Construction Of Pemetrexed Long Circulating Liposomes And Its In Vitro Antitumor Activity

Objective: To target tumor cells,a novel Pemetrexed(PEM)formulation,the PEM-loaded estrogen receptor targeted and sterically stabilized liposome(ES-SSL-PEM),was prepared and characterized.The preparation conditions were optimized,and its biocompatibility was studied.The anti-tumor effect of ER expressing positive tumor cell A549 and MCF-7 was studied in vitro.Methods:1.The synthesis of DSPE-PEG2000-ES: First,ES and SAA to generate carboxylate estrone ES-COOH,and then to generate amide coupling with DSPE-PEG2000-NH2 molecule to generate targeting fragment DSPE-PEG2000-ES.The structures of ES-COOH and DSPE-PEG2000-ES were identified by IR and NMR.2.Preparation and characterization of ES-SSL-PEM: PEM liposomes were prepared by thin film dispersion method.The effects of reaction conditions on the preparation of liposomes were studied,such as phospholipid-cholesterol ratio,hydration temperature and drug lipid ratio to confirm the feasibility of the optimal prescription and process.The particle size and Zeta potential of liposomes were detected by nano-particle size analyzer,the drug loading rate,encapsulation rate andin vitro release of liposomes were detected by ultraviolet spectrophotometry(UV).3.The biocompatibility of ES-SSL-PEM: The cytotoxic effect of ES-SSL-PEM on vascular endothelial cells(ECs cells)was detected by MTT.The adsorption of BSA by PEM liposomes was detected.The hemolysis rate of red blood cell suspension was determined by different concentrations of PEM liposomes.4.Anti-tumor effect of ES-SSL-PEM in vitro: ES-SSL-PEM in lung cancer cells with ER overexpression was selected.The proliferation inhibition of free PEM,SSL-PEM and ES-SSL-PEM on A549 cells was observed by MTT method,and the inhibition of the drug on the migration and motor ability of A549 cells was verified by scratch experiment.Results: Through IR and NMR,confirmed the ES-COOH and DSPE-PEG2000-ES were success of the synthesis.By single factor experiment,when the rate of PC and Chol 6:1,hydration temperature of 37 ?,medicine fat ratio 1:4,the ES-SSL-PEM was preparated with optimization.The average particle size of ES-SSL-PEM was 149.8±2.49,Zeta potential was-18.3±0.74 mv,DL was 6.13±0.287%,EE was52.3±0.99%.The results of in vitro release and stability study showed that ES-SSL-PEM had no significant change in particle size and Zeta potential for 12 days when stored at 2-8?,showing good stability.The drug release rate of ES-SSL-PEM was 68.3±3.3% after 24 hours in aconstant temperature oscillator at 37?.MTT showed that ES-SSL-PEM had little effect on normal cell ECs proliferation activity,the cell survival rate was no less than 90% at high drug concentration.Protein adsorption experiments showed that the BSA adsorption rate of the ES-SSL-PEM group was lower than that of the free PEM group,and the effect over time was lower than that of the free PEM group.Hemolysis experiments showed less than 5% hemolysis rate at different concentrations of ES-SSL-PEM.The anti-tumor effect of ES-SSL-PEM on the proliferation of A549 cells and MCF-7 cells were investigated in vitro and showed a strong and concentration-dependent inhibitory effect.The cell scratch experiment showed that ES-SSL-PEM had a better inhibitory effect on the free PEM of A549 cells.Conclusion: The long circle liposome ES-SSL-PEM was obtained successfully,which showed excellent physicochemical characterizations and biocompatibility.ES-SSL-PEM was able to improve the targeted delivery of PEM to ER overexpressing tumor cells,which provides new experimental data for the treatment of tumor by PEM.In the future,ES-SSL-PEM has a potential to achieve clinical practice.