靶向血栓诊疗纳米粒的初步实验研究

Preliminary experimental study of thrombus-targeted theranostic nanoparticles

目的制备靶向血栓的多功能纳米粒,探究其理化性质、靶向作用、光声成像能力及对血栓的溶解效果。方法以聚乳酸-羟基乙酸共聚物(PLGA)、近红外荧光碘化物(DiR)、全氟戊烷(PFP)、cCGPRPPC环肽等为原料,采用双乳化法制备及碳二亚胺法修饰从而获得cCGPRPPC-DiR-PFP-PLGA纳米粒(TNP)。用光学显微镜、激光粒径仪观测其形态、粒径、zeta电位及分散性,用紫外分光光度计测定DiR包封率,用共聚焦显微镜、流式细胞仪检测纳米粒的环肽携带情况,用共聚焦显微镜、荧光显微镜观察TNP的靶向性,用光声成像实验评估TNP的成像能力,用低能聚焦超声仪(LIFU)辐照TNP进行体外、体内溶栓实验。结果本实验制备的TNP为圆形,平均粒径为(262.67±23.46)nm、zeta电位为(-1.97±0.68)mV、多聚分散系数为0.06±0.05,DiR包封率为(82.00±0.03)%,纳米粒的环肽携带率为(99.58±0.47)%。TNP的体外光声信号强度具有浓度依赖性。TNP对体外和体内血栓都具有靶向作用。LIFU辐照30 min时TNP的体外溶栓率达(71.43±1.00)%;在体内溶栓实验中,血栓部位呈现的光声信号强度随着LIFU辐照时间的延长而降低。结论本实验成功制备了靶向血栓的造影剂TNP,它具有体内外光声成像能力和溶栓能力。

Objective To prepare multifunctional nanoparticles targeting thrombus,and to explore the physico-chemical properties,targeting effect,photoacoustic imaging ability and thrombolytic effect of the nanoparticles.Methods cCGPRPPC-DiR-PFP-PLGA nanoparticles(TNP)were prepared by double emulsification method and modified by carbodiimide technique,with poly(lactic-co-glycolic acid)(PLGA),near-infrared fluorescent iodide(1,1’-dioctadecyl-3,3,3’,3’-tetramethylindotricarbocyanine iodide,DiR),perfluoropentane(PFP)and cCGPRPPC cyclic peptide.The morphology,particle size,zeta potential and polydispersity were observed by optical microscope and laser particle size analyzer;the encapsulation rate of DiR was detected by ultraviolet spectrophotometer;the conjugation of cyclic peptide with nanoparticles was detected by confocal microscopy and flow cytometry;the targeting properties of TNP were observed by confocal microscopy and fluorescence microscopy;the imaging ability of TNP was analyzed by photoacoustic imaging experiments;and TNP were used for thrombolysis in vitro and in vivo with low-intensity focused ultrasound(LIFU)irradiation.Results The particle was round,the size of TNP was(262.67±23.46)nm,the zeta potential was(-1.97±0.68)mV,the polydispersity index was 0.06±0.05,the DiR encapsulation rate was(82.00±0.03)%,and the conjugation rate of peptide with nanoparticles was(99.58±0.47)%.The photoacoustic signal intensity of TNP in vitro was concentration dependent.TNP could target thrombus both in vitro and in vivo.In vitro,the thrombolytic rate was(71.43±1.00)%at 30 min after LIFU irradiation.In vivo thrombolysis experiment,the photoacoustic signal intensity of thrombus decreased with the prolongation of LIFU irradiation time.Conclusion Thrombus targeting contrast agents,TNP,have been successfully prepared,which exhibiting photoacoustic imaging ability and thrombolytic effect in vitro and in vivo.