靶向纤维蛋白多肽纳米探针在增强静脉血栓磁共振显像的研究

Study of targeting fibrin polypeptide nanoprobes in enhanced MRI for venous thrombosis

目的静脉血栓栓塞患者的个性化治疗与其良好的预后密切相关,而治疗方案的正确制订迫切需要精准的诊断,由于四氧化三铁和聚乳酸羟基乙酸良好的生物安全性,本研究设计了四氧化三铁-聚乳酸羟基乙酸-五肽(Fe_(3)O_(4)-PLGA-CREKA)纳米探针(nanoprobe,NP),通过靶向纤维蛋白去提高血栓的检测能力。材料与方法通过双乳化法和碳二亚胺法制备Fe_(3)O_(4)-PLGA-CREKA纳米探针,检测其理化特性;用T2WI及T2 mapping序列对Fe_(3)O_(4)-PLGA-CREKA纳米探针进行体外MRI,分析纳米探针浓度与T2弛豫率的相关性;采集大鼠静脉血制作冰冻切片,分别与Fe_(3)O_(4)-PLGA/DiI-CREKA NPs(靶向组)、Fe_(3)O_(4)-PLGA/DiI NPs(非靶向组)孵育,经倒置荧光显微镜观察不同纳米探针对血栓的靶向性;细胞上验证探针的安全性后,在SD大鼠的颈静脉建立静脉血栓模型,尾静脉分别注射Fe_(3)O_(4)-PLGA-CREKA NP(靶向组)、Fe_(3)O_(4)-PLGA NP(非靶向组),经T2WI和T2 mapping序列扫描后采集数据;同时收集主要器官,通过H&E染色验证纳米探针的安全性。结果本研究成功制备出粒径为(255.3±56.0)nm的Fe_(3)O_(4)-PLGA-CREKA纳米探针,其表面电位为(-18.90±5.84)mV;TEM结果显示该纳米探针为均匀球体,分散性良好;具备增强MR信号的能力;与非靶向组纳米探针比较,靶向组纳米探针可很好地靶向静脉血栓,并具备良好的生物安全性;非靶向组打药前、打药后;靶向组打药前、打药后的T2弛豫率分别为(17.33±2.25)s^(-1)、(49.00±6.66)s^(-1)、(19.00±3.90)s^(-1)、(72.83±6.68)s^(-1)。结论本研究制备的Fe_(3)O_(4)-PLGA-CREKA纳米粒具备良好的生物安全性和MRI能力,对静脉血栓有较高的特异性,有望为静脉血栓栓塞诊疗提供有效的帮助。

Objective:The individualized treatment of patients with venous thromboembolism is closely related to their good prognosis,and accurate diagnosis is necessary for the correct formulation of treatment plans.Given the high biological safety of ferric oxide and polylactic hydroxyacetic acid(PLGA),we designed a nanoprobe(NP)of ferric oxide polylactic hydroxyacetic acid pentapeptide(Fe_(3)O_(4)-PLGA-CREKA)to improve the detection ability of thrombus through targeting fibrin.Materials and Methods:Fe_(3)O_(4)-PLGA-CREKA nanoprobes were made by double emulsification and carbodiimide,and their physicochemical properties were tested.In-vitro magnetic resonance imaging(MRI)of Fe_(3)O_(4)-PLGA-CREKA nanoprobe was performed with T2WI and T2 mapping sequences to analyze the correlation between nanoprobe concentration and T2 relaxation rate.The venous blood of rats was collected to make frozen sections and incubated with Fe_(3)O_(4)-PLGA/DiI CREKA NPs(targeted group)and Fe_(3)O_(4)-PLGA/DiI NPs(non-targeted group)respectively.The targeting of different nanoprobes to thrombus was observed by inverted fluorescent microscope.After the safety verification of the nanoprobe on cells,a venous thrombosis model was established in the jugular vein of SD rats.Fe_(3)O_(4)-PLGA-CREKA NP(targeted group)and Fe_(3)O_(4)-PLGA NP(non-targeted group)were injected into the caudal vein respectively.Data were collected after T2WI and T2 mapping sequence scanning.At the same time,major organs were collected,and the safety of the nanoprobe was verified by H&E staining.Results:In this study,Fe_(3)O_(4)-PLGA-CREKA nanoprobes with a particle size of(255.3±56.0)nm were successfully prepared,and their surface potential was(-18.90±5.84)mV;The TEM results showed that the nanoprobe was a homogeneous sphere with good dispersion;The nanoprobes had the ability to enhance MR signal;Compared with the non-targeted group,the targeted group's nanoprobes can better target venous thrombosis,which has good biosafety;the T2 relaxation rates of non-targeted group and the targeted group before and after administration were(17.33±2.25)s^(-1),(49.00±6.66)s^(-1),(19.00±3.90)s^(-1)and(72.83±6.68)s^(-1),respectively.Conclusions:The Fe_(3)O_(4)-PLGA-CREKA nanoprobes prepared in our study possess good biosafety and MRI imaging ability with high specificity for venous thrombosis,which is expected to help with venous thromboembolism diagnosis and treatment in the future.