Construction And Evaluation Of Ginsenoside Biomimetic Liposomal Drug Delivery System

Currently,drug chemotherapy possesses a primary role in clinical treatment of tumors.However,most drugs have poor solubility,induce tumor drug resistance,and lack tumor targeting capabilities,resulting in a suboptimal efficacy of drug chemotherapy.The targeted drug delivery system carries the promise of enhancing treatment efficacy of chemotherapy and reducing toxicity,but its clinical transformation is hindered due to its complex compositions and relatively cumbersome preparation process.Therefore,development of a convenient,affordable and highly efficient targeted drug delivery system is of great significance in the field of tumor treatment.Inspired by the multi-functional sugar chains on the surface of cell membrane,a biomimetic structure(Ginposome)was constructed in this study by replacing the cholesterol molecules with ginsenoside Rg5 containing lipophilic steroid skeleton and hydrophilic glycosyl group.With paclitaxel(PTX)as a model drug,paclitaxel-loaded ginsenoside Rg5 liposome(Ginposome-PTX)was obtained,which displayed the following characteristics:(1)optimal drug loading capacity(PTX concentration of over5 mg/m L could be reached);(2)prolonged circulation time;(3)GLUT1 mediated active targeting,which is not affected by serum glucose levels;(4)enhanced tumor suppression and retained the ability of ginsenoside Rg5 to reverse acquired drug resistance;(5)reliable preclinical safety.This technology platform can overcome adversities faced by the development of drug delivery systems,provide efficient encapsulation and safe delivery for various anti-cancer drugs,and allow for clinical translation due to its simple biomimetic structure.1.Screening of ginsenosidesGinsenosides Rh3,Rg5,Rh4,Rf,F1,Rg1,F2 and Rb1 with glucosyl groups at the3,6,or 20-position were used to screen for the ideal cholesterol substitute.The effect of ginsenosides on the fluidity of the lipid membrane was detected by DPH fluorescence probe.Rg5 with glycosyl moiety at the 3-position of the skeleton have better stability on the lipid membranes than cholesterol.According to molecular dynamic calculation,glycosyl orientation towards water molecules can only be ensured when cholesterol is replaced with ginsenoside containing glycosyl moiety at the 3-position.Glycosyl group at an alternative carbon position,such as 6-position,results in transverse stretching and disruption of phospholipid arrangement,which reduces the structural stability of the liposome membrane.Meanwhile,the binding affinity between Rg5 and GLUT1 was analyzed,which was not subjected to the interference by high level of free glucose.Results were further verified at a cellular level under high-glucose medium.Therefore,Rg5 is preferred as a cholesterol substitute for the construction of the liposomes.2.Preparation and characterization of Ginposome-PTX(G-PTX)To develop a drug-loaded preparation,paclitaxel,one of the most widely used chemotherapy drugs in clinical practice,was selected as the model drug.Molecular dynamics simulations showed that Rg5 formed intensive hydrogen bonds with POPC and PTX,which further constituted hydrogen bond networks to stabilize the entire system.The stability of G-PTX tended to optimize when the ratio reached 3:1,with increasing the molar ratio of Rg5 to PTX and was superior compared to that of drug-loaded liposomes containing the same proportion of cholesterol.When the ratio reached 4:1 with increasing the molar ratio of Rg5 to PTX,its in vitro targeting function was not subjected to the interference by free glucose.Apropos the optimal drug loading ability and targeting function,the molar ratio of Rg5 to PTX in G-PTX was 4.5:1(4:1,mass ratio).G-PTX had a spherical shape with an average particle size of approximately110 nm and was highly stable with increased concentration of PTX.In vitro drug release results showed that G-PTX has an obvious trend of sustained drug release compared to that of conventional cholesterol liposome encapsulating paclitaxel(L-PTX).G-PTX could be freeze-dried into powder form and the particle size and drug content were minimally altered after redissolution.molecule dynamic simulation of 200 ns further confirmed the marked improvement in integral stability of G-PTX.3.Evaluation of the long circulation and active targeting function of G-PTXOn the basis of the high drug-loading concentration and high stability of G-PTX,we further investigated its expected long circulation and active targeting function in vivo.The glycosyl increased the surface hydrophilicity of G-PTX,which can reduce the content of protein corona adsorbed on the surface of G-PTX and avoid phagocytosis by phagocytic cells to a certain extent.The results of pharmacokinetics also showed that G-PTX significantly increased the blood concentration and prolonged the circulation time of PTX compared with L-PTX and Abraxane(Abr)at the same given dose.In vivo and in vitro fluorescence imaging showed that G-PTX was increased at HGC-27 subcutaneous and orthotopic tumor site.Similar trends were observed in two other subcutaneous tumor models(A549 and MCF-7),demonstrating a broad-spectrum tumor-targeting ability of G-PTX.The tissue distribution of mice under the free-feeding conditions revealed that the drug content of G-PTX in the subcutaneous tumor of HGC-27 was significantly higher than the other 3 groups(L-PTX,Abraxane and Taxol).On the other hand,the drug content in the liver and spleen of the G-PTX group was significantly lower than the L-PTX group.A decrease of drug content in muscle and heart tissues was also observed in the G-PTX group,which demonstrated high efficacy and low toxicity of G-PTX.Meanwhile,the active tumor targeting of G-PTX was mainly achieved through GLUT1-mediated mechanism,in which G-PTX could be further endocytosed by both clathrin-and caveolae-dependent pathways.4.Preliminary evaluation on the anti-tumor efficacy and safety of G-PTXFurther studies were conducted to investigate the comprehensive anti-tumor efficacy and preclinical safety of G-PTX.In vitro anti-tumor experiments showed that the IC50 values of G-PTX were lower than those of L-PTX and Abraxane in various tumor cell lines,especially in drug-resistant(PTX)cell lines.After initially ensuring the preclinical safety of G-PTX through hemolysis and acute toxicity test,in vivo experiments of G-PTX in tumor-bearing mouse models were conducted.On HGC-27/T,HGC-27,A549 and A549/T tumor-bearing mice models,G-PTX performed better than Abr.Meanwhile,on patient-derived tumor xenograft(PDX)intrinsic drug resistance models,G-PTX continued to display tumor suppression.In addition,G-PTX proved to impede the generation of drug resistance.A continuous low-dose PTX formulation stimulation of HGC-27 subcutaneous tumor was performed.The results showed that the sensitivity of tumor cells to paclitaxel did not change significantly after the stimulation by G-PTX,while the sensitivity of tumor cells to paclitaxel decreased after stimulated by Abraxane.The above results indicated that G-PTX had an obvious superiority in enhancing drug efficacy,reversing existing drug resistance and reducing the emergence of drug resistance.Also,the changes in body weight,haemocyte counts,blood biochemical indicators,and histopathology monitored in the in vivo experiments further confirmed the application safety of the G-PTX preparation.5.Effects of G-PTX on tumor cell autophagy and P-glycoprotein functionIn view of G-PTX had a comprehensive anti-tumor efficacy,a preliminary research was conducted on its mechanism of action.The results confirmed that G-PTX reversed existing drug resistance and reduced the emergence of drug resistance by inhibiting the efflux function of P-glycoprotein(P-gp)and regulating autophagy in tumor cells.In HGC-27/T cells,G-PTX significantly increased the intracellular content of P-gp substrates Rh 123 and PTX,which verified Rg5 in G-PTX could competitively bind to P-gp and inhibit the P-gp efflux function.The changes of autophagy-related proteins in HGC-27/T cells after G-PTX administration were also detected.The LC3-II and p62 protein levels were increased to varying degrees compared with control group,while LAMP2 protein expression was decreased,indicating that G-PTX blocked the late stage of autophagy by inhibiting lysosomal function and failing fusion of autophagosomes and lysosomes.This conclusion had also been verified in solid tumors,confirming that G-PTX inhibited autophagy flux.In conclusion,based on the structural similarity between ginsenosides and cholesterol,a biomimetic liposome was constructed with PTX as a model drug.It is preliminary proved that the drug delivery system has optimal drug loading capabilities,high anti-tumor efficacy,and reliable preclinical safety.The preparation obtained according to the strategy allows for simple compositions,easy manufacture and low costs,with the prospect for clinical translation.