壳聚糖/透明质酸复合物纳米粒子的制备及其在siRNA递送中的应用

Fabrication of chitosan/hyaluronic acid complex nanoparticles for effective siRNA delivery

摘要:

发展高效安全的递送载体是提高siRNA药物体内稳定性、推进siRNA药物的临床应用的关键。壳聚糖(chitosan,CS)是一类天然阳离子聚合物，在核酸药物的递送中具有巨大的应用前景。为了优化CS/siRNA纳米粒子(nanoparticles,NPs)的物理化学性质，提高其siRNA递送效率，本研究在CS中加入透明质酸(hyaluronic acid,HA)，通过静电作用形成稳定的复合物纳米粒子，同时利用HA对肿瘤细胞表面CD44的靶向作用，实现siRNA的高效递送。首先，系统探究了CS和HA的分子量和质量比对CS/HA NPs的物理化学性质的影响。结果显示，混合体系在HA:CS的质量比为5:5和6:4左右可以形成粒径较小、粒径分布较窄且存储稳定性较高的复合物纳米粒子。在其他条件相同的情况下，CS/HA NPs的尺寸随着CS和HA分子量的提高而增大。在此基础上，选择合适的条件制备CS/HA NPs用于siRNA的递送。细胞实验结果显示，通过引入HA可以有效降低CS递送体系的细胞毒性，提高了纳米粒子的细胞摄取，相关CS/HA/siRNA NPs可以沉默HeLa-Luc细胞中50%–60%荧光素酶基因。与纯CS相比，CS/HA NPs可以与siRNA形成更小的纳米粒子，并由HA介导与肿瘤细胞的特异性相互作用，从而实现siRNA的高效递送。研究结果为天然高分子复合物纳米粒子的构建及siRNA递送提供了有益的参考。

Abstract:

The development of safe and effective carriers is crucial for improving the in vivo stability of siRNA drugs and facilitating their clinical translation. Chitosan (CS), a natural cationic polymer, shows great potential in nucleic acid drug delivery. To optimize the physicochemical properties of CS/siRNA nanoparticles (NPs) and increase their siRNA delivery efficacy, in this study, hyaluronic acid (HA) was added into CS to form stable complex NPs through electrostatic interactions. The HA component is able to target the CD44 receptors on the surface of tumor cells, facilitating efficient siRNA delivery. First, we systematically investigated the effects of the molecular weights and mass ratio of CS and HA on the physicochemical properties of CS/HA NPs. The results showed that at HA:CS mass ratios of approximately 5:5 and 6:4, the complex NPs exhibited small particle sizes, narrow size distribution, and high storage stability. Under similar conditions, the size of CS/HA NPs increased with the increase in the molecular weights of CS and HA. Based on these findings, suitable conditions were selected to prepare CS/HA NPs for siRNA delivery. Cell experiments demonstrated that the introduction of HA effectively reduced the cytotoxicity of the CS delivery system and enhanced the NP uptake. The CS/HA/siRNA NPs achieved 50% to 60% silencing of the luciferase gene in HeLa-Luc cells. CS/HA NPs formed smaller nanoparticles with siRNA than pure CS and mediated specific interactions with tumor cells via HA, leading to efficient siRNA delivery. These findings provide valuable insights into the construction of natural polymer composite nanoparticles for application in siRNA delivery.