Department of Pharmaceutics and Biopharmaceutics, University of Marburg, Robert-Koch-Str. 4, 35037, Marburg, Germany.
Punjab University College of Pharmacy, University of the Punjab, Allama Iqbal Campus, Lahore, 54000, Pakistan.
Sci Rep. 2020 Dec 8;10(1):21446. doi: 10.1038/s41598-020-78123-6.
Clinical success of effective gene therapy is mainly hampered by the insufficiency of safe and efficient internalization of a transgene to the targeted cellular site. Therefore, the development of a safe and efficient nanocarrier system is one of the fundamental challenges to transfer the therapeutic genes to the diseased cells. Polyamidoamine (PAMAM) dendrimer has been used as an efficient non-viral gene vector (dendriplexes) but the toxicity and unusual biodistribution induced by the terminal amino groups (-NH) limit its in vivo applications. Hence, a state of the art lipid modification with PAMAM based gene carrier (lipodendriplexes) was planned to investigate theirs in vitro (2D and 3D cell culture) and in vivo behaviour. In vitro pDNA transfection, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) generation, cellular protein contents, live/dead staining and apoptosis were studied in 2D cell culture of HEK-293 cells while GFP transfection, 3D cell viability and live/dead staining of spheroids were performed in its 3D cell culture. Acute toxicity studies including organ to body index ratio, hematological parameters, serum biochemistry, histopathological profiles and in vivo transgene expression were assessed in female BALB/c mice. The results suggested that, in comparison to dendriplexes the lipodendriplexes exhibited significant improvement of pDNA transfection (p < 0.001) with lower LDH release (p < 0.01) and ROS generation (p < 0.05). A substantially higher cellular protein content (p < 0.01) and cell viability were also observed in 2D culture. A strong GFP expression with an improved cell viability profile (p < 0.05) was indicated in lipodendriplexes treated 3D spheroids. In vivo archives showed the superiority of lipid-modified nanocarrier system, depicted a significant increase in green fluorescent protein (GFP) expression in the lungs (p < 0.01), heart (p < 0.001), liver (p < 0.001) and kidneys (p < 0.001) with improved serum biochemistry and hematological profile as compared to unmodified dendriplexes. No tissue necrosis was evident in the animal groups treated with lipid-shielded molecules. Therefore, a non-covalent conjugation of lipids with PAMAM based carrier system could be considered as a promising approach for an efficient and biocompatible gene delivery system.
基因治疗的临床成功主要受到将转基因有效内化到靶向细胞部位的安全性和效率不足的阻碍。因此，开发安全高效的纳米载体系统是将治疗基因转染到病变细胞的基本挑战之一。聚酰胺胺（PAMAM）树枝状大分子已被用作有效的非病毒基因载体（树枝状聚合物），但其末端氨基（-NH）引起的毒性和异常的生物分布限制了其在体内的应用。因此，计划用基于 PAMAM 的基因载体（脂树状聚合物）进行最新的脂质修饰，以研究其在体外（2D 和 3D 细胞培养）和体内的行为。在 HEK-293 细胞的 2D 细胞培养中研究了 pDNA 转染、乳酸脱氢酶（LDH）释放、活性氧（ROS）生成、细胞蛋白含量、死活染色和细胞凋亡，而 GFP 转染、球体的 3D 细胞活力和死活染色则在其 3D 细胞培养中进行。在雌性 BALB/c 小鼠中进行了急性毒性研究，包括器官与体重比、血液学参数、血清生化、组织病理学特征和体内转基因表达。结果表明，与树枝状聚合物相比，脂树状聚合物显著提高了 pDNA 转染效率（p<0.001），同时降低了 LDH 释放（p<0.01）和 ROS 生成（p<0.05）。在 2D 培养中还观察到细胞蛋白含量显著增加（p<0.01）和细胞活力更高。脂质修饰的纳米载体系统显示出很强的 GFP 表达和改善的细胞活力谱（p<0.05），表明 3D 球体用脂树状聚合物处理后。体内研究结果显示，脂质修饰的纳米载体系统具有优越性，与未修饰的树枝状聚合物相比，肺部（p<0.01）、心脏（p<0.001）、肝脏（p<0.001）和肾脏（p<0.001）中的绿色荧光蛋白（GFP）表达显著增加，血清生化和血液学特征得到改善。用脂质屏蔽分子处理的动物组中没有观察到组织坏死。因此，PAMAM 基载体系统的非共价偶联可以被认为是一种有效的、生物相容性的基因传递系统的有前途的方法。
