Biosynthesis of ecofriendly antibacterial nanoparticles with healing effects in a murine diabetic skin infection model.

Diabetes mellitus is a global concern with complications including recurrent skin infections and poor wound healing. The involvement of multidrug-resistant bacteria (MDR) in skin infections renders them challenging. Nanoparticles such as silver, zinc oxide, and chitosan-based nanoparticles offer a promise for combating the growing threat of MDR bacteria by employing unique mechanisms that bypass recognized antibiotic resistance pathways. Therefore, this study reports the eco-friendly synthesis of silver (AgNPs), zinc oxide (ZnONPs), and chitosan-tripolyphosphate nanoparticles (Cs-TPP-NPs) using green tea extract (GTE) and gamma irradiation. AgNPs exhibited the highest antibacterial and antibiofilm efficacy among the tested preparations, as reflected by their lowest minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values against four bacterial strains associated with diabetic foot ulcers, including Pseudomonas aeruginosa, Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), and Streptococcus pyogenes. The killing activity of AgNPs was fast acting against all tested bacteria with > 10 log10 CFU/mL reduction in 2-6 h. The AgNPs-GTE loaded Cs-TPP-NPs combination was assessed for possible mechanisms of action, which revealed its ability to inhibit biofilm formation and compromise bacterial membrane integrity in MRSA and E. coli, leading to increased permeability, intracellular ion leakage, and nucleic acid release. Notable morphological damage to bacterial cells was confirmed by transmission electron microscope (TEM).  In vivo, the prepared combination hydrogel promoted healing of skin lesions in a CD1  mouse model of diabetic skin infection lesion within six days, demonstrating strong antibacterial activity with a 4 Log10 unit reduction in bacterial load compared with the untreated group, and significant decrease in inflammatory marker NF-kB, restoring it to healthy uninfected control levels. Histopathological evaluation of treated tissues showed full epidermal regeneration, enhanced fibroblast proliferation, increased angiogenesis, and the presence of well-organized mature collagen fibers in the dermis, compared to untreated controls. In conclusion, synthesized AgNPs-GTE loaded Cs-TPP-NPs combination offered an effective therapeutic approach for enhancing diabetic skin infection healing.