Multivariate chemometric design of nitric oxide-releasing chitosan nanoparticles for skin-related biomedical applications.

Nitric oxide (NO) is a critical signaling molecule with significant therapeutic potential for biomedical applications, particularly in wound healing, antimicrobial activity, and tissue repair. However, its clinical translation is hindered by its instability and rapid degradation in biological environments. In this study, we employed chemometric techniques to optimize the synthesis of glutathione-loaded chitosan nanoparticles (GSH-CSNPs) produced via ionotropic gelation. GSH serves as a precursor molecule for S-nitrosoglutathione (GSNO), a key NO donor. A multivariate experimental design was applied to systematically investigate eight synthesis parameters, optimizing particle size, polydispersity index (PDI), zeta potential (ZP), stability, storage conditions, and NO release kinetics. The optimized nanoparticles exhibited a hydrodynamic diameter of 77.1 ± 1.5 nm, a PDI of 0.209 ± 0.010, and a ZP of +15.3 ± 2.1 mV, ensuring considerable colloidal stability for at least 60 days at room temperature. NO release kinetics demonstrated a sustained and controlled release profile from GSNO-CSNPs compared to free GSNO, enhancing NO availability. Franz permeation cell assays revealed efficient GSNO permeation through synthetic skin membranes, and in vitro cytotoxicity assays using human fibroblast cells confirmed the biocompatibility of GSNO-CSNPs up to a NO donor concentration of 250 μmol L-1. Additionally, S-nitrosylated protein quantification in FN1 cells showed that GSNO-CSNPs at 500 μM induced a significant increase in S-nitrosylation levels, approximately 3 fold-higher than free GSNO at the same concentration, without a corresponding increase in cytotoxicity. This suggests that CSNPs enhance intracellular GSNO delivery, facilitating protein S-nitrosylation while maintaining cell viability. These findings highlight the pivotal role of Design of Experiments (DoE)-driven optimization in fine-tuning nanoparticle properties, providing a deeper understanding of how synthesis parameters influence their characteristics, and ultimately enhancing NO delivery systems for biomedical applications, particularly in skin-related therapies.