Preparation And Evaluation Of GA Grafted Low Molecular Weight Heparin Self-assembly Micelles

Object:In this subject, gambogic acid grafted natural hydrophilic polysaccharide low molecular weight heparin(GA-LMWH) were perpared to improve solubility of GA and self-assembled into amphiphilic polymer micelle in aqueous solution. The analysis method of GA-LMWH in vivo were established, and it’s tissue distribution and targeting tendency were determined, The anti-tumor effect of GA-LMWH in hepatocellular carcinoma(HCC)model mice were evaluated.Methods:(1) Preparation and characterization of GA-LMWH: Utilzing gambogic acid reacted with low molecular weight heparin, we prepared a novel amphiphilic copolymers GA-LMWH; The optimal prescription was established on single factor test and orthogonal test; The degree of substitution was detected by UV and FTIR, DSC, SEM, TEM, particle size, potential, and CMC were used to characterize GA-LMWH.(2) The pharmacokinetics of GA-LMWH micelles in mice: The stability of micelles in vitro plasma were inverstigated, and the targeting effect of GA-LMWH micelles were evaluated in mice, we gained epipolic GA-LMWH micelles labeled with FITC which particle size was around 200 nm; GA-LMWH were injected in caudal vein of mice,micelles content at different time points were detected by fluorospectro photometer, the AUC, MRT, et al were caluculated using pharmacokinetic software.(3) Antitumor effect of GA-LMWH: Orthotopic transplantation model of liver cancer with H22 ascites tumor cells were established, the antitumor effect of GA-LMWH and GA water solution were eluvated by liver fuction and biopsy staining method.Results:(1) Preparation and characterization of GA-LMWH: The optimal prescription of GA-LMWH was established by single factor test and orthogonal test, the preparetion conditions as follows: the temperature of activated intermediate ester was 0 oC, ethanediamine was 200 m L(GA: 0.3mol), The mole ratio of reactants was n(heparin sodium):n(GA):n(EDCI)=1:20:10. The results of FTIR and DSC showed that GA-LMWH was successfully prepared, the degree of substitution was(27.45±0.22913)%, SEM and TEM were used to observed the uniform morphology of micelles, the particle size was 200 nm and potential was-30 m V, and it showed that micelles were stable; Fluorescence detection instrument was used to calculating CMC which was(0.0024 ± 0.00019)mg × m L-1, it indicated that micelle can not been damaged when it was diluted in blood.(2) The pharmacokinetics of GA-LMWH micelles in mice: Plasma stability test showed that micelles can be stable in plasma in 24 h, and amide linkage was not broke.FITC labeled LMWH to react with GA to generate GA-LMWH-FITC; After injection of GA-LMWH-FITC in caudal vein of mice, they were enriched in the liver, and MRT has been lengthened significantly compared with GA.(3) Antitumor effect of GA-LMWH: The results showed that there are significant difference in the liver fuction and survival time between LMWH group and model group.Although GA-LMWH group and GA group could not reduce malignant ascites,GA-LMWH group were better in reduction of the growth of ascites and liver surface nodules, liver fuction, and survival time than GA treatment.Conclusion:Gambogic acid was grafted onto the Low molecular weight heparin by chemical synthesis to prepare amphiphilic copolymers, it could form nano micelle in water.GA-LMWH micelle not only retained antitumor activity of GA but also lengthened MRT,GA-LMWH has better antitumor effect than GA in the orthotopic transplantation tumor experiment.