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

Author(s)

Nunes, Renan S. ; Cinel, Victor D. P. ; Pieretti, Joana C. ; Mariano, Kelli C. F. ; dos Reis, Roberta A. ; Halfeld, Morgana ; Seabra, Amedea B.

• Document type: Journal article
• Source: NANOSCALE; v. 17, n. 21, p. 17-pg., 2025-05-08.

Abstract

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 mu mol L-1. Additionally, S-nitrosylated protein quantification in FN1 cells showed that GSNO-CSNPs at 500 mu M 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. (AU)

• FAPESP's process: 22/14645-2 - Nitric oxide releasing hydrogels and vascular prosthesis for cardiovascular applications
• Grantee: Marcelo Ganzarolli de Oliveira
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 24/04079-5 - Nanocomposite biomaterials for sustained release of nitric oxide: modeling and optimization of personalized platforms changing biomedical applications
• Grantee: Renan da Silva Nunes
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 24/20778-0 - Alginate-based biomaterials for nitric oxide delivery
• Grantee: Morgana Halfeld Andrade
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• FAPESP's process: 20/03646-2 - Impact of nanoplatforms allied to nitric oxide and chemotherapeutics on cytotoxicity and sensitization of resistant tumor cells
• Grantee: Joana Claudio Pieretti
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 23/16363-7 - Duo-gasotransmitters for biomedical applications: bioformulation of nanoparticles containing Aspirin-NOSH
• Grantee: Roberta Albino dos Reis
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 24/12112-2 - Nanoparticles combined with gaseous transmitters for biomedical and agricultural applications
• Grantee: Amedea Barozzi Seabra
• Support Opportunities: Regular Research Grants