cRGD修饰的PLGA纳米粒靶向血栓磁共振显像

发布时间：2018-04-18 10:23

本文选题：氧化铁 + PLGA　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的:制备c RGD修饰的载Fe_3O_4靶向血栓的MR分子探针(即Fe_3O_4-PLGA-c RGD纳米粒),并对其理化性质、生物毒性、体内分布、体内外靶向血栓能力及体内MR显像能力进行验证。方法:采用双乳化溶剂挥发法(水/油/水,W/O/W)以端羧基聚乳酸羟基乙酸(PLGA-COOH)及Fe_3O_4为壳材料,制备出Fe_3O_4-PLGA纳米粒,采用碳二亚胺法将环状精氨酸-甘氨酸-天冬氨酸肽段(cyclic arginine-glycine-aspartic,c RGD)连接于Fe_3O_4-PLGA纳米粒表面,制备Fe_3O_4-PLGA-c RGD纳米粒。光学显微镜观察纳米粒的形态、表面及分散性,透射电镜观察纳米粒的内部结构,激光粒度仪对纳米粒的粒径、多分散系数(polydispersity index,PDI)和Zeta电位进行检测。c RGD采用异硫氰酸荧光素(Fluorescein isothiocyanate,FITC)标记,激光共聚焦显微镜观察纳米粒表面c RGD的连接,流式细胞仪测定c RGD的携带率。原子吸收光谱法测定Fe_3O_4的携带率。通过分析SD大鼠的肝肾功、血细胞分析及观察主要脏器病理切片来评价纳米粒的体内毒性。离体血栓冰冻切片证实纳米粒对离体血栓的靶向能力。7.0T MR扫描仪观察SD大鼠腹主动脉非闭塞性附壁血栓构建情况、纳米粒在SD大鼠体内的分布以及体内靶向血栓显像的情况,并结合病理切片验证腹主动脉非闭塞性附壁血栓的形成、纳米粒体内靶向血栓的能力。结果:Fe_3O_4-PLGA-c RGD纳米粒构建成功,轮廓规则呈球形,分散性好,表面光滑,大小较均一,平均粒径为368.2±4.5 nm,PDI为0.081±0.058,Zeta电位为-9.43±0.86 m V,透射电镜下见Fe_3O_4黑色小颗粒较均匀分布于纳米粒壳上。激光共聚焦显微镜观察Fe_3O_4-PLGA-c RGD纳米粒呈绿色环状荧光,表明c RGD成功连接于纳米粒表面,流式细胞仪测得c RGD携带率为99.93±0.06%。原子吸收光谱法测得Fe_3O_4携带率为64.67±0.9%。SD大鼠肝肾功、血细胞分析及病理切片结果提示纳米粒没有明显体内毒性作用。靶向离体血栓冰冻切片见大量Fe_3O_4-PLGA-c RGD纳米粒粘附于血栓表面及血管内皮下。纳米粒体内分布实验可观察到注射纳米粒之后肝脾T2信号明显降低,肝脾信噪比明显低于注射前,而肾脏信噪比较注射前有轻微降低,提示纳米粒主要被肝脾吞噬,少许可能经肾脏代谢。体内靶向血栓实验结果显示SD大鼠腹主动脉非闭塞附壁血栓构建成功,注射Fe_3O_4-PLGA-c RGD纳米粒之后10 min腹主动脉周围信号明显降低,并随时间逐渐增加直到50 min,病理切片观察到纳米粒附着在血栓表面以及血管内皮下,说明纳米粒具有较好的体内靶向血栓能力及良好的磁共振显像效果。结论:Fe_3O_4-PLGA-c RGD纳米粒构建成功,轮廓规则并大小适宜,c RGD及Fe_3O_4携带率较高,没有明显体内毒性,对离体血栓和体内血栓均具有良好的靶向性,同时具备良好的MR负性对比增强效应,有望成为早期诊断血栓的一种高效、敏感、特异的磁共振对比剂。
[Abstract]:Aim: to prepare c RGD modified Mr molecular probe (Fe_3O_4-PLGA-c RGD nanoparticles) modified with Fe_3O_4 targeting thrombus, and to verify its physicochemical properties, biotoxicity, in vivo distribution, in vivo and in vitro targeting thrombus ability and in vivo Mr imaging ability.Methods: Fe_3O_4-PLGA nanoparticles were prepared by double emulsified solvent volatilization (water / oil / water W / O / W) with carboxyl polylactic acid (PLGA-COOH) and Fe_3O_4 as shell materials.The cyclic arginine-glycine-aspartic acid peptide segment of cyclic arginine-glycine-aspartic acid (Fe_3O_4-PLGA) was connected to the surface of Fe_3O_4-PLGA nanoparticles by carbodiimide method to prepare Fe_3O_4-PLGA-c RGD nanoparticles.The morphology, surface and dispersion of nanoparticles were observed by optical microscope, the internal structure of nanoparticles was observed by transmission electron microscope, and the particle size of nanoparticles was observed by laser particle size analyzer.Polydispersity index (PDI) and Zeta potential were measured. C RGD was labeled with fluorescein isothiocyanate. The connection of c RGD on the surface of nanoparticles was observed by laser confocal microscopy. The carrying rate of c RGD was measured by flow cytometry.The carrying rate of Fe_3O_4 was determined by atomic absorption spectrometry.The in vivo toxicity of the nanoparticles was evaluated by analyzing the liver and kidney function of SD rats and observing the pathological sections of the main organs.In vitro frozen section of thrombus confirmed the ability of nanoparticles to target thrombosis in vitro. 7.0T Mr scanner was used to observe the construction of non-occlusive mural thrombus in abdominal aorta of SD rats, the distribution of nanoparticles in SD rats and the target thrombus imaging in vivo.Pathological sections were used to verify the ability of non-occlusive thrombus in abdominal aorta and the ability of nanoparticles to target thrombus in vivo.Results the RGD nanoparticles were successfully constructed, with regular shape, good dispersibility, smooth surface and uniform size. The average particle size was 368.2 卤4.5nmPDI 0.081 卤0.058 MV. the Fe_3O_4 black particles were found to be more evenly distributed on the nanoparticles shell under TEM.The green ring fluorescence of Fe_3O_4-PLGA-c RGD nanoparticles was observed by laser confocal microscopy, which indicated that c RGD was successfully connected to the surface of nanoparticles. The carrying rate of c RGD was 99.93 卤0.06 by flow cytometry.The Fe_3O_4 carrying rate was 64.67 卤0.9%.SD rat liver and kidney function measured by atomic absorption spectrometry. The results of blood cell analysis and pathological section showed that the nanoparticles had no obvious toxicity in vivo.A large number of Fe_3O_4-PLGA-c RGD nanoparticles adhered to the surface of thrombus and vascular endothelium.The distribution of nanoparticles in vivo showed that the T2 signal of liver and spleen was obviously decreased, the signal-to-noise ratio of liver and spleen was lower than that of before injection, but the SNR of kidney was slightly lower than that before injection, suggesting that the nanoparticles were mainly swallowed by liver and spleen.A few may be metabolized through the kidney.The results of in vivo targeted thrombus test showed that SD rats were successfully constructed with non-occlusive adnexal thrombosis of abdominal aorta, and the signal intensity around abdominal aorta decreased significantly 10 min after injection of Fe_3O_4-PLGA-c RGD nanoparticles.It was observed that the nanoparticles were attached to the surface of thrombus and the vascular endothelium after 50 mins. The results showed that the nanoparticles had a good ability to target thrombus in vivo and a good effect of magnetic resonance imaging (MRI).Conclusion\\\% Fe3O4-PLGA-c RGD nanoparticles have been successfully constructed, with a regular profile and a suitable size for carrying c RGD and Fe_3O_4, no obvious in vivo toxicity, good targeting to both in vitro and in vivo thrombus, and good Mr negative contrast enhancement effect.It is expected to be an effective, sensitive and specific magnetic resonance contrast agent for the early diagnosis of thrombus.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R445.2;R543

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