Experimental Study On Enhanced HIFU Ablation Of Loaded Sonosensitizer Liquid Fluorocarbon Nanoparticles With Pre-targeting

High intensity focused ultrasound is to accumulate ultrasound energy at the focal region,produces transient high temperature and high pressure locally,which causes the tissue to produce irreversible coagulation necrosis.In order to increase the efficiency and reduce the complications of HIFU ablation,scholars try to use different synergist,such as iodine oil,hydroxyapatite,microbubbles,liquid fluorocarbon nanoparticles and mesoporous silica and sonosensitizer etc.Liquid fluorocarbon nanoparticles is considered as a very potential synergist,because the liquid fluorocarbon is liquid under normal temperature,temperature rise or ultrasonic irradiation causes liquid fluorocarbon to change from liquid to gas,so nanoparticles become microbubbles,thereby increasing the cavitation effect of HIFU treatment.At the same time,some scholars combined sonosensitizer with polymer materials to prepare the nanoparticle synergist loaded sonosensitizer,which organically combines the HIFU technology with the sonodynamic chemistry therapy(SDT)to increase the therapeutic effect of HIFU.If further more,liquid fluorocarbon nanoparticles and sonosensitizer are combined to prepare a liquid fluorocarbon nanoparticles loading sonosensitizer,under the irradiation of HIFU,occurs liquid-gas phase transition and nanoparticles become microbubbles,which not only change the tissue acoustic environment,but also increase the cavitation effect and activate the acoustic sensitizer,so as to achieve synergistic reaction between HIFU synergism and SDT.In order to make more precise treatment with synergist,it is necessary to modify the synergist so as to achieve targeted properties.Biotin-avidin targeting technology is derived from radioimmunoimaging and treatment of tumor.In the two step pretargeting technology,the first step is to inject the target molecule into the body,when it reaches its peak at the tumor site,then inject the effector molecule in the second step,which has the advantages of high sensitivity,high specificity and wide adaptability;At the same time,antibodies and effectors are only modified with biotin and avidin,that is relatively simple.This technology has been applied in other fields of imaging,such as MRI molecular imaging and ultrasound molecular targeting research.Therefore,the purpose of this project is to prepare a liquid fluorocarbon nanoparticles loading sonosensitizer,then modify to labeled streptavidin,and finally using pre-targeting technology targeting tumor and study the effect of HIFU ablation in vitro and in vivo,so as to provide a new idea for the preparation of synergist.The main contents are as follows:Objective1.Preparation of poly(lactic-co-glycolic acid)nanoparticles loaded hematoporphyrin monomethyl ether and liquid fluorocarbon(PFP),and detect its general properties and enhanced HIFU ablation effec in vitro.2.Preparation of streptavidin liquid fluorocarbon polymer nanoparticles loaded hematoporphyrin monomethyl ether(HMME+PFP/PLGA-SA),detect its general properties and in vitro targeting capability.3.To observe the effect of biotinylated VEGFR2 antibody mediated liquid fluorocarbon nanoparticles loaded hematoporphyrin monomethyl ether on the ablation of subcutaneous breast cancer xenografts in nude mice,and to explore the mechanism of HIFU synergism.Methods1.HMME+PFP/PLGA was prepared by single milk method.The morphological characteristics and distribution were observed by inverted microscope.The size of particle size and Zeta potential were measured by Malvern laser particle size measuring instrument,and the encapsulation rate was measured;Ligt microscope and ultrasonic imaging observation on temperature rise and ultrasonic irradiation on its phase transition;and the effect of enhance HIFU ablation on bovine liver was observed,fresh bovine liver was divided into:(?)double distilled water,(?)PLGA,(?)PFP/PLGA,(?)HMME/PLGA and(?)HMME+PFP/PLGA).The 200?l double steamed water and the different kinds of nanoparticles were injected into the bovine liver.At the injection point,HIFU ablation was performed with different power(90 W,120 W,150 W)and different irradiation time(2 s,5 s,10 s),and the target region gray scale change,the volume of coagulation necrosis and the energy efficiency factor were measured to evaluate the synergistic effect of the HIFU ablation.2.On the basis of the preparation of HMME+PFP/PLGA nanoparticles,HMME+PFP/PLGA-SA was prepared by carbodiimide method.The morphological characteristics and distribution were observed by microscope.The size of particle size and Zeta potential were measured by Malvern laser particle size measuring instrument,and the encapsulation rate was measured.The connection between the streptavidin and the nanoparticles was detected by laser scanning confocal microscopy and flow cytometry,and the targeting capability of nanoparticles to cells in the flow state was evaluated by parallel plate flow cavity method.3.?70 tumor bearing nude mice were divided into the following 7 groups randomly:HIFU group,PLGA + HIFU group,PFP/PLGA + HIFU group,HMME/PLGA + HIFU group,HMME+PFP/PLGA + HIFU group,HMME+PFP/PLGA-Ab + HIFU group and HMME+ PFP/PLGA-SA group,with 10 mice in each group.HIFU group was given only HIFU irradiation;the pre-targeting group(HMME+PFP/PLGA-SA + HIFU),the fist step was injection of biotinylated VEGFR2 antibody through the tail vein,after 24h 200 ?l HMME+PFP/PLGA-SA was injected in the second step;The remaining treatment groups were injected 200?l PLGA,HMME/PLGA,PFP/PLGA,HMME+PFP/PLGA and HMME+PFP/PLGA-Ab via tail vein,respectively;and all groups were treated by HIFU with the same parameters(150W,5s).After ablation the change of target gray scale,coagulative necrosis volume and energy efficiency factor were observed.HE staining was used to observe the change of organization structure,immunohistochemistry was used to detect the expression of CD34 and PCNA in peripheral tumor tissues of coagulative necrosis.TUNEL kit was used to detect the apoptosis of peripheral tumor cells of coagulative necrosis.?12 tumor bearing nude mice were randomly divided into 2 groups,HIFU group and pre-targeting group(HMME+PFP/PLGA-SA),6 mice in each group.With the same treatment parameters and methods,the weight,survival time and tumor volume of nude mice after treatment were observed.Results1.HMME+PFP/PLGA was a uniform coffee emulsion,dispersed evenly.Light microscopy and scanning electron microscopy showed that the morphology was spherical,smooth,well dispersed and uniform in size.Laser scanning confocal microscopy showed that HMME+ PFP/PLGA emitted red fluorescence.The average diameter was 381.5 �75.3 nm,the Zeta potential was-(14.1 � 0.4)mV.The average entrapment rate of porphyrin monomethyl ether and liquid fluorocarbon(PFP)were 52.52 �3.54%and 44.07 � 1.24%,respectively.When the temperature rises above 45? or HIFU irradiation,it can promote HMME+PFP/PLGA sundergo a phase transition to become microbubbles,and enhance ultrasound imaging.In the HIFU ablation experiment in vitro,compared with the other groups,the HMME+PFP/PLGA group was the largest in the gray scale change and coagulative necrosis volume,and the energy efficiency factor was the smallest(P<0.05).2.HMME+PFP/PLGA-SA was a uniform coffee emulsion,dispersed evenly.Light microscopy and scanning electron microscopy showed that the morphology was spherical,smooth,well dispersed and uniform in size.The average diameter was 477.8�81.8 nm,the Zeta potential was-(13.9�0.5)mV.The average entrapment rate of porphyrin monomethyl ether and liquid fluorocarbon(PFP)were 46.64�3.34%and 40.08�2.61%,respectively.Compared with HMME+PFP/PLGA,its general characteristics have not changed significantly.With laser scanning confocal microscopy,the HMME+PFP/PLGA nanoparticles were red,FITC labeled streptavidin was green,yellow fluorescent can be seen on the nanoparticles surface in the fusion picture.The binding efficiency of nanoparticles to streptavidin was 95.48�3.51%with flow cytometry.By parallel plate flow cavity method,the pre-targeting group(HMME+PFP/PLGA-SA)can be seen that a large number of nanoparticles bind to the peripheral and surface of the cell.The direct target group(HMME+PFP/PLGA-Ab)can be seen only a small amount of nanoparticles binding to the cell surface;the HMME+PFP/PLGA group,the HMME/PLGA group,the PFP/PLGA group,and the antibody blockage group have almost no nanoparticles binding to the cell surface.3.The change of the gray scale in the ablation zone and the volume of coagulation necrosis were the largest and the energy efficiency factor was the smallest in the pre-targeting group(HMME+PFP/PLGA-SA),and the difference was statistically significant compared with the other groups(P<0.05).The MVD and PCNA decreased and AI increased in pre-targeting group(HMME+PFP/PLGA-SA),and the difference was statistically significant compared with the other groups(P<0.05).The survival time of the pre-targeting group(HMME+PFP/PLGA-SA + HIFU)in nude mice was significantly longer than that in HIFU group,the difference was statistically significant(P<0.05).Conclusion1.HMME+PFP/PLGA nanoparticles were uniform size and good dispersity,high encapsulation rate,the temperature rise or HIFU irradiation can transformation,in vitro the bovine liver experiments showed that HMME+PFP/PLGA can effectively enhance HIFU ablation.2.The HMME+PFP/PLGA-S A nanoparticles prepared by carbodiimide method have good general properties,and the binding rate of streptavidin and nanoparticles is high.A large number of nanoparticles are visible on the peripheral and surface of the cell,and have high targeting capability.3.Biotinylated VEGFR2 mediated HMME+PFP/PLGA-SA can enhance HIFU treatment effectively,while MVD and PCNA decreased and AI increased in the tumor tissues around the ablation.