一种载cRGD肽相变型光声/超声双模态多功能纳米分子探针的制备及鉴定

Preparation and identification of a multifunctional and dual-modality photoacoustic/ultrasound phase-shift nanomolecular probe coated with cRGD peptide

目的:制备一种载cRGD肽相变型光声/超声双模态多功能纳米分子探针(cRGD-GNR-PFP),联合低强度聚焦超声(LIFU)辐照,体外评价其相变性能、光声/超声成像效果及靶向性。方法:以聚乳酸-羟基乙酸共聚物(PLGA)为载体,采用双乳法及碳二亚胺法制备表面载cRGD肽并包裹金纳米棒(GNR)和全氟戊烷(PFP)的纳米分子探针cRGD-GNR-PFP,利用光学显微镜、扫描及透射电镜、粒径及电位仪对探针进行相关表征,共聚焦显微镜与流式细胞仪检测结合率,体外研究探针超声和光声成像性能、LIFU辐照下的相变情况,及对新鲜动脉血栓的靶向效果,CCK8法检测其体外生物安全性。结果:制备的载cRGD肽并包裹GNR及PFP相变型双模态纳米分子探针形态规则,大小均匀,分散性好,具有较好的稳定性,平均粒径(225.87±1.56) nm,表面电位(9.59±0.22) mV,透射电镜下可见GNR被成功包载,LIFU辐照后探针可发生相变并能增强超声显影效果,纳米探针对光声成像具有很好的增强作用,随着其浓度的增加,光声信号也逐渐增强,cRGD肽连接率为92.79%,可介导分子探针靶向新鲜体外动脉血栓。与人脐静脉内皮细胞(HUVECs)孵育12 h及24 h后细胞存活率均在90%以上,具有良好的生物安全性。结论:成功制备出cRGD肽并包裹GNR及PFP相变型双模态纳米分子探针cRGD-GNR-PFP,具备良好的光声/超声成像能力及体外声致相变性能,靶向性好及生物安全性高,有望实现对动脉粥样硬化易损斑块精确识别及血栓精准防治。

Objective: To prepare a multifunctional and dual-modality photoacoustic/ultrasound phase-shift nanomolecular probe coated with cRGD peptide(cRGD-GNR-PFP) and observe the phase transition,imaging effect by photoacoustic/ultrasound and the targeted ability in vitro under low intensity focused ultrasound(LIFU) irradiation. Methods: Nanomolecular probes containing gold nanorods(GNR) and perfluoropentane(PFP) and coated with cRGD peptide were prepared by double emulsion and carbodiimide method with PLGA as the carrier. The probes were characterized by optical microscope, scanning and transmission electron microscope, particle size analyzer and potentiometer. The binding rate was measured by confocal microscopy and flow cytometry. The ultrasonic and photoacoustic imaging performance, phase transformation under LIFU irradiation and targeting effect on fresh arterial throm-bosis were studied in vitro. And the biosafety in vitro was detected by CCK8 method. Results: The cRGD-GNR-PFP had regular morphology, uniform size, good dispersion, and high stability. The average particle diameter was(225. 87±1. 56) nm, and the surface potential was(9. 59±0. 22) mV. Under the transmission electron microscope, GNR were successfully encapsulated. After LIFU irradiation,the cRGD-GNR-PFP could undergo phase transition, which enhanced ultrasound and photoacoustic imaging effect. With the increasing of its concentration, the photoacoustic signal gradually enhanced. The connection rate of cRGD peptide was 92. 79%, which can effectively mediate the probes targeting fresh arterial thrombosis in vitro. The survival rate of human umbilical vein endothelial cells(HUVECs) was above 90% after 12 h and 24 h incubation with the cRGD-GNR-PFP, which showed a good biosafety of the probes. Conclusion: The cRGD-GNR-PFP nanomolecular probes were successfully prepared,with good ultrasonic and photoacoustic imaging capabilities and acoustic droplet vaporization performance. Meanwhile, the nanomolecular probes have excellent targeting ability and high biosafety in vitro, and can provide the potential of precise identification of atherosclerotic vulnerable plaques for precise prevention and treatment of thrombosis.