目的 利用高密度脂蛋白(HDL)作为siRNA载体和多聚体胶束的靶向配体,制备HDL偶联多聚体胶束共递送siRNA和多柔比星(doxorubicin,Dox)载药系统,评价共转运递药系统逆转肿瘤多药耐药,发挥基因与化疗药物协同抗肿瘤的效果。方法 将HDL与经胆固醇修饰的siRNA (chol-siRNA)复合形成siRNA/HDL复合物,再与载Dox胶束(Dox/micelle)共价偶联,形成HDL偶联多聚体胶束共递送siRNA和Dox载药系统(siRNA/HDL-Dox/micelle)。考察多聚体胶束在不同pH缓冲液中的粒径变化和稳定性;利用HepG2/ADM耐药肿瘤细胞模型,考察多聚体胶束的细胞摄取、亚细胞分布和体外抗肿瘤活性;运用RT-PCR与Western blot技术评价siRNA在mRNA水平与蛋白水平上对靶基因的沉默效果。结果 HDL能有效包载siRNA并保护其免被酶解。透射电镜显示,siRNA/HDL-Dox/micelle多聚体胶束外观圆整,粒径分布均匀;并且多聚体胶束在pH 5.3缓冲液中粒径增大,电位升高,稳定性下降。siRNA/HDL-Dox/micelle 通过HDL包载siRNA的方式,将siRNA递送进肿瘤细胞并在mRNA水平及蛋白水平上有效下调P-gp的表达,从而使细胞内Dox蓄积量升高,抗肿瘤活性增强。结论 siRNA/HDL-Dox/micelle多聚体胶束能够有效递送siRNA与Dox进入肿瘤细胞,成功沉默mdr1基因的表达,从而逆转肿瘤耐药,增强Dox的抗肿瘤活性。
Abstract
OBJECTIVE To prepare siRNA/HDL modified Dox/micelle multimeric polymer(siRNA/HDL-Dox/micelle) by using HDL as a siRNA carrier and a targeting ligand and to realize the effective co-delivery of siRNA and antitumor drug. METHODS HDL was incubated with chol-siRNA to prepare siRNA/LDL complex, then coupled with Dox/micelle to form siRNA/HDL modified Dox/micelle multimeric polymer (siRNA/HDL-Dox/micelle). The particle size and stability were investigated in different medium. HepG2/ADM with P-glycoprotein(P-gp) over-expressed were used to study the cell uptake, sub-cellular localization and anti-tumor efficacy in vitro. The ability of siRNA to silence target genes at mRNA and protein level was examined by RT-PCR and Western blot. RESULTS HDL exhibited an efficient binding ability for siRNA and protected siRNA from RNase. The size and surface morphology of siRNA/HDL-Dox/micelle confirmed by TEM showed that most of the micelles were compact and spherical, exhibited a narrow size distribution and good dispersion. The particle size and Zeta potential increased with increasing incubation time in pH 5.3 PBS. The siRNA was efficiently delivered into the cells by encapsulation into HDL, and the expression of P-gp is effectively down-regulated at the mRNA level and the protein level, thereby increasing the accumulation of intracellular Dox and enhancing the antitumor activity. CONCLUSION siRNA/HDL-Dox/micelle could effectively deliver siRNA and Dox into tumor cells, thereby exerting gene silencing, reversing tumor drug resistance and enhancing anti-tumor effect.
关键词
多柔比星 /
多药耐药 /
共转运胶束 /
高密度脂蛋白 /
胞内蓄积
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Key words
doxorubicin /
multidrug resistance /
co-delivery micelle /
high-density lipoprotein /
intracellular accumulation
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中图分类号：
R944
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脚注
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基金
苏州市科技局科研项目资助(SYSD2018232,SYSD2019177)
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