【摘要】 目的 制备靶向血栓纤维蛋白诊疗一体化的多功能明胶纳米粒，在体外探究其双模态显像潜能与光热增效药物溶栓作用，并在体内观察其对血栓的主动靶向能力。方法 采用二次去溶剂法及碳二亚胺法制备靶向纤维蛋白多肽（Cys-Arg-Glu-Lys-Ala，CREKA）介导的载阿替普酶（rt-PA）及四氧化三铁（Fe₃O₄）的磁性明胶纳米粒（CREKA-Fe-rt-PA-Gel）。观察该纳米粒形态，检测其理化性质及体外安全性；验证其体外光声（PA）及磁共振（MR）显像特征；检测其光热效能；采用纤维蛋白及血红蛋白指标评估光热增效药物的溶栓效果及破坏纤维蛋白网状结构的能力；构建颈动脉血栓动物模型，观察该纳米粒的体内血栓靶向能力。结果 所制备的磁性明胶纳米粒CREKA-Fe-rt-PA-Gel大小均一，粒径（254.79±6.83） nm，多分散系数（0.09±0.05），表面电位（5.48±4.60） mV；成功连接CREKA且连靶率为98.66%，rt-PA包封率为23.03%±0.05%，Fe₃O₄包封率为92.78%±0.57%，磁滞曲线显示其具有良好的顺磁性。体外细胞毒性试验及溶血试验显示该纳米粒具有良好的生物相容性。在体外PA及MR成像中该纳米粒表现出优异的成像能力。经激光辐照后纳米溶液呈浓度依赖性快速升温；与血栓孵育后经近红外辐照，溶液中血红蛋白及纤维蛋白指标均明显升高（_P<0.01），且纳米粒释放药物联合光热能够显著损毁纤维蛋白网状结构；大鼠颈动脉血栓HE染色显示该纳米粒有良好的体内血栓靶向能力。结论 构建的CREKA-Fe-rt-PA-Gel纳米粒具备双模态（PA/MR）显像潜能、良好的光热效力、优异的血栓（纤维蛋白）靶向能力，且联合光热有较强的溶栓作用，有望用于临床血栓性疾病的早期诊断与多形式治疗。更多还原

【Abstract】 Objective To prepare fibrin-targeted,integration of diagnosis and treatment and multi-functional gelatin nanoparticles,explore their potential of dual-mode imaging and the mechanism and effect of photothermal synergistic drug thrombolytic therapy in vitro,and observe the active-targeting ability of thrombus in vivo.Methods Loaded with alteplase （rt-PA） and Fe₃O₄ mediated by targeting fibrin polypeptide （Cys-Arg-Glu-Lys-Ala,CREKA）,the magnetic gelatin nanoparticles （CREKA-Fe-rt-PA-Gel） were prepared by two-step desolvation and carbodiimide method.The morphology of CREKA-Fe-rt-PA-Gel was observed,and its physical and chemical properties and in vitro safety were detected.Its photoacoustic （PA） and magnetic resonance （MR） imaging features were also verified in vitro.The photothermal performance was monitored,the fibrin and hemoglobin indexes were used to evaluate the thrombolytic effect of photothermal therapy and the ability to damage fibrin network.Carotid artery thrombosis model was constructed with rats and HE staining section was performed to observe the targeting thrombus ability in vivo of the nanoparticles.Results CREKA-Fe-rt-PA-Gel were with uniform particle size,diameter of （254.79±6.83） nm,polydispersity coefficient of 0.09±0.05,surface potential of （5.48±4.60） mV.The CREKA peptide was successfully connected with the target connection rate of 98.66%,the encapsulation efficiency of rt-PA was 23.03%±0.05%,and of Fe₃O₄ was 92.78%±0.57%,the magnetization curve?showed that it has good paramagnetism.The cytotoxicity and hemolysis test in vitro confirmed that CREKA-Fe-rt-PA-Gel had appropriate biological compatibility.At the same time,CREKA-Fe-rt-PA-Gel showed excellent imaging ability of PA and MR imaging in vitro.After laser irradiation,the nano solution heated up rapidly in a concentration-dependent manner,hemoglobin and fibrin in solution increased significantly after incubation with thrombus by near infrared irradiation （P<0.01）,and nanoparticles released drugs combined with photothermal therapy could damage the fibrin network.HE staining of carotid artery thrombosis in rats showed good thrombus targeting ability of the nanoparticles in vivo.Conclusion The constructed CREKA-Fe-rt-PA-Gel have potential in dual-mode （PA/MR） imaging,good photothermal properties,outstanding targeting ability for thrombus （fibrin）,and excellent thrombolytic ability when combined with photothermal,which is expected to be used in early diagnosis and multi-form treatment in clinical thrombotic diseases.更多还原