基于壳聚糖的还原敏感型siRNA纳米递送系统的构建及性质研究

Construction and properties of reduction-sensitive siRNA nano delivery system based on chitosan

目的:基于还原敏感型壳聚糖(chitosan-disulfide-nona-arginine,CsR)构建小干扰RNA(small interfering RNA,siRNA)纳米递送系统并考察其保护siRNA稳定性的能力和还原敏感响应性能。方法:采用离子胶凝法制备CsR纳米粒(nanoparticles,NPs),考察处方因素对NPs粒径、Zeta电位及包封率的影响,并采用透射电子显微镜观察NPs形态。采用琼脂糖凝胶电泳考察CsR NPs与siRNA的结合能力、抗核酸酶降解能力和血清稳定性。将CsR/siRNA NPs与不同浓度的还原型谷胱甘肽(glutathione,GSH)孵育以评价其还原敏感响应性能。结果:当CsR与siRNA质量比为20∶1,三聚磷酸钠浓度为0.5 mmol/L时,制备得到球形NPs,粒径为(238.36±7.72)nm,Zeta电位为(7.57±1.22)mV,包封率为(93.21±4.12)%。琼脂糖凝胶电泳结果表明,CsR NPs与siRNA紧密结合,保护其免受核酸酶降解,维持siRNA血清中稳定至少24 h,并可响应高浓度GSH释放siRNA。结论:CsR NPs具有压缩、保护siRNA的能力和还原敏感响应性能,可作为优良的纳米载体实现siRNA的体内有效递送。

Objective:To construct siRNA nano delivery system based on chitosan-disulfide-nona-arginine(CsR)and investigate its ability to protect siRNA stability and reduction-sensitive responsiveness.Methods:CsR nanoparticles(NPs)were prepared by ionic gelation method and the effects of prescription factors on particle size,Zeta potential and encapsulation efficiency were investigated.The morphology of CsR NPs was observed by transmission electron microscope.The binding ability of CsR NPs to siRNA and their ability to protect siRNA from the degradation of nuclease and serum were investigated by agarose gel electrophoresis.CsR/siRNA NPs were incubated in reduced glutathione(GSH)with different concentrations to evaluate their reductive response.Results:When the mass ratio of CsR to siRNA was 20∶1 and the concentration of sodium triphosphate was 0.5 mmol/L,the spherical CsR NPs were successfully prepared.The particle size was(238.36±7.72)nm,the Zeta potential was(7.57±1.22)mV and the encapsulation efficiency was(93.21±4.12)%.The results of agarose gel electrophoresis experiments showed that CsR NPs could tightly bind to siRNA,effectively protect siRNA against nuclease degradation and stabilize siRNA in serum for at least 24 h.Furthermore,CsR NPs could response to high concentration of GSH for releasing siRNA.Conclusion:CsR NPs have the ability to protect siRNA stability and reduction-sensitive responsiveness,which can be used as an excellent nanocarrier to realize effective siRNA delivery in vivo.