载内皮抑素壳聚糖纳米粒的制备及其抗肿瘤作用的研究
发布时间：2018-08-04 17:58
【摘要】：目的:制备载内皮抑素壳聚糖纳米粒,并对其抗血管和抗肿瘤作用进行研究。方法:利用离子凝胶法制备载内皮抑素壳聚糖纳米粒(ES-NPs),探究负载内皮抑素量的不同对纳米粒粒径、包封率、载药量的影响,选出最佳的制备方案。利用体外透析法,检测ES-NPs的释药特点。并观察ES-NPs在不同温度下血清中的稳定性。利用MTT法和Transwell实验,观察ES-NPs和游离ES对脐静脉内皮细胞(HUVECs)增殖、迁徙的影响。随后建立Lewis肺癌移植瘤模型,随机平均分组,采用PBS、内皮抑素注射液、ES-NPs和空纳米粒对移植瘤进行治疗。每2天测量每只小鼠肿瘤的体积,绘制肿瘤生长曲线。21天观察结束后,计算抑瘤率,收集肿瘤组织和血清,免疫组化法检测各组小鼠移植瘤的微血管密度(MVD),ELISA法检测各组小鼠血清中内皮抑素和VEGF浓度。结果:我们利用离子凝胶法成功制备出负载不同质量内皮抑素的ES-NPs,通过对各批次纳米粒包封率和载药量的比较,我们发现,在40 mL壳聚糖溶液(1 mg/mL)中加入500μL ES(11.2mg/mL)制成的ES-NPs在保证较高包封率的同时也具有合适的载药量和粒径,能满足治疗的需要,并且通过加入5%海藻糖冻干后能够稳定保存。纳米粒外观呈球形,分布均匀,在体外具有明显的缓释性,7d累积释放量达到(60.22±2.58)%,并且在血清中能够稳定存在至少48h。MTT实验显示,ES-NPs对HUVECs的生长有抑制作用。在药物作用的最初24h,ES-NPs对HUVECs的抑制作用与游离ES相差不大(p=0.577)。但随着作用时间的延长,ES-NPs的作用效果明显优于游离ES(p0.05)。同样,在transwell实验中,ES-NPs对HUVECs的迁徙的抑制作用也要明显强于游离ES,差异具有统计学差异(p0.05);并且作用时间越长,两者的作用效果的差距越大。在Lewis肺癌移植瘤模型上,我们观察到ES-NPs能明显抑制移植瘤生长。甚至,ES-NPs每7天注射1次的效果都优于游离ES连续注射14天。而ES-NPs连续注射14天在我们的实验中取得了最好的治疗效果,对移植瘤的抑制率高达63.32%。免疫组化CD31染色结果显示,ES-NPs连续注射14天组肿瘤组织的MVD最低,明显低于对照组(p0.001),游离ES组(p0.001)。与MVD结果对应,ES-NPs连续注射14天组小鼠血清中的内皮抑素水平明显高于其它各组(p值均0.05),而VEGF水平明显低于其它各组(p值均0.05)。证明了ES-NPs主要通过抑制肿瘤血管生成而起到抗肿瘤作用。结论:1)壳聚糖是制备内皮抑素纳米合适的材料,用壳聚糖制备的ES-NPs具有较高的包封率、载药量和明显的缓释性,能满足治疗的需要。2)载内皮抑素壳聚糖纳米粒在体外能明显抑制HUVECs的增殖和迁徙,作用效果优于游离内皮抑素。3)在Lewis肺癌模型上,载内皮抑素壳聚糖纳米粒能增强内皮抑素的治疗作用,通过抑制血管生成而明显抑制肿瘤生长。
[Abstract]:Aim: to prepare endostatin-loaded chitosan nanoparticles and study their anti-vascular and anti-tumor effects. Methods: endostatin loaded chitosan nanoparticles (ES-NPs) were prepared by ionic gel method. The effects of different amount of loaded endostatin on particle size, encapsulation efficiency and drug loading were investigated. The drug release characteristics of ES-NPs were detected by in vitro dialysis. The stability of ES-NPs in serum at different temperature was observed. The effects of ES-NPs and free es on the proliferation and migration of umbilical vein endothelial cells (HUVECs) were observed by MTT and Transwell assay. Then the transplanted tumor model of Lewis lung cancer was established, and the transplanted tumor was treated with PBSs, endostatin injection (ESNPs) and empty nanoparticles. Tumor volume of each mouse was measured every 2 days, tumor growth curve was drawn after 21 days observation, tumor inhibition rate was calculated, tumor tissue and serum were collected. The concentrations of endostatin and VEGF in the serum of mice were detected by immunohistochemical method, and the microvessel density of transplanted tumor in each group was detected by (MVD) Elisa. Results: we successfully prepared ES-NPs loaded with different mass endostatin by ionic gel method. By comparing the encapsulation efficiency and drug loading of different batches of nanoparticles, we found that, The ES-NPs prepared by adding 渭 L ES (11.2mg/mL into 40 mL chitosan solution (1 mg/mL) not only guaranteed high entrapment efficiency, but also had suitable drug loading and particle size, which could meet the need of treatment. The ES-NPs could be stably preserved by adding 5% trehalose into the freeze-dried solution. The nanoparticles were spherical in appearance and uniform in distribution, and the cumulative release amount reached (60.22 卤2.58) in vitro after 7 days of sustained release. At least 48h.MTT showed that GES NPs could inhibit the growth of HUVECs in serum. The inhibitory effect of ES-NPs on HUVECs was similar to that of free es at the first 24 h of drug action (p0. 577). However, with the prolongation of the action time, the effect of GES NPs was obviously better than that of free es (p0.05). Similarly, in transwell experiment, the inhibitory effect of GES NPs on migration of HUVECs was significantly stronger than that of free ESs, the difference was statistically significant (p0. 05), and the longer the action time, the greater the difference of effect between them. In Lewis lung cancer transplant model, we observed that ES-NPs can significantly inhibit the growth of transplanted tumor. Even the effect of once every 7 days was better than that of free es for 14 days. ES-NPs injection for 14 days achieved the best therapeutic effect in our experiment, and the inhibition rate of transplanted tumor was as high as 63.32. The results of immunohistochemical CD31 staining showed that the MVD of tumor tissue was the lowest in the control group (p0.001) and the free es group (p0.001) after 14 days of continuous injection. The serum levels of endostatin and VEGF were significantly higher in mice of 14-day continuous injection group than those in other groups (p < 0.05), but the levels of VEGF were significantly lower than those in other groups (p < 0.05). It is proved that ES-NPs plays an anti-tumor role mainly by inhibiting tumor angiogenesis. Conclusion: chitosan is a suitable material for preparing endostatin nanoparticles. ES-NPs prepared by chitosan has high encapsulation efficiency, drug loading and obvious slow-release. Endostatin chitosan nanoparticles could significantly inhibit the proliferation and migration of HUVECs in vitro, and the effect was better than that of free endostatin 3) on Lewis lung cancer model. Endostatin-loaded chitosan nanoparticles can enhance the therapeutic effect of endostatin and inhibit tumor growth by inhibiting angiogenesis.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R73-36
[Abstract]:Aim: to prepare endostatin-loaded chitosan nanoparticles and study their anti-vascular and anti-tumor effects. Methods: endostatin loaded chitosan nanoparticles (ES-NPs) were prepared by ionic gel method. The effects of different amount of loaded endostatin on particle size, encapsulation efficiency and drug loading were investigated. The drug release characteristics of ES-NPs were detected by in vitro dialysis. The stability of ES-NPs in serum at different temperature was observed. The effects of ES-NPs and free es on the proliferation and migration of umbilical vein endothelial cells (HUVECs) were observed by MTT and Transwell assay. Then the transplanted tumor model of Lewis lung cancer was established, and the transplanted tumor was treated with PBSs, endostatin injection (ESNPs) and empty nanoparticles. Tumor volume of each mouse was measured every 2 days, tumor growth curve was drawn after 21 days observation, tumor inhibition rate was calculated, tumor tissue and serum were collected. The concentrations of endostatin and VEGF in the serum of mice were detected by immunohistochemical method, and the microvessel density of transplanted tumor in each group was detected by (MVD) Elisa. Results: we successfully prepared ES-NPs loaded with different mass endostatin by ionic gel method. By comparing the encapsulation efficiency and drug loading of different batches of nanoparticles, we found that, The ES-NPs prepared by adding 渭 L ES (11.2mg/mL into 40 mL chitosan solution (1 mg/mL) not only guaranteed high entrapment efficiency, but also had suitable drug loading and particle size, which could meet the need of treatment. The ES-NPs could be stably preserved by adding 5% trehalose into the freeze-dried solution. The nanoparticles were spherical in appearance and uniform in distribution, and the cumulative release amount reached (60.22 卤2.58) in vitro after 7 days of sustained release. At least 48h.MTT showed that GES NPs could inhibit the growth of HUVECs in serum. The inhibitory effect of ES-NPs on HUVECs was similar to that of free es at the first 24 h of drug action (p0. 577). However, with the prolongation of the action time, the effect of GES NPs was obviously better than that of free es (p0.05). Similarly, in transwell experiment, the inhibitory effect of GES NPs on migration of HUVECs was significantly stronger than that of free ESs, the difference was statistically significant (p0. 05), and the longer the action time, the greater the difference of effect between them. In Lewis lung cancer transplant model, we observed that ES-NPs can significantly inhibit the growth of transplanted tumor. Even the effect of once every 7 days was better than that of free es for 14 days. ES-NPs injection for 14 days achieved the best therapeutic effect in our experiment, and the inhibition rate of transplanted tumor was as high as 63.32. The results of immunohistochemical CD31 staining showed that the MVD of tumor tissue was the lowest in the control group (p0.001) and the free es group (p0.001) after 14 days of continuous injection. The serum levels of endostatin and VEGF were significantly higher in mice of 14-day continuous injection group than those in other groups (p < 0.05), but the levels of VEGF were significantly lower than those in other groups (p < 0.05). It is proved that ES-NPs plays an anti-tumor role mainly by inhibiting tumor angiogenesis. Conclusion: chitosan is a suitable material for preparing endostatin nanoparticles. ES-NPs prepared by chitosan has high encapsulation efficiency, drug loading and obvious slow-release. Endostatin chitosan nanoparticles could significantly inhibit the proliferation and migration of HUVECs in vitro, and the effect was better than that of free endostatin 3) on Lewis lung cancer model. Endostatin-loaded chitosan nanoparticles can enhance the therapeutic effect of endostatin and inhibit tumor growth by inhibiting angiogenesis.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R73-36
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