Polymeric Nucleic Acid Vehicles Exploit Active Interorganelle Trafficking Mechanisms

Materials that self-assemble with nucleic acids into nanocomplexes (e.g. polyplexes) are widely used in many fundamental biological and biomedical experiments. However, understanding the intracellular transport mechanisms of these vehicles remains a major hurdle in their effective usage. Here, we investigate two polycation models, Glycofect (which slowly degrades via hydrolysis) and linear polyethyleneimine (PEI) (which does not rapidly hydrolyze), to determine the impact of polymeric structure on intracellular trafficking. Cells transfected using Glycofect underwent increasing transgene expression over the course of 40 h and remained benign over the course of 7 days. Transgene expression in cells transfected with PEI peaked at 16 h post-transfection and resulted in less than 10% survival after 7 days. While saccharide-containing Glycofect has a higher buffering capacity than PEI, polyplexes created with Glycofect demonstrate more sustained endosomal release, possibly suggesting an additional or alternative delivery mechanism to the classical “proton sponge mechanism”. PEI appeared to promote release of DNA from acidic organelles more than Glycofect. Immunofluorescence images indicate that both Glycofect and linear PEI traffic oligodeoxynucleotides to the Golgi and endoplasmic reticulum, which may be a route towards nuclear delivery. However, Glycofect polyplexes demonstrated higher co-localization with the ER than PEI polyplexes, and co-localization experiments indicate the retrograde transport of polyplexes via COP I vesicles from the Golgi to the ER. We conclude that slow release and unique trafficking behaviors of Glycofect polyplexes may be due to the presence of saccharide units and the degradable nature of the polymer, allowing more efficacious and benign delivery.

能够与核酸自组装形成纳米复合物（如聚复合物(polyplexes)）的材料，已被广泛应用于诸多基础生物学与生物医学实验当中。然而，阐明此类基因递送载体的细胞内转运机制，仍是制约其高效应用的核心瓶颈。本研究选取两种聚阳离子模型展开探究：分别为可通过水解缓慢降解的Glycofect，以及无法快速水解的线性聚乙烯亚胺（linear polyethyleneimine, PEI），以明确聚合物结构对细胞内转运过程的影响。采用Glycofect转染的细胞，其转基因表达水平在40小时内持续上调，且在7天的观察周期内始终保持良好的细胞存活状态，无明显毒性反应。而采用PEI转染的细胞，其转基因表达水平在转染后16小时达到峰值，且在转染7天后细胞存活率不足10%。尽管含糖基修饰的Glycofect相较于PEI具备更高的缓冲能力，但由Glycofect构建的聚复合物，其介导的内体释放过程更为持久，这或许提示其存在不同于经典“质子海绵机制（proton sponge mechanism）”的额外或替代递送途径。相较而言，PEI更易促进DNA从酸性细胞器中释放。免疫荧光成像结果显示，Glycofect与线性PEI均可将寡脱氧核苷酸(oligodeoxynucleotides)转运至高尔基体与内质网（endoplasmic reticulum），这或许是实现核递送的潜在途径之一。但相较PEI聚复合物，Glycofect聚复合物与内质网的共定位水平更高；共定位实验结果表明，聚复合物可通过COP I囊泡(COP I vesicles)从高尔基体逆向转运至内质网。本研究最终得出结论：Glycofect聚复合物的缓慢释放特性与独特的转运行为，或源于其分子中含糖基单元以及聚合物本身的可降解性，这使其能够实现更为高效且安全的基因递送效果。