基于聚乙烯亚胺的还原响应型胶束提高反义寡核苷酸转染效率及其体内抗肿瘤作用研究

Study on the Improvement of Transfection Efficiency of Antisense Oligonucleotides and Its in Vivo Anti-tumor Effect by Reduction Responsive Micelle Based on Polyethyleneimine

关键词:  聚乙烯亚胺  还原响应  反义寡核苷酸  转染  抗肿瘤

Key words:  polyethyleneimine  reduction responsive  antisense oligonucleotide  transfection  anti-tumor

摘要:

目的 在支化聚乙烯亚胺(polyethyleneimine，PEI)基础上，合成一种含有二硫键的聚合物PEI-ss-PEG₂₀₀₀-DSPE，并将其制备成阳离子胶束(P-ss-PD)，在人乳腺癌细胞株中考察P-ss-PD胶束降低细胞毒性，提高反义寡核苷酸(Antisense oligonucleotides，ASO)转染效率的能力，并对P-ss-PD胶束在裸鼠体内的抗肿瘤效果进行研究。方法 以二硫键为连接臂，将PEG₂₀₀₀-DSPE接枝到支化PEI上合成PEI-ss-PEG₂₀₀₀-DSPE。采用乙醇注入法制备P-ss-PD胶束，与ASO结合后得到P-ss-PD/ASO纳米复合物。采用激光粒度分析仪测定不同质量比的P-ss-PD/ASO纳米复合物的粒径和zeta电位。采用琼脂糖凝胶阻滞试验考察P-ss-PD/ASO纳米复合物的结合度，确定最佳质量比；同时考察P-ss-PD胶束的还原响应能力。采用CCK8试剂盒检测P-ss-PD胶束的细胞毒性。使用流式细胞仪和高内涵细胞成像分析系统考察P-ss-PD胶束在MDA-MB-231细胞中的转染效率。建立MDA-MB-231荷瘤裸鼠模型，考察P-ss-PD胶束的抑瘤活性。结果 当质量比为300∶1时，P-ss-PD/ASO纳米复合物粒径最小且稳定性较好，平均粒径为(58.90±4.08)nm，平均zeta电位值为(16.80±1.23)mV，形态为均一的球形。P-ss-PD/ASO纳米复合物具有还原响应力，在高度还原性条件下能够释放装载的ASO。在体外细胞实验中，与未修饰的PEI相比，P-ss-PD胶束的细胞毒性显著降低，细胞转染效率显著提高。在体内动物实验中， P-ss-PD/ASO纳米复合物对荷瘤裸鼠的肿瘤生长抑制率>50%。结论 本研究制备的P-ss-PD胶束是一种低毒高效的非病毒载体，具有还原响应释放特性，在ASO药物递送方面具有应用前景。

Abstract:

OBJECTIVE To synthesize a polymer PEI-ss-PEG₂₀₀₀-DSPE containing disulfide bond and to prepare as cationic micelle(P-ss-PD) based on branched polyethyleneimine(PEI). To investigate the ability of P-ss-PD micelle to reduce cytotoxicity and improve the transfection efficiency of antisense oligonucleotide(ASO) in human breast cancer cell lines, and to study the anti-tumor effect of P-ss-PD micelle in nude mice. METHODS PEI-ss-PEG₂₀₀₀-DSPE was synthesized by grafting PEG₂₀₀₀-DSPE onto branched PEI with disulfide bond as a connecting arm. P-ss-PD micelle was prepared by ethanol injection method and P-ss-PD/ASO nanocomplex was obtained by combining P-ss-PD micelle with ASO. The particle size and zeta potential of P-ss-PD/ASO nanocomplex at various mass ratios were determined by laser particle size analyzer. Agarose gel retardation assay was used to investigate the binding degree of P-ss-PD/ASO nanocomplex and determine the optimal mass ratio. At the same time, the reduction responsive ability of P-ss-PD micelle was investigated. The cytotoxicity of P-ss-PD micelle was detected by CCK8 kit. The transfection efficiency of P-ss-PD micelle was investigated by flow cytometry and high content cell imaging analysis system in MDA-MB-231 cells. The anti-tumor effect of P-ss-PD micelle was investigated by tumor-bearing nude mice models.RESULTS When the mass ratio was 300∶1, the particle size of P-ss-PD/ASO nanocomplex was the smallest and had a good stability. The average particle size was (58.90 ± 4.08)nm, the average zeta potential was (16.80 ± 1.23)mV, and the morphology was uniform spherical. P-ss-PD/ASO nanocomplex had the reduction responsive ability and could release ASO under highly reductive conditions. In vitro, compared with unmodified branched PEI, the cytotoxicity of P-ss-PD micelle was significantly reduced and the transfection efficiency was significantly increased. In vivo, the tumor growth inhibition rate of P-ss-PD/ASO nanocomplex in tumor-bearing nude mice was more than 50%. CONCLUSION The P-ss-PD micelle prepared in this study is a kind of low toxicity and high transfection efficiency non-viral vector, which has the characteristics of reduction responsive releasing, and shows a promising application in ASO drug delivery.