Effects of storage on the stability and aerosolization efficiency of dry powder inhaler formulation of plasmid DNA-Chitosan nanoparticles

Dry powder inhaler (DPI) formulations of small nucleic acids (e.g. pDNA, siRNA) have been established as the preferred delivery mode for pulmonary gene therapy. While numerous studies have demonstrated the in vivo gene activity and in vitro aerosolization efficiency of the DPI formulations, few studies have examined their storage stability. Herein we investigated the effects of storage at 4 °C and 25 °C for 1 and 3 months on the in vitro stability and aerosolization efficiency of DPI of pDNA-chitosan nanoparticles prepared by spray-freeze-drying. The DPI of pDNA-chitosan nanoparticles exhibited sustained pDNA release profile and minimal cytotoxicity towards human lung epithelium cells. It lost approximately 10% and 20% of its activity after 3-month storage at 4 °C and 25 °C, respectively. As expected, the DPI formulation exhibited higher storage stability than the nanoparticle suspension form (40–50% loss). While the effect of raising the storage temperature to 25 °C on the pDNA activity was not very significant, it greatly affected the aerosolization efficiency, where the particle fraction suitable for deposition in the deep lungs decreased from 17% to 7%. The present results reaffirmed the previous studies performed on naked pDNA and pDNA polyplex that storage at 4 °C was generally adequate for DPI of pDNA.