携rtPA靶向血栓多功能MR分子探针的构建及表征
发布时间：2018-11-29 14:05
【摘要】：目的：制备携带溶栓药物重组组织型纤溶酶原激活剂（Recombinanttissue plasminogen activator, rtPA）的靶向血栓MR分子探针，并对其一般理化性能、体内外靶向血栓能力、溶栓能力及MR显影能力进行验证。 方法：采用碳二亚胺法将环状精氨酸-甘氨酸-天冬氨酸肽段（Cyclicarginine-glycine-aspartic, cRGD）连接于壳聚糖（Chitosan, CS）膜表面，得到CS-cRGD膜；双乳化溶剂挥发法（水/油/水法[W/O/W]）制备携带rtPA及载Fe3O4的聚乳酸羟基乙酸（Poly [lactic-co-glycolic acid],PLGA）纳米粒，通过制备条件的调整，分别得到Fe3O4-PLGA、Fe3O4-PLGA-rtPA、Fe3O4-PLGA-rtPA/CS及Fe3O4-PLGA-rtPA/CS-cRGD纳米粒。用光学显微镜、透射电镜及激光粒度仪检测纳米粒的形态、分散性、结构、粒径、粒径分布及Zeta电位，激光共聚焦显微镜及流式细胞仪验证CS-cRGD膜对纳米粒表面的被覆，Fe3O4携带率及rtPA包封率分别用原子吸收光谱法及比色法测得，rtPA的体外释放活性用生色底物法检测。经体外靶向血栓病理切片及靶向溶栓实验证实纳米粒对体外血栓的靶向溶解能力后，分别用1.5T及3.0T MR扫描仪验证纳米粒的弛豫性能及对体内外血栓的靶向溶解显影能力。 结果：各组纳米粒均成功制得，形态规则，表面光滑呈球形，分散性好，粒径较均一，透射电镜可见Fe3O4颗粒均匀镶嵌于纳米粒壳上，Fe3O4-PLGA-rtPA/CS-cRGD纳米粒的平均粒径为395.2±12.2nm，粒径分布（多分散系数[polydispersity index, PDI]）为0.163±0.024，Zeta电位为29.0±1.5mV。激光共聚焦显微镜显示Fe3O4-PLGA-rtPA/CS-cRGD纳米粒可见环状绿色荧光，即异硫氰酸荧光素（Fluoresceinisothiocyanate, FITC）标记的cRGD成功被覆于纳米粒表面，流式细胞仪测得cRGD携带率为84.27%。Fe3O4携带率及rtPA包封率分别为49.3±3.4%，63.7±1.5%，，体外释放实验显示Fe3O4-PLGA-rtPA/CS及Fe3O4-PLGA-rtPA/CS-cRGD纳米粒中释放的rtPA的酶活性明显高于Fe3O4-PLGA-rtPA纳米粒。体外靶向血栓病理切片可见大量Fe3O4-PLGA-rtPA/CS-cRGD纳米粒聚集于血栓周边，体外靶向溶栓实验示Fe3O4-PLGA-rtPA、 Fe3O4-PLGA-rtPA/CS和Fe3O4-PLGA-rtPA/CS-cRGD组溶栓率在60分钟时分别为游离rtPA溶液的1.98、2.41、3.05倍。在MRI扫描中各组载Fe3O4的纳米粒均可显著降低T2*信号，与纯Fe3O4溶液相比，其横向弛豫率（Transverse relaxation rate, R2*）及信噪比（Signal-to-noise ratio, SNR）无显著差异。3.0T MR扫描仪示Fe3O4-PLGA-rtPA/CS-cRGD纳米粒对体内外血栓均有靶向显影能力。 结论：Fe3O4-PLGA-rtPA/CS-cRGD纳米粒形态规则，粒径适宜，带正电荷，Fe3O4携带率及rtPA包封率较高，可较好的保留rtPA的酶活性；对体内外血栓具有良好的靶向性，并能显著增强rtPA的溶栓性能；具备良好的MR对比增强效应，有望在MR监测下实现对血栓的早期诊断及治疗。
[Abstract]:Aim: to prepare the target MR probe carrying recombinant tissue plasminogen activator (Recombinanttissue plasminogen activator, rtPA) for thrombolytic drugs, and to verify its general physical and chemical properties, in vivo and in vitro targeting thrombus ability, thrombolytic ability and MR development ability. Methods: the cyclic arginine glycine aspartic acid peptide segment (Cyclicarginine-glycine-aspartic, cRGD) was connected to the surface of chitosan (Chitosan, CS) membrane by carbodiimide method to obtain CS-cRGD membrane. Double emulsified solvent volatilization (water / oil / water method [W/O/W]) was used to prepare Poly [lactic-co-glycolic acid], PLGA) nanoparticles carrying rtPA and Fe3O4. Fe3O4-PLGA, was obtained by adjusting the preparation conditions. Fe3O4-PLGA-rtPA,Fe3O4-PLGA-rtPA/CS and Fe3O4-PLGA-rtPA/CS-cRGD nanoparticles. The morphology, dispersion, structure, particle size distribution and Zeta potential of the nanoparticles were examined by optical microscope, transmission electron microscope and laser particle size analyzer. The coating of CS-cRGD film on the surface of nanoparticles was verified by laser confocal microscopy and flow cytometry. The Fe3O4 carrying rate and rtPA entrapment rate were determined by atomic absorption spectrometry and colorimetry respectively. The release activity of rtPA in vitro was determined by chromogenic substrate method. The in vitro thrombolytic ability of nanoparticles was confirmed by pathological section and thrombolytic assay. The relaxation properties of nanoparticles and their ability to dissolve thrombus in vivo and in vitro were verified by 1.5T and 3.0T MR scanners. Results: all the nanoparticles were successfully prepared with regular morphology, smooth surface, good dispersion and uniform particle size. Transmission electron microscopy showed that Fe3O4 particles were uniformly embedded on the nanoparticles shell. The average particle size of Fe3O4-PLGA-rtPA/CS-cRGD nanoparticles is 395.2 卤12.2 nm, and the particle size distribution (polydispersity index, PDI) is 0.163 卤0.024 渭 mol / ml. The Zeta potential is 29.0 卤1.5 MV. Laser confocal microscopy showed that ring green fluorescence could be seen in Fe3O4-PLGA-rtPA/CS-cRGD nanoparticles. CRGD labeled with fluorescein isothiocyanate (Fluoresceinisothiocyanate, FITC) was successfully coated on the surface of nanoparticles. The 84.27%.Fe3O4 carrying rate and rtPA encapsulation rate of cRGD were 49.3 卤3.4 and 63.7 卤1.5, respectively. In vitro release assay showed that the enzyme activity of rtPA released in Fe3O4-PLGA-rtPA/CS and Fe3O4-PLGA-rtPA/CS-cRGD nanoparticles was significantly higher than that in Fe3O4-PLGA-rtPA nanoparticles. In vitro, a large number of Fe3O4-PLGA-rtPA/CS-cRGD nanoparticles were found to gather around the thrombus. The in vitro targeted thrombolytic assay showed Fe3O4-PLGA-rtPA,. The thrombolytic rates of Fe3O4-PLGA-rtPA/CS and Fe3O4-PLGA-rtPA/CS-cRGD groups were 1.98 ~ 2.41 ~ 3.05 times as much as that of free rtPA solution at 60 minutes, respectively. In MRI scanning, the T 2 * signal was significantly decreased by Fe3O4 loaded nanoparticles. Compared with the pure Fe3O4 solution, the transverse relaxation rate (Transverse relaxation rate, R 2 *) and the signal-to-noise ratio (Signal-to-noise ratio,) of each group of Fe3O4 nanoparticles were significantly lower than those of the pure Fe3O4 solution. There was no significant difference in SNR. 3.0T MR scanner showed that Fe3O4-PLGA-rtPA/CS-cRGD nanoparticles had the ability to target thrombosis in vivo and in vitro. Conclusion: the morphology of Fe3O4-PLGA-rtPA/CS-cRGD nanoparticles is regular, the particle size is suitable, the positive charge, the carrying rate of Fe3O4 and the entrapment efficiency of rtPA are higher, and the enzyme activity of rtPA can be better preserved. It has good targeting to thrombus in vivo and in vitro, and can significantly enhance the thrombolytic performance of rtPA, and has a good contrast enhancement effect of MR, which is expected to realize the early diagnosis and treatment of thrombus under the monitoring of MR.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R445.2
本文编号：2365187
[Abstract]:Aim: to prepare the target MR probe carrying recombinant tissue plasminogen activator (Recombinanttissue plasminogen activator, rtPA) for thrombolytic drugs, and to verify its general physical and chemical properties, in vivo and in vitro targeting thrombus ability, thrombolytic ability and MR development ability. Methods: the cyclic arginine glycine aspartic acid peptide segment (Cyclicarginine-glycine-aspartic, cRGD) was connected to the surface of chitosan (Chitosan, CS) membrane by carbodiimide method to obtain CS-cRGD membrane. Double emulsified solvent volatilization (water / oil / water method [W/O/W]) was used to prepare Poly [lactic-co-glycolic acid], PLGA) nanoparticles carrying rtPA and Fe3O4. Fe3O4-PLGA, was obtained by adjusting the preparation conditions. Fe3O4-PLGA-rtPA,Fe3O4-PLGA-rtPA/CS and Fe3O4-PLGA-rtPA/CS-cRGD nanoparticles. The morphology, dispersion, structure, particle size distribution and Zeta potential of the nanoparticles were examined by optical microscope, transmission electron microscope and laser particle size analyzer. The coating of CS-cRGD film on the surface of nanoparticles was verified by laser confocal microscopy and flow cytometry. The Fe3O4 carrying rate and rtPA entrapment rate were determined by atomic absorption spectrometry and colorimetry respectively. The release activity of rtPA in vitro was determined by chromogenic substrate method. The in vitro thrombolytic ability of nanoparticles was confirmed by pathological section and thrombolytic assay. The relaxation properties of nanoparticles and their ability to dissolve thrombus in vivo and in vitro were verified by 1.5T and 3.0T MR scanners. Results: all the nanoparticles were successfully prepared with regular morphology, smooth surface, good dispersion and uniform particle size. Transmission electron microscopy showed that Fe3O4 particles were uniformly embedded on the nanoparticles shell. The average particle size of Fe3O4-PLGA-rtPA/CS-cRGD nanoparticles is 395.2 卤12.2 nm, and the particle size distribution (polydispersity index, PDI) is 0.163 卤0.024 渭 mol / ml. The Zeta potential is 29.0 卤1.5 MV. Laser confocal microscopy showed that ring green fluorescence could be seen in Fe3O4-PLGA-rtPA/CS-cRGD nanoparticles. CRGD labeled with fluorescein isothiocyanate (Fluoresceinisothiocyanate, FITC) was successfully coated on the surface of nanoparticles. The 84.27%.Fe3O4 carrying rate and rtPA encapsulation rate of cRGD were 49.3 卤3.4 and 63.7 卤1.5, respectively. In vitro release assay showed that the enzyme activity of rtPA released in Fe3O4-PLGA-rtPA/CS and Fe3O4-PLGA-rtPA/CS-cRGD nanoparticles was significantly higher than that in Fe3O4-PLGA-rtPA nanoparticles. In vitro, a large number of Fe3O4-PLGA-rtPA/CS-cRGD nanoparticles were found to gather around the thrombus. The in vitro targeted thrombolytic assay showed Fe3O4-PLGA-rtPA,. The thrombolytic rates of Fe3O4-PLGA-rtPA/CS and Fe3O4-PLGA-rtPA/CS-cRGD groups were 1.98 ~ 2.41 ~ 3.05 times as much as that of free rtPA solution at 60 minutes, respectively. In MRI scanning, the T 2 * signal was significantly decreased by Fe3O4 loaded nanoparticles. Compared with the pure Fe3O4 solution, the transverse relaxation rate (Transverse relaxation rate, R 2 *) and the signal-to-noise ratio (Signal-to-noise ratio,) of each group of Fe3O4 nanoparticles were significantly lower than those of the pure Fe3O4 solution. There was no significant difference in SNR. 3.0T MR scanner showed that Fe3O4-PLGA-rtPA/CS-cRGD nanoparticles had the ability to target thrombosis in vivo and in vitro. Conclusion: the morphology of Fe3O4-PLGA-rtPA/CS-cRGD nanoparticles is regular, the particle size is suitable, the positive charge, the carrying rate of Fe3O4 and the entrapment efficiency of rtPA are higher, and the enzyme activity of rtPA can be better preserved. It has good targeting to thrombus in vivo and in vitro, and can significantly enhance the thrombolytic performance of rtPA, and has a good contrast enhancement effect of MR, which is expected to realize the early diagnosis and treatment of thrombus under the monitoring of MR.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R445.2
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