背景与目的:选择合适的载体是基因治疗成功的关键之一,近年来一种新型多聚阳离子化合物——聚乙烯亚胺(polyethylenimine,PEI)作为高效低毒的基因转导载体得到广泛重视。本研究应用光化学法制备系列不同粒径的PEI纳米凝胶,初步探讨其转导效率与粒径之间的关系,筛选理想的肿瘤基因转导载体。方法:光化学法制备PEI,光相干光谱仪测定粒径,并用扫描电子显微镜和原子力显微镜表征;以PEI为载体将增强型绿色荧光蛋白(enhanced green fluorescence protein,EGFP)报告基因分别转入Bel7402细胞和A549细胞,荧光显微镜下计数和流式细胞仪检测转染率。结果:光相干光谱仪检测PEI粒径38～200nm,扫描电子显微镜和原子力显微镜检测其形貌多为球形。荧光显微镜下计数和流式细胞仪检测86.9nmPEI(4μg)介导EGFP基因(2μg)时转染率最高,Bel7402细胞分别为(32.75±1.01)%、(32.40±1.41)%,A549细胞分别为(29.81±1.84)%、(30.00±1.86)%;与脂质体相比差异无统计学意义(P>0.05)。结论:光化学法合成的PEI是有效的基因转导载体,PEI粒径为86.9nm转染最为有效。
In this paper, we report a novel method to synthesize polyethylenimine (PEI) nanogels in the range of 80— 200nm with narrow size distribution by photo-chemistry at room temperature in aqueous solution. The nanogels’ size, size distributions and zeta potential were determined by photo correlation spectroscopy (PCS). Spherical morphology of the nanogels was characterized by scanning electron microscopy (SEM) and confirmed by atomic force microscopy (AFM). The nanogels are of high stability, low toxicity and low immunogenicity, as having been confirmed in in vivo tests with mice as animal model, and in vitro tests with human lung and liver cancer cells as well. Efforts are being made for further studies on synthesis and applications of the nanogels.
Photo-induced Fenton reaction was utilized in this paper to control the sizes of polyethylenimine hydrogels （M-PEIs,M,Fe atom） as gene delivery vector.H2O2 and FeCl2 were mixed with PEI prepolymer in water in a 3-neck quartz flask, and the mixture was stirred vigorously.M-PEIs nanogels dispersed very well in water could be prepared under illumination by use of low-pressure Hg lamps at room temperature.This system was protected from oxygen with N2.By changing reaction conditions,such as the pH value,reaction time and concentrations of solutes,sizes of M-PEIs nanogels could be controlled.It was found that the sizes of M-PEIs decreased with the pH value and the concentrations of FeCl2.The increasing of the initiator （H2O2） concentrations would make the particles more tighten,but might result in crosslinking or degradation of particles.A suitable irradiation time ensured formation of homogeneous nanogel products,but prolonged irradiation would cause degradation of nanogels.After photo crosslinking,the products were purified with membrane filters of 0.45 μm and treated further by dialysis bags with size exclusion below 10 kDa in order to make them more stable for storage.
