PREPARATION OF MIXED-LIGAND IMMOBILIZED ARTIFICIAL MEMBRANES FOR PREDICTING DRUG-BINDING TO MEMBRANES

Mixed ligand immobilized artificial membranes (IAMs) are surfaces that contain at least two immobilized membrane phospholipids which are des ignated as either the primary phospholipid or the secondary phospholip id. The primary immobilized phospholipid refers to the immobilized pho spholipid that has the highest surface density. For this work, the pri mary immobilized phospholipid was a single-chain ether phosphatidylcho line (PC) analog. Four mixed-ligand IAMs were prepared by use of immob ilized PC as the primary immobilized phospholipid. The secondary immob ilized phospholipid ligand was either phosphatidylethanolamine, phosph atidylserine, phosphatidylglycerol, or phosphatidic acid. All of these secondary phospholipids are bonded at approximately 6-10 mol % relati ve to the molar amount of immobilized PC. Each secondary phospholipid contains functional groups in the polar head group region that require protecting groups during the immobilization process. The four-step sy nthetic strategy to prepare mixed-ligand IAMs involves (i) immobilizat ion of the PC analog at high density to silica propylamine (SPA), (ii) immobilization of the second phospholipid (PL) analog at low density, (iii) end capping residual amines with a long-chain anhydride followe d by end capping with a short-chain anhydride, and (iv) deprotection o f the polar head group protecting groups; The surface density of the m ixed PLs bonded to the silica support was approximately 130 mu mol of PLs/g of SPA. High-performance liquid chromatography using these mixed lipid IAMs can be exploited to rapidly predict the membrane binding p roperties of drugs.