细胞外囊泡协同递送维替泊芬/TRAIL诱导口腔鳞癌细胞凋亡及增殖抑制实验研究

Experimental study on apoptosis-inducing and proliferation inhibition by synergistic delivery of viteporfen/TRAIL by extracellular vesicles in oral squamous cell carcinoma cells

目的制备细胞外囊泡(EVs)共载体系,以协同递送肿瘤坏死因子相关凋亡诱导配体(TRAIL)/维替泊芬(VPF)组合用于诱导口腔鳞癌细胞凋亡及增殖抑制。方法通过超速离心、过滤法分离纯化TRAIL/VPF共载EVs(MSCT-EVs/VPF)。通过BCA法检测CD63、CD9和TRAIL表达,确认EVs来源。用高效液相法检测VPF载药量,绘制体外释放曲线。使用MTT法检测细胞毒性和半数抑制浓度(IC50)。通过流式细胞仪检测细胞凋亡,最后Western blot被用于检测MSCT-EVs/VPF对SCC25细胞中凋亡相关蛋白和YAP表达的影响。结果MSCT-EVs/VPF颗粒圆整,分散性良好,直径约为100 nm;纳米体系载药量为(15.43±0.44)%,在10 h内释放57.8%的VPF,45 h释放约82.5%;MSCT-EVs和VPF均可抑制SCC25肿瘤细胞生长,呈剂量依赖性,MSCT-EVs和VPF的质量比(wt%)在10∶1~5∶1的比例范围内显示出最佳抑制效果;在100∶5~100∶15(wt%)比例下,MSCT-EVs/VPF的半抑制浓度(IC50)低于游离MSCT-EVs+游离VPF组(P<0.05),显示出更高效的抑制作用。MSCT-EVs/VPF对鳞癌细胞的高效抑制作用部分源自对Caspase-3、Bax、Bcl-2、mTOR、p-mTOR和YAP的调控。结论通过EVs递送固定比例的TRAIL/VPF,对口腔鳞癌细胞展现出高效抑制作用,为多药耐药肿瘤的治疗提供了新思路。

Objective To develop an extracellular vesicles(EVs)co-loading system by synergistically deliver tumor necrosis factor related apoptosis inducing ligand(TRAIL)/verteporfin(VPF)combination to induce apoptosis and inhibit proliferation of OSCC cells.Methods TRAIL/VPF co-loaded EVs(MSCT-EVs/VPF)was purified and collected though ultracentrifugation and dialysis.The expression of CD63,CD9 and TRAIL was detected by BCA to confirm the origin of EVs.High performance liquid chromatography(HPLC)was used to detect the drug loading of VPF and draw the release curve in vitro.Cytotoxicity and half inhibitory concentration(IC_(50))were detected by MTT assay.The apoptosis rate was detected by flow cytometry.Finally,Western blot was used to detect the effects of MSCT-EVs/VPF on the expression of apoptosis related proteins and Yap in SCC25 cells.Results MSCT-EVs/VPF particles were round and well dispersed with a diameter of about 100 nm.The drug loading of the nano system was about(15.43±0.44)%,57.8%of VPF was released in 10 h and 82.5%in 45h;MSCT-EVs and VPF could inhibit the growth of SCC25 tumor cells in a dose-dependent manner,showing good synergistic effect in the ratio of 10:1-5:1(CI<1,wt%).At the ratio of 100:5~100:15(Mass ratio of MSCT-EVs to VPF,wt%),the IC_(50) of MSCT-EVs/VPF was significantly lower than that of free MSCT-EVs+free VPF group(P<0.05),and showed a more effective inhibition.The high inhibitory effect of MSCT-EVs/VPF on squamous cell carcinoma cells was partly due to the regulation of Caspase-3,Bax,BCL-2,mTOR,p-mTOR and YAP.Conclusion EVs delivery of a fixed proportion of TRAIL/VPF shows high inhibitory effect on oral squamous cell carcinoma cells,which provides a new idea for the treatment of multidrug-resistant tumors.