还原性多肽共载基因和化学治疗药载体的构建与体外评价

Preparation and in vitro evaluation of redox-sensitive polypeptide vector for co-delivery of gene and chemotherapeutic drugs

目的制备一种共载基因和化学治疗药的硫辛酸(LA)修饰的以非内吞机制进入细胞的固有无序蛋白-细胞质定位的内化肽6(CL)的纳米复合物(LA-CL),并考察其在人胚肾细胞系HEK293细胞的转染效率、细胞摄取情况及其体外释放规律。方法以不同比例(2.5%、5%、10%、20%)半胱氨酸作为交联剂,合成4种不同交联度的LA-CLss,分别命名为LA-CLss1~LA-CLss4,并用核磁共振氢谱(1HNMR)和凝胶色谱鉴定。取增强型绿色荧光蛋白质粒(pEGFP)和LA-CLss以不同氮磷比(N/P,2.5、5、10、20、40、80)自组装形成纳米复合物,用激光粒度测定仪测定复合物的粒径和zeta电位,琼脂糖凝胶电泳测定载体LA-CLss对pEGFP的包裹能力和保护能力。用超声乳化法制备载多西他赛(DTX)的载药胶束,并用芘荧光探针光谱法测定LA-CLss3的临界胶束浓度。将LA-CLss/pEGFP纳米复合物与HEK293细胞共同培养,考察不同交联度复合物的细胞转染情况。结果 1HNMR结果确定LA-CLss合成成功。N/P=40时,HEK293细胞对LA-CLss3/p EGFP的转染效率高于LA-CL、LA-CLss1、LA-CLss2、LA-CLss4与pEGFP形成的复合物。超声乳化法制备的载药胶束包封率为(85.25±0.04)%,载药量为(8.81±0.02)%。细胞摄取结果表明,该载体可以有效地将基因递送至细胞内。体外释放实验结果表明,该多肽胶束具有还原性条件敏感释药行为。结论制备的LA-CLss/DTX/p EGFP纳米复合物有望成为一种高效的共载基因和化学治疗药的载体。

Objective To prepare a lipoic acid（LA） modified intrinsically disordered protein-cytosol-localizing internalization peptide 6（CL） nanocomplex（LA-CL） entering cells by non-endocytosis mechanism for co-delivery of gene and chemotherapeutic drugs, and to investigate its transfection efficiency and cellular uptake on human embryonic kidney cell line HEK293 cells and its release behavior in vitro. Methods We synthesized four disulfide cross-linked lipoic acid modified LA-CLss（1-4） at different cross-linked degrees using different mass fractions（2.5%, 5%, 10% and 20%） of cysteine as cross-linking agent. The construction of LA-CLss was characterized by 1 H nuclear magnetic resonance（1 HNMR） and gel permeation chromatography. The LA-CLss/plasmid enhanced green fluorescent protein（p EGFP） nanocomplexes were selfassembled with LA-CLss and p EGFP at different nitrogen/phosphorus（N/P） ratios（2.5, 5, 10, 20, 40 and 80）. The size and zeta potential of LA-CLss/p EGFP nanocomplexes were determined by particle size analyzer, and the pEGFP enrichment capacity of LA-CLss was determined by agarose gel electrophoresis. The docetaxel（DTX）-loaded micelles were prepared by ultrasonic emulsification, and the critical micelle concentration of LA-CLss3 was determined by pyrene fluorescence probe spectroscopy. The LA-CLss/p EGFP nanocomplexes were co-cultured with HEK293 cells, and the transfection efficiencies of LA-CLss/p EGFP nanocomplexes at different cross-linked degrees were investigated. Results 1 HNMR results showed the LA-CLss was successfully synthesized. When N/P ratio was 40, the transfection efficiency of LA-CLss3/pEGFP nanocomplex by HEK293 cells was significantly higher than that of LA-CL/p EGFP, LA-CLss1/p EGFP, LA-CLss2/p EGFP and LACLss4 nanocomplexes. The encapsulation efficiency and drug loading of docetaxel-loaded micelles prepared by ultrasonic emulsification were（85.25±0.04）% and（8.81±0.02）%, respectively. Cellular uptake test showed that the gene could be effectively delivered into the HEK293 cells by the LA-CLss micelles. In vitro release experiments showed that the LA-CLss micelles had redox-responsive drug release behavior. Conclusion The prepared LA-CLss/DTX/p EGFP nanocomplex is expected to become an efficient vector for co-delivery of gene and chemotherapeutic drugs.