Preparation Of Chitosan Coated Nanoliposome Loaded Betanin And Research On Its Antiproliferative Activity

Betanin is a water-soluble natural pigment rich in plants,which is often used as food colorants.Betanin possesses lots of biological activities,but it is easy to be degraded under external conditions such as enzyme,oxygen,high temperature and light,resulting in low stability.Besides,it has low bioavailability and is hard to be absorbed by the body.To improve its stability and bioavailability,liposome modified by chitosan with negative potential via electrostatic interaction was produced to encapsulate betanin,which was chitosan coated nanoliposome loaded betanin(CS-NL).It can improve the stability of liposome,prevent the interactions of nanoliposome and food components,and also can maintain drug activity and prolong and control drug release.Then,the structure characterization and stability analysis of CS-NL were evaluated.Finally,the antioxidant effects and antiproliferative effects of CS-NL were explored.The main results of this work were as follows:(1)The encapsulation efficiency of liposomes prepared by reverse phase evaporation,thin film dispersion,ethanol injection and ether injection were compared,which indicated that liposome loaded betanin produced by the reverse phase evaporation method had the highest encapsulate efficiency up to 21.29 ± 1.37%.The optimum conditions for NL preparation were as blow: the concentration of lectin,60mg/m L;the ratio of betanin and lectin,1:18;the ratio of lectin and cholesterol,6:1;ultrasound time,8 min.The encapsulation efficiency of NL was 42.67 ± 1.67%,and the drug loading was 2.63 ± 0.42%.The optimum concentration of chitosan binding to liposomes was 0.6%.The average size,PDI and zeta potential of CS-NL were 194.60 nm,0.29 and 5.80 mv,respectively.(2)The structures of NL and CS-NL were characterized by Transmission Electron Microscopy(TEM)and Fourier transform infrared spectroscopy(FTIR).The drug release behavior,stability and antioxidant activity of liposomes were investigated.TEM results indicated that liposomes showed regular sphericity,and chitosan was covered on the outer layer of the liposome.FTIR results showed that chitosan was bound to liposome via electrostatic interaction.The in vitro release study showed that the cumulative release rate of NL and CS-NL reached up to 28.98 ± 0.32% and 15.79 ±0.20% respectively,which showed that NL and CS-NL had good slow-release effect.p H stability experiment showed that NL and CS-NL were susceptible to p H.With the increasing of p H,the average size of NL had slight changes,but the average size of CSNL first increased and then reduced;the zeta potential of NL and CS-NL both decreased.The in vitro simulated digestion study indicated that NL and CS-NL were relatively stable in simulated salivary fluid and simulated gastric fluid,but were both digested in simulated intestinal fluid.(3)The PSC and ORAC value of NL and CS-NL increased compared to betanin,and CS-NL had the highest PSC and ORAC value.Meanwhile,The CAA value of NL and CS-NL were significantly higher than betanin,which was because that betanin exerted its antioxidant effects via attaching the cell membrane surface,but NL and CSNL could enter the cell and play the role of antioxidants.The cytotoxicity and antiproliferation activity of CS-NL were evaluated by methylene blue assay.Flow cytometry and RT-q PCR were used to explore the possible antiproliferative mechanism of CS-NL.The results indicated that CS-NL and betanin could inhibit the growth of MDA-MB-231,Hep G2 and A375 cells,and CS-NL showed stronger inhibitory effects and more cytotoxicity than betanin against three cancer cell lines.CS-NL had the best inhibitory effect on A375 cells,followed by MDA-MB-231 and Hep G2 cells,but the cytotoxicity of CS-NL to A375 cells was also high.CS-NL arrested the MDA-MB-231 cells in G2/M phase,increased the rate of apoptotic cells in a dose-dependent manner,significantly decreased the mitochondrial membrane potential,increased the mitochondrial membrane permeability and reduced the cellular ROS levels in MDAMB-231 cells.CS-NL could downregulate bcl-2 and bcl-xl,upregulate bax,cytochrome c and caspase 3 and increase the ratio of bax/bcl-2,which indicated that CS-NL could regulate the Bcl-2 family,resulting in cytochrome C release into the cytoplasm to activate the caspase cascade reaction,thus regulate the mitochondria-mediated apoptosis pathway and inhibit MDA-MB-231 cells.Meanwhile,CS-NL downregulated cyclin b1 and cdc2,thereby reduced the activity of cyclin b1-cdc2 complex preventing cells from entering the M phase,and inhibited the proliferation of MDA-MB-231 cells by blocking cells in the G2/M phase.