Chitosan-stabilized sulfur nanoparticles tailored via chemometric design: physicochemical properties, biocompatibility, and seed priming assessments

Sulfur nanoparticles (SNPs) have emerged as multifunctional nanomaterials with antimicrobial properties, potential as hydrogen sulfide (H₂S) donors, with wide applicability in biomedicine and sustainable agriculture. However, their translation into practice remains limited due to the poor solubility, aggregation, and cytotoxicity of bulk sulfur, in addition to the reliance on strong mineral acids in conventional synthesis protocols that often fail to ensure stability and biocompatibility. In this study, chitosan-stabilized sulfur nanoparticles (SNPs@CS) were produced via thiosulfate disproportionation in acetic acid, a mild and biocompatible alternative, and synthesis conditions were systematically optimized using chemometric design of experiments. The optimized formulation yielded nanoparticles with spherical morphology, nanometric size confirmed by multiple techniques (114.4 ± 70.6 nm by HRTEM, 174.6 ± 41.0 nm by NTA, and 258.4 ± 1.3 nm by DLS), low polydispersity (PDI 0.189 ± 0.012), and a positive zeta potential (+ 13.1 ± 0.7 mV), demonstrating the crucial stabilizing role of the chitosan coating. Additional characterization by XRD revealed the amorphous nature of the sulfur phase embedded within the polymeric matrix, while FTIR confirmed interactions between chitosan functional groups and sulfur, validating effective capping and surface modification. Biological assays further highlighted the advantages of chitosan stabilization: in human keratinocytes (HaCaT), SNPs@CS maintained high viability up to 200 µg/mL, whereas uncoated SNPs were strongly cytotoxic. In Medicago sativa (alfalfa plants), seed priming with low-to-intermediate SNPs@CS concentrations enhanced shoot elongation, reduced abnormal seedlings, and increased root branching, while higher doses triggered phytotoxic responses. Together, these findings establish a sustainable, reproducible, and biocompatible route for the synthesis of stable sulfur nanostructures, demonstrating that the combination of polymer stabilization and chemometric optimization is key to controlling physicochemical properties and biological performance. SNPs@CS are thus positioned as promising candidates for future biomedical and agricultural applications, bridging nanotechnology and sustainability.