| Background:Chemotherapy,as well as surgery and radiotherapy,is currently one of the three major methods of cancer treatment.It develops chemical drugs into the human body in order to kill tumor cells..The effect of chemotherapy depends primarily on the concentration and duration after the drugs reach the tumor target region.Despite curative effect,chemotherapy has toxic side-effect to other human systems.For better chemotherapy effect,systemic dose of chemotherapy drugs is usually increased in order to ensure enough drugs into the tumor target area.We have successfully developed carboplatin-loaded PLGA microspheres(CPMS).In several studies they were used in several tumor models combined with ultrasound and microbubble.The result is encouraging that it works against the tumor that the tumor size grows slower compared with control group,but tumor cells didn't apoptosis in situ completely.Purpose:In this study,rabbit VX2 transplanted tumor was subjected.Ultrasound-guided intratumor injection of carboplatin sustained-release microspheres was performed.Meanwhile,ultrasound irradiation was carried out on the tumor area with intravenous infusion of microbubble.Ultrasound targeted microbubble destruction produces biological effects of which improves the permeability of tumor cell membrane.With this method,bioavailability of carboplatin is expected to be improved in order to achieve the best therapeutic effect,and to reduce the toxic side effect.The morphological changes of tumor cells were observed by transmission electron microscopy(TEM).The changes of tumor growth-related factors Hif-1? and ICam-1 were also investigated.The biological effects of ultrasound irradiation microbubbles changed tumor microenvironment and its impact on intratumor injection of CPMS was explored.Methods:1.Establishment of CPMS and a rabbit model of VX2 subcutaneous transplantation.Based on the previous study of CPMS preparation,we decrease the particle size,change the active compounds of PLGA concentration,alter emulsification time and temperature.In condition of drug loading rate,we are aiming to produce smaller CPMS which is more suitable to arrive at the target area.Ten experimental rabbits were inoculated with granule inoculation method and tissue homogenate method,respectively.The tumor formation time,tumor size and tumor number were studied to find the appropriate modeling method.2.Curative effect of diagnostic ultrasound combined with microbubbles and intratumor carboplatin microspheres on subcutaneous VX2 tumor.Forty subcutaneous VX2 tumor bearing rabbits were randomized into four groups: group A,normal control,not processed;group B,Ultrasound combined with microbubble irradiation;group C,intratumoral injection of carboplatin microspheres;group D,intratumoral injection of carboplatin microspheres,intravenous injection of microbubble and ultrasound exposure.12 h after group experiment,tumors from 5 rabbits in each group were obtained for H.E.and TUNEL staining.Pathologic examination and apoptosis index was performed for qualitative and quantitative analysis,and the effect of different treatment on the apoptosis of tumor cells was discussed.The size of sparing tumors was measured in the seventh,fourteenth and twenty-first day after treatment.The tumor size was quantitatively analyzed to evaluate the long-term curative effect.3.Observation of microenvironment in VX2 tumor by different ultrasound combined with microbubble irradiation Carboplatin Sustained Release Microspheres.24 VX2 rabbit subcutaneous transplantation tumor were divided into 4 groups: group Control:control group;group CPMs: cbpms group;group S2000: S2000 ultrasound combined with microbubbles + carboplatin microspheres group;group AP-170: AP-170 ultrasound therapy combined with microbubble + carboplatin sustained-release microspheres group.Immediately after the experiment,each group of 1 rabbit was randomly selected for tumor tissue fixation,and the changes of tumor cell membrane were observed by transmission electron microscope.The remaining 5 rabbits in each group were used to observe the temperature of the tumor area immediately after using DH-2010 A far infrared thermography.After 6h,the tumor tissues of each group were collected.The expression of Hif-1 and ICam-1 were detected by IHC,RT-PCR and WB.At the same time,the drug release of carboplatin microspheres in vitro was studied.Results:1.The production of cbpms white powder sphere,5-100 ?m in diameter,the encapsulation rate was 74.8%,while the drug loading rate was 10.7%.The microspheres were smooth,spherical integrity,good mobility.In the rabbit VX2 tumor model,the tumor length by particle method was 0.76 cm ± 0.17cm(n=7),and 1.08 cm + 0.23cm(n=10)by homogenate method at the 15 th day.2.In group D,the tumor apoptosis index was 14.9 ± 1.59%,significantly higher than that of group C(10.8 ±2.13%),group B(5.16±0.42%)and group A(3.0±0.63%)(P<0.05).Pathological examination showed patchy necrosis and the injury was most serious.The tumor size was 525.22±57.89mm3 in the seventh day,930.87±102.34mm3 in the fourteenth day,and 1294.88±235.25mm3 in the twenty-first day.The size growth rate was significantly lower than that of group A,group B and group C(P<0.05).3.(1)Transmission electron microscopy of VX2 tumor cells.In the Control group,the tumor cells showed complete cell structure,smooth and continuous cell membrane.The membrane continuity of CPMs group was still acceptable.In the S2000 group and the AP-170 group,the Golgi and mitochondria of the tumor cells were edematous.The cell membrane was not uniform,with some thinner areas,poor continuity,and small pores.The lesion was even more obvious in the AP-170 group.(2)HIF-1?,ICAM-1 expression of VX2 tumor tissueThe expression of HIF-1 in tumor tissues of each group was gradually reduced,while ICam-1 expression increased by IHC,RT-PCR,WB,et al.(3)Temperature changes of VX2 tumor tissue.The temperature of rabbit tumor region in Control group was 37.04 ± 0.55°C,37.38± 0.46° C in CPMs group,and 36.52 ± 0.48 °C in S2000 group.No significant difference of tumor body temperature was found among the three groups.The tumor temperature in AP-170 group increased to 40.24±0.64°C,which is significantly different from that of Control group?S2000 group and CPMs group(p <0.05).(4)Drug release in vitroAt 37°C,the optical density in CPMs group was 0.1074±0.0027,0.128±0.0044 in S2000 group,0.1394±0.0024 in AP-170 group.The absorbance of the solution in the three groups showed a rising distribution.But there was no significant difference between S2000 group?CPMs group?AP-170 group.At 40°C,the optical density in AP170 group was 0.1932±0.0069,significant different from that in all groups at 37°C(p <0.05).Conclusion:1.By adjusting the particle size of carboplatin,the concentration of PLGA,and changing the emulsifying temperature and so on,on the basis of keeping the drug loading rate,the particle size of CPMS is 5-100?m and the drug loading rate is 10-13%.It is easier to be transported to the target tissue and organ.2.Diagnostic ultrasound exposure on intratumor carboplatin microspheres combined with microbubbles could promote curative effect of VX2 tumor.3.Ultrasound combined with microbubble irradiation VX2 tumor tissue can change the permeability of cell membrane,mitochondria,Golgi cell edema.It is better in the AP-170 group than in the S2000 group.4.The expression of Hif-1? in the tumor tissue gradually decreased and the ICam-1 increased with ultrasound combined with microbubble irradiation in carboplatin microspheres VX2.5.The temperature of VX2 tumor tissue changed by AP-170 ultrasound combined with microbubble irradiation and can increased to 40 °C.6.In vitro release experiments,elevated temperature can promote carboplatin sustained release microspheres chemotherapy drug release. |
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