Effect of N/P ratios on physicochemical stability, cellular association, and gene silencing efficiency for trimethyl chitosan/small interfering RNA complexes

Abstract

N,N,N-Trimethyl chitosan (TMC) with 40% quaternization was used as a vector for small interfering RNA (siRNA) delivery. Nano-sized complexes were formed in water by mixing siRNA with TMC; the smallest particle sizes were obtained at a N/P ratio of 10. The complexes had a positive surface charge that increased with increases in the N/P ratio and leveled off at +20 mV with N/P ratios > 10. The majority of particles had a diameter <100 nm under transmission electron microscope (TEM). When the N/P ratio was >10, the binding efficiency of TMC with siRNA was >90%. In 25% fetal bovine serum, the TMC/siRNA complexes with N/P ratios of 10 and 20 were intact for 12 and 48 h, respectively. TMC/siRNA complexes with an N/P ratio > 5 efficiently entered the human embryonic kidney (HEK) 293 cells and trapped initially in the lysosomes, which could then relocate in the cytoplasm. Gene silencing, tested by using enhanced green fluorescent protein (EGFP), was reduced to ~60% by the complexes with N/P ratios of 10 and 20. Specific silencing was confirmed by dose dependency and nonsilencing effect of sequence-mismatch siRNA. No significant cytotoxicity was detected for the TMC/siRNA complexes. In this study, the influence of the N/P ratio on TMC/siRNA complexes was systematically investigated and TMC was found to be an effective vector for siRNA delivery using optimized formulations.