The In Vitro And In Vivo Study Of The Radiosensitization Effect Of Nanoliposomal Cisplatin (NLDDP)

IntroductionMalignant tumor is a common disease threatening heath and life.The main treatment methods include surgery,radiotherapy,chemotherapy,biologic therapy and so on.Among them,Radiotherapy palys an important role,since more than 70 percent patients should receive radiation during their treatment. Thus,to improveme radiotherapy effect of cancer is very important.There are two directions through which we can improve radiaton effect,radiaton physilolgy and radiation bilogly.As to the radiation biology,the study of radiosensitizer,that can enhance the sensitivity of malignant tumor to radiation, has attracted a lot of attention during the past 50 years.Cisplatin is a kind chemothrepy drug with the potential of radiosensitization commonly used in clinic works,but its severe side effects limit its application.Therefore,if it can be made into a kind of delay-action and target-distribution preparation,the problem will probably disappear.In this study,Nanoliposomal cisplatin (NLDDP) was prepared in order to improve the circulating time and targeted distribution of the cispaltin in the body.The pharmokinetics of NLDDP was studied and the radiationsensitization effect was explored both in vitro and in vivo.This study will provide experiment references to the clinical study.ObjectiveTo observe the pharmacokinetics of nanoliposomal cisplatin(NLDDP) in mice bearing Lewis lung cancer.To explore the in vitro and in vivo anti-tumor effect of N/DDP when combined with radiation.To explore the radiation biological effect of nanoliposomal cisplatin(NLDDP) combined with radiotherapy in Lewis mice bearing lung carcinoma.Material and methods 1.NLDDP was irradiated by X ray,and then the drug release speed from the liposome was detected.As to the pharmacokinetics,Eighty C57 BL/6N mice bearing Lewis cancer were administrated with cisplatin solution(CDDP) and NLDDP respectively through their tail veins with dosage of 6 mg/kg.At different time points,five mice were killed after their blood was collected from eyepit, then their lungs,livers,kidneys and tumors were taken out.The content of free platinum in these plasmas was detected by HPLC,while the total platinum contents in tumor,liver,lung and kidney tissues were detected by Graphite Furnace Atomic Absorption Spectrometry.2.In vitro cytotoxicity was studied in A549 lung cancer cells.The inhibition to cell activity was detected by MTT test.Anti-tumor effect combined with radiation was evaluated through colony-forming experiment.3.C57BL/6N mice bearing Lewis lung carcinoma or melanoma were treated with single dose of radiation alone,NLDDP alone,CDDP alone,NLDDP followed by local single dose radiation or CDDP followed by single dose radiation after the tumor size reached 10mm in diameter.The drug was administrated through tail vein injection.Tumor size was measured three times a week.The tumor volume growth delay(TGD) time of each tumor was expressed as the difference between the tumor volume quintupling time(time of tumor volume to reach five times the original pretreatment volume,T5V₀) of treated tumors compared with the T5V₀ of untreated control tumors.The tumor cell cycle distribution in tumor tissue of the mice after NLDDP and CDDP injection was detected by flow cytometry.4.C57BL/6N mice bearing Lewis lung carcinoma were treated with single dose of radiation alone,NLDDP followed by local single dose radiation or CDDP followed by single dose radiation,drug was administrated through tail vein injection at a dosage of 6 mg/kg,and there were 6 levels of radiation dose for each group,including 0Gy,2Gy,6Gy,16Gy,28Gy and 40Gy.Tumor size were measured three times a week,The tumor volume growth delay(TGD) time of each groups was got,and the dose-effect curve was got through Compertz model,SER was the dose ratio of radiation alone group to the group combined with drug which was needed to reach same tumor growth delay. The damage enhancement of the drugs combined with radiation to intestinal epithelium was also observed.C57BL/6N mice were divided into 3 main groups,and then into 5 subgroups.The mice were irradiated at the whole abdomen with 0Gy,8Gy,10Gy,12Gy and 14Gy each subgroup 72 hours after NLDDP or CDDP injection through tail veins.All mice were sacrificed 3.5 days after irradiation and segments of jejunum were removed and transverse histological slice were cut and stained with haematoxylin and eosin,then the number of regenerating crypts per circumference was counted under microscope.Then,linear-quadratic model was got,and the SER was the ratio ofβvalue of combined group to radiation alone group.Results1.The drug release speed from liposome was not different with or without X ray irradiation in this study.In CDDP group,peak concentration of free platinum in plasma,3.24μg/ml,was achieved soon after drug injection,but it decreased rapidly,and no free platinum could be detected after two hours.In NLDDP group,the peak concentration reached 13.79μg/ml in 1 hour,4 times of which in CDDP group.In the tumor,kidney,liver and lung tissues,the contents and the AUC values of total platinum of NLDDP group were significantly higher than that of CDDP group.The biggest difference was in tumor tissue and the smallest was in kidney tissue.2.Lipid used in the preparation of NLDDP was proved no in vitro cytotoxicity. NLDDP got greater inhibition to A549 cells than CDDP,the IC50 of CDDP was almost 2.24 times of NLDDP.NLDDP combined with radiation provided the highest inhibition to colony forming.3.NLDDP produced better anti-tumor effect than CDDP both in Lewis lung cancer mice and B16 melanoma mice.Local radiation after NLDDP administration produced longer TGD time than radiation alone(p＜0.05).When combined with local radiation of 6Gy,administration of NLDDP before radiation delivery produced an longest TGD time of 15.29 days,significantly longer than the 6.65 days owing to the combination of CDDP followed by radiation with the same intervals(p=0.003).In the combination groups with NLDDP,radiation performed 72 hours after NLDDP injection caused greater inhibition of tumor growth delay than performed 1 hour after drug administration,the difference is statistically significant(P＜0.05).Better survival were achived in NLDDP combined with radiation group than CDDP(P=0.0001). Cell cycle distribution showed no difference in both groups after drug anmistration.4.Both NLDDP and CDDP produced radiosensitization effect in Lewis lung cancer mice,with the SER of 4.92 and 3.22 respectively.Also greater damage effects in jejunum tissue were got both in NLDDP and CDDP group than in radiation alone group,with the SER of 1.154 and 1.192 respectively。NLDDP proved a higher TGF(4.263) than CDDP(2.071) in the combination with radiation.Conclusions1.The drug release speed from liposome was not affected by X ray irradiation in this study.NLDDP prepared for radiosensitization in this study significantly extended the circulation time of free cisplatin in blood,brought more cisplatin into the tumor tissues,and markedly increased the effective bioavailability of cisplatin.2.NLDDP produces greater in vitro antitumor effect than CDDP,but the mechanism needs more investigation.3.NLDDP can provided a longer TGD time with or without combination with radiotherapy in Lewis lung carcinoma bearing mice and B16 melanoma mice,which is more effective than that of CDDP.The interaction between NLDDP and radiation maybe time dependent,which needs more investigation,as well as the mechanism.4.Both NLDDP and CDDP have radiosensitization effect,and NLDDP provided a better TGF than CDDP when combined with radiation for the anti-tumor effect without more damage to normal tissue.