携带超抗原SEA基因的白蛋白纳米载体的构建及其治疗膀胱癌潜能研究

Potential of human serum albumin nanoparticles as a novel gene vector to deliver SEA gene for targeting therapy of bladder cancer

目的 构建一种非病毒载体基因投递系统-携带超抗原葡萄球菌肠毒素A（SEA）基因的白蛋白纳米载体,观察白蛋白纳米粒表征并探讨其潜在的靶向基因投递作用和强大的抗瘤机制.方法 采用去溶剂化法制备白蛋白纳米粒,平均粒径（253.1±11.9）nm,Zeta电位（-34.0±4.5）mV,PDI 0.43±0.04;抽提SEA基因质粒（pSEA）260/280：1.84±0.02,pSEA浓度：（85.54±1.43）mg/L;经生物活性及基因测序鉴定后耦合SEA基因质粒和白蛋白纳米载体,并观察耦合物的稳定性及白蛋白纳米载体对SEA基因的保护作用.结果 成功构建携带超抗原SFA基因的白蛋白纳米载体,平均粒径（118.9±4.8）nm,Zeta电位（-43.9±10.5）mV,PDI 0.19±0.02,基因转载率为（97.61±0.06）%,性质稳定、分散性较好,体外实验表明白蛋白纳米载体能保护SEA基因免受DNase Ⅰ的降解.结论 获得符合超抗原SEA基因转染要求的白蛋白纳米载体.

Objective To assess the characteristics of human serum albumin nanoparticles （HSA-NP） as a nonviral vector system for delivery staphylococcal enterotoxin A （SEA） gene and probe into its potential targeted antitumor mechanism.Methods HSA-NP and plasmid containing SEA gene （pSEA）encapsulated in HSA （pSEA-HSA-NP） were prepared by a desolvation-crosslinking method.HSA-NP had a mean size of （253.1 ± 11.9） nm,zeta potential of （ -34.0 ±4.5） mV,polydispersity index of 0.43 ±0.04.The superantigen SEA gene was extracted by the Endo-Free Plasmid Maxi Kit,260/280 of pSEA was 1.84 ±0.02 and the concentration of pSEA was （ 85.54 ± 1.43 ） mg/L.pSEA was verified by sequencing and biological activity survey,the stability of pSEA-HSA-NP was investigated by laying for 10 days at room temperature,and the size and zeta potential were remeasured and contrasted with the samples 10 days before.HSA-NP protecting pSEA from degradation of DNase I was detected by gel electrophoresis.Results Electrophoretic mobility analysis and fluorescent labeling revealed that pSEA-HSA-NP was successfully constructed.PSEA-HSA-NP had a mean size of （ 118.9 ±4.8） nm,zeta potential of （ -43.9 ± 10.5 ） mV,polydispersity index of 0.19 ±0.02 and pSEA encapsulation efficiency of （97.61 ±0.06）%.The characteristics of pSEA-HSA-NP solution laying for 10 days at room temperature indicated a better stability and polydispersity.HSA-NP stabilizing pSEA against DNase I in vitro was also testified by gel electrophoresis.Conclusion pPSEA-HSA-NP served the transfecting needs of superantigen SEA gene.