载siRNA的眼用三甲基壳聚糖修饰纳米粒的制备及其跨角膜作用评价  

Preparation of ophthalmic trimethyl chitosan-modified nanoparticles loaded with siRNA and the transcorneal ability evaluation

作　　者：何瑶 赖梦琴 李翔[2] 刘骏 周雄 金一 张婧 HE Yao;LAI Meng-qin;LI Xiang;LIU Jun;ZHOU Xiong;JIN Yi;ZHANG Jing(Key Laboratory of Modern Preparation of TCM,Ministry of Education,Jiangxi University of Chinese Medicine,Nanchang 330004,China;China State Key Laboratory of Innovative Drug and Efficient Energy-Saving Pharmaceutical Equipment,Jiangxi University of Chinese Medicine,Nanchang 330006,China)

机构地区：[1]江西中医药大学现代中药制剂教育部重点实验室,南昌330004 [2]江西中医药大学创新药物与节能降耗制药设备国家重点实验室,南昌330006

出　　处：《中国新药杂志》2022年第13期1315-1321,共7页Chinese Journal of New Drugs

基　　金：国家自然科学基金资助项目(81760639);江西省教育厅科技项目(GJJ190663,GJJ190666);江西省青年井冈学者及1050人才计划(张婧,李翔);江西省百千万人才工程(李翔,张婧);江西省自然科学基金(20212ACB206004,20202ACBL216015,20202BABL206157);江西省大学生创新创业训练计划(S202010412074)。

摘　　要：目的:构建三甲基壳聚糖修饰非对称脂质双层PARP-1 siRNA基因递送系统(trimethyl chitosanmodified nanoparticles with asymmetric lipid bilayers loading with poly ADP-ribose polymerase-1 siRNA,TMC-siNPs),并对其理化性质及其角膜透过能力进行评价。方法:采用qRT-PCR实验筛选最佳沉默效果的PARP-1 siRNA;采用兔眼部刺激性实验考察TMC安全性;采用钙磷沉淀-微乳法、薄膜分散法,并通过静电吸附制备TMC-siNPs;采用透射电子显微镜、粒度仪、琼脂糖凝胶电泳法对其形态、粒径、Zeta电位、包载能力进行测定;通过流式细胞仪考察人晶状体上皮细胞(HLECs)对TMC-siNPs的摄取能力;采用离体兔角膜透过实验结合激光共聚焦扫描显微镜成像考察TMC-siNPs的角膜透过能力。结果:确定最佳PARP-1 siRNA序列,并确定TMC最终浓度为0.1%。TMC-siNPs粒子形态呈球形且分散性良好;平均粒径为(57.3±0.6)nm,粒径分布良好;Zeta电位为(19.8±0.7)mV,显著高于TMC未修饰组,且琼脂糖凝胶电泳表明siRNA能稳定地包裹于纳米粒中。细胞摄取结果表明,相同时间下TMC-siNPs荧光强度明显强于Free-siRNA组,且随着时间增加,荧光逐渐增强。角膜透过性实验结果表明,与Free-siRNA组相比,siNPs和TMC-siNPs渗透至角膜深层的距离分别是Free-siRNA组的4和6倍,且主要通过细胞旁路通道途径发生角膜透过。结论:TMC修饰非对称脂质双层纳米粒可高效包载PARP-1 siRNA、促进基因角膜透过,有望提高基因药物的眼内疗效。Objective:To prepare trimethyl chitosan-modified nanoparticles with asymmetric lipid bilayers loaded with poly ADP-ribose polymerase-1 siRNA(TMC-siNPs)and evaluate its physicochemical properties and transcorneal ability.Methods:qRT-PCR technology was used to screen the optimal PARP-1 siRNA sequence.The safety of TMC was investigated by rabbit eye irritation test.Calcium-phosphorus precipitation-microemulsion method and the thin film dispersion method were applied to prepare siNPs.siNPs were further coated with TMC by electrostatic adsorption.The morphology,particle size,zeta potential and loading capacity were determined by transmission electron microscope,Nanosizer and agarose gel electrophoresis.The cellular uptake of TMC-siNPs by HLECs was investigated by flow cytometry.The corneal permeability of TMC-siNPs was investigated by in vitro rabbit corneal permeability experiment and laser confocal scanning microscope imaging.Results:The optimal PARP-1 siRNA sequence was determined and the optimal concentration of TMC was 0.1%.TMC-siNPs particles were spherical and well dispersed.The average particle size was(57.3±0.6)nm and the particle size distribution was good.The Zeta potential was(19.8±0.7)mV,significantly higher than that of siNPs.Agarose gel electrophoresis showed that siRNA can be loaded in the nanoparticles.The results of cellular uptake showed that the fluorescence intensity of TMC-siNPs was significantly stronger than that of Free-siRNA group at the same time point,and the fluorescence gradually increased with the time.The results of corneal permeability showed that the penetration distance of siNPs and TMC-siNPs into the deep layer of cornea was 4 times and 6 times that of the Free-siRNA group,respectively.The corneal permeability occurs mainly through paracellular pathways.Conclusion:TMC-modified nanoparticles with asymmetric lipid bilayer can facilitate the efficient encapsulation of PARP-1 siRNA and significantly increase its corneal permeability to improve ocular therapeutic effect of gene drugs.

关 键 词：PARP-1 siRNA 三甲基壳聚糖 纳米粒 角膜透过 

分 类 号：R943[医药卫生—药剂学]