Influence And Mechanism Of Cavitation On Phase-shift Perfluorohexane Nanoemulsion-enchanced Focused Ultrasound Ablation

High-intensity focused ultrasound(HIFU)has been successfully used in clinical practice for the noninvasive treatment of a variety of solid tumor,such as uterine fibroids,liver cancer,breast cancer,pancreatic cancer and kidney cancer.With development of HIFU technology and clinical applications,previous studies have revealed that due to the ribs,rich blood supply and deep location of treated tumors,HIFU energy at the focus is not sufficient for ablation,leading to unsatisfactory treatment outcomes.On the other hand,high power and long-period HIFU exposures increase energy deposition in normal tissues on focused ultrasound(US)pathway,resulting in serious complications of HIFU treatment.Therefore,how to increase local deposition of ablative energy and reduce the incidence of complication has become one of the most difficult problems that need to be emergently solved in HIFU clinical applications.HIFU-induced inertial caviation generates broadband noise emission that can increase thermal effect while absorbed in the treated tissues.Increased local temperature can also boost local cavitation effect conversely.Therefore,both heat and caviation can interact with each other,enhancing HIFU ablation of tumor.Recently,some researchers have found that through cavitation and thermal effects,phase-shift perfluorocarbon nanoemulsions can significantly increase heat deposition and ablation size of HIFU exposure,and subsequently reduce the incidence of complications.The present study intends to explore the ablative effects of pulsed-wave high intensity focused ultrasound(p HIFU)combined with phase-shift perfluorohexane(PFH)nanoemulsions,and compare the ablative efficiency between p HIFU and continuous-wave high intensity focused ultrasound(c HIFU).The goal of the study is to investigate whether cavitation could significantly influence the PFH-enhanced HIFU ablation and its potential mechanism.It will provide important data and experimental evidence for clinical use of a novel HIFU strategy.Part I Influence of Cavitation on Perfluorohexane Nanoemusion-Enhanced HIFU Ablation: An in Vitro Study of Tissue-Mimicking PhantomObjectives Using a tissue-mimicking phantom,the study is to investigate the effects of cavitation on perfluorohexane nanoemusion-enhanced HIFUAblation in vitroMaterials and Methods Perfluorohexane(PFH)was encapsulated by mesoporous silica nanocapsule(MSNC)to form a nanometer-sized droplet(MSNC-PFH).It was added into a tissue-mimicking phantom;whereas phosphate buffered saline(PBS)was added as a control(PBS-control).HIFU exposures(Pac=150W,t=5s/10s)were performed in both MSNC-PFH and PBS-control phantoms.Duty cycle(DC)was 2%,5%,10% and 100% respectively.Real-time US images were obtained before and after HIFU.HIFU-induced lesions in both phantoms were measured and calculated after HIFU exposures.Results Compared to the PBS-control,MSNC-PFH nanoemulsion could significantly increase the volume of HIFU-induced lesion(P < 0.01),with an earlier occurrence of a hyperechoic region on US imaging in the MSNC-PFH phantom.p HIFU could significantly change the lesion volume in the MSNC-PFH phantom while compared with c HIFU.The mean volume of lesion induced by 5s exposure was significantly larger at a DC of 10%(P < 0.005),but smaller at a DC of 2%(P < 0.005).Conclusions MSNC-PFH nanoemulsion can significantly enhance HIFU ablation in the phantom.Appropriate p HIFU exposure can significantly increase the lesion volume and reduce total US energy required for HIFU ablation.Compared to c HIFU exposure,the mean volume of lesion inducedby 5s exposure at a DC of 10% is significantly increased.Part Ⅱ Effects of Blood Flow and Perfusion on Perfluorohexane Nanoemusion-Enhanced HIFU Ablation and Its Potential Mechanism: A Study of Isolated Perfused Porcine LiverObjectives Using an isolated perfused porcine liver,the study is to investigate the effects of blood flow and perfusion on HIFU ablation,and explore whether perfluorohexane nanoemusion could enhance HIFU ablation as well as its potential mechanism.Materials and Methods The fresh porcine liver was used to establish an isolated perfused liver model in vitro.The perfusion liquid flow rates of the portal vein were 0(no flow),100,200,400 and 800ml/min respectively.This study was divided into three parts,and HIFU parameters used for each part were as follows:(1)acoustic power was 100 W and 200 W,duty cycle was 100%,and exposure time was 1,2,3,4 and 6s;(2)acoustic power and duty cycle were 200 W 100% and 400 W 50% respectively,exposure time was 1,2,3,4 and 6s;(3)MSNC-PFH was evenly added in the perfusion liquid,acoustic power was 150 W,duty cycle was 10%,20%,50% and 100%,and exposure time was 3,5 and 10 s.Real-time US imaging was used to observe and record B-mode USchanges before and after HIFU procedure.Passive cavitation detection(PCD)was used to record broadband and subharmonic noise during HIFU exposures.The band-pass filtered root mean squared(RMS)value of the mitral pressure signal was used as a measure of cavitation intensity.After HIFU exposure the liver was cut into slices,and each lesion was measured and calculated.The marginal tissues between the treated and untreated regions were collected for histological study.Results(1)Perfusion liquid flow rate could significantly influence the size of HIFU-induced lesion in the isolated perfused porcine liver.Using 100 W exposure an increase of the perfusion flow rates could significantly reduce the lesion volume at exposure of 4s and 6s(P < 0.001),though no effect was observed at exposures of 1,2 and 3s in the perfused liver.When acoustic power increased up to 200 W,a negative relationship was significantly found between the perfusion flow rate and lesion volume in all exposures(P < 0.001).In addition,PCD revealed both broadband and subharmonic noise at the focus during HIFU exposures.The inertial cavitaion signal was weak at exposures of 100 W,and there was no effects of the perfusion flow on the RMS values.However,when acoustic power increased up to 200 W,the effect of the perfusion flow on the RMS values was significantly observed.With an increase of the perfusion flow rate,theRMS values were significantly decreased(P < 0.01).(2)Compared to c HIFU exposure at 200 W,an increase of the perfusion flow rates could significantly reduce the lesion volume at p HIFU(400W,50% DC)exposure of 4s and 6s(P < 0.05),though no effect was observed at exposures of 1,2 and 3s in the perfused liver.Broadband and subharmonic noise was detected during p HIFU.It presented that RMS rose up to the peak value at the beginning of p HIFU exposure.However,there was no effect of the perfusion flow on the RMS values.(3)Ablative effects of p HIFU were directly dependent on the DC.Compared to c HIFU exposure,the mean volume of lesion at a DC of 10% and 20% was significantly smaller(P<0.05),except for lesion volume in 20%,10 s exposure(P>0.05).The mean volume of lesion induced by 10 s exposure at a DC of 50% was significantly larger than that at a DC of 100%(P<0.05).No significant difference of the lesion volume in 3s and 5s exposure was observed between the exposures at the DC of 100% and 50%(P>0.05).Conclusion Blood flow and perfusion can significantly influence the volume of HIFU-induced lesion in the isolated perfused porcine liver.With an increase of perfusion flow rates,the decrease of lesion volume was observed in the perfused liver.MSNC-PFH nanoemulsion can significantlyenhance c HIFU ablation in the perfused liver.In the p HIFU exposure this enhanced effect depends on the duty cycle.Compared to c HIFU exposure,lesion volume was reduced in the p HIFU with low DC.However,with an increase of DC and exposure time,the lesion volume became larger gradually,and a significant increase of lesion volume was observed at 50% DC and 10 s exposure.Appropriate pulsed-HIFU exposure could significantly increase the volume of lesion and reduce total US energy required for HIFU ablation.Part Ⅲ Influence of Cavitation on Perfluorohexane Nanoemusion Enhanced HIFU Ablation of Tumor: An in Vivo Study of Animal Tumor ModelObjectives Using subcutaneous implanted VX2 tumor model in rabbit,this study is to investigate whether MSNC-PFH nanoemulsion could enhance HIFU ablation for tumor,and cavitation would play an important role in the enhancement.Materials and Methods A subcutaneous implanted VX2 tumor model was established in rabbits.Fourteen days after tumor implantation,15 rabbits were randomly into control group and two experimental groups.In the experimental groups the rabbits received MSNC-PFH(4 mg/m L)injectionvia the ear vein,but the rabbits in the control received intravenous injection of the same amount of PBS solution.Twenty seconds after the injection HIFU exposure was performed in all rabbits.Therapeutic parameters were 150 W in acoustic power and 5s in exposure time.But DC was various,with 100% for the control and one experimental group and 2% for the remaining one.Real-time US imaging was obtained before and after HIFU treatment.One and three days after HIFU ablation,the rabbits in both control and experimental groups were sacrificed.The treated tumors were dissected,and HIFU-induced lesion was measured and calculated.The marginal tissue between the treated and untreated areas were collected,fixed and stained for electronic and light microscopy.The apoptosis and PCNA expression of the tumor cells in the untreated area were examined using TUNEL method and immunohistochemistry respectively.(1)Pathological changes: Under light microscope a typical coagulation necrosis was observed in both groups,with a clear margin between the treated and untreated tumor.Electronic microscopy showed serious damage on cell membrane with continuity disappearance,vacuoles in the cytoplasm,continuity loss of nuclear membrane with rupture and shrinkage of chromatin,and apoptotic bodies.There were no obvious differences of pathological changes between the control and experimental groups.(2)A hyperechoic change was observed on B-mode US imaging in the treatedtumor.Compared to the control,the hyperechoic area and lesion volume were significantly bigger(P < 0.01)in both experimental group,but there was no significant difference between two experimental groups.(3)Compared to the control group,apoptosis index of the untreated tumor cells was significantly increased,with a decreased expression of PCNA in both experimental groups.There were statistical differences between the control group and experimental groups(P < 0.01).However,no difference was observed between two experimental groups.Conclusion: MSNC-PFH nanoemulsion can significantly enhance HIFU ablation of tumor.It can also induce apoptosis and inhibit proliferative capacity in the untreated tumor cells.Part Ⅳ Mechanism of Cavitation in Perfluorohexane Nanoemusion-Enhanced HIFU AblationObjectives To investigate the mechanism of perfluorohexane nanoemulsion-enhanced HIFU ablation and the potential role of cavitation involvedMaterials and Methods Materials are same as Part one.HIFU exposures were performed in MSNC-PFH phantom.Acoustic power was 150 W,exposure time was 5s,and DC was 100%,10%,5% and 2% respectively.Temperature rise and inertial cavitation emissions were detected and recorded at the focus during HIFU exposure.Results Peak temperatures were 78.16±5.64℃ at a DC of 100%,70.17±6.43℃ at 10%,53.17±4.54℃ at 5%,and 42.00±5.55℃ at 2% respectively.Inertial cavitation was much stronger in the pulsed-HIFU than that in the continuous-wave HIFU exposure,with the strongest one at a DC of 10%.Conclusions Cavitation can play a significant role in MSNC-PFH enhanced HIFU ablation,which may be concerned with the increasing of evaporation of phase-shift perfluorohexane nanoemulsion.