氧化还原型交联硫辛酸纳米载体的构建及靶向抗肿瘤作用研究
发布时间：2018-08-25 16:34
【摘要】：肿瘤纳米靶向化疗(nanotarget chemotherapy)是指将化疗药物利用纳米技术靶向输送到肿瘤部位,以在发挥最大的疗效同时减少化疗药物的毒副作用,而肿瘤纳米靶向治疗的关键在于纳米载体材料。良好的纳米载体能够保护药物在体内循环不被过早的降解,而在到达靶部位后可顺利释药,同时自身降解代谢为无毒代谢物。因此开发新型良好的纳米药物载体材料存在一定的挑战,且具有非常重要的意义。本课题基于肿瘤细胞的微环境及相关研究,合成了一种新型的可生物降解的高分子载体材料---交联硫辛酸。此材料成分单纯,制备工艺简单可控,稳定性较好,毒性较低,且具有肿瘤微环境敏感性释药、经济性好等优势,因此具有工业化、商品化的价值。体内外研究结果表明,该载体材料能够保护药物在体内循环时不被过早的降解,且将化疗药物高效地靶向递送至肿瘤部位,且材料自身降解,保持低毒性。本课题的第一部分利用半胱氨酸催化激活硫辛酸五元环断裂后相互交联,并对交联合成条件进行筛选,后采用超声乳化法合成制备硫辛酸纳米粒。对载体材料进行紫外验证及GPC分子量检测均证实合成成功。对纳米粒进行特征性评价,结果显示纳米粒径在110nm左右,电位约为-35mv,呈近似球形。纳米粒稳定性良好,且多西他赛载药量达4.51%±0.49%,并具有肿瘤微环境敏感性释药特性。本课题的第二部分对硫辛酸纳米粒进行了体外细胞学评价,以肺癌A549细胞为目标细胞,结果显示空白纳米材料细胞毒性较低,细胞摄取率相比于单体明显增强,约为其10倍,且与PLGA纳米粒相当,细胞摄取途径主要为网格蛋白及质膜微囊介导的内吞作用,且细胞摄取定位表明,硫辛酸纳米粒可部分逃逸溶酶体。CCK-8、细胞凋亡及周期研究结果均证实包载多西他赛的纳米粒其体外肿瘤细胞杀伤性药效明显增强,其IC50值相对于单体的277.84 ng/ml,降低至79.62 ng/ml,且相比于PLGA-DTX纳米粒的135.61 ng/ml亦有明显减少。本课题的第三部分对硫辛酸纳米粒进行了体内研究。结果表明硫辛酸纳米粒具有良好的肿瘤靶向性,其经尾静脉注射后能较好的在肿瘤部位发生蓄积,且相比于PLGA纳米粒可以有效地减少脾脏、肺脏的毒性作用。体内抗肿瘤药效评价亦证实其抗肿瘤药效明显增强,其肿瘤抑制率由单体的64.8%上升至81.62%,且相比于PLGA-DTX纳米的76.96%亦有上升。
[Abstract]:Tumor nano-targeted chemotherapy (nanotarget chemotherapy) refers to the delivery of chemotherapeutic drugs to tumor sites using nanotechnology in order to maximize the efficacy and reduce the side effects of chemotherapeutic drugs. The key of nano-targeted tumor therapy lies in nano-carrier materials. Good nano-carriers can protect the drug from premature degradation in vivo, and release the drug smoothly after reaching the target site, and self-degradation metabolism as non-toxic metabolites. Therefore, the development of new good nano-drug carrier materials has some challenges, and has a very important significance. Based on the microenvironment of tumor cells and related studies, a novel biodegradable polymer carrier material, crosslinked lipoic acid, was synthesized. This material has the advantages of simple composition, simple and controllable preparation process, good stability, low toxicity, and has the advantages of sensitive release of tumor microenvironment, good economy and so on, so it has the value of industrialization and commercialization. The results in vivo and in vitro showed that the carrier material could protect the drug from premature degradation in vivo circulation, and deliver chemotherapeutic drugs to tumor site efficiently, and the material itself degraded and kept low toxicity. In the first part of this paper, the cysteine was used to catalyze the catalytic activation of the quaternary ring of lipoic acid and cross-linking each other, and the conditions of cross-linking synthesis were screened, and then the phacoemulsification method was used to synthesize the lipoic acid nanoparticles. UV verification and GPC molecular weight detection of the carrier material confirmed that the synthesis was successful. The characteristics of the nanoparticles were evaluated. The results showed that the particle size was about 110nm and the potential was about -35 MV, which was approximately spherical. The nanoparticles were stable, and the drug loading of docetaxel was 4.51% 卤0.49, and the drug release was sensitive to tumor microenvironment. In the second part of this study, the in vitro cytological evaluation of lipoic acid nanoparticles was carried out. The results showed that the cell toxicity of the blank nano-materials was lower, and the uptake rate of the cells was about 10 times higher than that of the monomers. In the same way as PLGA nanoparticles, the main pathway of cell uptake was endocytosis mediated by grid protein and plasma membrane microencapsulation, and the location of cell uptake showed that, Lipoic acid nanoparticles could escape lysosome. CCK-8. The results of apoptosis and cell cycle study confirmed that doxetaxel nanoparticles could significantly enhance the killing effect of tumor cells in vitro. The IC50 value decreased to 79.62 ng/ml, relative to 277.84 ng/ml, of monomers and significantly decreased compared with 135.61 ng/ml of PLGA-DTX nanoparticles. In the third part of this paper, the in vivo study of lipoic acid nanoparticles was carried out. The results showed that lipoic acid nanoparticles had good tumor targeting ability and could accumulate well in tumor site after injection through tail vein. Compared with PLGA nanoparticles, it could effectively reduce the toxicity of spleen and lung. The evaluation of anti-tumor effect in vivo also confirmed that its anti-tumor effect was obviously enhanced, its tumor inhibition rate increased from 64.8% of monomer to 81.62%, and it also increased compared with 76.96% of PLGA-DTX nanoparticles.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943;R96
，
本文编号：2203480
[Abstract]:Tumor nano-targeted chemotherapy (nanotarget chemotherapy) refers to the delivery of chemotherapeutic drugs to tumor sites using nanotechnology in order to maximize the efficacy and reduce the side effects of chemotherapeutic drugs. The key of nano-targeted tumor therapy lies in nano-carrier materials. Good nano-carriers can protect the drug from premature degradation in vivo, and release the drug smoothly after reaching the target site, and self-degradation metabolism as non-toxic metabolites. Therefore, the development of new good nano-drug carrier materials has some challenges, and has a very important significance. Based on the microenvironment of tumor cells and related studies, a novel biodegradable polymer carrier material, crosslinked lipoic acid, was synthesized. This material has the advantages of simple composition, simple and controllable preparation process, good stability, low toxicity, and has the advantages of sensitive release of tumor microenvironment, good economy and so on, so it has the value of industrialization and commercialization. The results in vivo and in vitro showed that the carrier material could protect the drug from premature degradation in vivo circulation, and deliver chemotherapeutic drugs to tumor site efficiently, and the material itself degraded and kept low toxicity. In the first part of this paper, the cysteine was used to catalyze the catalytic activation of the quaternary ring of lipoic acid and cross-linking each other, and the conditions of cross-linking synthesis were screened, and then the phacoemulsification method was used to synthesize the lipoic acid nanoparticles. UV verification and GPC molecular weight detection of the carrier material confirmed that the synthesis was successful. The characteristics of the nanoparticles were evaluated. The results showed that the particle size was about 110nm and the potential was about -35 MV, which was approximately spherical. The nanoparticles were stable, and the drug loading of docetaxel was 4.51% 卤0.49, and the drug release was sensitive to tumor microenvironment. In the second part of this study, the in vitro cytological evaluation of lipoic acid nanoparticles was carried out. The results showed that the cell toxicity of the blank nano-materials was lower, and the uptake rate of the cells was about 10 times higher than that of the monomers. In the same way as PLGA nanoparticles, the main pathway of cell uptake was endocytosis mediated by grid protein and plasma membrane microencapsulation, and the location of cell uptake showed that, Lipoic acid nanoparticles could escape lysosome. CCK-8. The results of apoptosis and cell cycle study confirmed that doxetaxel nanoparticles could significantly enhance the killing effect of tumor cells in vitro. The IC50 value decreased to 79.62 ng/ml, relative to 277.84 ng/ml, of monomers and significantly decreased compared with 135.61 ng/ml of PLGA-DTX nanoparticles. In the third part of this paper, the in vivo study of lipoic acid nanoparticles was carried out. The results showed that lipoic acid nanoparticles had good tumor targeting ability and could accumulate well in tumor site after injection through tail vein. Compared with PLGA nanoparticles, it could effectively reduce the toxicity of spleen and lung. The evaluation of anti-tumor effect in vivo also confirmed that its anti-tumor effect was obviously enhanced, its tumor inhibition rate increased from 64.8% of monomer to 81.62%, and it also increased compared with 76.96% of PLGA-DTX nanoparticles.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943;R96
，
本文编号：2203480
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