包载治疗基因的聚合物纳米粒子:I.纳米粒子制备及动物模型基因治疗实验研究

以可生物降解材料聚乳酸聚乙醇酸共聚物(Poly-dl-lactic-co-glycolic,PLGA)为原料,采用多相乳化技术制备载VEGF纳米粒子。并对纳米粒子的粒径,VEGF含量,体外释放等进行了测定。VEGF纳米粒子和VEGF裸质粒被注射到兔下肢缺血模型的缺血部位,通过RT-PCR,免疫组化和血管造影等技术来验证基因治疗的效果,评价VEGF纳米粒子作为基因载体在动物模型基因治疗中的效率。制备的VEGF纳米粒子的平均粒径约为300nm,包埋效率在96%以上,纳米粒子中VEGF含量约4%。可在体外维持恒定释放约两周。两周基因注射结果表明VEGF-NP治疗组与裸质粒VEGF治疗组的毛细血管密度明显高于对照组,VEGF纳米粒子组(81.22permm2),对照组(29.54mm2),两者有显著性差异(P<0.05)。RT-PCR结果显示VEGF纳米粒子组表达(31.79au*mm)明显高于VEGF裸质粒组(9.15au*mm)。在动物模型中VEGF纳米粒子是比裸质粒DNA更好的基因载体系统,结果显示了纳米粒子可望在人类基因治疗中得到很好的应用。

Polymeric Nanoparticles with Therapeutic Gene for Gene Therapy: I. Preparation and In Vivo Gene Transfer Study

VEGF nanoparticle(VEGF-NP) was prepared by a multi-emulsification technique using a biodegradable poly-dl-lactic-co-glycolic (PLGA) as matrix material?The nanoparticles were characterized for size, VEGF loading capacity, and in vitro release. VEGF-NP and naked VEGF plasmid were intramuscularly injected into the ischemia site of the rabbit chronic hindlimb ischemia model and the efficiency of VEGF-NP as gene delivery carrier for gene therapy in animal model was evaluated. Gene therapuetic effect was assessed evaluated by RT-PCR, immunohistochemistry and angiography assay. The average size of VEGF-NP was around 300 nm. The encapsulation efficiency of VEGF was above 96%. Loading amount of VEGF in the nanoparticles was about 4%. In vitro, nanoparticles maintained sustained-release of VEGF for two weeks. Two weeks post gene injection the capillary density in VEGF-NP group (81.22 per mm2) was significantly higher than that in control group (29.54 mm2). RT-PCR results showed greatly higher VEGF expression in VEGF-NP group (31.79au*mm) than that in naked VEGF group (9.15 au*mm). As a carrier system for gene therapy in animal model, VEGF-NP is much better than naked DNA plasmid. The results demonstrate great possibility of using NP carrier in human gene therapy.