[关键词]
[摘要]
目的 评价CD133抗体修饰的紫草素微乳(anti CD133 antibody-modified shikonin-loaded microemulsion,Anti CD133Ab-SKN-MEs)治疗三阴性乳腺癌的可行性及优势。方法 利用经典的成乳工艺制备紫草素微乳(SKN-MEs),再通过EDC/NHS缩合技术制备Anti CD133Ab-SKN-MEs,以粒径、Zeta电位、包封率为指标优化投料比、抗体修饰密度等工艺参数;采用MTT法考察三阴性乳腺癌MDA-MB-231细胞增殖行为,利用异硫氰酸荧光素(FITC)作为探针定性定量考察细胞摄取行为,借助膜联蛋白V-PE/7-氨基放线菌素D(Annexin V-PE/7-AAD)试剂盒考察给药组诱导细胞凋亡行为;借助悬浮培养技术富集MDA-MB-231干细胞,通过各给药组孵育观察细胞成球性以及CD133+细胞率;建立裸鼠MDA-MB-231异位移植瘤模型,设生理盐水、紫草素(SKN)、SKN-MEs、Anti CD133Ab-SKN-MEs,4 mg/kg隔天iv 5次,观察肿瘤体积、裸鼠生存时间、抑瘤率和CD133+细胞比率。结果 Anti CD133Ab-SKN-MEs的最佳质量比为1.0%,抗体密度为0.025%,粒子形态圆整,粒径为(31.4±2.1)nm,电位为(-18.7±2.5)mV,包封率为(93.6±2.8)%;Anti CD133Ab-SKN-MEs对MDA-MB-231细胞的IC50为(1.53±0.43)μg/mL,细胞摄取显著高于SKN和SKN-MEs,孵育8 h可诱导(67.9±4.2)%细胞凋亡;Anti CD133Ab-SKN-MEs可以显著抑制MDA-MB-231干细胞的成球性,明显降低CD133+细胞率;Anti CD133Ab-SKN-MEs体内抑瘤率为78.5%,69 d后仍有12.5%裸鼠存活,瘤组织CD133+细胞率显著降低。结论 与SKN相比,Anti CD133Ab-SKN-MEs在治疗三阴性乳腺癌方面优势明显,机制可能与降低肿瘤细胞干性作用有关。
[Key word]
[Abstract]
Objective To evaluate the feasibility and advantages of therapy of triple-negative breast cancer with Anti CD133 antibody-modified shikonin-loaded microemulsion (Anti CD133Ab-SKN-MEs). Methods Anti CD133Ab-SKN-MEs were prepared by a classic EDC/NHS conjugation technique. The drug loading efficiency and density of modified antibody were optimized using average particle size, Zeta potential and entrapment efficiency as indicators. The cell proliferation of MDA-MB-231 cells was investigated by MTT method. The cellular uptake of various formulations was qualitatively and quantitatively investigated using FITC as a probe. MDA-MB-231 cellular apoptosis induced by various treatments was evaluated by the Annexin V-PE/7-amino actinomycin D (Annexin V-PE/7-AAD) assay kit. MDA-MB-231 breast cancer stem cells (MDA-MB-231 CSC) was enriched by a suspension culture technique, and the cell morphology and proportion of CD133-positive cells were studied after treatment with various SKN formulations. The model of MDA-MB-231 tumor-bearing nude mice was established, and then injected five times every other day with saline, shikonin (SKN), SKN-MEs, and Anti CD133Ab-SKN-MEs at a dose of 4 mg/kg, to observe the tumor volume, survival time, tumor inhibition and CD133+ cells ratio during/after the treatment. Results The optimal mass ratio of SKN to total carrier was 1.0% in the preparation of Anti CD133Ab-SKN-MEs, and the optimal density of modified antibody was 0.025%. The particle was spherical with a particle size of (31.4 ±2.1) nm, a potential of (-18.7 ±2.5) mV and an encapsulation efficiency of (93.6 ±2.8)%. The IC50 of Anti CD133Ab-SKN-MEs against MDA-MB-231 cells was (1.53 ±0.43) μg/mL, the cell uptake of Anti CD133Ab-SKN-MEs was significantly higher than that of SKN-MEs and SKN, and 8 h incubation induced (67.9 ±4.2)% cell apoptosis. Anti CD133Ab-SKN-MEs can significantly inhibit the globularity of MDA-MB-231 CSC, with a decrease in the number of CD133-positive cells. The in vivo tumor inhibition rate of Anti CD133Ab-SKN-MEs-treated mice was 78.5%, and 12.5% of tumor-bearing nude mice still survived at day 69. Moreover, the ratio of CD133-positive tumor cells within the tumor tissues was significantly reduced. Conclusion Anti CD133Ab-SKN-MEs has obvious advantages in treatment of triple-negative breast cancer, which might be related to the inhibition of tumor cells differentiation.
[中图分类号]
R283.6
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