Li Zhenhua, Ning Wen, Wang Jumei, Choi Augustine, Lee Pui-Yan, Tyagi Pradeep, Huang Leaf
Center for Pharmacogenetics, School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA.
Pharm Res. 2003 Jun;20(6):884-8. doi: 10.1023/a:1023887203111.
Currently, most pDNA delivery systems based on synthetic polymers are either nonbiodegradable or not sensitive to the release environment. The primary objective of this study was to develop and evaluate an aqueous-based, thermosensitive, biodegradable and biocompatible triblock copolymer to control pDNA delivery in vitro and in vivo.
The triblock copolymers, poly[ethylene glycol-b-(D, L-lactic acid-co-glycol acid)-b-ethylene glycol] (PEG-PLGA-PEG), were synthesized as previously described. The molecular weight and polydispersity of PEG-PLGA-PEG were monitored by gel permeation chromatography (GPC). The cytotoxicity of PEG-PLGA-PEG was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The release of 32P-labeled pDNA entrapped in aqueous dispersion of PEG-PLGA-PEG in 0.1 mol/L sodium phosphate buffer solution (pH 7.4) was studied at 37 degrees C under agitation. Gene transfection efficiency was evaluated in a skin wound model in CD-1 mice.
The aqueous dispersion of PEG-PLGA-PEG flows freely at room temperature but form a gel at 37 degrees C body temperature. The in vitro degradation of PEG-PLGA-PEG lasted for more than 30 days. The cytotoxicity of PEG-PLGA-PEG evaluated in HEK 293 cells was significantly lower than that of poly-L-lysine hydrochloride. The release profile of supercoiled pDNA from the polymer followed the zero-order kinetics up to 12 days. Maximal gene expression of luciferase was at 24 h in the skin wound of CD-1 mice and by 72 h, the expression dropped by nearly 94%.
These results suggest hydrogel formed by PEG-PLGA-PEG could be a promising platform for delivery of pDNA, which represents a novel strategy that may serve as a non-viral vector for gene therapy in wound healing.
目前，大多数基于合成聚合物的质粒DNA（pDNA）递送系统要么不可生物降解，要么对释放环境不敏感。本研究的主要目的是开发和评估一种水基、热敏、可生物降解且生物相容的三嵌段共聚物，以在体外和体内控制pDNA的递送。
按照先前描述的方法合成三嵌段共聚物聚[乙二醇-b-（D，L-乳酸-共-乙醇酸）-b-乙二醇]（PEG-PLGA-PEG）。通过凝胶渗透色谱法（GPC）监测PEG-PLGA-PEG的分子量和多分散性。通过3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐法评估PEG-PLGA-PEG的细胞毒性。在37℃搅拌条件下，研究了包裹在PEG-PLGA-PEG水分散体中的32P标记的pDNA在0.1mol/L磷酸钠缓冲溶液（pH 7.4）中的释放情况。在CD-1小鼠的皮肤伤口模型中评估基因转染效率。
PEG-PLGA-PEG的水分散体在室温下自由流动，但在37℃体温下形成凝胶。PEG-PLGA-PEG的体外降解持续超过30天。在HEK 293细胞中评估的PEG-PLGA-PEG的细胞毒性明显低于聚-L-赖氨酸盐酸盐。超螺旋pDNA从聚合物中的释放曲线在12天内遵循零级动力学。荧光素酶的最大基因表达在CD-1小鼠的皮肤伤口中于24小时出现，到72小时，表达下降了近94%。
这些结果表明，由PEG-PLGA-PEG形成的水凝胶可能是一种有前途的pDNA递送平台，这代表了一种新的策略，可作为伤口愈合中基因治疗的非病毒载体。
