靶向血栓诊疗纳米粒的初步实验研究

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.

Preliminary experimental study of Thrombus-Targeted Theranostic Nanoparticles

Abstract] Objective To prepare multifunctional nanoparticles targeting thrombus, and investigate the preparation, physico-chemical properties, targeting ability, photoacoustic imaging ability and thrombolytic effect of the nanoparticles. Methods The thrombus-targeted nanoparticles (cCGPRPPC-DiR-PFP-PLGA NPs, TNPs) were prepared by double emulsification method and modified by carbodiimide technique. The particle morphology, size, zeta potential and polydispersity index (PDI) were investigated by using optical microscope and Malvern particle size analyzer, the encapsulation rate of DiR in TNPs was detected by using ultraviolet spectrophotometer, the conjugation of cyclic peptide with nanoparticles was investigated by using confocal microscope and flow cytometer. Confocal microscope and fluorescence microscope were used to observe the targeting properties of TNPs. The photoacoustic intensity of nanoparticles in vitro or thrombus in vivo was determined by using photoacoustic imager, and then the data was analyzed to evaluated photoacoustic imaging capabilities or thrombolysis effect of TNPs. TNPs was used to thrombolysis in vitro and in vivo, with low-intensity focused ultrasound (LIFU) irradiation. Results The particle size of TNPs was 262.67 ± 23.46 nm, the potential was -1.97 ± 0.68 mV, the PDI was 0.06±0.05, the DiR encapsulation rate in TNPs was 82.00±0.03%, and the conjugation rate of peptide with nanoparticles was 99.58±0.47%. The intensity of photoacoustic signal increased along with the increase concentrations of TNPs in vitro. TNPs possess the ability of targeting thrombus both in vitro and in vivo. In vitro, the thrombolytic rate is 73.15±1.00 % at 30 min after LIFU irradiation. In vivo, the photoacoustic signal gradually weakened with increasing LIFU irradiation time. Conclusion Thrombus targeting contrast agents, TNPs, was successfully prepared, exhibiting excellent photoacoustic imaging ability and thrombolytic effects in vitro and in vivo.