摘要
化疗药物普遍缺乏特异性,开发一种可主动靶向递送化疗药物、对生物体无免疫原性的载体越来越受到人们的关注。本研究通过滚环扩增技术(rolling circle amplification,RCA)将合成的具有串联重复序列的DNA长单链与几条包含适配体AS1411的短链DNA杂交来构筑含有多价适配体的核酸载体(multivalent aptamer,Multi-Apt),利用其双螺旋结构大量负载抗肿瘤药物阿霉素(doxorubicin,Dox),用于靶向治疗小鼠黑色素瘤细胞(B16细胞)。通过琼脂糖凝胶电泳优化了RCA产物与短链的结合比例,利用酶标仪探究了Dox的负载及释放,并采用荧光显微镜、流式细胞术、酶标仪、CCK-8法和划痕实验考察了Multi-Apt-Dox对B16细胞的靶向性和生长抑制情况。实验结果显示,RCA产物与3条短链的最佳物质的量比为1∶50。荧光显微镜照片、流式细胞术和酶标仪实验结果表明,每个Multi-Apt可负载约200个Dox分子,而且其对B16细胞的亲和性是单价适配体的46倍。细胞实验表明,Multi-Apt在细胞内降解并释放药物后诱导产生选择性细胞毒性,从而极大降低Dox对正常细胞的毒性,为肿瘤治疗的靶向给药提供一种新的策略。
Chemotherapeutic drugs generally lack specificity,and so the development of a carrier that can actively target delivery of chemotherapy drugs without immunogenicity to organisms has attracted increasing attention.In this work,a multivalent aptamer(Multi-Apt)was constructed by hybridizing a long single-stranded DNA(ssDNA)with tandem repeated sequences synthesized by rolling circle amplification(RCA)with several ssDNA encoding aptamer AS1411 sequences.The double-helix structure was used to load the anti-tumor drug doxorubicin(Dox)for targeted treatment of B16 cells.The binding ratio of RCA product to ssDNA was optimized by agarose gel electrophoresis,and the loading and release of Dox were explored using a microplate reader.The targeting and growth inhibition of Multi-Apt-Dox on B16 cells were investigated by fluorescence microscopy,flow cytometry,microplate reader,CCK-8 assay and wound healing assay.The results showed that the optimal molar ratio of RCA product to ssDNA was 1:50.Fluorescence microscopic pictures,flow cytometry analysis and microplate reader experimental results showed that each Multi-Apt could load approximately 200 Dox molecules,and the affinity of Multi-Apt for B16 cells was 46-fold higher than that of free AS1411.Cell experimental results demonstrated that Multi-Apt induced selective cytotoxicity after intracellular degradation and drug release,thereby greatly reducing the adverse reactions of Dox to normal cells and providing a new strategy for targeted drug delivery in tumor treatment.
作者
张如燕
张子辰
张国栋
张志庆
ZHANG Ruyan;ZHANG Zichen;ZHANG Guodong;ZHANG Zhiqing(College of Chemistry and Chemical Engineering,China University of Petroleum(East China),Qingdao 266580,China)
出处
《中国药科大学学报》
北大核心
2025年第3期312-320,共9页
Journal of China Pharmaceutical University
基金
山东省自然科学基金项目(ZR2022MB148,ZR2023MB148)
中国石油大学(华东)研究生教改项目。
关键词
滚环扩增
多价适配体
靶向递送
肿瘤细胞
AS1411
rolling circle amplification
multivalent aptamer
targeted delivery
cancer cells
AS1411
