CD133抗体修饰的紫草素微乳的制备及抗三阴性乳腺癌研究
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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)m V,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/m L,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。
Objective To evaluate the feasibility and advantages of therapy of triple-negative breast cancer with Anti CD133 antibody-modified shikonin-loaded microemulsion(Anti CD133 Ab-SKN-MEs). Methods Anti CD133 Ab-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 CD133 Ab-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 CD133 Ab-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) m V and an encapsulation efficiency of(93.6 ± 2.8) %. The IC50 of Anti CD133 Ab-SKN-MEs against MDA-MB-231 cells was(1.53 ± 0.43) μg/m L, the cell uptake of Anti CD133 Ab-SKN-MEs was significantly higher than that of SKN-MEs and SKN, and 8 h incubation induced(67.9 ± 4.2)% cell apoptosis. Anti CD133 Ab-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 CD133 Ab-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 CD133 Ab-SKN-MEs has obvious advantages in treatment of triple-negative breast cancer, which might be related to the inhibition of tumor cells differentiation.
Objective To evaluate the feasibility and advantages of therapy of triple-negative breast cancer with Anti CD133 antibody-modified shikonin-loaded microemulsion(Anti CD133 Ab-SKN-MEs). Methods Anti CD133 Ab-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 CD133 Ab-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 CD133 Ab-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) m V and an encapsulation efficiency of(93.6 ± 2.8) %. The IC50 of Anti CD133 Ab-SKN-MEs against MDA-MB-231 cells was(1.53 ± 0.43) μg/m L, the cell uptake of Anti CD133 Ab-SKN-MEs was significantly higher than that of SKN-MEs and SKN, and 8 h incubation induced(67.9 ± 4.2)% cell apoptosis. Anti CD133 Ab-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 CD133 Ab-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 CD133 Ab-SKN-MEs has obvious advantages in treatment of triple-negative breast cancer, which might be related to the inhibition of tumor cells differentiation.
引文
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[3]Farrugia M K,Wen S,Jacobson G M,et al.Prognostic factors in breast cancer patients evaluated by positronemission tomography/computed tomography before neoadjuvant chemotherapy[J].World J Nucl Med,2018,17(4):275-280.
[4]Sang M M,Liu F L,Wang Y,et al.A novel redox/p H dual-responsive and hyaluronic acid-decorated multifunctional magnetic complex micelle for targeted gambogic acid delivery for the treatment of triple negative breast cancer[J].Drug Deliv,2018,25(1):1846-1857.
[5]Xiao W,Zheng S,Yang A,et al.Incidence and survival outcomes of breast cancer with synchronous hepatic metastases:A population-based study[J].J Cancer,2018,9(23):4306-4313.
[6]Kim H J,Hwang K E,Park D S,et al.Shikonin-induced necroptosis is enhanced by the inhibition of autophagy in non-small cell lung cancer cells[J].J Transl Med,2017,15(1):123.
[7]Bi Y,Zhu Y,Zhang M,et al.Effect of shikonin on spinal cord injury in rats via regulation of HMGB1/TLR4/NFk B signaling pathway[J].Cell Physiol Biochem,2017,43(2):481-491.
[8]柴冰阳,陈泽慧,张闪闪,等.4种细胞毒活性方法评价紫草素体外肿瘤细胞抑制作用效果[J].中草药,2019,50(1):172-177.
[9]Lu B,Gong X,Wang Z Q,et al.Shikonin induces glioma cell necroptosis in vitro by ROS overproduction and promoting RIP1/RIP3 necrosome formation[J].ActaPharmacol Sin,2017,38(11):1543-1553.
[10]Zhao X Y,Zhu Y N,Hu J H,et al.Shikonin inhibits tumor growth in mice by suppressing pyruvate kinase M2-mediated aerobic glycolysis[J].Sci Rep,2018,8(1):14517.
[11]Liu J,Qu C B,Xue Y X,et al.Mi R-143 enhances the antitumor activity of shikonin by targeting BAG3expression in human glioblastoma stem cells[J].Biochem Biophys Res Commun,2015,468(1/2):105-112.
[12]Thakur R,Trivedi R,Rastogi N,et al.Inhibition of STAT3,FAK and Src mediated signaling reduces cancer stem cell load tumorigenic potential and metastasis in breast cancer[J].Sci Rep,2015,14(5):10194.
[13]Min H S,Kim H J,Ahn J,et al.Tuned density of anti-tissue factor antibody fragment onto si RNA-loaded polyion complex micelles for optimizing targetability into pancreatic cancer cells[J].Biomacromolecules,2018,19(6):2320-2329.
[14]Li M,Chen X,Hu S,et al.Determination of blood concentrations of main active compounds in Zi-CaoCheng-Qi decoction and their total plasma protein binding rates based on hollow fiber liquid phase microextraction coupled with high performance liquid chromatography[J].Chromatogr B Analyt Technol Biomed Life Sci,2018,1072:355-361.
[15]Liu M,Shen S,Wen D,et al.Hierarchical nanoassemblies-assisted combinational delivery of cytotoxic protein and antibiotic for cancer treatment[J].Nano Lett,2018,18(4):2294-2303.
[16]Abramson V G,Lehmann B D,Ballinger T J,et al.Subtyping of triple-negative breast cancer:Implications for therapy[J].Cancer,2015,121(1):8-16.
[17]Qu D,Guo M,Qin Y,et al.A multicomponent microemulsion using rational combination strategy improves lung cancer treatment through synergistic effects and deep tumor penetration[J].Drug Deliv,2017,24(1):1179-1190.