Jilin University, School of Life Sciences, Changchun, Jilin, China.
Jilin University, School of Life Sciences, Changchun, Jilin, China; Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH, USA.
Int J Pharm. 2019 Jun 10;564:214-224. doi: 10.1016/j.ijpharm.2019.04.049. Epub 2019 Apr 17.
Gene therapy strategies for liver cancer have broad application prospects but still lack a stable and efficient delivery vehicle. To overcome this obstacle, we designed a multifunctional gene delivery vector, sTPssOLP, which was based on oleylamine (OA)-modified disulfide-containing polyethylenimine (PEI) and incorporated into lipids to prepare a lipid nanoparticle. sTPssOLP consisted of the core of PEI derivative and cationic lipids bound to siRNA. The modified polyethylene glycol (PEG) and transferrin (Tf) were partially embedded in the phospholipid bilayer through the lipid and the other as the outer shell. The aim was to use the redox responsiveness of disulfide to trigger siRNA release in cytoplasm to enhance transfection efficiency. Pegylated lipids and Tf focus on increasing cycle life in the body and increasing accumulation at the tumor site of the carrier. In addition, two vectors were prepared as controls, one based on a PEI derivative containing no disulfide bond (POLP) and the other on the surface of the carrier not linked to Tf (PssOLP). PEI derivatives effectively avoid the toxicity problems caused by the use of PEI alone (25 kDa). Meanwhile, it was confirmed by gel retardation experiments that in the presence of dithiothreitol (DTT), the disulfide bond can indeed be reduced and the siRNA entrapped in the vector can be released. Both HepG2 and SMMC had significant uptake of sTPssOLP. The results of intracellular and lysosomal co-localization indicated that sTPssOLP achieved lysosomal escape. RT-PCR and Western blot results also confirmed that sTPssOLP had the best gene silencing activity. In vivo, the tumor inhibition rate of sTPssOLP in nude mice carrying HepG2 xenografts was 56%, which was significantly greater than that of the saline control group. In vivo imaging results showed that fluorescently labeled siRNA loaded in sTPssOLP was able to deliver more to the tumor site. At the same time, it was observed that sTPssOLP did not show significant damage to normal tissues. Therefore, this multifunctional gene delivery vector warrants further investigation.
用于肝癌的基因治疗策略具有广阔的应用前景，但仍缺乏稳定高效的递药载体。为了克服这一障碍，我们设计了一种多功能基因递药载体 sTPssOLP，它以油胺（OA）修饰的含二硫键聚乙二烯亚胺（PEI）为基础，并与脂质结合以制备脂质纳米粒。sTPssOLP 由 PEI 衍生物的核心和与 siRNA 结合的阳离子脂质组成。修饰的聚乙二醇（PEG）和转铁蛋白（Tf）通过脂质部分嵌入磷脂双层，另一个作为外壳。目的是利用二硫键的氧化还原响应在细胞质中触发 siRNA 释放，以提高转染效率。聚乙二醇化脂质和 Tf 旨在增加载体在体内的循环寿命并增加载体在肿瘤部位的积累。此外，还制备了两种对照载体，一种基于不含二硫键的 PEI 衍生物（POLP），另一种基于载体表面未与 Tf 连接（PssOLP）。PEI 衍生物有效避免了单独使用 PEI 引起的毒性问题（25kDa）。同时，凝胶阻滞实验证实，在二硫苏糖醇（DTT）存在的情况下，二硫键确实可以被还原，并且包裹在载体中的 siRNA 可以被释放。HepG2 和 SMMC 对 sTPssOLP 均有明显的摄取。细胞内和溶酶体共定位的结果表明 sTPssOLP 实现了溶酶体逃逸。RT-PCR 和 Western blot 结果也证实 sTPssOLP 具有最佳的基因沉默活性。在体内，携带 HepG2 异种移植瘤的裸鼠中 sTPssOLP 的肿瘤抑制率为 56%，明显高于生理盐水对照组。体内成像结果表明，负载在 sTPssOLP 中的荧光标记 siRNA 能够更多地递送到肿瘤部位。同时，观察到 sTPssOLP 对正常组织没有明显的损伤。因此，这种多功能基因递药载体值得进一步研究。
