Nebulized anionic guanidinylated O-carboxymethyl chitosan/N-2-hydroxypropyltimehyl ammonium chloride chitosan nanoparticles for siRNA pulmonary delivery: preparation, characterization and <i>in vitro</i> 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 <i>in vitro</i> 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.本研究开发了一种由胍基化O-羧甲基壳聚糖（guanidinylated O-carboxymethyl chitosan, GOCMCS）与N-2-羟丙基三甲基氯化铵壳聚糖（N-2-hydroxypropyltrimethyl ammonium chloride chitosan, N-2-HACC）组成的pH响应型阴离子递送系统，用于雾化给药后高效递送小干扰RNA（small interfering RNA, siRNA）至肺部。通过O-甲基异脲辅助修饰，约16.8%的胍基基团被引入O-羧甲基壳聚糖（O-carboxymethyl chitosan, OCMCS）中。凝胶电泳（gel electrophoresis）结果显示，siRNA被成功包封于粒径150~180 nm、ζ电位（zeta potential）约-17 mV的纳米颗粒中。相较于其氨基类对照载体，搭载GOCMCS的纳米颗粒展现出更优异的转染性能。体外（in vitro）评价结果表明，该纳米颗粒通过能量依赖型内吞作用（endocytosis）被A549细胞摄取，随后借助GOCMCS的pH响应特性，通过胍基基团的直接跨膜穿透以及纳米颗粒的溶胀行为实现内体逃逸（endosomal escape）。使用GOCMCS/N-2-HACC/siSurvivin纳米颗粒可将生存素基因（survivin gene）的mRNA水平下调至6.9%。该纳米颗粒介导的生存素siRNA可实现30%的细胞生长抑制，并诱导19.45%的细胞凋亡（cell apoptosis），其效果与Lipofectamine 2000相当。对载siRNA纳米颗粒进行雾化处理后，siRNA的稳定性得以维持，经两级撞击器（two-stage impinger）检测，其细粒级分（fine particle fractions）占比超过60%。上述结果表明，GOCMCS/N-2-HACC纳米颗粒有望成为安全高效的siRNA肺部递送载体。