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目的制备一种胰腺癌靶向的纳米级超声造影剂并评价其体外靶向性效果。方法用聚乳酸-羟基乙酸共聚物(PLGA)、N-羟基琥珀酰亚胺(NHS)、聚乙二醇(PEG)合成高分子聚合物PLGA-PEG-NHS并利用核磁共振氢谱检测;用PLGA-PEG-NHS和全氟溴辛烷(PFOB)以乳化挥发法制备包裹PFOB的PLGA纳米造影剂,然后连接Hedgehog抗体。透射电镜观察纳米造影剂的形态,动态光散射法测定其粒径、电位;气相色谱-质谱法测定纳米造影剂的包封率及载药量;透析法测定纳米造影剂的体外释放特性。用添加了纳米造影剂的培养液培养人胰腺癌细胞株SW1990和CFPAC-1,利用荧光显微镜和流式细胞仪定性和定量验证纳米造影剂的体外靶向能力。结果所制得的胰腺癌靶向纳米超声造影剂平均粒径为198.9nm,Zeta电位为-31.8mV,包封率(63.7±3.9)%,载药量(14.3±0.9)%,48h释药量为85.3%;体外细胞实验显示靶向造影剂与高表达Hedgehog抗原的SW1990细胞大量结合,而与不表达Hedgehog抗原的CFPAC-1细胞未见特异性结合。结论通过乳化挥发法制备的胰腺癌靶向纳米超声造影剂各项表征符合要求,并能特异性靶向Hedgehog高表达的胰腺癌细胞,有望成为胰腺癌诊断的超声造影剂。
Objective To prepare a pancreatic cancer targeted nanoscale ultrasound contrast agent and evaluate its in vitro targeting effect. Methods The polymer PLGA-PEG-NHS was synthesized by PLGA, NHS and PEG. The PLGA-PEG-NHS and perfluorooctyl bromide (PFOB) were used to prepare PFOB-encapsulated PLGA nanofumages by emulsion evaporation followed by attachment of Hedgehog antibodies. The morphology of nano-contrast agent was observed by transmission electron microscope. The particle size and potential were measured by dynamic light scattering. The encapsulation efficiency and drug loading of nano-contrast medium were determined by gas chromatography-mass spectrometry. The in vitro release characteristics of nano-contrast agent were determined by dialysis method. Human pancreatic cancer cell lines SW1990 and CFPAC-1 were cultured in culture medium supplemented with nano-contrast agent, and the targeting ability of nano-contrast agent was verified qualitatively and quantitatively by fluorescence microscopy and flow cytometry. Results The average particle size of the targeted nano-contrast agent for pancreatic cancer was 198.9nm, the Zeta potential was -31.8mV, the entrapment efficiency (63.7 ± 3.9)%, drug loading (14.3 ± 0.9)%, Amount of 85.3%. In vitro experiments showed that the targeted contrast agent strongly bound to SW1990 cells highly expressing Hedgehog antigen, but not to CFPAC-1 cells that did not express Hedgehog antigen. CONCLUSIONS: The NPC-targeted nano-contrast agents prepared by the emulsion evaporation method meet the requirements and can specifically target Hedgehog-overexpressing pancreatic cancer cells, which is expected to become a diagnostic ultrasound contrast agent for pancreatic cancer.