System Construciton And Theoretical Analysis Of Cucurbitacin B Liposome Assisted With Tcd Components For Active Targeting To Hcc

As a high mortality and obstinate illness,hepatoma threats the human’s health seriously.Drug therapy plays an important role on the cancer treatment,however,the toxicity of chemotherapy drugs and the resistance of tumor cells limits the effectiveness.It is important to choose the drugs with low toxicity,synergistic application,MDR reversing and specific targeting.Under the guidance of traditional Chinese medicine(TCM)theory,people are keen to adopt high-efficiency and low-toxicity components to treat hepatoma from traditional Chinese drugs(TCD).In this paper,under the guidance of TCM theory,active targeting liposome was designed with TCD components,such as cucurbitacin B,curcumin and glycyrrhetinic acid.The preparation was tumor-target,sustain-release,membrane-adherence and biocompatibility on the basis of the activity of cucurbitain B,the auxiliary of curcumin and the tendency of glycyrrhetinic acid.The anti-hepatoma principle of different components and the delivery,location and release mechanisms of liposome were investigated in the designed course.The active targeted cucurbitacin B liposome was constructed with good stability,exact effect,little toxicity,weak resistance and obvious hepatoma-targeting.The synergistic effect of cucurbitacin B in combination with curcumin was studied on hepatoma BEL7402/5-Fu cells.The synergistic anticancer activity of the 2 compounds involved 2 mechanisms.Firstly,curcumin synergistically enhanced the apoptosis of the cells induced by cucurbitacin B in the optimal mass ratio of 2:1(cucurbitacin B:curcumin).The mechanism may result from the cell arresting in different phases of cell cycles,activation of Caspase 3 and the apoptotic change of ultrastructure in BEL7402/5-Fu cells.Secondly,curcumin reversed the multidrug resistance(MDR)caused by cucurbitacin B in the optimized concentration of 25μg/ml.The mechanism was associated with the Rho123 axxumulation,P-gp reduction,ΔΨm collapse and mitochondrial colocalization in BEL7402/5-Fu cells.18β-glycyrrhetinic acid(18β-GA)、18α-glycyrrhetinic acid(18α-GA)、3-acetyl glycyrrhetinic acid(3-Aceyl-GA)、11-deoxy glycyrrhetinic acid(11-Deoxy-GA)and fluorescence labeled glycyrrhetinic acid(FITC-GA)were obtained as glycyrrhetinic acid derivations.In human hepatoma HepG2 cells,the specific binding saturation of glycyrrhetinic acid showed the dissociation constant(Kd)and binding sites was about 7.457±2.122 pmol/L and 2.385±0.175 pmol/2.5×106 cells respectively.Using fluorescence detection and confocal laser scanning microscope(CLSM)examination,we showed the receptor competitiveness of different glycyrrhetinic acid derivations was 3-Aceyl-GA≈18β-GA>11-Deoxy-GA>18α-GA.The targeting tendency was closely related to the construction of glycyrrhetinic acid.βconfiguration mainly contributed to the receptor specific binding,but a configuration was weak.The results indicated that the C3-hydroxyl or C11-carbonyl group had little or certain influence on the targeting ability,too.A series of DSPE-PEGlyated glycyrrhetinic derivations,18β-GA-PEG-DSPE,18α-GA-PEG-DSPE,3-Aceythl-GA-PEG-DSPE and 11-Deoxy-GA-PEG-DSPE were synthesized as guidance compounds to prepare coumarin 6(Cou6)liposomes.In HepG2 cells,the results of uptake indicated that the liposomes with 18β-GA-PEG-DSPE and 3-Aceythl-GA-PEG-DSPE showed better affinity to the cells membrane.DiR was loaded in the liposomes with guidance compounds as fluorescent probe.In H22 tumor-bearing nude mice,the results of in vivo fluorescence imaging revealed that the targeting activity of glycyrrhetinic acid affect more on transport speed,but less on transport quantity and further proved the relation of the guidance tendency with the structure of glycyrrhetinic acid to hepatoma cells.The transport processes of glycyrrhetinic acid and modified liposome labeled(FITC-GA and Cou6-liposome)by fluorescence were studied in the following study in HepG2 cells.We have demonstrated that the uptake FITC-GA of the cells included passive diffusion and active transport process,and the efflux FITC-GA from the cells approached to exponential decay process.We have also demonstrated that caveolin mediated the Cou6-liposome uptake,and second order fitted the efflux Cou6 process of the cells.The Cou6 contents were detected by HPLC-FLD in blood,liver and tumor tissues after treated with Cou6-liposome to H22 tumor-bearing nude mice.The pharmacokinetics equations showed that the glycyrrhetinic acid mediated liposome expressed significant active targeting tendency to liver and hepatoma with higher bioavailability.And then,the compound liposome of cucurbitacin B and curcumin mediated with glycyrrhetinic acid was prepared and evaluated.Our results supported that the compound liposome inhibited the proliferation of hepatoma tumor and reversed the resistance of hepatoma cells effectively in hepatoma cells in vitro and in tumor-bearing nude mice in vivo.The compound liposome mediated with 18β-GA showed toxicity reducing and efficacy enhancing.Anti-tumor,gaining weigh,prolonging survival and prefect living condition were proved in tumor-bearing nude mice in vivo.We have focused a road to treat in HEPATOMA,and to provide reference to disease of liver cancer and other malignant tumors for comprehensive treatment in TCD components via the research.Furthermore,We hope to illustrate the guidance of TCM theory to modern drug delivery system design.