Abstract

Purpose: Cystic fibrosis (CF) is a life-threaten disease caused by mutations in CFTR gene. The emergency of gene-editing technology such as CRISPR/Cas9 system provides a promising way to fundamentally cure CF. Different administration routes to lung have been investigated, including intravenous, intratracheal and inhalation routes. In present study, we developed PEGylated oligo chitosan (PEG-OC)/DNA nanocomplexes with different N/P (amine/phosphate) ratios by electrostatic interaction. Methods: Aerogen Solo nebulizer was employed to aerosolize the nanocomplex formulations. Agarose gel electrophoresis was used to detect the loading efficiency of DNA and the integrity of nanocomplex after nebulization. Zetasizer was used to detect the particle size and zeta potential of the formulation with or without nebulization process. Results: The electrophoresis results showed the formation of nanocomplexes and the complete loading of DNA was reached at N/P ratio of 10. The integrity of the nanocomplexes was not disrupted by the nebulization stresses when N/P ratio reach 2. The size of PEG-OC/DNA nanocomplexes without nebulization changed in the range of 291.3 ± 4.1 nm at N/P ratio of 1, to 172.3 ± 4.2 nm at N/P ratio of 5. The size of PEG-OC/DNA nanocomplexes after nebulization were in the range of 832.4 ± 95.1 nm at N/P ratio of 1 to 165.7 ± 2.9 nm at N/P ratio of 20. The zeta potential of mPEG-OC/DNA without nebulization switched from -13.0±2.5 mv at N/P ratio of 0.5 to 17.2±1.1 mv at N/P ratio of 20, while the zeta potential of mPEG-OC/DNA nanocomplexes after nebulization changed from 0.522±0.7 mv at N/P ratio of 0.5 to 14.0±1.0 at N/P ratio of 30. The transfection assay demonstrated that the nanocomplexes showed that optimal transfection efficiency at N/P ratio of 20 in HEK cells and the nebulization process did not reduce the delivery capability of the nanocomplexes. Conclusion: In conclusion, the PEGylated chitosan has the potential to deliver CRISPR/Cas9 system in the format of DNA for the treatment for CF.