Evaluation Of Membrane Permeability Of Small-molecule Drugs By Liposome Capillary Electrophoresis

Small-molecule drugs need to penetrate a series of biofilms to enter the body and exert their effects.Therefore,the prediction of drug membrane permeability is of great significance in the process of drug development.Liposomes can simulate the structure of natural biological membranes,and have good stability and cell affinity.They were often used as pseudo-stationary phases or stationary phase in capillary electrophoresis（CE）analysis.Liposome capillary electrophoresis with liposome solution as running medium,can be used as a simple,effective,stable and feasible in vitro drug screening model to study the interaction between drugs and biological membranes.In this study,liposome electrokinetic chromatography（LEKC）using liposomes as pseudo-stationary phase and immobilized liposome capillary column chromatography（ILCCC）using covalently bond liposomes in the inner wall of capillary as stationary phase,to study the interaction between small-molecule drugs and biomimetic membranes,and further evaluated the membrane permeability of the drugs.There are four chapters in this paper,including two chapters of experimental research.The first chapter is a literature review.The common evaluation methods of drug permeability was constructed firstly,briefly including the definitions,principles,advantages and disadvantages of these methods and related literature research.Secondly,the sources,advantages,formation principles of lipoeomes and factors affecting the preparation of liposomes on liposome capillary electrophoresis method were introduced in detail.The theory principle of this method and its application status and shortcomings in the prediction of drug membrane permeability were described.Then,the application of the linear free energy relationship（LFER）related to the membrane permeability and physicochemical properties of drugs,and the d-value analysis method between different evaluation methods of drug permeability were briefly introduced.Finally,the content and significance of this thesis were put forward.In the second chapter,the liposomes were prepared from natural phosphatidylcholine（soybean lecithin,SPC）or synthetic phosphatidylcholine（1,2-distearoyl-sn-glycero-3-phosphocholine,DSPC）and cholesterol（Chol）,respectively,and their morphology were characterized.A stable and effective LEKC method was established to determine the logarithm of lipid water partition coefficients(log K_L/W)of neutral and ionic drugs.The log K_L/W measured by natural or synthetic phospholipids were further compared by LEKC method.The linear relationship of log K_L/W between SPC/Chol and DSPC/Chol liposomes was good（R²=0.89）.Furthermore,the linear relationships between LEKC system and other related research systems（octanol-water system,immobilized artificial membrane（IAM）,Caco-2 cell model and software prediction）were analyzed.The results illustrated that DSPC/Chol liposomes or SPC/Chol liposomes had a good linear relationship with Caco-2 cell model,R² were0.81 and 0.72,respectively.The LFER results showed that the main drug property parameters driving drug partition in liposomes were solute volume and hydrogen bond basicity,and the two types of liposomes LEKC systems showed very close normalization coefficients with other chromatographic systems through LFER.Therefore,the LEKC method established in this chapter is an effective and feasible method to evaluate the membranes permeability of drugs.In the third chapter,a new method for the preparation of covalently bonded liposome capillary column was proposed.Firstly,through mixing SPC or DSPC with Chol and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine（PE）,respectively,SPC/PE/Chol or DSPC/PE/Chol liposomes were prepared.Secondly,the liposomes with amino groups were covalently fixed on the inner wall of capillary column by using the adhesion of polydopamine（PDA）membrane and the cross-linking property of glutaraldehyde（GA）.A CE based ILCCC method was successfully constructed to study the retention behavior of drugs in column chromatography.The liposomes were covalently bonded to the inner wall of the capillary tube in the form of spherical particles,which were characterized by FT-IR,SEM,calcein leakage and optical fluorescence microscopy.The electrophoretic mobility of analyte was determined by ILCCC method,showed that the column had good repeatability and stability.Then the retention factors（log k）of seventeen drugs were determined by immobilized SPC/PE/Chol or DSPC/PE/Chol liposome capillary columns.The results showed that the log k measured by the two ILCCC had a good linear fitting（R²=0.86）.In addition,the linear relationship between ILCCC system and other related research systems（octanol-water system,IAM,LEKC and software prediction）was analyzed.It was found that SPC/PE/Chol ILCCC,DSPC/PE/Chol ILCCC and IAM system had good fitting results,R² values were 0.86 and 0.78,respectively.These results indicated that the two systems were similar in the study of drug membrane interaction.Finally,the normalization coefficients of ILCCC and IAM systems obtained by LFER analysis were close and the d value was small.The ILCCC method established in this chapter is a simple and feasible method for the study of drug membrane permeability,which has the advantages of reusability,low sample consumption and high stability.The fourth chapter is the summary and prospect of this thesis.The LEKC and ILCCC methods developed in this paper had the unique advantages of liposome membrane anisotropy and fluidity,as well as easy automation,rapid analysis and low sample consumption of CE.They could be used to study the liposomes formed by different types of phospholipid components for the study membrane interaction between drugs and liposomes,and provided a simple and feasible idea for the study of different types of drugs.These methods could further provide more reference information for evaluating the membrane permeability of drugs.