c(RGDyk)环肽修饰纳米胶束逆转脑胶质瘤对多西他赛的耐药性

doi:10.3969/j.issn.2095-9400.2019.11.001

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摘要目的：构建c(RGDyk)环肽修饰的纳米胶束包载多西他赛（DTX），体外评价其逆转人脑胶质瘤U87细胞对DTX耐药性。方法：以泊洛沙姆-188（PL-188）为原材料，经琥珀酸酐羧基化后，键合c(RGDyk)环肽，合成c(RGDyk)-PL-188靶向材料并进行结构确证；c(RGDyk)-PL-188为包裹材料，利用纳米共沉淀法制备载DTX的纳米胶束[c(RGDyk)DTX-NPs]并进行处方优化筛选；透射电镜（TEM）、差示扫描量热法（DSC）以及动态透析法对c(RGDyk)DTX-NPs形态、药物晶型和体外药物释放行为进行详细表征；体外CCK8细胞存活率检测、细胞摄取以及肿瘤球生长抑制，评价c(RGDyk)DTX-NPs对DTX耐药的人脑胶质瘤U87细胞逆转效果。结果：1H-NMR和FT-IR表明成功合成c(RGDyk)-PL-188材料，该材料能自发组装成粒径为115.6 nm的胶束；c(RGDyk)-PL-188/DTX质量比10:1时，该胶束对DTX载药量可达7.88%±0.02%，包封率高达85.50%±2.78%，粒径稍增大，为（159.2±0.2）nm，Zeta电位为（-19.2±0.4）mV，TEM显示具有球形微观结构；体外释放表明c(RGDyk)DTX-NPs展现缓释释放行为，48 h内DTX累积释放仅为78%；细胞摄取表明c(RGDyk)-NPs能特异靶向耐药的人脑胶质瘤U87细胞，显著提高荧光探针ICG在细胞内荧光分布，其靶向性被游离c(RGDyk)竞争抑制；与DTX溶液和DTX-NPs相比，c(RGDyk)DTX-NPs对DTX耐药的人脑胶质瘤U87细胞具有更强的细胞毒性；体外U87人脑胶质瘤细胞球模型研究表明c(RGDyk)DTX-NPs能更有效渗透至肿瘤球深部，抑制肿瘤球生长。结论：c(RGDyk)环肽修饰纳米胶束能有效装载DTX，具有较高的载药量和包封率，能特异性靶向人脑胶质瘤U87细胞，逆转其对DTX的耐药性。

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	吴其隆
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	陈斌

关键词 ：多西他赛, 胶束, 泊洛沙姆, 神经胶质瘤, 细胞球, 多药耐药

Abstract：

Objective: To prepare c(RGDyk) cyclic peptide modified Nano-micelles encapsulating docetaxel (DTX) and to evaluate its ability to reverse the resistance of U87 glioma cells against DTX. Methods: Using Poloxamer 188 (PL-188) as raw material, after carboxylation of succinic anhydride, the c(RGDyk) cyclic peptide was bonded to synthesize c(RGDyk)-PL-188 targeting material and its structure was confirmed by 1H-NMR and FT-IR. c(RGDyk)-PL-188 was used as the coating material; the Nano-micelles encapsulating DTX [c(RGDyk)DTX-NPs] were prepared by Nano coprecipitation method and the prescription was optimized. Transmission electron microscope (TEM), differential scanning calorimetry (DSC) and dynamic dialysis method were used to characterize the morphology, drug crystal form and drug release behavior of c(RGDyk)DTX-NPs in vitro. CCK8 cellular viability assay in vitro, cellular uptake and U87 cells spheroids growth inhibition test were applied to evaluate the reversal effect of c(RGDyk)DTX-NPs on human glioma U87 cells resistance to DTX. Results: 1H-NMR and FT-IR showed that c(RGDyk)-PL-188 was successfully synthesized, which could be spontaneously assembled into micelles with mean particle diameter of (115.6±0.2) nm. When the mass ratio of c(RGDyk)-PL-188/DTX is 10:1, drug loading content and drug loading efficiency for DTX-loaded micelles were high up to 7.88%±0.02% and 85.5%±2.78%, respectively. DTX-loaded micelles had a mean diameter of (159.2±0.2) nm and zeta potential of (-16.8±0.8) mV, exhibiting the spherical shape determined by TEM. In vitro DTX release showed that c(RGDyk)DTX-NPs exhibited sustained-release behavior and the cumulative release of DTX was only 78% within 24 hours. Cellular uptake indicated that c(RGDyk)-NPs could specifically target drug-resistant human glioma U87 cells, which significantly increased the intracellular fluorescence distribution of fluorescent probe ICG, and its targetability was inhibited by free c(RGDyk) solution. Compared with DTX solution and DTX-NPs, c(RGDyk)DTX-NPs had the stronger cytotoxicity to drug-resistant human glioma U87 cells. The study of U87 human glioma cells spheroids model in vitro showed that c(RGDyk)DTX-NPs could penetrate into the deep part of the tumor spheroids more effectively and inhibit the growth of the tumor spheroids. Conclusion: DTX is efficiently encapsulated in c(RGDyk) cyclic peptide modified Nano-micelles with high drug loading content and drug loading efficiency, specifically targeting U87 human glioma cells and reversing their resistance against docetaxel

Key words： docetaxel micelles poloxamer glioma;cells spheroids;multi-drugs resistance

收稿日期: 2019-04-09

基金资助:浙江省自然科学基金资助项目（LY17H180008）；国家自然科学基金资助项目（81603036）。

通讯作者: 陈斌，教授，硕士生导师，Email：doctorchbe@126.com。

作者简介: 吴其隆（1993-），男，浙江乐清人，硕士生。

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https://xb.wmu.edu.cn/CN/10.3969/j.issn.2095-9400.2019.11.001 或 https://xb.wmu.edu.cn/CN/Y2019/V49/I11/781

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