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Title: Development of Polymeric Hydrogel Nanocomposites for Antibacterial Application
Authors: Dhiman, Navneet Kaur
Supervisor: Agnihotri, Shekhar
Reddy, M. Sudhakara
Keywords: water disinfection;immobilization;disinfection model, cytotoxicity;silver nanoparticles, antibacterial mechanism, antimicrobial coatings, hydrogel,
Issue Date: 15-Jan-2024
Abstract: The invasion of bacterial pathogens has becoming a pervasive and challenging issue for public health and environmental well-being with far-reaching implications. The survival and success of all such pathogens share a common requirement: a conducive niche that allows initial attachment, colonization, and replication. Once established, they become resilient to eradicate, develop multi drug-resistance capacity and facilitate further spread. Drinking water and biomedical implants serve as prominent examples of such niches that grapple with bacterial contamination, bearing huge socioeconomic burdens with a high mortality rate. Inherent limitations in conventional disinfection technologies necessitate novel strategies in these two sectors to counteract the challenges posed by such ‘smart’ enemies. The present work, thus is an attempt to develop polymer-based antimicrobial therapeutics aimed at disrupting the ‘niche-resistance’ feature of bacteria, utilizing nanotechnology as a tool with multimodal functionalities. Polymeric nanocomposites, in the form of hydrogels and thin films, were fabricated for biocidal applications in both water disinfection and biomedical implants. All such chitosan-based polymeric nanocomposites were synthesized using eco-friendly and straightforward protocols. These materials served as a template for immobilizing nano-antimicrobials, specifically silver nanoparticles (AgNPs) or graphene oxide (GO), with the primary objective of enhancing their antibacterial efficacy while limiting their release into the surrounding environment. The research includes a comprehensive analysis of material characteristics, antibacterial performance against environmentally and clinically relevant strains, elucidation of antibacterial mechanisms, modelling disinfection kinetics, in vitro cytotoxicity assessment, hemocompatibility testing, and evaluation of soil biodegradability. Under environmentally relevant conditions, the improper disposal of biomedical waste presents hazardous consequences for natural water resources, as contamination can occur through various leakage pathways into waterways. This study successfully demonstrates the disinfection potential of nano-silver loaded chitosan-PVA (CS/PVA/Ag) hydrogel to completely eradicate biomedical contaminants (S. aureus; S. epidermidis) coexisting with natural contaminants (E. aerogenes; E. coli) in environmental samples for the first time. The polymeric networks of hydrogel served dual role for in situ synthesis and immobilization of silver nanoparticles (AgNPs) simultaneously. Porous Ag-loaded hydrogels elicited a temperature-dependent swelling behaviour and exhibited an improved mechanical strength (Young's modulus, 12.36±0.29 MPa; elongation at break, 180%) by effectively distributing the external stress and restored its structural integrity. A complete disinfection (100% killing) could be achieved within 4 h against all four tested contaminants, demonstrating a distinct strain-specific biocidal activity. Being a diffusion-controlled process, the oxidative dissolution of AgNPs, deeply buried in interiors of hydrogel architectures was adversely affected on repeated use and restricted a maximum silver release of 38.8±5.6 μg g-1 hydrogel in aqueous suspension over seven days. Correlating reusability and silver release kinetics, a predominant contact-active role of hydrogel was envisaged via ‘capture and kill’ over silver ions leaching for rapid water disinfection. The Ag-loaded hydrogels also severely inhibited biofilm formation of Escherichia coli and Staphylococcus aureus till 48 h. Finally, hydrogels could completely disinfect the natural water samples i.e., river, ground and tap water with inherited microbiota and biomedical contaminants in 2 h under real test conditions. Another nanocomposite hydrogel, consisting of chitosan-graphene oxide loaded with silver nanoparticles (CS/GO/Ag) was evaluated for on-demand disinfection ability against natural contaminants and biomedical pathogens in natural waters. In situ synthesized silver nanoparticles (AgNPs) with fine-size of ~4.2 nm were subsequently immobilized within 3D polymeric hydrogel network. The hydrogel nanocomposites displayed time & temperature swelling behaviour with low AgNPs release concentration (87.4 ppb) and rate (0.07%) over 96 h. A definite species-specific biocidal activity exhibiting 100% kill rate could be achieved within 2 h against E. aerogenes, E. coli, S. aureus, and S. epidermidis. Nano-silver loaded hydrogels were equally effective with ≥87.8% growth inhibition at higher bacterial loads of ~104-106 CFU mL-1. CS/GO/Ag hydrogels fabricated in a continuous-flow reactor (bed height, 8cm; flow-rate, 2.6 mL min-1) disinfected river and rain water within 20 minutes with >90% reusability over five cycles. The modelling kinetics demonstrated that bacterial inactivation followed the non-linear Weibull model. Comprehensive analysis of the disinfection mechanism revealed that membrane destruction and elevated oxidative stress play pivotal roles in initiating the bactericidal process. The CS/GO/Ag nanocomposite indicated superior mechanical strength, soil biodegradability (30.2% wt. loss) and significant biocompatibility towards various mammalian cells including peripheral blood mononuclear cells (PBMCs), Vero, and human hepatocellular carcinoma (HepG2). In healthcare, Biomaterial-associated infections (BAIs) pose a major hindrance to the successful integration of biomedical implants during regenerative surgeries. An alternative antimicrobial therapeutics to diminish bacterial attachment by modifying implant surface via passive coatings is proposed here. A uniform, thin-film of chitosan/polyvinyl alcohol/graphene oxide (CS/PVA/GO) was coated on 316L stainless steel (SS) surface through spread casting followed by solvent evaporation. The abundant anchoring sites available at macromolecular interfaces of chitosan/PVA matrix facilitated a smooth, dense loading of GO. The effect of GO content on drying process parameters, physicochemical features, antibacterial potential, and biocompatibility of coatings was thoroughly studied. The residual solvent at drying equilibrium increased with GO content and by altering the initial coating thickness from 700 to 1400 µm. The hybrid films displayed good adhesion behavior, and UV-protection ability with desired mechanical and thermal stability when coated on SS surface. Coatings manifested a 1.5-1.7 fold rise in antibacterial efficacy against Staphylococcus epidermidis and Staphylococcus aureus while exhibiting a permanent biocidal response after 6 h. We investigated excessive ROS generation as the predominant antibacterial mechanism, which diminishes bacterial integrity by inducing membrane permeability and suppressing respiratory chain activity as secondary mechanisms. All coatings with varying GO content appeared non-hemolytic (<2%) with ultra-low cytotoxicity (<29.08%) against human hepatocellular carcinoma (HepG2) and peripheral blood mononuclear cells (PBMCs). The degradation rate of coatings in simulated body fluid exhibited a higher stability, indicated by a lower weight loss (69-78%) and a decrease in pH values as the GO content in coatings increased from 0.05 to 0.15 wt.%. Such an anti-infective coating could be a step forward in inhibiting bacterial colonization on SS surfaces to extend its lifespan. In conclusion, the use of polymeric nanocomposites featuring silver nanoparticles and graphene oxide holds immense promise for point-of-use water disinfection and as protective coatings for stainless steel biomedical implants. The cost-effectiveness, accessibility to materials, and straightforward fabrication process, all without posing any toxicological concerns, make these nanocomposites an exceptionally practical and impactful solution for various antimicrobial applications.
Description: Ph.D. thesis
Appears in Collections:Doctoral Theses@DBT

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