Study On The Arterial Embolization Hyperthermia Mediated By Carbonyl Iron Powder/Lipiodol Suspension

Background Hepatocellular carcinoma(HCC) is one of the most common fatal malignant tumors world wide.70%-80%of tumors are inoperable because diagnosis is established in the late stages. Transcatheter arterial embolization(TAE) has been established as the preferred nonsurgical treatment for HCC, due to its higher tumor response rate and fewer adverse effects comparing with other interventional therapy. But some disadvantages still exists, for the performance of the majority of tumor necrosis is incomplete, and repeated treatments are needed. Magnetic arterial embolization hyperthermia (MAEH) is a combined therapy of arterial embolization and magnetic hyperthermia. It is based on selective arterial embolization of liver tumors with magnetic embolic agents. An external alternating magnetic field is then applied to produce heating in the embolized particles. The common mediators for MAEH are ferromagnetic particles.Magnetic mediators play critical role in the MAEH, especially on the size and size distribution concern. However, most of the works about mediators in MAEH are focused on the magnetic nanoparticles. It has been proposed that particles with smaller size (nano-scaled) may pass through the venous circulation, and the ideal hyperthermia medium particle size is about10microns. Also the safety of nanoscale ferrites remains unclear. However, seldom research was carried out on developing an ideal mediator of micro-size for MAEH.ICP (with trade name of Ferronyl(?), ISP pharmaceuticals) has been approved by American Food and Drug Administration (FDA) for dietary iron supplement by oral administration due to its low toxicity and excellent bioavailability. It is elemental iron with high iron content and highly resistant to oxidation. The particle size ranges from2to10μm, and the average size is about5microns, which potentiates its further application as mediator for AEH. Our previous in vitro and in vivo experiments showed that carbonyl iron powder was safe. In the presented work, we develop ICP as magnetic mediator, to make an embolic agent by mixed with lipiodol. and the in vivo biocompatibility, inductive heating characteristics, as well as inhibitory effect of MAEH on VX2carcinoma-induced rabbit liver tumor model were studied. The deductions from the experiments may have clinical significance for application of MAEH mediated by CIP in liver cancer treatment.Part I In vitro evaluation of cytotoxicity of carbonyl iron powder and the inductive heating characteristics in alternating magnetic fieldObjective To assess the cytotoxicity of carbonyl iron powder and its potential by heated in alternating magnetic field, to evaluate the feasibility of its application to the MAEH.Methods By application of the magnetic induction hyperthermia instrument developed by our department, the elevated temperature properties of CIP-lipiodol suspension with concentrations of80,120,160,200,240mg/mL were measured by magetic field strength of45Gs, and the elevated temperature properties of CIP-lipiodol suspension with concentrations of160mg/mL were measured by magetic field strength of25,35,45,55,65Gs. Sterilized CIP samples were added to RPIM-1640cultural medium in a proportion of1cm2/mL and incubated for24h at37℃. Mouse fibroblast L-929cells were cultured in the medium of extraction of carbonyl iron powder with a concentration of100%,75%,50%and25%for24,48and72hours. Cell growth was tested by CCK-8assay and the cell relative growth rate (RGR) was calculated. The cytotoxicity was graded by generally accepted standard.Results The rate of the heating depend strongly on the CIP contents, higher concentration can ensure a higher temperature upon equilibrium finally, and higher field strength results in a greater increase in the temperature. Suspensions of160mg/mL had the ideal therapic temperature above60℃by heated within10min under45Gs of the alternative magnetic field. During the culture period, L929cells cultured in the extraction with a concentration of50%,25%showed normal morphology and proliferation compare with the control,50%and25%, extraction were demonstrated as non-toxic. While extraction with a concentration of100%、75%appeared mild cytotoxity, with a cytotoxicity level of1at24,48and72hours after incubation.Conclusions CIP has ideal inductive heating characteristics which can guarantee treatment temperature under the alternative magnetic field of500kHZ. Suspension of160mg/ml in concentration has the ideal therapic temperature in alternative magnetic field of45Gs. The CIP particles showes mild cytotoxicity, excellent biocompatibility for further investigation. Part II Feasibility of arterial embolization hyperthermia mediated by CIP/lipiodol suspensions on rabbit VX2liver tumor model and the effect on tumor growthObjective To access the feasibility and security of CIP-lipiodol suspensions mediated arterial embolization hyperthermia on rabbit VX2liver tumor model and investigate the effect of it on growth of hepatic tumor.Methods Eigthteen days after tumor implantation, the rabbit bearing VX2liver tumor were randomly divided into five groups, including the pseudo-treatment control group(Group I), lipiodol embolization group(Group II), CIP/lipiodol suspensions embolization group(Group III) and CIP/lipiodol suspensions embolization hyperthermia group(Group IV). The rabbits were exposed to alternating magnetic field three days after embolization, and heated at43-45℃for30mins. Three rabbits in group IV were conducted abdominal CT scanning immediately, three days and fourteen days after the embolization procedure respectively. Blood routine and their function of liver and kidney were tested before the embolization procedure, and at0,3and10days after the embolization procedure respectively. All animals were sacrificed on the14th day after embolization. Tumor size was measured, volume and growth rates of tumors were calculated. The changes of tumors were observed macroscopically and microscopically.Results (1) The temperature of tumor core and rim both rose rapidly and reached the objective temperature above43℃in10min in magnetic field with a strength of30-55Gs, and was maintained within43-45℃by modulating the magnetic field strength.(2) The limited higher density imaging of intratumoral CIP/lipiodol suspension deposits compared to the surrounding tissue was observed clearly by CT scanning, the density was even higher in the tumor rim than the core.(3) ALT and AST values of rabbits in group Ⅱ, Ⅲ, Ⅳ were higher than group Ⅰ (P <0.05) on the day after transcatheter embolization, but without significant difference among those three treated groups. AST in group IV was elevated after hyperthermia, with significance difference from other groups (P<0.05). ALT and AST values in all groups went down close to the normal range10days after embolization, without significant difference(P>0.05).(4)14days after embolization, the tumor volume in group Ⅱ, Ⅲ, Ⅳ were significantly smaller than group Ⅰ (P<0.01), and those in group IV were even smaller than group Ⅱ and Ⅲ (P<0.05).(5) Either on macroscopically or microscopically observeion, the tumor growth were inhibited obviously in group Ⅱ and Ⅲ comparing with group Ⅰ, and the lung metastases in the formal were less and smallar than those in the later. Microscopically, deposition of carbonyl iron powders and iodized oil in arteriole could be seen in group Ⅲ and Ⅳ, large necrotic area for group Ⅱ, Ⅲ and almost complete necrosis for group Ⅳ were observed. There were a small amount of carbonyl iron powder and iodized oil remained in the normal liver tissue arterioles of rabbits in group Ⅲ and Ⅳ, litter liver tissue surrounding embolized arterioles occured atrophy and degeneration. There were tiny amount of remained carbonyl iron in pulmonary arterioles in1rabbit of group Ⅲ and Ⅳ respectively, but lung tissue structure kept being normal.Conclusions CIP/lipiodol suspension mediated hepatic arterial embolization hyperthermia is effective in suppresses the growth of VX2liver tumors. Arterial embolization induces a reversible liver function injury, while CIP makes no burden on the liver function, hyperthermia may increase injury on the liver function. Distribution of CIP-lipiodol suspension depends on the distribution of blood vessels within the tumors, which is advantageous for the treatment of hypervascular liver tumors. CIP rare flow into the body circulation, CIPs in this size have a relatively high security, a mild increase in the particle size of CIP may be appropriate in order to further improved security in clinical trials. Part Ⅲ Comparative study of magnetic arterial embolization hyperthermia and direct injection hyperthermia mediated by CIP/lipiodol suspension in treatment of hepatic rabbit VX2carcinomaObjective To compare the effect of direct injection hyperthermia (DIH)and hepatic artery embolization hyperthermia (AEH) mediated by CIP/lipiodol suspension, and offer an experimental basis for selecting a suitable implant method of magnetic medium in the magnetic induction hyperthermia for liver cancer.Methods18days tumor-bearing rabbits were divided into two groups as AEH group and DIH group. The rabbits in AEH group were treated by hyperthermia three days after the artery embolization operation, and the rabbits in DIH group were treated by hyperthermia just after the direct injection of suspension, the two groups were both heated in43-45℃for30min. Reguard the artery embolization surgery day as the first day, all tumor-bearing rabbits had blood routine tests and liver and kidney function tests in the zero,4th, and11th days. All rabbits were sacrificed in the14day, tumor size was measured, volume and growth rates of tumors were calculated. The changes of tumors were observed macroscopically and microscopically. Results (1) The intratumoral temperature of all the rabbits can be rised to above43℃in10min during the hyperthermia. Temperature of tumor could be maintained between43-45℃in magnetic field with a strength of30-55Gs in AEH group and25-35Gs in DIH group; The temperature was higher at the tumor core than the rim in the DIH group, in contrast to the AEH group. The temperature of normal liver tissues1cm away from the tumor marginin in both groups did not exceed40℃.(2) Compare with the DIH group, white blood cell count, ALT and AST values of rabbits were higher in AEH group after hyperthermia, with significant difference (P<0.05). Till the11th day, the white blood cell count, ALT and AST values were restored to near normal levels in both groups of experimental animals, without significant difference (P>0.05).(3) The tumor volumes of rabbits in AEH group were smaller than the DIH group in the14th day, and the difference was statistically significant (P<0.05). Tumor growth of rabbits in DIH group was more active than the rabbits in AEH group both in macroscopically and microscopically observeion. All the rabbits occurred lung metastases in the DIH Group, while only two experimental rabbits in the AEH group occurred lung metastases, and the metastasis was not as large as the DIH group.(4) There were a small amount of carbonyl iron powder and iodized oil remained in the normal liver tissue arterioles of rabbits in the AEH group, and a very small number of liver tissue surrounding embolized arterioles occured atrophy and degeneration. There were tiny amount of deposited carbonyl iron powders in pulmonary arterioles, but lung tissue structure kept being normal. No carbonyl iron powder was observed in the normal liver tissues outside the tumors or in lung tissues in the DIH group.Conclusions More excellent target effect of CIP is conformed in DIH than AEH, the required magnetic field strength for the objective treatment temperature is also smaller. While the tumor inhibition effect of AEH is superior to DIH in the same treatment temperature as43-45℃. Part IV Effect of CIP-lipiodol suspensions mediated arterial embolization hyperthermia on rabbit VX2liver tumor vessels.Objective To investigate the effect of CIP-lipiodol suspensions mediated arterial embolization hyperthermia on rabbit VX2liver tumor vessels.Methods The rabbits bearing VX2liver tumor were randomly divided into five groups, including control group(Group Ⅰ), liPiodol embolization group(Group Ⅱ), CIP/lipiodol suspensions embolization group(Group Ⅲ) and CIP/lipiodol suspensions embolization hyperthermia group(Group Ⅳ). Two weeks after the embolization, expression of CD34and VEGF of tumors were detected by Super Vision immunohistochemical methods. MVD calculation was based on the number of CD34positive blood vessels.Results The MVD in tumor tissues and the positive rate of VEGF in two embolization groups were significantly lower than that in the control (P<0.01), and those in the AEH group was even more lower than in two embolization groups (P<0.01)Conclusion CIP-lipiodol suspensions mediated arterial embolizati-on hyperthermia at43-45℃can effectively injury tumor vessels and inhibit the positive expression of VEGF, which may play an important role in treatment of tumor.