Department of Materials, University of California, Santa Barbara, CA 93106, USA.
Biomaterials. 2012 Jun;33(19):4928-35. doi: 10.1016/j.biomaterials.2012.03.038. Epub 2012 Apr 1.
Cationic liposome-DNA (CL-DNA) complexes are being pursued as nonviral gene delivery systems for use in applications that include clinic trials. However, to compete with viral vectors for systemic delivery in vivo, their efficiencies and pharmacokinetics need to be improved. The addition of poly (ethylene glycol)-lipids (PEGylation) prolongs circulation lifetimes of liposomes, but inhibits cellular uptake and endosomal escape of CL-DNA complexes. We show that this limits their transfection efficiency (TE) in a manner dependent on the amount of PEG-lipid, the lipid/DNA charge ratio, and the lipid membrane charge density. To improve endosomal escape of PEGylated CL-DNA complexes, we prepared an acid-labile PEG-lipid (HPEG2K-lipid, PEG MW 2000) which is designed to lose its PEG chains at the pH of late endosomes. The HPEG2K-lipid and a similar but acid-stable PEG-lipid were used to prepare PEGylated CL-DNA complexes. TLC and dynamic light scattering showed that HPEG2K-CL-DNA complexes are stable at pH 7.4 for more than 24 h, but the PEG chains are cleaved at pH 5 within 1 h, leading to complex aggregation. The acid-labile HPEG2K-CL-DNA complexes showed enhanced TE over complexes stabilized with the acid-stable PEG-lipid. Live-cell imaging showed that both types of complexes were internalized to quantitatively similar particle distributions within the first 2 h of incubation with cells. Thus, we attribute the increased TE of the HPEG2K-CL-DNA complexes to efficient endosomal escape, enabled by the acid-labile HPEG2K-lipid which sheds its PEG chains in the low pH environment of late endosomes, effectively switching on the electrostatic interactions that promote fusion of the membranes of complex and endosome.
阳离子脂质体-DNA（CL-DNA）复合物作为非病毒基因传递系统，正在被研究用于临床试验等应用。然而，为了与病毒载体在体内进行系统传递竞争，需要提高它们的效率和药代动力学性质。聚乙二醇化脂质（PEGylation）的添加延长了脂质体的循环寿命，但抑制了 CL-DNA 复合物的细胞摄取和内涵体逃逸。我们发现，这以依赖于 PEG 脂质的量、脂质/DNA 电荷比和脂质膜电荷密度的方式限制了它们的转染效率（TE）。为了提高 PEG 化 CL-DNA 复合物的内涵体逃逸，我们制备了一种酸不稳定的 PEG 脂质（HPEG2K-脂质，PEG MW 2000），该脂质在晚期内涵体的 pH 值下会失去其 PEG 链。使用 HPEG2K-脂质和类似但酸稳定的 PEG 脂质来制备 PEG 化 CL-DNA 复合物。TLC 和动态光散射表明，HPEG2K-CL-DNA 复合物在 pH 7.4 下稳定超过 24 小时，但在 pH 5 下 1 小时内 PEG 链被切断，导致复合物聚集。酸不稳定的 HPEG2K-CL-DNA 复合物的 TE 高于用酸稳定的 PEG 脂质稳定的复合物。活细胞成像显示，在与细胞孵育的前 2 小时内，两种类型的复合物都以定量相似的颗粒分布被内化。因此，我们将 HPEG2K-CL-DNA 复合物的 TE 增加归因于有效的内涵体逃逸，这得益于酸不稳定的 HPEG2K-脂质，它在晚期内涵体的低 pH 环境中失去其 PEG 链，有效地开启了促进复合物和内涵体膜融合的静电相互作用。
