牢固附着中性磷脂表面膜的Lipopolyplex核酸载体的自组装及通用性考察

While synthetic carriers of nucleic acids are superior over viral vectors in terms of safety and production efficiency, their transfection efficiency is far behind. To optimizing the composition and load of functional components, the artificial methods seem to be less perfect than the evolution in viruses. To address the issue, we propose to take siRNA or mRNA, which have simple intra-cellular pathway, as the example, to establish thermodynamically self-regulated assembly to to construct an easily functionalizing universal carrier as a research tool for biopharmaceutical reseatchers. We plan to simulate the structure of lantivirus, a popular viral vector, as its neutral phospholipid membrane surface and nucleic acid core, but avoid using the charged lipids seen in lipopolyplex which was reported by Huang et al. In details, we synthesized an amphiphilic molecule with a large cross-section multi-charged head and a small cross-section hydrophobic chain, and let the molecule to strongly adsorb on polyplex in the form of altered micelles. The tension generated from this altered shape may serve as a thermodynamic driving force to facilitate the hydrophobic chain to insert into the phospholipid membrane matrix to fuse consequently added liposomes to to surface as anchored membrane. The stability and flexibility in functionalization wil be examined by immobilizing variety of functional components on it.

核酸合成载体虽安全性和量产便捷性占优，递送效率不如病毒载体；其中，人为地优化功能基团的组合与含量不及病毒载体的进化来得精准和完备。对此， 我们以细胞内路径简单的siRNA或mRNA的递送为应用，以热力学自洽机制代替合成载体构建常用的随机过程，构建便于医药研究人源精准地进行表面功能化的通用性合成载体。我们模拟常用的慢病毒载体的磷脂膜表面+核酸内涵的结构，参考Huang等人提出的具有类似结构的lipopolyplex不得不使用的带电脂质对细胞摄取的干扰的不足，以静电吸附+疏水“锚定”相结合的机制，将自然形态的中性磷脂膜牢固地附着在polyp lex表面。做法上，我们合成大截面多电荷亲水端和小截面疏水链两亲性分子，使之以变形胶束强吸附于polyplex。变形张力热力学驱动疏水链插入随后添加的脂质体中性磷脂膜，使之在表面融合为“锚定”的磷脂膜；其稳定性和功能化便捷性将通过负载各类细胞靶向基团验证。

将组成细胞或一些病毒的外膜类似磷脂附着于聚阳离子与核酸凝聚的纳米颗粒（polyplex）表面，使合成载体具有病毒的一些递送功能是基因合成载体的研究者尝试实现的目标。本项目之前，文献报道的方法包括：随机混合；共价键接枝；共价键接枝+疏水自组装；带电荷磷脂的静电自组装。这些方法分别存在着如下缺陷：没有磷脂形成表面膜的化学机制；在纳米颗粒表面进行化学反应的不便；诱导疏水自组装的“锚化合物”合成过程繁琐；以及带电荷磷脂对靶向递送的干扰。本课题设计了一种将不带电荷的磷脂不通过共价键接枝反应，牢固地附着在聚阳离子与核酸凝聚的polyplex表面的方法：以多电荷两亲性分子形成的胶束吸附在polyplex表面作的锚基团为融合并固定磷脂双层膜，并通过此双层膜疏水固化细胞靶向分子。计划中的物理化学表征、生物学考察、以及初步的动物实验验证了本课题设计的技术思路的可行性，完成了提出的目标。随后，全国陷入新冠疫情，课题团队做了些将本课题相关的合成载体用于病毒学和RNA疫苗的尝试。