Effects And Mechanisms Of Hyaluronidase On Intratumoral Drug Penetration And Therapeutic Efficacy Of Transarterial Chemoembolization In Liver Cancer

PART ?: Cytotoxicity assay of hyaluronidase and doxorubicinObjective: To study the toxicity of hyaluronidase and doxorubicin to Hep G2 cells and decide the best drug concentration and processing time.Materials and Methods: The Hep G2 cells in the logarithmic growth phase were cultured in a blank medium and a medium containing different concentrations of hyaluronidase or doxorubicin for different times.After the predetermined culture time of the cells,the 450 nm light absorption value was measured by a microplate reader.The solvent-treated cells were used as a control group,and the cell-free medium was a blank group.The inhibition rate of the drug on the cells was calculated according to the formula.Results: There were no significant differences in the inhibitory rates of hyaluronan different drug concentrations and duration of process on Hep G2 cells.The IC50 at 0,30,60,and 120 minutes were 536.743?M,483.112?M,425.019?M,and 416.206?M,respectively.The inhibitory rates of doxorubicin at different concentrations and duration of process on Hep G2 cells showed that IC50 at 0,30,60,90,and 120 minutes were 4622.167?M,1273.328?M,678.042?M,329.502?M,and 183.767?M,respectively.Conclusion: Under different hyaluronidase drug concentration and processing time,there is no obvious toxic effect on tumor cells;combined with the experimental results,the concentration of hyaluronidase was 160?M in subsequent cell experiments,the process time was 60 minutes.Meanwhile,because the follow-up experiment involves the amount and penetration distance of doxorubicin drug fluorescence in multi-cell spheroids,the cell survival rate is required to be above 80%.The optimal combination of doxorubicin drug concentration is 160?M,the process time was 60 minutes.PART ?: Effect of hyaluronidase on the distribution of doxorubicin in tumor multicellular spheroidsObjective: To study the effect of hyaluronidase on the distribution of doxorubicin in multicellular spheroids through cell experiments.Materials and Methods: The multi-cell spheroids of human Hep G2 hepatoma cells were cultured in vitro by using the flask agar method.They were divided into two groups and treated with doxorubicin,doxorubicin and hyaluronidase respectively.The concentration of hyaluronidase was 160?M and the processing time was 60 minutes.After treatment,the distribution of doxorubicin drug fluorescence in tumor multicellular spheroids was observed by fluorescence microscope,and the amount and penetration distance of doxorubicin drug fluorescence in multicellular spheres were calculated by Image Pro Plus software.Results: There was no significant difference in the diameter of Hep G2 multicellular spheroids between the two groups.The doxorubicin with hyaluronidase group was significantly higher than the doxorubicin group in the fluorescence area,fluorescence intensity and fluorescence penetration distance of doxorubicin.Conclusion: Hyaluronidase can significantly promote the penetration of doxorubicin in tumor multicellular spheroids.PART ?: Effect of hyaluronidase on tumor interstitial fluid pressureObjective: Compare the effects of hyaluronidase on tumor interstitial fluid pressure with different routes of administration and decides the best hyaluronidase administration schedule and processing time.Materials and Methods: Purchased 15 New Zealand white rabbits,male or female,weighing 2.5~3.0kg.Fifteen experimental rabbit VX2 liver cancer models were established by transplantation,and the tumors were externally located in the left outer lobe of the liver.Tumor size was measured by MRI on the 17 th day after surgery.15 tumor-bearing rabbits were divided into 3 groups(5 in each group),group A(blank control group);group B(IV group)was injected with hyaluronidase solution through the rabbit ear vein(10ml,10mg/ml);group C(hepatic arterial infusion group,TAI group)was perfused with hyaluronidase(10ml,10mg/ml)through the hepatic artery;At first,we embed the pressure catheter into the experimental rabbit VX2 tumor and observe the change of pressure in real time,After the pressure become stabilized,the hyaluronidase was perfused and the interstitial fluid pressure was recorded.After the experiment,the time-interstitial fluid pressure curve was drawn according to the relevant values.Results: The rate of change in the intratumoral interstitial hydraulic TAI group was the highest,followed by the IV group,and the control group was the smallest.After 40 minutes of administration,the tumor interstitial fluid pressure dropped to the “platform stage”.Conclusion: Hyaluronidase can effectively reduce the interstitial fluid pressure in the tumor.The route of applying hyaluronidase through hepatic artery by intervention is more advantageous.The tumor interstitial fluid pressure tends to be stable after 40 minutes of administrationPART ?: Efficacy and mechanism of transcatheter interventional therapy combined with hyaluronidase in the treatment of liver cancerObjective: To evaluate the effect of TACE combined with hyaluronidase on drug penetration and efficacy of transcatheter interventional therapy for hepatocellular carcinoma,and to explore the mechanism of combined therapy method.Materials and Methods: Twenty rabbit VX2 liver cancer models were established and divided into two groups(10 in each group)receiving doxorubicin via hepatic artery injection + PVA embolization(TACE alone)and hyaluronidase through hepatic artery injection + doxorubicin via hepatic artery injection + PVA Embolization(TACE combined with hyaluronidase group);5 animals in each group were tested for tumor interstitial fluid pressure by microtip catheter pressure sensor 0 to 1 hour after treatment,and were sacrificed 4 hours,then using immunofluorescence technique to detect drug penetration.The other 5 animals in each group were sacrificed 7 days after treatment.The tumor necrosis rate was evaluated by HE staining.The interstitial differences in tumor interstitial fluid pressure,tissue drug penetration and concentration and necrosis rate were compared,and the relationship between them was analyzed.Results: There was a significant difference in interstitial fluid pressure between the two groups.TACE combined with hyaluronidase group(58.7%±9.8%)was significantly greater than c TACE group(35.8%±8.3%)(P=0.004);TACE combined with hyaluronic acid The fluorescence points of doxorubicin in the enzyme group and c TACE group were 3390.91±945.73 and 1564.38±633.99,respectively(P<0.01).The relative permeation distances of doxorubicin were 38.06±8.72 ?M and 29.51±4.76 ?M(P<0.05),respectively.The number of fluorescent spots and the penetration distance of doxorubicin in the hyaluronidase group were significantly higher than those in the simple doxorubicin group.Comparison of TACE combined with hyaluronidase group and c TACE in the concentration of doxorubicin showed that the concentration of doxorubicin in the tumor was lower than that in the tumor at 4 hours after procedure.Meanwhile,The concentration of doxorubicin in the TACE combined with hyaluronidase group was significantly higher than that in the c TACE group,and the difference was statistically significant(P=0.021,P=0.0004);the tumor necrosis rate in the TACE combined with hyaluronidase group(90.61%±5.38%)was also significantly higher than the c TACE group(77.49%±7.11%)(P=0.011).Conclusion: The hyaluronidase can promote the penetration of doxorubicin into the tumor parenchyma by reducing the interstitial fluid pressure in the tumor,improving the drug distribution,increasing the drug concentration in the tumor parenchyma,and thereby improve the therapeutic effect of TACE.