Experimental Study Of Enhancing Delivery Of Chemotherapy Drug Of VX2 Tumor In Rabbits By Increasing Tumor Blood Flow With Ultrasound And Microbubbles

Objective:Anticancer drugs gain access to solid tumors via the circulatory system and must open the blood tumor barrier.We study whether the pulsed low frequency ultrasound combined the microbubbles can open the blood tumor barrier and increase the blood flow in VX2 tumor temporarily.To determine the change of the adriamycin penetration in VX2 tumor.Methods:1.Experimental modeling: The New Zealand white rabbits of healthy adult females were used to prepare the model of superficial muscular transplantation tumor in hind limbs;2.Experimental Group and experimental intervention: In this study,all of the 66 tumor-bearing rabbits were divided into 6 groups randomly(n=11 per group).These 6 groups contains adriamycin therapy together with ultrasound combined with microbubbles treatment group(Ad-US-MB treatment group),US-MB treatment group,US treatment group,MB treatment group,Adriamycin treatment group(Ad-treatment group)and blank controlled group(BC group).Ad-US-MB treatment group: The animals were intravenous injected with adriamycin,and then the tumor of the animals were disposed by low-intensity ulrtasound and mirobubbles for 10 minutes in order to temporary open the blood-tumor barrier;US-MB treatment group: The animals were intravenous injected with the same volume of saline solution,and then the tumor of the animals were disposed by low-intensity ulrtasound and mirobubbles for 10 minutes;US treatment group: The animals were intravenous injected with the same volume of saline solution,and then the tumor of the animals were disposed by low-intensity ulrtasound for 10 minutes;MB treatment group: The animals were intravenous injected with the same volume of saline solution,and then were intravenous injected with the same volume of microbubbles,meanwhile the tumor of the animals were disposed by sham ulrtasound irradiation for 10 minutes.Ad treatment group: The animals were intravenous injected with adriamycin,and then were intravenous injected with the same volume of saline solution,meanwhile the tumor of the animals were disposed by sham ulrtasound irradiation for 10 minutes.The contrast-enhanced ultrasound(CEUS)of tumor tissues in rabbits were perfomed before and after intervention.Then 8 rabbits per group were choose radomly and these rabbits in each group were sacrificed immediately after treatment.Tumors tissue were divided into two equal parts.Then take one part of the tumor tissue randomly to detected the concentration of doxorubicin by the fluorometric analysis.And take the other part of the tumor tissue for H&E staining and immunofluorescence staining.The rest rabbits were reared until 28 days after the first intervention.The rest rabbits in each group were give the intervention as mentioned before every 7 days.The growth of the rabbits were minitored by ultrasound system with B-mode.3.CEUS : Contrast-enhanced ultrasonic perfusion imaging of the tumor was performed on two occasions: before treatment,immediately after treatment.Then the tumor perfusion was estimated by visual observation and AUC value.4.Fluorometric analysis:(960MC)Fluorescence spectrophotometer was used to detect the concentration of adriamycin in tumor tissues by Ag NO3 spectrofluorimetry.5.Immunofluorescence staining:Immunofluorescence staining was used to detect the distribution of adriamycin and the tumor microvasculature.6.H&E staining: After euthanasia,the tumor tissues were soaked in the polyformaldehyde solution,then stained with HE.The pathological changes were observed under light microscope.7.Observation of tumor volume: Tumor volume was assessed every 7 days until 28 days using B-mode ultrasound imaging at a mechanic index of 0.08.In each of the B-mode images,the length and width of the tumor were measured.And then tumor volume was calculated.Results:1.CEUS:The visual strength of CEUS in Ad-US-MB treatment group and US-MB treatment group was slightly stronger than that before treatment.There was no significant change before and after treatment in Ad treatment group,US treatment group,MB treatment group and BC treatment group.The AUC value of tumor perfusion in the Ad-US-MB treatment group and US-MB treatment group was raised immediately after treatment(p<0.01,p<0.05).The change of AUC value in the rest treatment groups have no statistical significance.2.Fluorometric analysis: Ultrasound combined with microbubbles increased the concentration of adriamycin in sample of Ad-US-MB treatment groups,compare to Ad treatment groups(p<0.05).3.Immunofluorescence staining:In immunofluorescent staining section,high concentrations of adriamycin were observed mainly around bloodvessels,and some even discovered in further area in Ad-US-MB treatment groups.While,the relatively low concentrations of adriamycin were observed only around blood vessels in Ad treatment groups,avascular and adjacent regions of the tumor did not contain detectable adriamycin.Also,there were a small number of blood vessels without surrounding adriamycin.4.H&E staining: The pathological results are basically the same between US group,MB group and BC group.Irregular nests were found in the tumor tissue.The tumor cells in different stages of proliferation were seen in the nest,densly arranged,large nuclei stained.The disorder blood vessels have no obvious wall injure,there are red blood cells within the lumen.Focal necrosis of tumor cells were found in the Ad group and US-MB group.In the injure area of US-MB group,the microvascular congestion and expansion,the shape of the vessel is round or oval,the structure of the wall is not complete and leakage of red blood cell can be seen.In the US-MB-Ad group,the most tumor vessels are structurally complete,but focal necrosis of tumor cells were found also,the microvascular congestion and expansion,the shape of the vessel is round or oval,the structure of the wall is not complete and leakage of red blood cell can be seen.5.Tumor growth: Growth delay is expressed as the number of days for tumors in Ad-US-MB treatment groups and Ad treatment groups as compared with other groups.With the prolongation of the time of treatment,the volume of tumor was significantly reduced in Ad-US-MB treatment groups and Ad treatment groups.Nevertheless,the volume of tumor of Ad-US-MB treatment groups is smaller than the volume of tumor of Ad treatment groups 24 days after treatment.Conclusion:The pulsed low frequency ultrasound combined with microbubbles and chemotherapy drug can increase the regional blood flow in the tumor temporarily and promote the effective transport of adriamycin in tumor.