Experimental Study Of Retinoblastoma Imaging By Nanoparticles Loading With ICG And ADM In Vitro

Objective:1.To prepare a kind of phase-change multifunctional lipid nanoparticles(ICG/FA/Pct-PFPNP: ADM)containing indocyanine green(ICG)and doxorubicin(ADM)targeting folate receptors,and to study the basic properties of the nanoparticles,and to detect its photothermal effect,photoacoustic and ultrasound dual-mode imaging capabilities.2.To explore the bio-safety of ICG/FA/Pct-PFPNP: ADM nanoparticles and their effectiveness in target-seeking of retinoblastoma cells in vitro,so as to provide a preliminary research for further realization of targeting dual-mode imaging of retinoblastoma in vivo.Methods:1.ICG/FA/Pct-PFPNP: ADM nanoparticles were prepared by double emulsification method,folic acid modified lipid was used as loaded with ICG,PFP and ADM.The particle size and surface potential of nanoparticles were analyzed by Malvern particle size analyzer.The encapsulation efficiency and drug loading of ICG and ADM were measured by ultraviolet spectrophotometer.The expression of folic acid on the surface of ICG/FA/Pct-PFPNP: ADM nanoparticles was observed by confocal laser microscopy and its expression rate was detected by flow cytometry.2.The photothermal effect of ICG/FA/Pct-PFPNP: ADM nanoparticles was detected by infrared imager and the photoinduced phase transition process was observed by optical microscope under 808 nm laser irradiation.3.The biological safety of ICG/FA/Pct-PFPNP nanoparticles was detected by CCK-8 method.The targeting ability in vitro of ICG/FA/Pct-PFPNP: ADM group(targeting group),ICG/Pct-PFPNP: ADM group(non-targeting group)and antibody blocking group to retinoblastoma Y79 cells was detected by confocal laser microscopy and flow cytometry.4.The photoacoustic effect and signal value of ICG/FA/Pct-PFPNP: ADM nanoparticles with different concentration wer detected by photoacoustic imager.The relationship between intensity and concentration of photoacoustic signal was analyzed.Under 808 nm laser irradiation,the enhancement effect of ICG/FA/Pct-PFPNP: ADM nanoparticles with different concentration on ultrasound development was observed in vitro,and the changes of ultrasonic intensity were analyzed by DFY software before and after laser induced phase transformation.Results:1.In this study,ICG/FA/Pct-PFPNP:ADM nanoparticles were successfully prepared.The average particle size was 374.4±23.48 nm and the average surface potential was-28±4.5 mV.The appearance of ICG/FA/Pct-PFPNP:ADM was spherical and uniformly distributed under optical and electron microscopes.The encapsulation efficiency of ICG was(91.85 ±2.98)%,drug loading was(9.19 ±0.29)%.The encapsulation efficiency of ADM was(62.04±5.1)% and drug loading was(6.2±0.51)%.The results showed that the expression of folic acid on the surface of nanoparticles was good,and the expression rate was(98.83±0.17)% by flow cytometry and confocal laser microscopy.2.ICG/FA/Pct-PFPNP:ADM nanoparticles have good photothermal conversion ability,and the photothermal efficiency was positively correlated with the concentration of nanoparticles and laser power.After laser irradiation,nanoparticles can rapidly transform into microbubbles.3.CCK8 method indicated that ICG/FA/Pct-PFPNP nanoparticles have a good bio-safety.Confocal microscopy showed that Y79 cells could bind to a large number of ICG/FA/Pct-PFPNP: ADM nanoparticles under the action of receptor-ligand due to the high expression of folate receptor on the surface of Y79 cells,while there were fewer nanoparticles around Y79 cells in non-target group and antibody blocking group.Flow cytometry showed that the connection rate between Y79 cells and target group was(96.57 +3.17)%.There was significant difference by comparing non-target group and antibody blocking group(F = 1537.6,P < 0.05).4.ICG/FA/Pct-PFPNP: ADM nanoparticles had a significant photoacoustic imaging ability,and the photoacoustic signal increased with the increase of nanoparticle concentration.Ultrasound signals of ICG/FA/Pct-PFPNP: ADM nanoparticles solution were enhanced under B-mode and Contentrast-enhanced ultrasound(CEUS)modes in ultrasound imaging system after 808 nm near infrared laser irradiation.Conclusions:1.Targeted phase change nanoparticles(ICG/FA/Pct-PFPNP: ADM)containing indocyanine green and doxorubicin were successfully prepared by double emulsification method.The nanoparticles have regular morphology,uniform particle size distribution,high drug loading,good targeting performance and high bio-safety.The phase transition and photothermal effect can be induced by 808 nm laser irradiation.2.The prepared ICG/FA/Pct-PFPNP: ADM nanoparticles have good photoacoustic / ultrasound dual-modal imaging ability.Combining with its good targeting ability,it provides a new possibility for the realization of dual-modal imaging of retinoblastoma.