负载自噬抑制小干扰RNA的聚精氨酸多肽纳米胶束抗肿瘤转移作用的初步研究  

Preliminary study on anti-tumor metastasis of polyarginine polypeptide nanomicelles loaded with autophagy inhibiting small interfering RNA

作　　者：胡楚玲 顾芬芬 宫春爱 夏清明 Hu Chuling;Gu Fenfen;Gong Chun′ai;Xia Qingming(Department of Pharmacy,Jiaxing Maternity and Child Health Care Hospital,Jiaxing 314001,China;Department of Pharmacy,Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine,Shanghai 200092,China;Department of Pharmacy,Changhai Hospital,Second Military Medical University,Shanghai 200433,China)

机构地区：[1]嘉兴市妇幼保健院药剂科,浙江嘉兴314001 [2]上海交通大学医学院附属新华医院药学部,上海200092 [3]海军军医大学附属长海医院药学部,上海200433

出　　处：《实用药物与临床》2023年第11期961-966,共6页Practical Pharmacy and Clinical Remedies

基　　金：浙江省医药卫生科技计划项目(2023RC100)。

摘　　要：目的 制备一种负载si-Beclin1的阿霉素聚精氨酸多肽纳米胶束,考察其对前列腺癌细胞的体外抗肿瘤转移能力,并探索其可能的作用机制。方法 采用多肽固相合成法合成聚精氨酸组氨酸(HR),再与阿霉素衍生物(DOX-EMCH)通过缩合反应合成DHR,产物与L-半胱氨酸反应并纯化得到DHRss。DHRss与si-Beclin1共孵育形成聚合物胶束DHRss-B。通过透射电镜观察形态,动态光散射法测定其粒径和电位,同时考察其稳定性;在人前列腺癌细胞株PC-3细胞中,采用流式细胞术考察细胞摄取情况;采用CCK-8法检测DHRss-B抗细胞增殖能力;采用荧光显微镜观察MDC染色自噬小体的生成;采用Western blot法观察Beclin1、LC3及Paxillin蛋白的表达;采用Transwell法观察纳米胶束抗细胞侵袭能力。结果 制备的聚精氨酸多肽胶束(DHRss-B)粒径大小合适,平均粒径为(129.9±2.5)nm,电位为(25.8±1.65)mV,形态分布均匀,2~8℃放置96 h后稳定性良好。流式细胞结果表明,DHRss-B具有较好的细胞摄取能力;CCK-8结果显示,DHRss-B具有较强的细胞毒性。此外,MDC染色法及Western blot结果显示,DHRss-B可显著抑制阿霉素(DOX)引起的细胞自噬。抗细胞侵袭试验表明,DHRss-B具有较强的抗肿瘤转移能力,可能与抑制Paxillin蛋白的表达有关。结论 负载si-Beclinl的阿霉素聚精氨酸多肽纳米胶束具有较强的抗肿瘤转移能力,具有良好的临床应用前景。Objective To prepare a si-Beclin1-supported doxorubicin polyarginine polypeptide nanoparticle micelle to investigate its in vitro anti-tumor metastasis ability on prostate cancer cells,and to explore its possible mechanism of action.Methods Polypeptide solid phase synthesis method was used to synthesize polyarginine histidine(HR),and then DHR was synthesized by condensation reaction with doxorubicin derivative(DOX-EMCH).The product was reacted with L-cysteine and purified to obtain DHRss.DHRss was incubated with si-Beclin1 to form polymer micelle DHRss-B.The morphology was observed by transmission electron microscopy,the particle size and potential were determined by dynamic light scattering method,and the stability was investigated.The uptake of DHRss-B in human prostate cancer cell line PC-3 cells was investigated by flow cytometry.The anti-cell proliferation ability of DHRss-B was detected by CCK-8 method.The formation of MDC stained autophagosomes was observed by fluorescence microscope.The expressions of Beclin1,LC3 and Paxillin were investigated by Western blot.Transwell method was used to investigate the anti-cell invasion ability of nano micelles.Results The prepared polyarginine polypeptide micelle(DHRss-B)had a suitable particle size,the average particle size was(129.9±2.5)nm,the potential was(25.8±1.65)mV,the morphology distribution was uniform,and the stability was good after being stored at 2~8℃ for 96 h.Flow cytometry results showed that DHRss-B had good ability of cell uptake,and CCK-8 results showed that DHRss-B had strong cytotoxicity.In addition,MDC staining method and Western blot results showed that DHRss-B significantly inhibited DOX-induced autophagy.Anti-cell invasion assay showed that DHRss-B had a strong ability of tumor metastasis,which might be related to inhibiting the expression of Paxillin protein.Conclusion The polyarginine polypeptide nano micelle has strong anti-tumor metastasis ability and has good clinical application prospect.

关 键 词：前列腺癌 自噬 阿霉素 抗转移 

分 类 号：R737.25[医药卫生—肿瘤]