| With the development of molecular biology and molecularimaging,ultrasound molecular imaging has been advanced rapidly.Thekey is the molecular probe.Ultrasound contrast agent(UCA) as a newmolecular imaging ultrasound probe,is the hot spot.UCA have beenused to increase the sensitivity and specificity of the detection ofdisease due to the increase of signal reflection from blood flow,it canenhance the backscattered signal and improve the resolution.The majorprinciple behind all ultrasound contrast agent is an impedancemismatch between the surrounding medium (blood and soft tissue) andthe agent(gas).As blood pool imaging agents,conventional contrast agents containmicrobubbles that can not permeate through blood vessel wall,whichlimits the research on ultrasound molecular imaging.Over recentyears,many nanoscale UCA including liposome,fluorocarbonemulsions have been developed.These nanoscale UCA represent the trend of developing highly efficient,miniaturized with sound targetingperformance in molecular imaging and therapy.It was shown that only particles with a diameter＜700nm canpermeate through the spaces between vascular endothelial cells in suchdiseases as tumor.Nanoscale UCA can permeate through vascularendothelium into tissue spaces,thus allowing for imaging ofextravascular target tissues.Oeffinger et al.demonstrated the feasibilityof using nanoscale microbubbles to enhance the performance ofultrasound contrast.Meanwhile,as the microbubble size is reduced tonanoscale,the molecular properties of UCA change substantially,withemergence of some unique properties,such as long half-lives,highsurface reactivity,strong absorbability,and resistance to enzymaticdegradation.All these properties make possible new applications ofnanoscale UCA in medicine.In the present study,after having successfully developed aconventional micrometer-grade UCA,we further optimized thepreparation process and method to develop the nanoscale lipid-coatedUCA through low-speed centrifugation.We prepared and preliminarilycharacterized a nanobubble.We researched it's effect of the echointensity,and the ablation effect of high intensity focused ultrasound,inbreast VX2tumor model of rabbit.On the other hand,the conventional ultrasonic imaging technology to real-time HIFU effect assessment mainly dependent on thegrey-scale,but this approach has many problems,such as lowsensitivity,subjective judgment.When the grey-scale is not evident,themisjudgment would be maked in HIFU ablation process,and lead toirradiation increased,treatment time increase,and unnecessarycomplications.There is an urgent need to seek a more sensitive,accurateand reliable evaluation method.Speckle refers to the irregular smallobjects formed in ultrasonic scattering.Researchers think speckle isalso a kind of information carrier,the speckle measurement techniquehas been born.Recently study shows that,the ultrasonic specklecorrelation function,the amura markings analysis based on wavelettransform,multi-resolution analysis index can be used to judge whetherthe target area of HIFU irradiation have coagulative necrosis,and betterthan the grey-scale values evaluation,is expected to become the newHIFU real-time effect assessment method.This subject was studied mainly from the following four parts. Objective:The purpose of the study is to prepare a nanobubble and toinvestigate it's characterization.Methods:Lipids(DSPC,DPPC,DPPA)and other constituents were mixed at appropriate ratios and prepared into a suspension.A1-ml suspension was sub-packaged into penicillinampules(actual volume3.5ml) and lyophilized using an optimizedprocess to enhance the tightness of lipid coating.The lipid bubbles wereself made,which was awarded by National Invention Patent of China in2005and widely applied in experimental studies in contrast imaging inliver,heart and kidney in animal study.1ml of the hydration solutioncontaining hyperosmotic sugar and surfactants was added into thelyophilized preparation,and then C3F8was slowly infused into thepenicillin ampule to replace air in the ampule top,followed by90s ofhorizontal mechanical vibration[working frequency≥4500vibrationsper minute,vibrationamplitude:(15±1)mm].The prepared microbubbleswere separated by low-speed centrifugation(300rpm,3min) into twofractions.The microbubbles in the upper fraction were larger than thosein the lower fraction.The upper fraction was discarded,and themicrobubbles in the lower fraction were further analyzed.Thedistribution and appearance of the microbubble were observed under alight microscope and a electron microscope.The diameter and the zetapotential of the nanobubble were measured using a Zeta SIZIER3000electric potential analyzer.HER2antibody had been mixed withmicrobubble.Results:Macroscopic observation,the suspension of themicrobubble was white,milky,viscose,and opaque,with no obviousfoams on the surface.Microscopic observation,the microbubble was found to be round,distribute evenly,and did not aggregate in normalsaline.The diameter of the microbubble was623.4nm on average(278.8nm-697.0nm),and the zeta potential was1.3mV on average(3.2mV-+9.5mV).Microbubble can be binding with antibody by staticadsorption method.Conclusions:Because perfluorocarbon-filled lipidmicrobubbles were sufficiently stable during circulating in thevasculature as blood pool agents,phospholipids were used as thematerials of forming membrane in the study.UCA injected parenterallymust be less than8μm in diameter in order to traverse the capillariesin the pulmonary bed,these UCA remain in the vasculature until theyare eliminated from the body by a variety of mechanisms.Targeting ofcells outside the capillaries requires UCA diameters＜700nm,andpreferably＜700nm to enable escape through the larger-than-usualpores that have been noted in the leaky vasculature of a tumor.UCAhave the potential to be used to directly target cells if the agent can bemade small enough to pass through the vessels and be modified toattach to specific sites in the targeted cells.We have demonstrated thatthe stable nanobubble can be prepared by machine vibration and lowspeed centrifugation.This study provides an important platform forminiaturizing and improving the targeting performance of ultrasoundcontrast agents. Objective:AHNP mix with nanobubble.We Research the targetfunction of the AHNP-Nanobubble compound in vitro and invivo.Methods:Preparation the nanobubble,add sugar solution andsurfactant shaken,add EDC-AHNP compound that marked fluorescentin normal atmospheric temperature for30min.After the oscillation byusing the mechanical vibration tester,choose appropriate power andtime,we can get initial targeting nanobubble.Rosette formationtest,rosette forming block experiment and immunofluorescence areused for observation targeting nanobubble in vitro.Rabbits with VX2tumors were provided by Chongqing Medical University.Tumor tissueswere resected from rabbit liver under aseptic conditions and washed inphysiological saline,and the tumor tissue was excised from the tumormargin and cut into small pieces with scissors.Thirty healthy femaleNew Zealand rabbits weighing2.5–3.0kg were used in the study.Underanesthesia,the bilateral breasts tissue was injected with tumor tissuesuspension (1ml per side).The tumors developed in rabbits breasts2weeks after the injection and they measured15mm in diameter using a5-10MHz linear array transducer(LOGIQ9),and randomized intogroups.The hair over the breast was shaved,and ultrasound couplingmedium was applied.Color ultrasonograph(LOGIQ9) was used for contrast-enhanced imaging with the probe frequency of7.0MHz,themechanical index of0.13,the dynamic range of51,and the soundoutput power of5%.The parameters of time gain compensation andfocus range were adjusted to the optimum.Bolus injections of thetargeting nanobubble,or sonovue,or PBS were given through a20-gauge catheter in an ear vein at the0.2ml/kg.The tubing wasflushed with1ml saline after injection.The dynamic and staticimageswere stored for further analysis.Results:The targeting nanobubble arelight yellow color in visual observation.In light microscopy,thetargeting nanobubble are uniform distribution.There are no obviousdifference between the targeting nanobubble and nomal microbubble inZETA SIZIER3000determination.In fluorescence microscope,thetargeting nanobubble are light yellow color,nomal microbubble can notbe detected,or an sporadic weak fluorescent.Rosette formation test rateof more than80%,conjunct ratio achieved60%.The targetingnanobubble can enhance the echo intensity of rabbit VX2breasttumor,8s after injection,the echo intensity of vessels was enhanced,and15s after injection,the echo intensity of parenchyma wasenhanced.Significant enhancement was still observed after15min.11safter injection of Sonovue,the echo intensity of vessels wasenhanced,and significant enhancement was still observed after20min.PBS group have not enhance effect.Conclusions:The penetrating potential of nanoscale UCA is stronger than that of micrometer-grade.The targeting nanobubble are prepared successfully,which havestrongly targeted and stability in vitro,and have better targeted contrastimaging performance in vivo.We should further enhance the imagingperformance, stability,and antibody binding of this nanobubble. Objective:Breast tumor is one of the most common malignancies inwomen.Surgical treatments including radical mastectomy,modifiedradical mastectomy and breast conservation are invasive.HIFU is aminimally invasive treatment tool for tumor.HIFU can penetrate theskin and focus low energy ultrasonic waves onto the target tumorarea,causing coagulation necrosis of the tumor tissue by means oftransient high temperature (above65℃),cavitation and mechanicaleffects,without obvious injury to the adjacent normal tissues.Thepotential advantage of therapeutic HIFU is not only to provide aminimally invasive treatment for malignant tumors,but also to sparebreast tissue.Ultrasonic wave energy attenuates with increasingtransmission distance in tissues.Ultrasound energy is unlikely toaccumulate in the target tissue due to uniform tissue texture andinsignificant differences in acoustic resistance between adjacent tissue interfaces.For achieve tumor ablation,large doses and long time periodsare usually employed in HIFU,increasing the risk of complicationssuch as burns.In order to increase the efficiency of HIFU and reducethe complications,researchers have attempted to change the intrinsicacoustic properties for increasing ultrasound energy accumulation inthe target tissues by changing their structure,density,bloodperfusion,and functional status(remodeling the acoustic environment oftissue,RAET).One way to mitigate the disadvantage of HIFU might beto use UCA.UCA have been applied in conventional ultrasoundimaging not only for differentiating malignant tumors from benignones,but also for improving the detection of small ill-defined tumors onconventional grayscale imaging.UCA are important potentiators forincreasing the tissue coagulation range following HIFU.They may alsoserve as ultrasound cavitation cores,which may increase tissueabsorption of ultrasonic wave energy and rise tissue temperature.UCAmay enhance the thermal effect and cavitation of HIFU,while reducingthe tissue injury threshold to HIFU.To further investigate the propertiesof nanobubble,we evaluated the effectiveness of combiningnanobubble with HIFU on the treatment of breast cancer VX2tumorsin rabbits with ablation.Methods:Eighty female rabbits with10mmVX2breast tumor(per side),weighing2.5–3.0kg,were used in thestudy,randomized into groups.The JC-200focused ultrasound system(Haifu Technology) was used for tumor treatment.Underanesthesia,the breast tumor rabbit was immobilized on the treatmentcouch.Tumors were located,PBS(1ml) or nanobubble(0.2ml/kg) wereinjected into the ear rim vein10s prior to HIFU irradiation.The focusof the treatment system was within the tumor,and the ultrasoundimages were acquired immediately following HIFU irradiations withdifferent settings (150W for3s,150W for5s,120W for3s,120W for5s and100W for5s).Images demonstrating changes of the grayscale inthe target area before and after HIFU irradiation were stored in thecomputer for further analysis.Pathologic examination after2days.Statistical analysis data of grayscale value and area ratio ofnecrosis.Results:The increase of grayscale value of ultrasoundimaging is the result of HIFU exposure induced cavitation and bubbleformation,which causes a rapid temperature rise in tissues andstructural changes in coagulative necrotic tissues.The grayscale changein the target area was significantly higher in the HIFU+NB group thanthe HIFU+PBS group after5s irradiation (120W or150W)(p<0.01),butthere was no significant difference between both groups after5s at100W irradiation.Macroscopic observation,the necrotic area wassignificantly larger in the HIFU+NB group than in the HIFU+PBSgroup(5s,150W).HE staining showed the area of coagulation necrosiswas significantly larger in the HIFU+NB group than the HIFU+PBS group(p<0.001).These findings demonstrated that nanobubbleincreased the HIFU effectiveness.In the HIFU+NB group,the area ofdamage after irradiation at150W was larger than after irradiation at120W,given the same irradiation time.The area of damage after HIFUfor5s was larger than that after HIFU for3s,given the same irradiationpower.These findings suggested that the area of coagulation necrosiswas positively correlated with the dose and duration of HIFUirradiation.Conclusions:Poliachik et al. found that microbubbles serveas cavitation cores in blood and reduce the cavitation threshold, thuspromoting cavitation.Bailey et al.found that microbubbles affected themorphology of the HIFU treated area in vivo experiments involving ahydrostatic pressure chamber.Yu et al.conducted HIFU treatment inrabbit kidneys and found that its efficacy was significantly enhanced inthe microbubble group when compared to the control group; the tissuenecrosis rate increased by a factor of3.1–3.4and no viable tissues wereidentified pathologically in the focused area in either group.Sokka etal.performed an in vitro study to investigate the thermal effect offocused ultrasound plus microbubbles in rabbits,and found thatmicrobubbles significantly accelerated tissue heating and expanded thearea of tissue damage by a factor of2–3.In the present study,it wasdemonstrated that HIFU-induced lesions were larger in tumors thatcontained nanobubble than in those that contained PBS.The area of tissue damage in VX2breast tumors in rabbits was increased,and thestrength and time of HIFU irradiation were reduced significantly in theHIFU+NB group.Therefore,the nanobubble could function as anenhancer of HIFU ablation.During HIFU treatment,real-timeevaluation of the grayscale value determines whether coagulativenecrosis has occurred.When the grayscale value reaches a certainvalue,it represents coagulative necrosis.We analyzed and calculated thedifference in the grayscale values between pre-HIFU and post-HIFUexposure.As a result, the difference in grayscale value in theHIFU+NB group was higher than in the HIFU+PBS group.Thissuggested that the effectiveness of treatment by means of the size anddegree of coagulative necrosis was significantly different between theHIFU+NB group and the HIFU+PBS group.Therefore,the utilization ofreal-time ultrasound to guide,locate, monitor and evaluate therapeuticefficacy of HIFU with nanobubble should be encouraged.Themechanism of the heating effect produced by microbubbles andnanobubbles presenting in the ultrasound field remains unclear.Twopossible factors are: Heating by oscillation and/or explosion ofmicrobubble contrast agents exposed to HIFU,and cavitation bubblesgenerated by HIFU exposure.However,in terms of generatingheat,which one plays more important role than the other betweencavitation and microbubble oscillation is still unknown.To understand the interactions between ultrasound and contrast agents,it is importantto compare the effectiveness of microbubbles with nanobubbles inHIFU treatment.Consequently,our study suggested that a nanoscaleultrasound contrast agents appears to be a useful enhancer not only forincreasing the detection of tumors,but also for improving theeffectiveness of treatment of tumors with HIFU. Objective:Texture analysis and correlation analysis are used to judgecoagulative necrosis after HIFU irradiation with nanobubble,andcompare with grey-scale values.Methods:One hundred and twentyhealthy female VX2breast tumor rabbits,weighing2.5–3.0kg,wereused in the study,randomized into groups.The JC-200focusedultrasound system was used for tumor treatment.Images demonstratingchanges of the grayscale in the target area before and after HIFU withnanobubble irradiation were stored in the computer for furtheranalysis.In Matlab software,extract four texture feature parametersfrom the wavelet ultrasonographic(kurtosis,skewness,mean,variance)as a Texture1.Another Tamura texture feature parameters extraction ofcoarseness,contrast and directionality,as a Texture2,and the correlation of the target area was computed.Establish decision surfaces by meansof support vector machine(SVM),and analyzing samples.Histology andmicroscopic examination were harvested2days after HIFU.Statisticalanalysis,compare with the gray scale.Results:Texture analysis ofcoagulative necrosis judgment,accuracy and sensitivity are better thangray evaluation(P<0.05).Accuracy rate of Correlation analysisis higherthan the accuracy rate of gray scale(P<0.05).Conclusions:Theaccuracy and sensitivity judgment of coagulation necrosis by means ofwavelet transformation texture analysis is higher than grey scaleevaluation.Correlation analysis data can be used to predict coagulationnecrosis of target tissues after HIFU irradiation with nanobubble,it ismore exact and sensitive than traditional gray-scale evaluation method.The next step,more samples should be use to confirmed these results.
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