攜rtPA靶向血栓多功能MR分子探針的構(gòu)建及表征
發(fā)布時(shí)間：2018-11-29 14:05
【摘要】：目的：制備攜帶溶栓藥物重組組織型纖溶酶原激活劑（Recombinanttissue plasminogen activator, rtPA）的靶向血栓MR分子探針，并對(duì)其一般理化性能、體內(nèi)外靶向血栓能力、溶栓能力及MR顯影能力進(jìn)行驗(yàn)證。 方法：采用碳二亞胺法將環(huán)狀精氨酸-甘氨酸-天冬氨酸肽段（Cyclicarginine-glycine-aspartic, cRGD）連接于殼聚糖（Chitosan, CS）膜表面，得到CS-cRGD膜；雙乳化溶劑揮發(fā)法（水/油/水法[W/O/W]）制備攜帶rtPA及載Fe3O4的聚乳酸羥基乙酸（Poly [lactic-co-glycolic acid],PLGA）納米粒，通過制備條件的調(diào)整，分別得到Fe3O4-PLGA、Fe3O4-PLGA-rtPA、Fe3O4-PLGA-rtPA/CS及Fe3O4-PLGA-rtPA/CS-cRGD納米粒。用光學(xué)顯微鏡、透射電鏡及激光粒度儀檢測(cè)納米粒的形態(tài)、分散性、結(jié)構(gòu)、粒徑、粒徑分布及Zeta電位，激光共聚焦顯微鏡及流式細(xì)胞儀驗(yàn)證CS-cRGD膜對(duì)納米粒表面的被覆，F(xiàn)e3O4攜帶率及rtPA包封率分別用原子吸收光譜法及比色法測(cè)得，rtPA的體外釋放活性用生色底物法檢測(cè)。經(jīng)體外靶向血栓病理切片及靶向溶栓實(shí)驗(yàn)證實(shí)納米粒對(duì)體外血栓的靶向溶解能力后，分別用1.5T及3.0T MR掃描儀驗(yàn)證納米粒的弛豫性能及對(duì)體內(nèi)外血栓的靶向溶解顯影能力。 結(jié)果：各組納米粒均成功制得，形態(tài)規(guī)則，表面光滑呈球形，分散性好，粒徑較均一，透射電鏡可見Fe3O4顆粒均勻鑲嵌于納米粒殼上，F(xiàn)e3O4-PLGA-rtPA/CS-cRGD納米粒的平均粒徑為395.2±12.2nm，粒徑分布（多分散系數(shù)[polydispersity index, PDI]）為0.163±0.024，Zeta電位為29.0±1.5mV。激光共聚焦顯微鏡顯示Fe3O4-PLGA-rtPA/CS-cRGD納米�？梢姯h(huán)狀綠色熒光，即異硫氰酸熒光素（Fluoresceinisothiocyanate, FITC）標(biāo)記的cRGD成功被覆于納米粒表面，流式細(xì)胞儀測(cè)得cRGD攜帶率為84.27%。Fe3O4攜帶率及rtPA包封率分別為49.3±3.4%，63.7±1.5%，，體外釋放實(shí)驗(yàn)顯示Fe3O4-PLGA-rtPA/CS及Fe3O4-PLGA-rtPA/CS-cRGD納米粒中釋放的rtPA的酶活性明顯高于Fe3O4-PLGA-rtPA納米粒。體外靶向血栓病理切片可見大量Fe3O4-PLGA-rtPA/CS-cRGD納米粒聚集于血栓周邊，體外靶向溶栓實(shí)驗(yàn)示Fe3O4-PLGA-rtPA、 Fe3O4-PLGA-rtPA/CS和Fe3O4-PLGA-rtPA/CS-cRGD組溶栓率在60分鐘時(shí)分別為游離rtPA溶液的1.98、2.41、3.05倍。在MRI掃描中各組載Fe3O4的納米粒均可顯著降低T2*信號(hào)，與純Fe3O4溶液相比，其橫向弛豫率（Transverse relaxation rate, R2*）及信噪比（Signal-to-noise ratio, SNR）無顯著差異。3.0T MR掃描儀示Fe3O4-PLGA-rtPA/CS-cRGD納米粒對(duì)體內(nèi)外血栓均有靶向顯影能力。 結(jié)論：Fe3O4-PLGA-rtPA/CS-cRGD納米粒形態(tài)規(guī)則，粒徑適宜，帶正電荷，F(xiàn)e3O4攜帶率及rtPA包封率較高，可較好的保留rtPA的酶活性；對(duì)體內(nèi)外血栓具有良好的靶向性，并能顯著增強(qiáng)rtPA的溶栓性能；具備良好的MR對(duì)比增強(qiáng)效應(yīng)，有望在MR監(jiān)測(cè)下實(shí)現(xiàn)對(duì)血栓的早期診斷及治療。
[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 鹵12.2 nm, and the particle size distribution (polydispersity index, PDI) is 0.163 鹵0.024 渭 mol / ml. The Zeta potential is 29.0 鹵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 鹵3.4 and 63.7 鹵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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R445.2
本文編號(hào)：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 鹵12.2 nm, and the particle size distribution (polydispersity index, PDI) is 0.163 鹵0.024 渭 mol / ml. The Zeta potential is 29.0 鹵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 鹵3.4 and 63.7 鹵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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R445.2
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