Mechanism And Safety Of Chemotherapy Enhanced By Ultrasound Mediated Tumor Perfusion Effect

BackgroundRecently,the therapeutic applications and researches of ultrasound-targeted microbubble destruction（UTMD）have been investigated.Previous studies found that UTMD combined with chemotherapy significantly increased the concentration of drugs in tumor,inhibited tumor growth and prolonged survival.In addition,clinical studies reported that Diagnostic ultrasound combined with microbubble enhanced chemotherapy of pancreatic cancer and prolonged survival time.The mechanism of ultrasound and microbubble enhance chemotherapy is not fully understood,and there are two possible factors:one contains inflammation induced by destruction of tumor microvascular related to cavitation.The other involves sonoporation caused by cavitaion.It causes mechanical perforation damage to vulnerable neovascular of tumor,and produce reversible acoustic holes in the tumor tissue and vessel walls,resulting in increase of chemotherapy drugs concentration.However,vasodilation,perfusion enhancement and permeability improvement may increase the risk of tumor metastasis and thus hinder its clinical application and promotion.Currently,there are few studies focusing on the effect of ultrasound combined with microbubbles on metastasis,especially the effect of DUS combined with microbubbles on metastasis.Based on previous experiments,mechanism and metastatic risk of chemotherapy enhanced by ultrasound mediated tumor perfusion effect were further investigated using the modified VINNO 70 integrated ultrasound theranostic system.Objective1.To quantitative assess the effect of ultrasound combined with microbubbles on tumor perfusion and drug concentration using a modified ultrasound theranostic system.2.To explore the mechanism of ultrasound combined with microbubble in enhancing chemotherapy preliminarily.3.To evaluate the efficacy and feasibility of ultrasound and microbubble for chemotherapy enhancement by observing tumor growth inhibition,metastasis and survival.Materials and methods1.Experiment equipment1.1 VINNO 70 integrated ultrasound theranostic system（VINNO technology Co.,Ltd）with X4-12L linear array probe.VINNO 70 has a Vflash mode in which therapeutic and diagnostic pulses were emitted intermittently.The modified ultrasound is capable of modulating cavitation and Mechanical index（MI）by multiple parameters.And the therapeutic beams were weakly electronic focused to a region-of-interest（ROI）.1.2 ECARE ultrasound machine（ECARE Electronics Science&Technology Co.,Ltd）.Therapeutic pulses were completely mixed within diagnostic sequences.1.3 Vevo 2100 ultrasound imaging system（FUJIFILM Visual Sonics,Inc.）was applied with a 21 MHz linear array probe.2.Experiment reagents2.1 Contrast agent:Zhifuxian,Sono Vue^?2.2 Doxorubicin3.Experiment animalsNew Zealand rabbits4.Experiment methodsExperiment 1:Mechanism and Drug Deliver of Chemotherapy Enhanced by Ultrasound mediated Tumor Perfusion Effect.（1）Measurements of microbubble destruction and Peak negative pressure（PNP）of VINNO 70 using hydrophone.（2）Thirty-three rabbits bearing VX2 tumor were randomly divided into 3 groups,each of11 rabbits:Control,VINNO ultrasound treatment 1（PRF=1000 Hz,MI=0.29）,VINNO ultrasound treatment 2（PRF=500 Hz,MI=0.34）.All rabbits underwent contrast-enhanced ultrasound before and after treatment.In addition,all groups received chemotherapy for 10min.After chemotherapy,ultrasound treatment group received ultrasound combined with microbubble treatment for 20 min,while control received chemotherapy only.（3）Measurement index:Peak intensity,area under curve,perfusion area before and after treatment;high efficiency liquid chromatography;doxorubicin fluorescence intensity;electron microscopy;Reactive oxygen species（ROS）fluorescence intensity.Experiment 2:Effect of ultrasound enhanced chemotherapy on metastasis and survival.（1）Forty-four rabbits were randomly divided into 3 groups:Control（n=16）,VINNO ultrasound treatment（MI=0.29,n=17）,ECARE ultrasound treatment（MI=0.33,n=11）.All rabbits received 3 times chemotherapy for 10 min.After each chemotherapy,ultrasound treatment group received ultrasound combined with microbubble treatment for 10 min,while control received chemotherapy only.（2）Measurement index:tumor growth rate;survival time;metastasis to liver,lung,brain and kidney;HE staining of metastasis and primary tumor.ResultsExperiment 1:Mechanism and Drug Deliver of Chemotherapy Enhanced by Ultrasound mediated Tumor Perfusion Effect.（1）Measurements of VINNO 70:Microbubbles inside the ROI were destructed,while microbubbles outside the ROI were not.In addition,actual MI was different from the displayed MI at 2cm or 5cm away from the probe,consequently it was necessary to rectify the parameters.（2）Perfusion effect:Peak intensity,area under curve and perfusion rate were significantly increased after treatment in ultrasound treatment groups（P<0.05）.（3）Drug concentration in tumor:According to high efficiency liquid chromatography,drug concentration in treatment 1 was 2.63 times of that in control,and treatment 2 was 2.07times（P<0.05）.The treatment groups observed stronger fluorescence of doxorubicin than control by frozen sections.（4）Mechanism of ultrasound and microbubble in enhancing chemotherapy:Mean fluorescence intensity of ROS in the treatment groups was significantly stronger than that in control（P<0.01）.Electron microscopy showed that the vascular structure of control was intact.The basement membrane of vessels in treatment 1 was destroyed completely,while that of treatment 2 was partially preserved.Experiment 2:Effect of ultrasound enhanced chemotherapy on metastasis and survival.（1）Metastasis:There were no statistic differences in metastasis stage and single organ metastasis among groups（P>0.05）.（2）Tumor growth rate:No difference was observed on the 3^rd day.The tumor growth rate on 5^th day for treatment groups was significantly lower than control（P<0.01）.（3）survival curve:VINNO treatment>ECARE treatment>control,but there was no statistical difference among groups（P>0.05）.Conclusion1.Ultrasound combined with microbubbles enhanced perfusion of rabbit VX2 tumor and significantly increased drug concentration.It may be associated with increased permeability of vascular wall and local inflammation induced by cavitation.2.Ultrasound combined with microbubbles and chemotherapy inhibited tumor growth,but showed no effect on survival and metastasis.