Department of Neurology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, 1665 Kongjiang Road, Shanghai 200092, China.
J Nanobiotechnology. 2014 Apr 6;12:13. doi: 10.1186/1477-3155-12-13.
A challenge in gene therapy is the efficient delivery of DNA/siRNA to the diseased cells. The physicochemical characteristics of siRNA, such as high molecular weight, negative charges and hydrophilic nature-prevent passive diffusion across the plasma membrane for most cells. A therapeutically feasible carrier for intra-cellular delivery of gene materials should accomplish a series of tasks such as: condensing nucleic acid, protecting nucleic acid from leaking in vivo, facilitating endosome escape and releasing DNA/siRNA to the target site. To meet these requirements, an efficient gene vector based on polycation synthesis for siRNA delivery both in vitro and in vivo was developed.
The polymer was synthesized by 1, 4-butanediol bis (chloroformate) and PEI 800 Da to form PEI-Bu which could condense siRNA at the N/P ratio of 38.35 or above. The size of the nanoparticles was 100-300 nm and zeta potential was in the range of 10-30 mV at different N/P ratios. The nanoparticles can achieve the ability of cellular uptake and the silencing efficiency was about 46.63% in SMMC-7721 cell line which was generated to stably express GL3 luciferase gene. The cytotoxicity of the polyplex nanoparticles was almost negligible on SMMC-7721 cells by MTT assay, indicating that the reduced luciferase expression was the effect of RNAi, not the influence of cytotoxicity of polyplexes. The polyplex nanoparticle formulated by PEI-Bu and siRNA at N/P ratio of 115.05 was injected into the SMMC-7721 tumor bearing mice locally and the expression of luciferase can reduce to 63.17% compared with control group.
Results in this study suggested that PEI-Bu polycation might provide a promising solution for siRNA delivery and had the potential in anti-tumor gene therapy.
基因治疗的一个挑战是将 DNA/siRNA 有效递送到病变细胞。siRNA 的理化特性，如高分子量、负电荷和亲水性，阻止了大多数细胞的被动扩散穿过质膜。一种用于细胞内基因物质递送的治疗可行载体应该完成一系列任务，如：凝聚核酸、防止核酸在体内泄漏、促进内涵体逃逸和将 DNA/siRNA 释放到靶位。为了满足这些要求，开发了一种基于聚阳离子合成的高效基因载体，用于体外和体内的 siRNA 递送。
该聚合物通过 1,4-丁二醇双（氯甲酸酯）和 PEI 800Da 合成形成 PEI-Bu，其在 N/P 比为 38.35 或以上时可以凝聚 siRNA。纳米粒的大小为 100-300nm，在不同的 N/P 比下，zeta 电位在 10-30mV 范围内。纳米粒可以实现细胞摄取的能力，并且在稳定表达 GL3 荧光素酶基因的 SMMC-7721 细胞系中，沉默效率约为 46.63%。MTT 测定表明，多聚物纳米粒对 SMMC-7721 细胞的细胞毒性几乎可以忽略不计，这表明降低的荧光素酶表达是 RNAi 的效果，而不是多聚物的细胞毒性的影响。PEI-Bu 和 siRNA 在 N/P 比为 115.05 下形成的多聚物纳米粒局部注射到 SMMC-7721 荷瘤小鼠体内，与对照组相比，荧光素酶的表达可降低至 63.17%。
本研究结果表明，PEI-Bu 聚阳离子可能为 siRNA 递送提供一种有前途的解决方案，并具有抗肿瘤基因治疗的潜力。
