载吲哚菁绿与阿霉素靶向纳米粒用于视网膜母细胞瘤体外成像的实验研究

Application of nanoparticle loaded with indocyanine green and adriamycin in in vitro imaging of retinoblastoma: An experimental study

目的:制备一种靶向叶酸受体的载吲哚菁绿(indocyanine green,ICG)及阿霉素(adriamycin,ADM)的相变型多功能脂质纳米粒(ICG/FA/Pct-PFPNP:ADM)并研究其基本性质,检测其靶向能力、光热效应、超声及光声双模态显像能力,为视网膜母细胞瘤的诊断提供新策略。方法:采用双乳化法制备ICG/FA/Pct-PFPNP:ADM纳米粒,透射电镜观察纳米粒形态,马尔文粒径分析仪检测粒径及电位,紫外分光光度计检测纳米粒的包封率、载药量;流式细胞术量化及激光共聚焦显微镜下观察纳米粒表面叶酸表达情况和体外寻靶能力;CCK-8法检测ICG/FA/Pct-PFPNP纳米粒的生物安全性;近红外激光(808 nm)激发ICG/FA/PctPFPNP:ADM纳米粒,检测其超声/光声双模态显像能力。结果:成功制备了ICG/FA/Pct-PFPNP:ADM纳米粒,其外观呈球形,平均粒径为(374.40±23.48)nm,平均电位(-28.0±4.5)mV。测得ICG包封率为(91.85±2.98)%,载药量为(9.19±0.29)%;ADM包封率为(62.04±5.10)%,载药量为(6.20±0.51)%。流式细胞术和激光共聚焦显微镜结果均显示纳米粒表面叶酸表达情况良好,靶向组与Y79细胞连接率为(96.57±3.17)%,与非靶向组、抗体封闭组相比差异具有统计学意义(F=1537.6,P<0.05)。CCK-8结果显示ICG/FA/Pct-PFPNP纳米粒没有明显的细胞毒性。ICG/FA/Pct-PFPNP:ADM纳米粒经激光辐照后能发生液气相变,增强超声显影并能使局部温度上升至60.8℃。光声信号随纳米粒浓度升高而增强,具有良好的线性关系。结论:成功制备了载吲哚菁绿与阿霉素的靶向相变型纳米粒,其靶向能力好,光热效应佳,增强超声及光声显像效果好,可以用于视网膜母细胞瘤的诊断。

Objective:To prepare the phase-change multifunctional lipid nanoparticle containing indocyanine green(ICG)and adriamycin(ADM)and targeting folate receptor(ICG/FA/Pct-PFPNP:ADM),to investigate its basic properties,targeting ability,photothermal effect,and ultrasonic/photoacoustic bimodal imaging ability,and to provide new strategies for the diagnosis of retinoblastoma.Methods:ICG/FA/Pct-PFPNP:ADM nanoparticles were prepared by the double emulsification method.A transmission electron microscope was used to observe the morphology of nanoparticles,the Malvern particle size analyzer was used to measure particle size and electric potential,and an ultraviolet spectrophotometer was used to measure the encapsulation efficiency and drug loading rate of nanoparticles.Flow cytometry and confocal laser scanning were used to observe the expression of folic acid on the surface of ICG/FA/Pct-PFPNP:ADM nanoparticles and targeting ability in vitro.CCK-8 assay was used to evaluate the biosafety of nanoparticles.Nearinfrared laser(808 nm)was used to excite ICG/FA/Pct-PFPNP:ADM nanoparticles and assess their ultrasonic/photoacoustic bimodal imaging ability.Results:ICG/FA/Pct-PFPNP:ADM nanoparticles were successfully prepared,with a spherical appearance,an average particle size of(374.40±23.48)nm,and an average potential of(-28.0±4.5)mV.The encapsulation efficiency and drug loading rate of ICG were(91.85±2.98)%and(9.19±0.29)%,respectively,and the encapsulation efficiency and drug loading rate of ADM were(62.04±5.10)%and(6.20±0.51)%,respectively.Both flow cytometry and laser confocal microscopy showed good expression of folic acid on the surface of the nanoparticles;the targeted group had a Y79 cell connection rate of(96.57±3.17)%,which was significantly different from that in the non-targeted group and the antibody blocking group(F=1537.6,P<0.05).The CCK-8 assay showed no obvious cytotoxicity of ICG/FA/Pct-PFPNP:ADM nanoparticles.The ICG/FA/Pct-PFPNP:ADM nanoparticles showed liquid-gas phase transition after laser irradiation,enhanced ultrasound imaging,and increased local temperature(60.8℃).The intensity of photoacoustic signal increased with the increase in nanoparticle concentration,and there was a good linear relationship between nanoparticle concentration and signal intensity.Conclusion:The phase-change nanoparticles loaded with ICG and ADM were successfully prepared,with good targeting ability and photothermal effect.Such nanoparticles can enhance the quality of ultrasonic/photoacoustic imaging.Therefore,it can be used in the diagnosis of retinoblastoma.