Biophysics and Interfaces Group, Applied Physics Department, Universidade de Santiago de Compostela, Spain; AFFINImeter-Software 4 Science Developments S.L. Edificio Emprendia s/n Campus Vida, Santiago de Compostela, Spain.
Cátedras CONACyT-Tecnológico Nacional de México/I. T. Tijuana, Centro de Graduados e Investigación en Química-Grupo de Biomateriales y Nanomedicina, Blvd. Alberto Limón Padilla S/N, 22510 Tijuana, BC, Mexico.
Colloids Surf B Biointerfaces. 2022 Feb;210:112219. doi: 10.1016/j.colsurfb.2021.112219. Epub 2021 Nov 17.
Polyethylenimine (PEI) has been demonstrated as an efficient DNA delivery vehicle both in vitro and in vivo. There is a consensus that PEI-DNA complexes enter the cells by endocytosis and escape from endosomes by the so-called "proton sponge" effect. However, little is known on how and where the polyplexes are de-complexed for DNA transcription and replication to occur inside the cell nucleus. To better understand this issue, we (i) tracked the cell internalization of PEI upon transfection to human epithelial cells and (ii) studied the interaction of PEI with phospholipidic layers mimicking nuclear membranes. Both the biological and physicochemical experiments provided evidence of a strong binding affinity between PEI and the lipidic bilayer. Firstly, confocal microscopy revealed that PEI alone could not penetrate the cell nucleus; instead, it arranged throughout the cytoplasm and formed a sort of aureole surrounding the nuclei periphery. Secondly, surface tension measurements, fluorescence dye leakage assays, and differential scanning calorimetry demonstrated that a combination of hydrophobic and electrostatic interactions between PEI and the phospholipidic monolayers/bilayers led to the formation of stable defects along the model membranes, allowing the intercalation of PEI through the monolayer/bilayer structure. Results are also supported by molecular dynamics simulation of the pore formation in PEI-lipidic bilayers. As discussed throughout the text, these results might shed light on a the mechanism in which the interaction between PEI and the nucleus membrane might play an active role on the DNA release: on the one hand, the PEI-membrane interaction is anticipated to facilitate the DNA disassembly from the polyplex by establishing a competition with DNA for the PEI binding and on the other hand, the forming defects are expected to serve as channels for the entrance of de-complexed DNA into the cell nucleus. A better understanding of the mechanism of transfection of cationic polymers opens paths to development of more efficiency vectors to improve gene therapy treatment and the new generation of DNA vaccines.
聚亚乙基亚胺（PEI）已被证明在体外和体内都是一种有效的 DNA 传递载体。人们普遍认为，PEI-DNA 复合物通过内吞作用进入细胞，并通过所谓的“质子海绵”效应从内涵体中逃逸。然而，对于多聚物在细胞内如何以及在何处解络合以进行 DNA 转录和复制，人们知之甚少。为了更好地理解这个问题，我们：（i）跟踪 PEI 在转染人类上皮细胞时的细胞内化；（ii）研究 PEI 与模拟核膜的磷脂层的相互作用。生物学和物理化学实验都为 PEI 与脂质双层之间的强结合亲和力提供了证据。首先，共焦显微镜显示，PEI 本身不能穿透细胞核；相反，它排列在细胞质中，并在核周围形成一种光环。其次，表面张力测量、荧光染料泄漏实验和差示扫描量热法表明，PEI 与磷脂单层/双层之间的疏水和静电相互作用的结合导致模型膜中稳定缺陷的形成，允许 PEI 通过单层/双层结构插入。分子动力学模拟也支持了 PEI-脂质双层中孔形成的结果。正如本文通篇讨论的那样，这些结果可能揭示了 PEI 与核膜相互作用在 DNA 释放中可能发挥积极作用的机制：一方面，PEI-膜相互作用有望通过与 DNA 竞争结合 PEI 来促进从多聚物中解离 DNA，另一方面，形成的缺陷有望作为进入细胞核的去络合 DNA 的通道。更好地理解阳离子聚合物转染的机制为开发更有效的载体以改善基因治疗和新一代 DNA 疫苗开辟了道路。
