aggregation initiator, NT₁₇ part of Httex1. Further, a biocompatible CLC loaded BSA nanoparticles were made which reduced the polyQ aggregates in HD-150Q cells.