Study On Preparation And Tissue Distribution Of Chlorambucil Liposomes

Objective To improve solubility and stability of chlorambucil,and to increase the possibility of transfer drugs into cells by prepared chlorambucil liposome.Liposome can change the tissue distribution of drug,reduce the side effects,improve the therapeutic index that provided a reference for the development of new dosage forms of chlorambucil.Methods Chlorambucil liposome was prepared by film-ultrasonic method which soybean phospholipid and cholesterol as materials.HPLC method for determination of chlorambucil was established.The formulation of liposomes was determined by single factor analysis and orthogonal optimization.The encapsulation efficiency was determined by microcolumn centrifugation-HPLC method.The particle size and distribution and Zeta potential of the liposome prepared by the best process were investigated with a laser particle size analyzer,the appearance of the liposomes were characterized by Transmission Electron Microscopy,and the in vitro release characteristics were investigated by the small cup method.Chlorambucil freeze-dried liposomes was prepared by freeze-drying technology.Taking the encapsulation efficiency as the main index,the redispersibility and appearance of liposomes were investigated in concert to screen the optimal freeze-drying process and the type and amount of protective agent.The content of chlorambucil in biological samples was determined by HPLC-MS method.Using kunming mice as experimental object.Chlorambucil solution and chlorambucil liposome was injected on the tail vein.Study on the distribution difference of chlorambucil in plasma,heart,liver,spleen,lung,and kidney tissues of mice.The Relative targeting efficiency?Re?and targeted efficiency?Te?as the evaluation index to evaluate targeting effect of chlorambucil liposome.Results Established the HPLC method for determination of chlorambucil,The best prescription condition was as follow:the ratio of cholesterol to phospholipid was 1:3,the ratio of drug to phospholipid was 1:10,the p H value of PBS was 7.4 and the concentration of phospholipid was0.3%.The encapsulation efficiency of the chlorambucil liposome prepared according to the prescription was 88.42%?n=3?,the average particle size was 149.3±4.58 nm?n=3?,polydispersed index?PDI?was 0.248±0.04?n=3?,zeta potential was 6.79±0.33-m V?n=3?,the particle was well distributed.In the in vitro release experiment,the cumulative release rate of liposomes was 82.36%at 24 h,which was better than the cumulative release rate of chlorambucil?68.46%?,and provided a theoretical basis for the in vivo study of the preparation.The freeze-dried liposomes was prepared by 15%mannitol-lactose?1:1?as protective agents,pre-frozened at-80?for 24 h and freeze-dried for 24 h.The freeze-dried liposome had good appearance and redispersibility.The average encapsulation efficiency was 87.98%?n=3?,the average particle size was 153.6±2.21 nm?n=3?,PDI was 0.339±0.05.A method for the determination of chlorambucil in biological samples by HPLC-MS was established.The pharmacokinetic results of mice plasma showed that the AUC of the liposome group was 198309.28±33669.49 ng·min/g,which was increased compared to the AUC of the solution 165740.10±42144.09 ng·min/g;The t_1/2was 26.598 minutes,which was longer than the solution group's t_1/2 of 5.967 minutes,which prolonged its half-life and increased the drug content in mice.According to the study of mice tissue distribution,liposomes can increase the concentration of chlorambucil in mice plasma and tissues.Liposomes have a certain targeting effect on mice plasma,heart,liver,spleen,lung and kidney,the Re of liver is 1.85,which had the best targeting effect.There was little difference in the Te between the two dosages in heart,spleen,lung and kidney,while the liposome was 0.70 in liver,which was significantly higher than that of solution agent and had the strongest selectivity to liver tissue.Conclusion In this study,chlorambucil liposome was successfully prepared.The preparation process used was simple and reproducible.The prepared chlorambucil liposomes have uniform particle size distribution and enhanced in vitro release rate;the freeze-drying technology achieved the storage stability of the chlorambucil liposome.This liposome showed certain liver targets in mice.In summary,this study provides a reference for the development of new formulations of chlorambucil.