载酞菁锌靶向新生血管相变纳米粒体外超声显像与光热治疗实验研究

Ultrasound imaging and photothermal therapy of ZnPc loaded angiogenesistargeting nanoparticles in vitro

目的制备一种新型靶向新生血管诊疗一体化的超声分子探针,体外评价其靶向性、增强超声显像及光热治疗能力。方法采用双乳化法制备搭载全氟己烷(perfluorohexane,PFH)和酞菁锌(zinc phthalocyanine,ZnPc)的PLGA纳米粒;用碳二亚胺法将纳米粒与血管内皮生长因子受体-2(vascular endothelial growth factor receptor-2,VEGFR-2)抗体相偶联制备靶向纳米粒;检测其一般特性、体外寻靶能力;经激光辐照后检测其增强超声显像以及光热治疗能力。结果成功制备出靶向纳米粒,粒径(256.40±57.14)nm;CCK-8检测结果表明纳米粒没有明显的细胞毒性(F=0.402,P=0.837);激光共聚焦显微镜及流式细胞仪结果均证明靶向纳米粒具有良好的靶向能力,流式细胞仪检测非靶向组、抗体封闭组、靶向组中,纳米粒与细胞连接率分别为(9.52±2.14)%、(9.92±1.62)%、(61.89±3.62)%,差异有统计学意义(F=463.7,P<0.05),非靶向组(q=40.21,P<0.05)、抗体封闭组(q=30.91,P<0.05)分别与靶向组比较差异有统计学意义。经激光(1 W/cm2,5 min)辐照后纳米粒能够发生相变,增强超声显像并能使局部温度升高超过42℃,对细胞具备光热治疗的能力,而靶向组诱导细胞凋亡比例[(79.49±2.22)%]明显高于非靶向组[(24.23±1.95)%,P<0.05]。结论成功制备了靶向新生血管诊疗一体化超声分子探针,其具有良好的靶向能力,可用于增强超声显像及光热治疗。

Objective To prepare a new type of angiogenesis-targeting theranostic molecular probe and investigate its targeting ability, contrast enhanced ultrasound imaging and photothermal therapy in vitro. Methods Perfluorohexane （PFH） and zinc phthalocyanine （ZnPc） loaded targeting nanoparticles were prepared by double emulsification method and conjugated with vascular growth factor receptor2 （VEGFR2） antibodies via carbodiimide technique. The basic features and targeting ability of nanoparticles were detected. Contrast enhanced ultrasound imaging was observed and cell apoptosis was detected after laser irradiation. Results Targeting nanoparticles were successfully prepared with an average diameter of 256.40±57.14 nm. No obvious cytotoxicity was observed by CCK-8 assay （F=0.402, P〉0.05）. Good targeting ability was observed by confocal laser scanning microscopy （CLSM） and flow cytometry （FCM）. FCM results showed that the connection rate was （9.52±2.14）%, （9.92±1.62）%, and （61.89±3.62）% respectively, in the un-targeting group, closed receptor group, and targeting group, with significant difference （F=463.7, P〈0.05）. And significant differences were also seen in the un-targeting group （q=40.21, P〈0.05） and the closed receptor group （q=30.91,P〈0.05） when compared with the targeting group. After laser irradiation （1 W/cm2, 5 min）, phase transition of PFH was excited, which resulting in contrast enhanced ultrasound imaging and regional temperature exceeding 42 ℃, and thus leading to photothermal ability. The apoptotic rate was significantly higher in the targeting group than the un-targeting group [（79.49±2.22）% vs （24.23±1.95）%, P〈0.05]. Conclusion PFH and ZnPc loaded targeting nanoparticles are successfully prepared, which show strongly specific affinity to VEGFR-2. The novel nanoparticles show a great potential to be used for ultrasound imaging and photothermal therapy.