Preparation And Study On Antitumor Activity Of Carboxymethyl Chitosan Coated Kaempferol Liposomes

Kaempferol has a wide range of pharmacological activities,and has broad prospects for the development and clinical application of preparations.However,the physiological properties of kaempferol have limited its further development and utilization.The purpose of this project is to develop a kaempferol liposome coated with a layer of hydrophilic substance,in order to reduce the external adsorption of internal drugs,improve its stability,and enhance the exertion of the pharmacological activity.Meanwhile,the in vitro release behavior of kaempferol and different liposomes was evaluated,and its antitumor mechanism was initially explored.The main contents and results are as follows:1.HPLC method for the determination of kaempferol and liposome was established in pre prescription study.The apparent oil-water partition coefficient of kaempferol showed that kaempferol is a poorly soluble drug.The solubility of kaempferol in 5% Tween-80 solution is the highest,and 5% Tween-80 solution could be chosen as the release medium of kaempferol in vitro release tests.2.With egg yolk lecithin and cholesterol as the main excipients,the conventional liposomes were prepared by thin film dispersion method.The ultrasound probe was used for 10 minutes to disperse the liposomes.Taking the particle size and entrapment rate of liposomes as the index,the optimal formulation of lipid preparation was selected by single factor test(kaempferol: lecithin: cholesterol: water and medium = 5:100:8.35:5).The cationic liposome was prepared by adding octadecylamine(octadecylamine: kaempferol = 1:40),dropping it into 0.2% carboxymethyl chitosan solution,stirring it at a constant speed for 30 min to form kaempferol liposome coated with carboxymethyl chitosan.3.Investigate the physical and chemical properties of the two kinds of liposomes.The morphologic characteristics of the both liposomes were regular spherical,no adhesion between the particles,and there was a layer of translucent substance on the surface of the carboxymethyl chitosan coated liposomes under the transmission electron microscope.The particle size and potential of CMCS-KLs were slightly larger than KLs.The entrapment efficiency of liposomes was 83.19±2.52% by Sephadex column method.The stability test showed that carboxymethyl chitosan coated with lipid could improve the stability of liposome nanoparticles in vitro.The preliminary safety evaluation of kaempferol liposomes was carried out in pre-and post-coated liposomes by vitro hemolysis and abnormal toxicity tests.The results showed that the high safety of the both liposomes although differences in existence.The abnormal toxicity tests were qualified though there was no abnormal phenomenon during the experimental observation period.4.The in vitro release behavior of the drug was investigated by dialysis bag method.The results showed that pH had no significant effect on the drug except CMCS-KLs release rate.The release rate of two liposomes under three pH conditions was significantly higher than that of kaempferol,and the pre release rate of modified liposomes was lower than that of KLs.It indicated that CMCS could block the release of liposomes to some extent.5.SCG-7901 cell line was used as the experimental object to investigate the antitumor mechanism of Kae,KLs and CMCS-KLs by cytotoxicity experiment,AO staining,colony formation experiment,cell cycle arrest and apoptosis experiment.The results demonstrated that CMCSKLs inhibited the proliferation and clone of SCG-7901 cells line obviously,and induced cell apoptosis.The early apoptosis rate was 57.19%,higher than Kae group 44.13% and 51.28% in KLs group.Cell cycle distribution showed that kae,KLs and CMCS-KLs could inhibit cell proliferation in S phase,and CMCS-KLs had the most significant effect.In this paper,liposomal preparation can significantly increase the solubility of kaempferol and improve the drug release behavior in vitro;the adjuvants used are safe and can be used for intravenous injection;enhance the anti-tumor activity of the drug and induce the apoptosis of SCG-7901 cells.