| BackgroundMicrobubble in fluid can produce transiently high temperature, high voltage,shock wave and jet stream during being oscillated, inflating, contracting and implosion mediated by ultrasound,such of energy liberation behaviors is called sonopration.Reseach have demonstrate that tumor cells in vitro would have pores in cell membrane and higher permeability mediated by physics stream produced during microbubble destruction, thereby the cells had higher medicine uptake, and cytotoxicity of medicine promating tumor cell to die was increased.That discover had ultrasound field’attention rapidly,and was tried to cure malignant tumor.Curing malignant tumor is always the emphasis and difficulty in clinic. Chemotherapy is one of main treatment for it,but muti-drug resistance usually happens,which affects chemotherapy’treat and become the strongest barrier.muti-drug resistance means tumor cells will generate resistance to other kinds of drugs that cells haven’t been exposed to after being exposing to one kind of drug,though it has different structure and effect mechanism from the others.Reseach indicated tumor cells’decreased intake of drug caused by multi-factors is one of the main biochemistry mechanism about MDR.Many scholars at home and abroad have tried to reverse human resistant cells using ultrasound and gained initial achievement which showed attractive prospect.Tumor cells in vivo are different from those in vitro because of capillary and interstitial space between former cells and microbubble,but the latter are close to microbubble,so that the distance is short between physics stream from microbubble mediated by ultrasound and cell,and the active force will be very strong.Through pure ultrasound could produce pores in tumor cell membrane in vitro and increace cytotoxicity of drug,pure ultrasound need high pressure to kill and wound cell because of no cavitation nucleation in organisms,which could have side effect,such as injuring normal tissue beside being difficult to control.Drug-loaded microbubble can decrease reaction between the curing drug and others or endoenzyme in vivo that has drug adhering to,integrating or inlaying shell of microbubble,even integrating in its interior. Drug-loaded microbubble mediated by ultrasound can release drug,increase drug lever in target area,which could cure lesions at lastest.However,drug-loading rate of these gas-nuclear micropaticles is usually low because of the limited cavity on shell for loading drug.Increasing shell width to enhance loading cavity was suggested to solve the problem.But this might increase the preparation complexity.Scholars set up subcutaneous hepatoma model of the rats,and observed that Evans blue extravasation in ultrasound-targeted microbubble destruction(UTMD)group was much higher than pure ultrasound group,confirming that UTMD was able to transiently increase capillary permeability in hepatomas,increased dispersion of chemotherapy drug to interstitial space and achieved treatment about tumor.But there is unknown question that whether microbubbles can pass through pores on capillary intima to interstitial space and produce twice even mult-cavitation,and whether sonopration in interstitial space can produce pores on tumor cell membrane in vivo,increace permeability of cell membrane even nuclear membrane and improve effect of tumor chemotherapy,which hasn’t been reported.ObjectiveThe purpose of this study is to investigate the change of permeability on VX2tumor cells in vivo using lanthanum nitrate electron microscopy observing the ultrastructure change of tumor cell membrane and nuclear membrane under different sound intensity,pulse width, insonated time of therapeutic ultrasound instrument and different microbubble dose,and whether ultrasoud-mediated microbubble can produce pores on cell membrane and nuclear membrane.On the other hand,to explore the appropriate acoustics parameters that can achieve the greatest permeability of tumor cell is based on investigating the effect of microbubble enhanced ultrasound cavitation on VX2tumor model of rabbits.Materials and methods1.MaterialsTherapeutic ultrasound instrument:CZ-906A model noninvasive-treating ultrasound devices with high power, and the diameter of probe is2.5cm,whose area is4.9cm2.The sound intensity of treating probe is between0.21W/cm2～1.09W/cm2,pulse width between1.21ms～6.05ms. Diagnostic ultrasound imaging system: Philips IU22ultrasound system,9L probe,eligible to perform contrast enhanced ultrasonography(CEUS)with low MI.Hitachi H-600model electron microscope.Microbubbles:Quanfuxian,a perfluoropropane filled albumin MB,with bubbles concentration of4～9×109/ml,average2.9μm in diameter,and98%of the MBs less than8μm in diameter.Other reagent:Sumianxin II for injection, Atropine,3%Pentobarbital, adriamycin, Sodium Chloride.Experiment animals:73healthy New Zealand white rabbits(2.0kg-2.5kg body weight) were bought from experimental animal center of Nanfang Hospital.VX2tumor tissue homogenate were supplied from experimental animal center of Sun Yat-sen University.2.Preparation of animal modelModel preparation of permeability change about VX2tumor cell membrane:a New Zealand white rabbit bearing intramuscular VX2tumor in its lateral hind limb were anesthetized with0.35mk/kg sumianxin Hand0.25ml/kg Atropine for intramuscular injection,and established intravenous access for injecting3%pentobarbital.The rabbit were maintained lateral position.The tumor local position and its1.5cm around were cleaned and sterilized,then the tumor tissue was taken out under condition, rejected the connective tissue and necrotic tissue quickly.The retained part was put in sterile sodium chloride,sheared into small pieces aboutl mm3,and transplanted into45healthy New Zealand white rabbits’muscular layer of its bilateral hind limbs.The models would be used for experiment after14days.Model preparation of chemotherapy effect on VX2tumor:the former part is same as (1),but after being sheared into small pieces about1mm3,the pieces were transplanted in28healthy New Zealand white rabbits’muscular layer of its right lateral hind limbs. The models would be used for experiment after11days.3.Experimental procedureExperimental procedure of permeability change about VX2tumor cell membrane:Thirty-eight New Zealand white rabbits bearing intramuscular VX2tumor according with experiment condition were randomly divided into four groups for the impact factors including sound intensity,pulse width, insonated time of ultrasound instrument and microbubble dose,inspecting every factor one by one..When inspecting levers of one factor,the other factors were fixed.Each lever repeated five times. Therapeutic ultrasound was delivered directly to the tumor surface during intravenous infusion of microbubbles. The tumor surface was cut using sharp blades after treatment,then fresh fishlike tissue of tumor rim was taken out and investigated in the permeability change of tumor cell membrane using lanthanum nitrate electron microscopy.Every lever in each factor was randomly choosen three tumors to accept pathological examination.The next factor was investigated after the former factor having gotten the appropriate lever.Experimental procedure of chemotherapy effect on VX2tumor:Twenty-eight New Zealand white rabbits bearing intramuscular VX2tumor according with experiment condition were randomly divided into four groups:drug group,drug and microbubble (Drug+MB) group, drug and ultrasound (Drug+US) group,drug and microbubble and ultrasound (Drug+MB+US) group. Drug+MB+US group was injected chemotherapy drug intravenously,then therapeutic ultrasound was delivered directly to the tumor surface for10min after2min,microbubbles were injected intravenously, simultaneously. Drug+US group was applied with saline injection instead of microbubble,and Drug+MB group wasn’t insonated,but drug group was applied with neither microbubble nor ultrasound. The procedure was repeated every2days until the13th day. Grey scale ultrasonography and contrast enhanced US were acquired before every treatment to get the tumor size,volume measurements and perfusion images.4.Data analysesThe number of lanthanum particles in tumor cell after treatment as outcome measures of permeability change about cell membrane was scored. The statistical were performed using SPSS13.0software. The lanthanum particles score in the same factor were initially subjected to a Kruskal-Wallis H test,which does not assume population variances are equal.If the total differencewas significant,a Mann-Whitney U test was then used to analyse the differences between any two groups.The tumor volume as outcome measures of hemotherapy effect on VX2tumor were expressed as Mean±SD,the comparisons of the tumor volume among groups in each time were subjected to One-Way ANOVA.A p value<0.05was deemed statistically significant.Results1.Results of permeability change about VX2tumor cell membrane:There was no lanthanum particles in tumor cell when ultrasoud wasn’t insonated.There were lanthanum particles when the sound intensity of ultrasound were0.21W/cm2、0.43W/cm2、0.65W/cm2,and lanthanum particles scores had statistical significance between0.43W/cm2condition and other levers(P<0.01).When pulse width was2.42ms its lanthanum particles scores was higher than other lever(Grade2, P<0.05).When insonated time was10min its lanthanum particles scores had statistical significance compared with other lever groups(P<0.05).The lanthanum particles scores after treatment displayed a upward trend as the gradually increased microbubble concentration,and when it was up to0.50ml/kg,the score was the highest(Grades2-3, P<0.05).There was no lanthanum particles in nucleus under all levers.2. Results of chemotherapy effect on VX2tumor:There average tumor volume of the Drug+MB+US, Drug+MB, Drug+US and Drug groups at the baseline were0.42±0.36cm3、0.70±0.62cm3、0.55±0.30cm3、0.53±0.42cm3respectively.It grew to0.48±0.44cm3、1.61±1.06cm3、1.89±0.81cm3.1.62±0.69cm3with a13d experimental period, respectively.The average tumor volume of Drug+MB+US group was significantly smaller than that of other groups (P<0.05),however, there were no statistical significance in average tumor volume between Drug+MB, Drug+US and Drug groups (P>0.05).The tumor growth rate of Drug+MB+US group was smallest than that of other groups. The tumor growth inhibition rate of Drug+MB+US, Drug+MB and Drug+US groups were70.4%,0.6%,-16.7%, compared with Drug group, respectively.Conclusionsl.Sonopration induced by microbubble-enhanced ultrasound can induce pores in capillary intima, and microbubbles can pass through pores and entre the interstitial space where the tumor cells is,following cavitatioin happening;or after a microbubble in capillary experiences the first cavitation,it will become smaller one that will pass through the enlarged pores in capillary intima and entre the tumor interstitial space, moreover,produce twice or multi-sonopration.2.The permeability change of tumor cell membrane in vivo has significant difference under different acoustics parameter about ultrasound and microbubble,which shows a certain regularity.3.Sonopration induced by microbubble-enhanced ultrasound can significantly increase the permeability of tumor cell membrane in vivo,enhance cell uptake of drug,so that it can reinforce tumor chemotherapy in sensitivity,which is expected to become a novel,noninvasive and repeatable method of tumor chemotherapy in vivo. |
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