本文選題：氧化鐵 + PLGA��； 參考：《重慶醫(yī)科大學》2017年碩士論文

【摘要】：目的:制備c RGD修飾的載Fe_3O_4靶向血栓的MR分子探針(即Fe_3O_4-PLGA-c RGD納米粒),并對其理化性質、生物毒性、體內分布、體內外靶向血栓能力及體內MR顯像能力進行驗證。方法:采用雙乳化溶劑揮發(fā)法(水/油/水,W/O/W)以端羧基聚乳酸羥基乙酸(PLGA-COOH)及Fe_3O_4為殼材料,制備出Fe_3O_4-PLGA納米粒,采用碳二亞胺法將環(huán)狀精氨酸-甘氨酸-天冬氨酸肽段(cyclic arginine-glycine-aspartic,c RGD)連接于Fe_3O_4-PLGA納米粒表面,制備Fe_3O_4-PLGA-c RGD納米粒。光學顯微鏡觀察納米粒的形態(tài)、表面及分散性,透射電鏡觀察納米粒的內部結構,激光粒度儀對納米粒的粒徑、多分散系數(shù)(polydispersity index,PDI)和Zeta電位進行檢測。c RGD采用異硫氰酸熒光素(Fluorescein isothiocyanate,FITC)標記,激光共聚焦顯微鏡觀察納米粒表面c RGD的連接,流式細胞儀測定c RGD的攜帶率。原子吸收光譜法測定Fe_3O_4的攜帶率。通過分析SD大鼠的肝腎功、血細胞分析及觀察主要臟器病理切片來評價納米粒的體內毒性。離體血栓冰凍切片證實納米粒對離體血栓的靶向能力。7.0T MR掃描儀觀察SD大鼠腹主動脈非閉塞性附壁血栓構建情況、納米粒在SD大鼠體內的分布以及體內靶向血栓顯像的情況,并結合病理切片驗證腹主動脈非閉塞性附壁血栓的形成、納米粒體內靶向血栓的能力。結果:Fe_3O_4-PLGA-c RGD納米粒構建成功,輪廓規(guī)則呈球形,分散性好,表面光滑,大小較均一,平均粒徑為368.2±4.5 nm,PDI為0.081±0.058,Zeta電位為-9.43±0.86 m V,透射電鏡下見Fe_3O_4黑色小顆粒較均勻分布于納米粒殼上。激光共聚焦顯微鏡觀察Fe_3O_4-PLGA-c RGD納米粒呈綠色環(huán)狀熒光,表明c RGD成功連接于納米粒表面,流式細胞儀測得c RGD攜帶率為99.93±0.06%。原子吸收光譜法測得Fe_3O_4攜帶率為64.67±0.9%。SD大鼠肝腎功、血細胞分析及病理切片結果提示納米粒沒有明顯體內毒性作用。靶向離體血栓冰凍切片見大量Fe_3O_4-PLGA-c RGD納米粒粘附于血栓表面及血管內皮下。納米粒體內分布實驗可觀察到注射納米粒之后肝脾T2信號明顯降低,肝脾信噪比明顯低于注射前,而腎臟信噪比較注射前有輕微降低,提示納米粒主要被肝脾吞噬,少許可能經腎臟代謝。體內靶向血栓實驗結果顯示SD大鼠腹主動脈非閉塞附壁血栓構建成功,注射Fe_3O_4-PLGA-c RGD納米粒之后10 min腹主動脈周圍信號明顯降低,并隨時間逐漸增加直到50 min,病理切片觀察到納米粒附著在血栓表面以及血管內皮下,說明納米粒具有較好的體內靶向血栓能力及良好的磁共振顯像效果。結論:Fe_3O_4-PLGA-c RGD納米粒構建成功,輪廓規(guī)則并大小適宜,c RGD及Fe_3O_4攜帶率較高,沒有明顯體內毒性,對離體血栓和體內血栓均具有良好的靶向性,同時具備良好的MR負性對比增強效應,有望成為早期診斷血栓的一種高效、敏感、特異的磁共振對比劑。
[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 鹵4.5nmPDI 0.081 鹵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 鹵0.06 by flow cytometry.The Fe_3O_4 carrying rate was 64.67 鹵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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R445.2;R543

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