Mechanism of Polyplex- and Lipoplex-Mediated Delivery of Nucleic Acids: Real-Time Visualization of Transient Membrane Destabilization without Endosomal Lysis
Mechanism of Polyplex- and Lipoplex-Mediated Delivery of Nucleic Acids: Real-Time Visualization of Transient Membrane Destabilization without Endosomal Lysis
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DOI：
10.1021/nn3049494
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发表时间：
2013-05-01
期刊：
影响因子：
17.1
通讯作者：
Zuhorn, Inge S.
中科院分区：
文献类型：
--
作者：
Rehman, Zia Ur;Hoekstra, Dick;Zuhorn, Inge S.
脂质复合物和聚合复合物作为非病毒基因传递载体被广泛应用。虽然它们的转染效率相当，但它们的递送机制，特别是在从核内体释放核酸的水平上，是不同的。因此，提出了脂质复合物介导的释放依赖于脂质混合，如在脂质复合物和内体靶膜之间发生的，随后的膜去稳定化导致核酸递送到胞质溶胶中。相比之下，聚合复合物，特别是那些显示高质子缓冲能力的聚合复合物，从内体释放其核酸货物的机制被认为依赖于所谓的“质子海绵效应”，本质上是内体膜的质子诱导的破裂。然而，尽管大量的间接见解支持这两种机制，但仍然缺乏直接证据。因此，为了进一步阐明这些机制，我们通过活细胞成像研究了脂质复合物和复合物与Hela细胞的相互作用。如在2小时的孵育期内监测的，我们的数据显示，与在脂质复合物介导的递送的情况下涉及许多纳米载体相反，只有非常有限数量的聚合复合物，即，每个细胞少至一个至四/五个纳米载体，每个细胞平均一个/两个，有助于核酸从内体释放并随后积累到细胞核中。值得注意的是，在两种情况下，都没有观察到内体完全破裂，也没有观察到完整的聚合复合物或脂质复合物释放到胞质溶胶中。相反，在内体逃逸时，聚合物及其遗传有效载荷分别喷射到细胞质中，推测是经由内体膜内的局部孔。具体而言，观察到核酸和载体（残余物）几乎瞬时且完全地从内体排出。在脂质复合物的情况下，数据表明在同一内体膜内随着时间的推移形成多个瞬时孔，通过这些孔，货物更逐渐地转移到胞质溶胶中。
Lipoplexes and polyplexes are widely applied as nonviral gene delivery carriers. Although their efficiencies of transfection are comparable, their mechanisms of delivery, specifically at the level of nudeic acid release from endosomes, are different. Thus, lipoplex-mediated release is proposed to rely on lipid mixing, as occurs between lipoplex and endosomal target membrane, the ensuing membrane destabilization leading to nucleic add delivery into the cytosol. By contrast, the mechanism by which polyplexes, particularly those displaying a high proton buffering capacity, release their nucleic acid cargo from the endosome, is thought to rely on a so-called "proton sponge effect", in essence an osmotically induced rupturing of the endosomal membrane. However, although a wealth of Indirect insight supports both these mechanisms, direct evidence Is still lacking. Therefore, to further clarify these mechanisms, we have investigated the interaction of lipo- and polyplexes with Hela cells by live cell imaging. As monitored over an incubation period of 2 h, our data reveal that in contrast to the involvement of numerous nanocarriers in case of lipoplex-mediated delivery, only a very limited number of polyplexes, that is, as few as one up to four/five nanocarriers per cell, with an average of one/two per cell, contribute to the release of nudeic acids from endosomes and their subsequent accumulation into the nucleus. Notably, in neither case complete rupture of endosomes nor release of intact polyplexes or lipoplexes into the cytosol was observed. Rather, at the time of endosomal escape both the polymer and its genetic payload are separately squirted into the cytoplasm, presumably via (a) local pore(s) within the endosomal membrane. Specifically, an almost Instantaneous and complete discharge of nucleic acids and carrier (remnants) from the endosomes is observed. In case of lipoplexes, the data suggest the formation of multiple transient pores over time within the same endosomal membrane, via which the cargo is more gradually transferred into the cytosol.