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Nebulized anionic guanidinylated O-carboxymethyl chitosan/N-2-hydroxypropyltimehyl ammonium chloride chitosan nanoparticles for siRNA pulmonary delivery: preparation, characterization and in vitro evaluation.

This study developed a pH-sensitive anionic system composed of guanidinylated O-carboxymethyl chitosan (GOCMCS) and N-2-hydroxypropyltimehyl ammonium chloride chitosan (N-2-HACC) for efficient siRNA delivery to the lungs following nebulization. About 16.8% of guanidine groups were incorporated into O-carboxymethyl chitosan (OCMCS) with the aid of O-methylisourea. Gel electrophoresis images demonstrated that siRNA was successfully encapsulated in nanoparticles ranging from 150 to 180 nm with zeta potential of about -17 mV. The nanoparticles containing GOCMCS existed superior transfection performance compared with their amino-based analogs. The evaluation in vitro revealed that nanoparticles were internalized into A549 cells by energy-dependent endocytosis, then achieved endosomal escape by direct transmembrane penetration of guanidine moieties as well as swelling behavior of nanoparticles due to the pH sensitivity of GOCMCS. The mRNA level of survivin gene was down-regulated to 6.9% using GOCMCS/N-2-HACC/siSurvivin NPs. The survivin siRNA mediated by nanoparticles caused 30% of cell growth inhibition and induced 19.45% of cell apoptosis, which was comparable to Lipofectamin2000. After nebulization of siRNA-loaded nanoparticles, the stability of siRNA was maintained and fine particle fractions were detected by two-stage impinger that accounted for more than 60%. These results suggested that GOCMCS/N-2-HACC nanoparticles possessed potential as safe and efficient carrier for siRNA pulmonary delivery.

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