Preparation And Performance Evaluation Of Agarose Gel Lipid Raft Stationary Phase

Aims: Lipid raft chromatography is a bioaffinity chromatographic model using lipid rafts as stationary phase loaded on the surface of silica spheres constructed in our group’s previous research,which has unique advantages in screening active drugs.In this project,Lipid Rafts@CNBr-Sepharose 4B stationary phase was constructed using agarose gel as the carrier material.The effect of functional group modification on the ability of agarose gels to couple lipid rafts was investigated;the specific adsorption performance of agarose gels was evaluated with the aid of high performance liquid chromatography to assess whether the constructed affinity chromatographic system has the ability of affinity adsorption;the study of elution conditions was carried out in order to construct a novel bioaffinity chromatographic screening system and provide new ideas for the online affinity screening of active ingredients.Methods: Lipid rafts from human glioma cells(U251 cells)were extracted by the descaler method combined with sucrose density gradient centrifugation and characterized by protein blotting(Western Blot),infrared spectroscopy(IR)and immunofluorescence(IF).The epoxy groups were modified on the surface of agarose microspheres,and the activation process of the activated epoxy groups was optimized by single-factor method to improve the modification density of the epoxy groups,and the successful coupling of lipid rafts was verified by immunoblotting and IR spectroscopy.Immunofluorescence and protein quantification were used to compare the effect of coupling lipid rafts by agarose gel microspheres after different group modifications,and to optimize the coupling time,number of rinses and coupling ratio.The specific adsorption of lipid raft-coupled agarose gels(Sepharose 4B)and cyanogen bromide modified agarose gels(CNBr-Sepharose 4B)was investigated using the Trk Atargeted drug gefitinib,the non-Trk A-targeted drug 5-fluorouracil(5-FU)and gemcitabine as model drugs to evaluate the specific adsorption of lipid raft-coupled agarose gels(Lipid Raft@ CNBr-Sepharose 4B)stationary phase to evaluate the affinity performance,elution effect and experimental reproducibility.Result: Lipid Rafts@CNBr-Sepharose 4B stationary phase enriched with Tk A protein was successfully constructed.The Lipid Rafts@CNBr-Sepharose 4B stationary phase showed higher fluorescence intensity and more uniform lipid raft wrapping.The agarose gel modified with cyanogen bromide moiety(CNBr-Sepharose 4B)coupled the lipid rafts best,and most of the lipid rafts could be coupled with it within 6 h.The coupling rate was 90%,and the reproducibility was good.In the experiments to evaluate the adsorption performance of blank agarose gels(Sepharose 4B and CNBr-Sepharose4B),the adsorption rate was only about 1%;Lipid Rafts@CNBr-Sepharose 4B stationary phase was effective in affinity adsorption.The results of the elution conditions of the positive drug showed that when the p H of the eluent was 6 and the ion concentration was 0.1 mol/L,the number of elutions was less,the recovery of the positive drug was higher,the reproducibility was good,and the damage to the lipid raft column was less.Conclusion: In this study,the effect of carrier material coupling lipid rafts,the coupling conditions between carrier material and lipid rafts,the adsorption performance of the affinity stationary phase and the elution conditions of positive drugs were investigated respectively.Among them,the agarose gels modified with cyanogen bromide moieties have strong binding ability to the lipid rafts,and the prepared agarose gel lipid raft stationary phase has good affinity adsorption effect.The elution conditions screened were mild and the recovery of positive drugs was high.The results of this study provide new ideas for the selection of carrier materials in the field of bioaffinity chromatography.