载基因/化疗药物双层纳米粒的制备及其体内外抗乳腺癌效应初步研究

Preparation and anti-breast neoplasms effect in vitro and in vivo evaluation of gene/chemotherapeutic drug loaded double-layer nanoparticles

目的以血管生成为靶点治疗肿瘤的假说验证后,抗肿瘤血管生成基因治疗因其明显的优势而受到关注,与化疗联合,既抑制肿瘤血管内皮细胞,亦杀灭肿瘤细胞,协同增强抗肿瘤效应。文中探讨了共载血管内皮生长因子(VEGF)的小型干扰RNA(siRNA)与化疗药物(EPI)的双层纳米粒的负载基因能力和体内外抑制乳腺癌初步效应。方法将合成的mPEG-g-CS与PLGA纳米粒通过超声透析法制备成双层纳米粒(DL NPs),外层为mPEG-g-CS,负载siRNA,内层为PLGA纳米粒,包载化疗药物EPI,研究双层纳米粒的理化性质,基因负载能力。将裸鼠随机数字表法分成4组,每组6只,重复3次:等渗盐水组,游离EPI组、mPEG-g-CS-siRNA NPs组以及DL NPs-siRNA组,EPI给药剂量为25 mg EPI/kg,mPEG-g-CS-siRNA以及DL-siRNA NPs给药剂量为65μg siRNA/kg,开始进行治疗。定期测量肿瘤长宽,22 d时处死裸鼠,取出肿瘤组织、称重。结果琼脂糖凝胶电泳阻滞实验表明mPEG-g-CS能有效地与pDNA结合形成稳定的复合物,且对细胞活性影响小,证明其可作为基因载体。siRNA浓度为100 nmol/L为最佳负载浓度。当N/P比例为5时,mPEG-g-CS-siRNA的粒径最小[(-12.70±0.42)nm],而对DL NPs-siRNA而言,当N/P比例为20时其粒径最小[(153.82±30.15)nm]。当N/P比例为0.1时,可以看到有少量的pDNA迁移,此时mPEG-g-CS不能完全缩合pDNA;当N/P比例为0.4及之上,pDNA完全被mPEG-g-CS阻滞,此时mPEG-g-CS已经完全缩合pDNA。以聚乙烯亚胺(PEI)为阳性对照,MCF-7细胞、HUVEC细胞与纳米粒共孵育24、48、72 h后,mPEG-g-CS-p DNA NPs都显示出较低的毒性。但是随着培养时间的延长,mPEG-g-CS-p DNA NPs的毒性增加。siRNA浓度为100 nmol/L时,MCF-7细胞存活率为78.66%,HUVEC的细胞存活率为85.69%,毒性更大。在孵育24 h时,DL NPs-siRNA浓度为0.5、50和500μg/mL时MCF-7细胞的平均存活率分别是92.4%、78.1%和51.4%,并且,随着孵育时间的延长,各组细胞存活率均降低。治疗22 d后,游离EPI组的肿瘤抑制率约为22.36%,mPEG-g-CS-siRNA组的肿瘤抑制率约为85.01%,相比之下,DL NPs-siRNA组肿瘤抑制率高达99.90%。22 d后,解剖小鼠,取出肿瘤组织,对照组的肿瘤体积和质量最大,DL NPs-siRNA组最小,肿瘤几乎完全消失。结论DL NPs可用于负载siRNA及化疗药物,有望成为一种新的抗肿瘤药物输送体系。

Objective After the hypothesis of treating tumors with angiogenesis as the target was verified,anti-tumor angiogenesis gene therapy has attracted attention due to its obvious advantages.Combined with chemotherapy,it can not only inhibit tumor vascular endothelial cells,but also kill tumor cells,and synergistically enhance the anti-tumor effect.In this study,we discussed the gene loading ability of the double-layer nanoparticles,as well as the preliminary effects of small interfering RNA(siRNA)co-loading vascular endothelial growth factor(VEGF)and chemotherapy drugs on breast cancer inhibition in vitro and in vivo.Methods The DL NPs comprising a nanoscale self-assembly siRNA-mPEG-g-CS envelope coating on a nuclear PLGA NPs loading Epirubicin.The physical/chemical properties,DNA-binding property and in vitro and in vivo anti-tumor activities were evaluated.Results The agarose gel electrophoresis experiment showed that when N/P was 0.4,mPEG-g-CS completely condensed pDNA.The cytotoxicity experiment showed that the nanocarrier had little effect on cell activity,which proved that the DL NPs could be used as siRNA vector.Conclusion In this study,a proper transfection concentration of siRNA(100 nM)was used.The results of in vivo anti-tumor effect in tumor-bearing nude mice showed that the DL NPs treatment group showed the most effective tumor growth inhibition.The DL NPs can be used novel carriers for gene therapy and combined chemotherapy.