Abstract
Gene-based therapy is crucial in treating various disorders and diseases by conferring genetic material to target cells. Calcium phosphate (Ca-P) nanoparticles show promise as a non-viral vector for targeted gene delivery due to cost-effectiveness and established biocompatibility, which enhances their application in nanomedicine. Our previous pre-clinical studies in rats and monkeys exhibited the safety of our Ca-P nanoparticle formulation by local and systemic administration, with a simple preparation. However, the tendency of plasmid DNA-encapsulated Ca-P (pDNA/Ca-P) nanoparticles to aggregate during prolonged storage at inappropriate temperatures is a significant challenge. To investigate the effects of temperature and storage duration on the physicochemical properties and gene delivery efficiency of pDNA/Ca-P nanoparticles, we exposed the pDNA/Ca-P nanoparticles to varied temperatures ranging from − 80 °C to 65 °C for 7 days after formulation and measured physicochemical properties, including size, shape, surface charge, pH level, and pDNA delivery efficiency. The results indicated that pDNA/Ca-P nanoparticles stored at 4 °C and 29 °C exhibited the highest gene expression while maintaining the smallest particle size. Continuous storage at a similar temperature for 5 months demonstrated that the nanoparticles retained their favourable physicochemical properties. These findings showed that the proper long-term storage temperature at 29 °C of the pDNA/Ca-P nanoparticles, without the need for specialised instruments, paves the way for extensive use of Ca-P nanoparticles for gene therapy in pre-clinical and clinical studies in resource-limited areas. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-025-33265-3.