Glycyrrhetinic Acid Receptor Mediated Triptolide Nanoparticles For The Treatment Of Orthotopic Liver Transplantation Cancer

Hepatic-targeted drug delivery systems have attracted much more attention recently for their promising roles in increasing the efficiency of pharmaceutical agents for liver,reducing drug doses and significantly decreasing the toxic side effects.Glycyrrhetinic acid-modified chitosan?GA-CTS?is used as liver-targeted carrier for drug delivery.In this paper,nanoparticles were prepared by ionic gelation process.The antineoplastic effect of the triptolide?TPL?-loaded nanoparticles was also investigated in vitro and in vivo.Chapter I:Preparation of GA-CTS/TPL nanoparticlesGlycyrrhetinic acid?GA?conjugate of chitosan?CTS?was synthesized.The structure of the product was confirmed by infrared spectroscopy and nuclear magnetic resonance spectroscopy techniques.The nanoparticles was prepared by ionic gelation method.The physicochemical properties of GA-CTS/TPL nanoparticles was determined.The particle size of GA-CTS/TPL nanoparticles was?197±9?nm and the polydispersity was?0.30±0.03?.Besides,the GA-CTS/TPL nanoparticles was observed with circular,uniform distribution and no reunion phenomenon by transmission electron microscopy?TEM?.The encapsulation efficiency?EE%?was?34.8±2.8?%,and the drug loading?DL%?was?0.220±0.011?%.In vitro drug release was performed by dialysis bag at pH 6.8,7.4 and pH 7.4 in plasma.The GA-CTS/TPL nanoparticles provided a better sustained-release system at pH 7.4 in PBS buffer,while the release curve was consistent with the monoexponential equation.ChapterII:The antineoplastic effect of GA-CTS/TPL nanoparticlesThe inhibitory effect of GA-CTS/TPL nanoparticles and TPL solvents on HepG2,L02,MCF and HL-60 cells were determined by MTT assay.In vitro data indicated that GA-CTS/TPL nanoparticles could inhibit the above tumor cells in dose-depended.The effective drug exposure time against hepatic cancer cells was increased in comparison with that observed with TPL solvents.But for normal liver L02 cells,the24 h inhibition rate of GA-CTS/TPL nanoparticles was less than TPL solvents.Endocytosis of GA-CTS nanoparticles by normal liver L02 cells and by hepatic cancer HepG2 cells was analyzed under confocal microscopy.Strong green fluorescence was observed in HepG2 cells following exposure to GA-CTS nanoparticles.While HepG2 cells exposed to CTS nanoparticles showed moderate intensity fluorescence.However,only weak intensity fluorescence was observed in L02 cells exposed to GA-CTS nanoparticles.GA-CTS nanoparticles targeted hepatic cancer cells and entered the cells through receptor-mediated endocytosis.The orthotopic liver transplantation in nude mice was successfully established.GA-CTS/TPL nanoparticles and TPL solvents were injected into the tail-vein of nude mice with the dose of 0.2 mg/kg everyday.In vivo tumor inhibitory rates of GA-CTS/TPL nanoparticles,TPL solvents,and 5-Fluorouracil?5-FU?groups?10mg/kg,q.3d,iv?were 58.3%,51.6%,and 48.7%,respectively.In the GA-CTS/TPL nanoparticles group,tumor weight were significantly with the control group?P<0.01?,but no differences were seen between the GA-CTS/TPL nanoparticles group and TPL solvents group?P>0.05?.Only the GA-CTS/TPL nanoparticles group liver indicators of Alanine aminotransferase/Aspartate aminotransferase?ALT/AST?ratio was higher than 1,the toxicity of GA-CTS/TPL nanoparticles in liver was less than the other groups.Chapter III:Pharmacokinetics study of GA-CTS/TPL nanoparticles in orthotopic liver transplantation in miceLiquid chromatograph-mass spectrometer?LC/MS?was established to determine the concentration of TPL in plasma and different tissues?liver,heart,kidney,lung,spleen?of mice.The pharmacokinetics and biodistribution of GA-CTS/TPL nanoparticles and TPL solvents were investigated.Orthotopic liver transplantation in mice was established.GA-CTS/TPL nanoparticles and TPL solvents were injected into the tail-vein of mice with the dose of 0.2 mg/kg.Pharmacokinetics parameters such as C_max,t_1/2,MRT?Mean Residence Time?_0-180min,AUC?Area under curve?_0-180min and AUC_0-?of GA-CTS/TPL nanoparticles were 1.03,2.16,1.76,1.55,and1.81 times to the TPL solvents in the plasma respectively.Among them,the t_1/2,Vz,CLz,AUC_{0-180 min},AUC_0-?between the two groups were statistically significant.GA-CTS/TPL nanoparticles had longer retention time and higher bioavailability than the TPL solvents in plasma.In tissue distribution,the AUC_{0-180 min} in liver and lungs were found to be a significant difference and the MRT in liver tumor was found to be a significant difference between the two groups.The liver targeting choice efficiency T%was selected as a target effect indicator.Compared with the solvents group,GA-CTS/TPL nanoparticles in liver and liver tumor had higher target efficiency,while lower in the other tissues.Conclusion:In order to enhance the anti-liver tumor effect and reduce the liver damage of TPL.The GA-CTS with liver targeting was synthesized.GA-CTS/TPL nanoparticles were prepared.The size of GA-CTS/TPL nanoparticles was small,and the GA-CTS/TPL nanoparticles were selectively on hepatic cancer cells.It had a good tumor inhibitory rates in liver orthotopic transplantation in nude mice.While the toxicity of GA-CTS/TPL nanoparticles in liver was lower than the other groups.Meanwhile,GA-CTS/TPL nanoparticles had a longer residence time and higher bioavailability in the plasma of orthotopic liver transplantation in mice.Pharmacokinetics datas were consist with the pharmacodynamics results.It targeted liver tumor and reduced the distribution in other tissues.To summarize,GA-CTS/TPL nanoparticles can enhance higher selectivity in liver tumor and reduce the liver damage of TPL,which can provide a new method in orthotopic liver transplantation therapy.