Study On The Application Of Gold Nanoparticles And Polymer Materials In Chiral Separation And Activity Evaluation Of Drugs

In the field of analytical science,chiral separation and activity evaluation of drugs have always been a hot research direction.Common chromatographic analysis methods include ultraviolet spectrophotometry(UV),fluorescence spectrometry and high performance liquid chromatography(HPLC).Compared with these chromatographic methods,the capillary electrophoresis has attracted more and more attention because of simple operation,less sample consumption,high separation efficiency and low environmental pollution.In addition,the diversification of separation models has expanded its application in analytical science.Especially it has very important theoretical and practical significance in the analysis of effective components of drugs,the separation of chiral drugs and the interaction between drugs and receptors.In this paper,the Au nanoparticles and polymer materials were used to investigate their applications in chiral drug separation and the interaction between drugs and receptors,with three analysis models,capillary zone electrophoresis(CZE),affinity capillary electrophoresis(ACE)and capillary electro chromatography(CEC).In the study of enantiomeric separation by CZE method,five acidic drugs,ibuprofen,mandelic acid,dipivlin,epinephrine and isoprenaline,were investigated using streptomycin as a new chiral selector.Streptomycin,as an aminoglycoside antibiotic,contains abundant active groups,such as amino and hydroxyl groups.It is easy to associate with carboxyl,hydroxyl and amino groups in the five drugs'structures by hydrogen bond.And there are many chiral centers in streptomycin and the five drugs,which also cause interaction between streptomycin and durgs.The experimental parameters such as the concentration of streptomycin,buffer concentration and p H value,operating voltage and column temperature were all investigated systematically in order to optimize the chiral sepatation.Except for the poor resolution of dipivlin,the results showed that the baseline separation of the other four drugs was achieved using streptomycin as chiral selector by CZE method.Gold nanoparticles(GNPs)have the characteristic that is easily modified by sulfhydryl groups.Therefore,the GNPs were modified with chiral molecular L-cysteine(L-Cys),which the complex of L-Cys-GNPs was synthesized.There was a large amount of L-Cys on the surface of the complex,which makes it have more active groups and chiral centers,and possess the structural characteristics of chiral selectors.In the next steps,the enantiomers of epinephrine,isoproterenol and norepinephrine were separated excellently by CZE using L-Cys-GNPs as a chiral selector for the first time.The experimental parameters such as the L-Cys bonding concentration,p H value of buffer solution,operating voltage and column temperature were all investigated systematically in order to optimize the chiral sepatation.The results showed that all three adrenaline drugs reached baseline separation using L-Cys-GNPs as chiral selector by CZE method.And the separation effect of isoproterenol was better than that of isoproterenol and norepinephrine.Enlightened by column chromatographic separation,a monolithic capillary electrochromatography column was prepared to investigate the resolution effect of chiral drugs.Firstly,the inner surface of capillary column was silylated by?-methacrylate oxypropyltrimethoxysilane(?-MAPS)under the technology of microwave-assisted.Then the reaction monomer glycidyl methacrylate(GMA),crosslinking agent ethylene glycol dimethyl acrylate(EDMA),mixed porogen cyclohexanol and dodecanol,and initiator AIBN were mixed in a three-neck bottle in a certain proportion.After removing oxygen by feeding nitrogen,the mixture was pushed into the capillary.As a result,the polymer-based GMA-EDMA stationary phase matrix was prepared by traditional in-situ polymerization.Thirdly,the bovine serum albumin(BSA)was bonded onto the surface of GMA-EDMA stationary phase matrix with epoxy group,and the Chiral Monolithic columns were prepared.The main factors affecting the polymerization,such as the ratio of monomer and crosslinking agent,the selection of pore-forming agent,the polymerization temperature and time,were discussed,and the polymer conditions of the mixture were optimized.Finally,the chiral separation effect of the monolithic column was evaluated by using isoproterenol as a sample,which provide a scientific basis for the separation of chiral drugs by CEC method.The interaction between drugs and?₂-adrenergic receptors(?₂-AR)was also studied by open-tube column of CEC.Firstly,the inner surface of capillary column was silylated by?--aminopropyl trimethoxysilane(APTS)under the technology of microwave-assisted.Then the open-tube column was prepared with?₂-AR as coating material by chemical bonding method.The binding effect of?₂-AR was characterized by infrared spectroscopy,scanning electron microscopy and fluorescence labeling.Secondly,the length of the coating was changed from controlling the injection time of?₂-AR.Finally,the adrenaline hydrochloride,norepinephrine bistartrate and propranolol hydrochloride were injected into open-tube column with different coating lengths respectively,and the fluidity changes were linearly fitted with the coating length.Based on the linear equation,the binding constants K_b of three drugs and(?₂-AR)-AR were obtained,and the order of binding constants was adrenaline hydrochloride>norepinephrine bistartrate>propranolol hydrochloride.As shown in experimentals,the?₂-AR bonded on the capillary wall by chemical bonding still maintains its original biological activity,and it also can interact with various drugs(ligands)effectively.This method can be used for drug activity evaluation and screening.In this paper,the interaction between norepinephrine(NE-B)and bovine serumalbumin(BSA)was also studied by ACE method.The mixture of BSA and N,N-dimethylformamide(DMF)was injected as sample,and the NE-B with different concentrations was added to the running buffer solution.Then the peak time of BSA was determined at 25?and 37?respectively.According to the relationship between the peak time of BSA and the concentrations of NE-B,linear fitting of reciprocal concentration and the peak time was carried out.Based on the linear equation,the binding constants K_b of NE-B and BSA were obtained at differret temperature.In order to further test the results that obtained by ACE method,the binding constants of NE-B and BSA were also determined by traditional UV method.The results showed that the binding constantsof the two methods are in good agreement,and the K_b value at 37?was greater than that at 25?.It was showen that the K_b value increases with the increase of temperature in a certain temperature range.Finally,a series of thermodynamic parameters were calculated according to the binding constants.It was concluded that the interaction between NE-B and BSA was hydrophobic mainly,and the reaction taked place spontaneously.