The study aimed to synthesize non-noxious, clean, reliable, and green sulfur nanoparticles (SNPs) from Citrus limon leaves. The synthesized SNPs were used to analyze particle size, zeta potential, UV-visible spectroscopy, SEM, and ATR-FTIR. The prepared SNPs exhibited a globule size of 55.32 ± 2.15 nm, PDI value of 0.365 ± 0.06, and zeta potential of -12.32 ± 0.23 mV. The presence of SNPs was confirmed by UV-visible spectroscopy in the range of 290 nm. The SEM image showed that the particles were spherical with a size of 40 nm. The ATR-FTIR study showed no interaction, and all the major peaks were preserved in the formulations. An antimicrobial and antifungal study of SNPs was carried out against Gram-positive bacteria (Staph. aureus, Bacillus), Gram-negative bacteria (E. coli and Bordetella), and fungal strains (Candida albicans). The study showed that Citrus limon extract SNPs exhibited better antimicrobial and antifungal activities against Staph. aureus, Bacillus, E. coli, Bordetella, and Candida albicans at a minimal inhibitory concentration of 50 μg/mL. Different antibiotics were used alone and in combination with SNPs of Citrus limon extract to evaluate their activity against various strains of bacteria and fungal strains. The study showed that using SNPs of Citrus limon extract with antibiotics has a synergistic effect against Staph.aureus, Bacillus, E. coli, Bordetella, and Candida albicans. SNPs were embedded in nanohydrogel formulations for in vivo (wound healing) studies. In preclinical studies, SNPs of Citrus limon extract embedded within a nanohydrogel formulation (NHGF4) have shown promising results. To be widely used in clinical settings, further studies are needed to evaluate their safety and efficacy in human volunteers.

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