Experimental Study On Pa/us Dual-modality Imaging And Photothermal Therapy Of ZnPc Loaded Angiogenesis-targeting Phase Transition Nanoparticles In Vitro

PART ? PREPARATION AND CHARACTERIZATIONS OF ZNPC/PFH LOADED ANGIOGENESIS-TARGETING NANOPARTICLESObjective To prepare a new type of zinc phthalocyanine(ZnPc)and perfluorohexane(PFH)loaded angiogenesis-targeting nanoparticles and investigate their general properties,encapsulation effiency of ZnPc(E.E.%),photothermal effect and optical droplet vaporization(ODV)in vitro.2Methods ZnPc/PFH loaded angiogenesis-targeting nanoparticles were prepared by dual-emulsion technology and conjugated with VEGFR-2 antibodies via carbodiimide technique.Their basic characterizations were detected by nano-size and zeta potential analyzer,TEM and UV-VIS spectrophotometer.Standard curve of ZnPc was drawn and encapsulation rate was calculated.Conjugation between nanoparticles and VEGFR-2 antibodies was observed by laser scanning confocal microscope(LSCM)and conjugation rate was detected by flow cytometry(FCM).Photothermal effect and ODV was observed by thermal imager and optical microscope after laser irradiation,respectively.Results The V-PLGA@ZnPc@PFH were spherical,with an average particle size of 256.4±57.1 nm and Zeta potential of-25.4±5.00 m V,respectively.A strong light absorption of V-PLGA@ZnPc@PFH is in the area of 600~680 nm.Encapsulation efficiency(E.E.%)of ZnPc were 83.5±3.7%.Conjugation between nanoparticles and VEGFR-2 antibodies was demonstrated by LSCM and conjugation rate was 82.73±2.02%.The temperature of nanoparticles increased with irradiation time and ODV was observed after laser irradiation.Conclusions ZnPc/PFH loaded angiogenesis-targeting nanoparticles were prepared successfully.It has uniform size,high connection rate with antibodies,excellent photothermal effect and ODV properties,which provide a experimental basis for further research of PA/US imaging,targeting and photothermal therapy(PTT).PART ? TARGETING,PA/US DUAL-MODALITY IMAGING AND PHOTOTHERMAL THERAPY CAPABILITIES OF ZNPC/PFH LOADED ANGIOGENESIS-TARGETING NANOPARTICLES IN VITROObjective To detect PA/US dual-modality imaging and evaluated cytotoxicity,targeting and PTT capabilities of ZnPc/PFH loaded angiogenesis-targeting nanoparticles in vitro.Methods The capabilities of PLGA@ZnPc@PFH for PA/US imaging was performed by PA imaging system and color doppler ultrasound diagnostic system after laser irradiation.PLGA@PFH was used as control.Cytotoxicity of ZnPc/PFH loaded angiogenesis-targeting nanoparticles was detected by CCK-8 method.Nanoparticles marked with Di I were prepared.Targeting capability to HUVEC of targeting group was observed by LSCM and conjugation rate was detected by FCM,and un-targeting group and closed-receptor group were acted as controls.The nanoparticles connected with HUVECs were irradiated by laser after incubated for 2 h,then cell apoptosis was detected by FCM after incubated for another 24 h.Pure laser irradiation was used as control.Results The capabilityies of PLGA@ZnPc@PFH for PA/US dual-modality imaging were observed and the signal intensity of was positively correlated with the concentration of nanoparticles.However,no signal of PLGA@PFH appeared both in PA and US imaging.The cell viability was 0.975±0.031%,0.95±0.078%,0.948±0.073%,0.946±0.073%,0.942±0.084% after incubated with various concentrations(0.2,0.4,0.6,0.8,1.0 mg/m L)of V-PLGA@ZnPc@PFH,indicating that V-PLGA@ZnPc@PFH have no obvious cytotoxicity to HUVECs(F=0.402,P>0.05).From the imaging of LSCM,A large amount of nanoparticles surrounded the cells in targeting group.However,a few nanoparticles were observed in un-targeting group and closed-recepor group.The result of FCM was consist with that of LSCM,connection between nanoparticles and HUVECs in targeting group was significant high than that of un-targeting group(q=40.21,P<0.05)and closed-receptor group(q=30.91,P<0.05),indicating the good targeting ability of V-PLGA@ZnPc@PFH.In the control group and the pure laser group,the proportion of cell apoptosis(<10%)and the differences between the two groups were negligible,indicating that pure laser irradiation could not cause cell apoptosis without the nanoparticles.And the cell apoptosis in targeting group was graeter that in un-targeting group(q=53.30,P<0.05),demonstrated that better photothermal therapy efficiency could be aquired in targeting group.Conclusions The angiogenesis-targeting nanoparticles loaded with ZnPc and PFH could target to HUVECs specifically and enhance PA/US dual-modality imaging significantly and improve PPT efficiency greatly,which provides a foundation for deeper research work.