纳米靶向递药系统AS1411-PLGA-DOX的构建及其对脑胶质瘤的治疗作用与机制研究

发布时间：2018-09-14 17:02

【摘要】：背景:恶性肿瘤给人类健康和社会发展带来了沉重的伤害。由于肿瘤呈侵袭性生长,手术难以完全根除,加之放疗、化疗的毒副作用大,因此,寻找最佳的是肿瘤综合治疗的手段迫在眉睫。随着材料学、生物医学及纳米科学的交叉融合,纳米靶向制剂逐渐引起科学家的重视。纳米靶向给药系统是目前研究的热点,它具有可靶向性,提高药物稳定性及缓释性等特点。聚乳酸-羟基乙酸共聚物(PLGA)被广泛用于载药系统的研究,主要是由于具有生物相容性、低毒性和可降解性等良好的特性。盐酸多柔比星(又称阿霉素,DOX),对多种肿瘤均具有显著的抑制作用,它但它的骨髓抑制、心脏损伤等不良反应成为DOX治疗肿瘤的瓶颈。核酸适配体(AS1411)具有特异性强的靶标结合能力、易于穿透肿瘤组织、易于体外修饰等优点,并特异性结合于核仁蛋白(C23),以发挥其抗肿瘤的重要作用。C23是细胞凋亡相关蛋白,它可调控细胞增殖生长过程,其中p38丝裂原活化蛋白激酶(p38MAPK)在细胞凋亡的过程所起的重要作用越来越来引起广大科学研究者的重视。目的:本课题以DOX为抗脑胶质瘤药物,以PLGA为纳米载体,AS1411偶联于PLGA纳米微球,以构成纳米靶向递药系统AS1411-PLGA-DOX,以发挥其特异性靶向抗脑胶质瘤的作用,并进一步明确其抗脑胶质瘤作用的分子机制。方法:采用复乳乳化溶剂挥发法及碳二亚胺两步缩合反应法进行AS1411-PLGA-DOX的制备;并对其进行表征;再从体内外两方面明确AS1411-PLGA-DOX抗脑胶质瘤的作用;最后从分子生物学方面,以肿瘤凋亡的分子机制为着眼点,深入研究AS1411-PLGA-DOX抗脑胶质瘤的内在分子机制。结果:1.AS1411-PLGA-DOX为球形的纳米粒,大小(245±42.6nm)分布较规整,表面较光滑;多分散系数 0.212±0.023,电位-36.2±1.8mV,LC%为 1.72±0.06%、EE%为 88.1±4.7%;体外释放性为 47.7±3.9.%(pH=7.4),57.0±4.6%(pH=5),其结果展现出具有pH响应性释放的特性;溶血率为0.244%以展现出良好的生物形容性。同时,AS1411-PLGA-DOX的荧光强度显著高于PLGA-DOX,且被细胞摄入内部并释药,细胞定量定位的结果表明其具有一定的靶向性。2.体外实验表明,AS1411-PLGA-DOX对U87细胞生长抑制率的变化呈现浓度依赖性,并可将细胞阻滞于G2/M,S期,其抑制肿瘤的机制可能与AS1411-PLGA-DOX 激活 p38MAPK 通道,上调 p38 的表达,下调 C23、Bcl-2、Bcl-XL的表达有关。3.体内实验表明,AS1411-PLGA-DOX可抑制皮下荷瘤裸鼠的瘤体生长,延长荷瘤裸鼠的生存时间,其进一步的分子生物学机制与细胞实验相一致。结论:本课题已成功构建具有靶向作用的纳米递药系统AS1411-PLGA-DOX,同时其体内外实验结果表明,AS1411-PLGA-DOX是一种有潜在开发价值的抑制脑胶质瘤的纳米制剂,其作用机制可能激活p38凋亡信号通路,造成p38蛋白的上调表达及C23、Bcl-2、Bcl-XL蛋白的下调表达。
[Abstract]:Background: malignant tumors have brought serious harm to human health and social development. Because of the aggressive growth of the tumor, the operation is difficult to completely eradicate, combined with radiotherapy, chemotherapy has great toxic side effects, so it is urgent to find the best method of comprehensive treatment of tumor. With the cross-fusion of materials science, biomedicine and nano-science, nano-targeted preparations have attracted more and more attention. Nano-targeted drug delivery system is a hot research topic at present. It has the characteristics of targeting, improving drug stability and slow release. Polylactic acid-glycolic acid (PLGA) copolymers have been widely used in drug delivery systems due to their good biocompatibility, low toxicity and degradability. Doxorubicin hydrochloride (doxorubicin), also known as doxorubicin, has a significant inhibitory effect on many kinds of tumors, but its adverse reactions, such as bone marrow suppression and heart injury, have become the bottleneck of DOX in the treatment of tumors. Nucleic acid aptamer (AS1411) has many advantages, such as specific target binding ability, easy penetration of tumor tissue, easy modification in vitro, and specific binding to nucleolar protein (C23) in order to play its important role in anti-tumor. C23 is a apoptosis-related protein. P38 mitogen-activated protein kinase (p38MAPK) plays an important role in the process of cell apoptosis. Objective: in this study, DOX was used as an anti-glioma drug and PLGA as a nano-carrier, AS1411 was coupled to PLGA nanospheres in order to form a nano-targeted delivery drug system (AS1411-PLGA-DOX,) to play its specific role in targeting glioma. The molecular mechanism of its anti-glioma action was further clarified. Methods: AS1411-PLGA-DOX was prepared by solvent volatilization and carbodiimide two-step condensation reaction, and characterized. The effects of AS1411-PLGA-DOX on glioma were confirmed in vivo and in vitro. Based on the molecular mechanism of tumor apoptosis, the intrinsic molecular mechanism of AS1411-PLGA-DOX antiglioma was studied. Results: 1. AS1411-PLGA-DOX was spherical, the size (245 卤42.6nm) was more regular and the surface was smooth, the polydispersity coefficient was 0.212 卤0.023, the potential was -36.2 卤1.8mVN LC1.72 卤0.06CV% was 88.1 卤4.7%, the in vitro release rate was 47.7 卤3.9.% (pH=7.4) 57.0 卤4.6% (pH=5), the results showed the characteristic of pH responsive release. The hemolysis rate was 0.244% to show good biological description. At the same time, the fluorescence intensity of AS1411-PLGA-DOX was significantly higher than that of PLGA-DOX,. The results of cell quantitative localization showed that AS1411-PLGA-DOX was targeted to some extent. In vitro experiments showed that AS1411-PLGA-DOX inhibited U87 cell growth in a concentration-dependent manner and could block the cells in G _ 2 / MN _ S phase. The mechanism of inhibition might be related to the activation of p38MAPK channel by AS1411-PLGA-DOX, upregulation of p38 expression and down-regulation of the expression of C23 ~ (23) Bcl-2O _ (Bcl-XL) in U87 cells. In vivo experiments showed that AS1411-PLGA-DOX could inhibit the tumor growth and prolong the survival time of nude mice bearing subcutaneous tumor, and its further molecular biological mechanism was consistent with the cell experiment. Conclusion: AS1411-PLGA-DOX, with targeting effect has been successfully constructed in this study. The results in vitro and in vivo indicate that AS1411-PLGA-DOX is a promising nanometer preparation for the inhibition of glioma. The mechanism may activate the p38 apoptosis signal pathway, resulting in the up-regulation of p38 protein expression and the down-regulation of C23Bcl-2Bcl-XL protein.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R943;R96

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