摘要
目的观察携带载缺氧诱导因子-1α(HIF-1α)短发卡式小干扰RNA(shRNA)质粒(pshHIF-1α)的纳米粒转染眼部组织的可行性。方法应用聚乳酸聚乙醇酸共聚物(PLGA)包载绿色荧光蛋白(GFP)表达的pshHIF-1α,制备纳米粒,检测其粒径、包封率、载药量及体外释放情况;将48只大鼠分为4组,每组12只(12只眼),分别向玻璃体内注入10μL磷酸盐缓冲液(PBS)、空白纳米粒(1.89mg/mL)、裸质粒pshHIF-1α(0.02mg/mL)和pshHIF-1α纳米粒(1.89mg/mL)。分别在注药后3、7、14、28d观察纳米粒的眼内穿透性和基因转染情况。通过组织病理学检查,观察注射后第3天眼内组织结构的改变。结果纳米粒的载药率和包封率分别为1.06%和60.2%,平均粒径为284nm,体外释药达4周。体内实验显示,载pshHIF-1α纳米粒定向聚集于视网膜色素上皮(RPE)层,GFP表达时间持续达28d。组织病理学检查眼内未见明显炎性细胞浸润。结论纳米粒可向眼底递送质粒形式的DNA,是眼部基因治疗安全、有效的载体之一。
Objective To evaluate the possibility of nanoparticle as a gene vector for hypoxia-inducible factor 1α （ HIF- 1α）shRNA plasmid （pshHIF-1α）. Methods Nanoparticles encapsulating HIF-1α short hairpin RNA （shRNA） plasmid （pshHIF-1α nanoparticles） encoding for green fluorescence protein （GFP） were formulated using poly （D, L-lactide-coglyeolide） （PLGA） by multiple emulsion technique. The envelopment efficiency, plasmid loading capacity, size and in vitro release of the nanoparticles were determined. Forty-eight eyes of 48 Brown Norway rats were divided into phosphate buffer solution （PBS, 10 μL） group,blank nanoparticles（ 10 μL, 1.89 rag/ mL） group ,free plasmid（ 10 μL,2.0 mg/mL） injection and pshHIF- 1 α NPs （ 10 μL, 1.89 mg/mL） injection group. The animals were sacrificed on day 3,7,14 and 28 after intravitreous injection, and immunofluoreseence was used to evaluate the location of GFP. Histologic analysis was performed to observe the tissue change in the eyes on day 3. Results The envelopment efficiency and the size of pshHIF-1α nanoparticles were 60.2% and 164-342 nm respectively. Loading rate of plasmid in the nanoparticles was 1.06%. In vitro, nanoparticles presented the sustained-release of plasmid for 4 weeks. Immunofluorescence showed that GFP was successfully transferred into the retina and choroid,showing the green fluorescence in the retinal pigment epithelium （RPE） layer at 7 days and lasted for 3 weeks with a significantly longer period than that of group free plasmid, pshHIF-1α nanoparticles did not induce any histology signs of ocular inflammation 3 days after their injection in the vitreous. Conclusion PLGA nanoparticles can act as a vector to transfer specific plasmid in vivo. It possesses the advantages of low drug toxicity,controlled release and prolonged action,and may be a novel therapeutic approach to ocular neovascularization diseases.
出处
《眼科研究》
CSCD
北大核心
2009年第6期462-467,共6页
Chinese Ophthalmic Research
基金
国家自然科学基金资助(30371516、30672291)
德国洪堡基金会仪器设备捐赠基金的资助
关键词
基因载体
PLGA纳米粒
质粒
RNA干扰
缺氧诱导因子-1Α
gene delivery
poly （D, L-lactic/glycolic acid） nanoparticles
plasmid
RNA interference
hypoxiainducible factor 1α
