Experimental Study Of Apatinib-loaded Microspheres For Embolization In Rabbit VX2 Liver Cancer Model

Purpose: Transcatheter arterial chemoembolization(TACE)is one of the most widely used treatment methods in intermediate-advanced liver cancer.However,incomplete embolization can increase angiogenesis of residual tumor,which affects seriously the prognosis of treatment.Apatinib mesylate inhibits tumor angiogenesis by selectively blocking the VEGF/VEGFR pathway,and our previous studies have demonstrated that its inhibition effect of angiogenesis is more significant under hypoxia than normoxic conditions.In addition,Calli Spheres Beads is a new embolic agent developed in China.It has the characteristics of well embolization effect,quick and large drug loading,and many types of drugs that can be loaded.Herein,the study aimed to construct the apatinib-loaded Calli Spheres Beads and investigate its application in TACE of a rabbit VX2 liver tumor model.The characteristics of drug loading and releasing would be evaluated.Then,the pharmacokinetics of apatinib delivered via the hepatic artery would be further explored.And the antitumor response and inhibition effect of tumor angiogenesis after treatment would be assessed.Materials and Methods:(1)100-300 μm Calli Spheres Beads were loaded with the fully-dissolved apatinib through ion exchange mechanism,and the process of drug releasing was simulated in vitro.The ultraviolet-visible spectrophotometry was used to detect and calculate the drug loading rate and cumulative release percentage.(2)Healthy New Zealand white rabbits were selected to establish rabbit VX2 liver cancer model by laparotomic route.15 days after the VX2 tumor tissue was implanted into the left lateral lobe of rabbit liver,the successful model was selected by enhanced CT scan.(3)A total of 100 tumor-bearing rabbits were randomly divided into 4 groups,25 rabbits in each group.After hepatic arteriography and superselective catheterization,the rabbits were respectively given: normal saline control group(NS group),apatinib perfusion group(APA group),blank Calli Spheres Beads embolization group(CB group),apatinib-loaded Calli Spheres Beads embolization group(CBAPA group).(4)The blood samples were collected at 5 min,10 min,30 min,1 h,3 h,6 h after treatment in the APA group and CBAPA group,respectively.In addition,some rabbits were sacrificed at 6h,3d and 7d after treatment in the two groups.We took liver tumor,adjacent normal liver tissue,heart,lung,kidney and spleen tissues,and grinded them thoroughly into the homogenate.The apatinib concentration was analyzed by high performance liquid chromatography–tandem mass spectrometry in the processed blood and tissue samples.(5)Blood samples were taken at 1d,3d and 7d after treatment in the above four groups,and liver and kidney function changes in each group were analyzed by biochemical tests.Then,we recorded and compared the survival time of tumor-bearing rabbits in each group.In addition,enhanced CT scan was performed to measure tumor volume at7 d after treatment in each group,and tumor growth rate was calculated by comparing with preoperative tumor volume.The rabbits were sacrificed immediately after CT scan,and tumor specimens were removed for pathological examination.To observe the tumor necrosis and microvessel density after different treatment.Results: The established stock solution containing 20,40 or 60 mg apatinib were fully mixed with 1 g Calli Spheres Beads for 2 hours,respectively.The drug loading rate reached the maximum at 30 min and was 70.7%,46.6% and 31.8% in the three groups,respectively.The result revealed 20 mg/g group can present highest apatinib loading efficiency.Further,as to apatinib release profile,the percentage gradually increased within 12 h in 20 mg/g group and then kept stable.The cumulative release percentage reached 47.2% within 24 h.In vivo,we successfully performed interventional therapy in different groups of tumor-bearing rabbits.The results showed that,compared with APA group,the apatinib concentration of plasma was significantly lower in CBAPA group,while the concentration in tumor was higher(P < 0.05).After embolization treatment of apatinib-loaded Calli Spheres Beads,the liver and kidney function were temporarily affected without substantial damage.Tumor growth rate in CBAPA group was significantly lower than that in other three groups,and survival time of CBAPA group was prolonged(P < 0.05).Pathological and immunohistochemical results showed that,CBAPA group can achieve higher tumor necrosis rate and fewer new vessels compared to other three groups(P < 0.05).Conclusions: Angiogenesis inhibitor apatinib could be combined effectively with Calli Spheres Beads and released steadily for a period of times.Furthermore,apatinibloaded Calli Spheres Beads can be used as an effective embolic agent for interventional embolization of rabbit VX2 liver cancer model,which can improve local intratumoral drug concentration with reducing systemic toxicity.In addition,the combination can inhibit tumor angiogenesis,slow tumor growth and improve survival time.These findings will provide a new potential approach for interventional treatment of liver cancer.