Preparation And Application Of Novel Phospholipids Functionalized Polymer-based Monolithic Column For Micro-HPLC

Immobilized artificial membrane(IAM) chromatography was widely used as a biomimetic membrane technology through chromatographic approach to mimic the drug- membrane interactions. To date, commercial silica-based IAM columns have been developed, which was prepared by linking phosphatidylcholine(PC) analogue covalently to silica-propylamine through double chain ester or single ether bond to obtain a stable surface of phosphatidylcholine at a high molecular surface density. Those commercial IAM columns have been applied successfully to predict drug membrane transportation, drug volume of distribution, drug induced phospholipidosis risk and protein purification. However, those commercial silica-based IAM columns have some shortcomings such as high cost, tedious preparation processes, limited p H range and lack of phospholipid species. Therefore, it is of great interest to develop a novel preparation process of IAM stationary phases. Monolithic columns have the advantages of high permeability, easy modification and simple preparation. It provides a very promising direction in preparing novel IAM columns. Based on our previous work, the present research aims to design and prepare different types of organic polymer based IAM monolithic columns. The optimized monoliths will be applied to drug screening and sample separation. These novel IAM monoliths would provide an useful tool for studying the drug-membrane interaction.In the first chapter, immobilized artificial membrane chromatography and its important application in several different areas, including drug- membrane interactions, prediction of drug membrane permeability, sample separation and purification, were systematically introduced. Moreover, the development of IAM and monolith were also reviewed. O n this basis, the research idea and scientific problem of this research were put forward.In the second chapter, the preparation and application of a novel acidic phospholipid functionalized monolith was investigated. The 12- methacryloyl dodecylphosphoserine containing acidic phospholipids was synthesized via a multi-step reaction. The poly(MDPS-co-EDMA) monolith was prepared by in situ co-polymerization of 12-methacryloyl dodecylphosphoserine(MDPS) and EDMA were used as functional monomer and crosslinker, respectively. The composition of the polymerization mixture was carefully optimized in order to obtain satisfactory performance with respect to column permeability, efficiency and selectivity. The optimized monolith was applied to the separations of small peptides and protein degest.In the third chapter, in order to better mimic the phospholipid environment of biomembranes, a novel mixed phospholipid functionalized monolithic column(poly(MDPC80PS20-co-EDMA)) was developed through a co-polymerization of MDPS and 12-methacryloyl dodecylphosphocholine(MDPC), which use EDMA as the crosslinker, iso-propanol(IBA) and 1,4-butanediol(BDO) as the porogenic solvents, AIBN as an initiator. Scanning electron microscopy, elemental analysis, FT-IR spectra, ζ-potential analysis and micro-HPLC were carried out to characterize the physicochemical properties and separation performance of the monolith. The obtained mixed phospholipid functionalized monolithic columns were applied to predict drug- induced phospholipidosis(DIDIPLD) risk. Moreover, the effect of the acidic phospholipid phosphatidylserine proportion on prediction accuracy was also investigated through comparison between mixed phospholipid monoliths with different content of MDPS.In the fourth chapter, in order to better mimic the acidic phospholipid environment of lysosome, poly(MDPC80PS20-co-EDMA) and poly(MDPC-co-EDMA) monolith were first applied to predict drug- induced DIPLD risk in the acidic environment. Moreover, poly(MDPC80PA20-co-EDMA) was developed through a co-polymerization of MDPC and 12-methacryloyl dodecylphosphatidic acid(MDPA), which use EDMA as the crosslinker, IBA and BDO as the porogenic solvents, AIBN as an initiator. The obtained poly(MDPC80PA20-co-EDMA) monolith was also applied to the prediction of DIDIPLD risk under the acidic condition. The results was also compared with DIDIPLD risk data measured using poly(MDPC80PS20-co-EDMA) andpoly(MDPC-co-EDMA), respectively.In the fifth chapter, all contents were summaried and the prospects of IAMC was discussed.