Bioengineered Silver Nanoparticle-Loaded Chitosan Hydrogel for Enhanced Wound Healing: Characterization, Antimicrobial Activity, and In Vivo Assessment in Rats

Abstract

Background: Chronic and infected wounds require advanced topical therapies capable of providing effective antimicrobial protection while accelerating tissue regeneration. Green-synthesized silver nanoparticles (AgNPs) incorporated into biocompatible hydrogels represent a promising strategy for enhancing wound healing.

Objective: The present study aimed to evaluate the physicochemical characteristics, antimicrobial efficacy, in vivo wound healing potential, and stability of a neem-mediated green-synthesized silver nanoparticle-loaded chitosan hydrogel developed using an optimized Quality by Design (QbD) approach.

Methods: Green-synthesized AgNPs were prepared using Azadirachta indica (Neem) leaf extract and optimized through a Box–Behnken Design. The optimized nanoparticles were incorporated into a chitosan hydrogel and evaluated for pH, viscosity, spreadability, occlusive properties, and silver nanoparticle content. Antibacterial activity was assessed against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa using the agar well diffusion method. Wound healing efficacy was investigated in Wistar rats using an excision wound model with four experimental groups: untreated control, marketed silver nitrate gel, optimized AgNP-loaded chitosan hydrogel, and neem hydrogel. Wound contraction was monitored for 21 days, followed by histopathological evaluation. Accelerated stability studies were conducted according to ICH guidelines.

Results: The optimized hydrogel exhibited a skin-compatible pH (5.87 ± 0.35), suitable viscosity (4208 ± 101.01 cPs), good spreadability (12.11 ± 3.11 g·cm/s), high occlusion factor (76.69 ± 5.18%), and excellent AgNP content (98.64 ± 0.71%). The formulation demonstrated superior antibacterial activity against all tested pathogens, producing inhibition zones of 24.8 ± 0.7 mm against S. aureus, 23.5 ± 0.8 mm against E. coli, and 22.6 ± 0.5 mm against P. aeruginosa, outperforming the marketed silver gel. In vivo studies showed significantly enhanced wound healing, with the optimized AgNP hydrogel achieving 97.01 ± 0.17% wound contraction by Day 21 compared with 90.01 ± 0.33% for the marketed gel, 81.92 ± 0.17% for neem hydrogel, and 78.78 ± 1.75% for the untreated control (p < 0.001). Histopathological observations confirmed enhanced re-epithelialization and tissue remodeling in the AgNP-treated group. The hydrogel remained physically and chemically stable during three months of accelerated storage, retaining more than 97% AgNP content.

Conclusion: The optimized green-synthesized silver nanoparticle-loaded chitosan hydrogel demonstrated excellent physicochemical characteristics, broad-spectrum antimicrobial activity, superior wound healing efficacy, and satisfactory stability. The synergistic combination of neem phytochemicals, silver nanoparticles, and chitosan significantly accelerated wound repair compared with the marketed silver gel, highlighting its potential as a safe, effective, and sustainable topical wound dressing for future clinical translation.