Experimental Studyonthe Safetyand Efficacyof Lenvatinib-loadedpoly(l-lactic Acid)microspheres Designedfor Transarterial Chemoembolization Therapy

There are two parts in our studyPart1.Preparation of Lenvatinib(LEN)-loaded poly(L-lactic acid)(PLLA) Microspheres(MSs)and Experiment of Arterial Embolization in a Rabbit Kidney ModelObject:1.To Prepare and characterize LEN-PLLA MSs.2.To evaluate safety and efficacy of LEN-PLLA MSs in rabbit renal artery embolization.Materials and methods: LEN-PLLA MSs were fabricated by microfluidic technology and evaluated though scanning electron microscopy(SEM),particle size analysis,the release of Lenvatinib and degradation in vivo.Ten New Zealand white rabbits were randomly divided into experimental group(LEN-PLLA MSs,100-300μm,n=5)and control group(PVAs,150-350μm,n = 5).Blood sampling was collected from the rabbit era veins before embolization and 1,3,7,14,and 21 days after embolization,and creatinine(Cr)and blood urea nitrogen(BUN)were measured to evaluate systemic toxicity of each group.All rabbits underwent abdominal enhanced CT scanning to observe the vascular in the embolization area at 1,2,3,4 weeks after embolization and were sacrificed after last CT scanning.Then both kidneys were removed from each rabbit and weighed.Finally,the embolized kidneys were collected for HE,MASSON and TUNEL staining respectively to evaluate the distribution of LEN-PLLA MSs and PVAs in the renal artery and its branches,the damage degree to vascular wall of both embolic agents,and the apoptosis rate of embolized renal tissue in each group.Results:1.LEN-PLLA MSs prepared by microfluidic technology had regular shape,uniform diameter and controllable size.The average diameter of the MSs was 195±40.1μm,and the most of them distributed was about 200μm.The cumulative release of Lenvatinib was about 60% within 28 days in vitro.2.The changes of renal function over time were similar in both groups.The levels of Cr and BUN increased after embolization within 1 or2 days and then decreased to the normal level.Arterial Recanalization can be observed in 2of LEN-PLLA MSs group and 3 of PVAs group from the abdominal enhanced CT until the4 th week,but the recanalization area was less than 50% of the whole renal cortical region.There was no statistically significant difference in the quality of each side kidney of both group after embolization with drug-loaded microspheres and PVAs(P>0.05).HE staining showed that LEN-PLLA MSs were significantly distributed in the proximal and distal ends of the renal artery,while PVAs were mostly concentrated in the proximal.Masson staining showed that LEN-PLLA MSs damaged the intima and media membrane of the vascular wall,while PVAs damaged the whole layer of blood vessels.TUNEL staining displayed the viable renal tissue was found in four embolized kidneys of LEN-PLLA MSs group and in all kidneys of the PVAs group.The average apoptosis rates of the two groups were80.90%±5.29% and 76.44%±0.44% respectively,with no significant difference(P > 0.05).Conclusion: Compared with PVAs,LEN-PLLA MSs had more complete embolization degree and less damage to blood vessels,which seem to be a safe and effective embolic agent.Part2.Experimental Study on the Treatment of Rabbit VX2 Liver Cancer with LEN-PLLA MSsObject:To investigate the safety and efficacy of LEN-PLLA MSs in the treatment of rabbit VX2 liver cancer.Materials and methods: A certain amount of VX2 tumor tissue was filled into the left lobe of rabbit liver by CT-guided percutaneous puncture.Two weeks later,CT enhanced scanning was performed to confirm the successful models of rabbit VX2 liver cancer.The tumor-bearing rabbits were randomly divided into three groups: A(control group),B(oral administration group)and C(LEN-PLLA MSs group).Rabbits were fed routinely without any treatment in Group A,while the ones were treated with the Lenvatinib solution(multiple dosing;1.5mg/kg/day dose)for 14 days in Group B,and treated by transcatheter arterial injection of LEN-PLLA MSs(single dosing;0.5mg/kg dose)in group C.Blood samples were obtained from the ear marginal vein of each rabbit before treatment and on the 1st,3rd and 7th day after treatment for liver function detection,and enhanced CT scanning was performed on the 14 th day after treatment to compare the volume of tumors among three groups.Then the animals were sacrificed after CT scanning,and the liver tumors were taken out to test the drug concentrations both in tumor regions and normal livers of group B and C.At the same time,the liver tumors of three groups were stained with HE to observe the necrosis of the tumor regions.Result: The successful rate of rabbit VX2 liver cancer model was 90%(18/20).Two of group C failed during the operation because of the abnormal liver arteries and the rupture of a blood vessel,meanwhile a rabbit of group C died on the third day after treatment.By the end of the experiment,a total of 15 rabbits underwent all procedures and survived.The changes of liver function among three groups at over time were different,but there was no significant change in the untreated group.Alanine aminotransferase(ALT)and Aspartate aminotransferase(AST)in group B began to rise slightly on the second day and then remained stable,while increased significantly on the first day after embolization and then returned to the normal level within 7 days in group C.CT enhanced scanning showed that the tumor growth rate among three groups was significantly different(P <0.05).The tumors grew rapidly in group A,while slowly in group B and were significantly inhibited in group C.The concentration ratios of Lenvatinib in tumor and normal liver of the group B and C were 0.33 and 14.4 respectively.Conclusion: Delivery of LEN-PLLA MSs into the target lesion can increase the drug concentration within tumor tissue and significantly inhibit the growth of VX2 liver cancer in rabbits,which maybe safe and effective.