Study On Immobilized Liposome Capillary Electrophoresis And Its Applications

Interest in liposome as model membrane for the study of interactions between membrane and drugs or biologically important molecules is rapidly increasing, since liposomal lipid bilayers are structurally similar to biomembrane. Immobilized liposome capillary electrophoresis (ILCE) is an important branch of liposome model. The purpose of this study was to investigate ILCE in terms of the preparation of immobilized liposome capillary column (ILCC) and the application of ILCE. The main contents studied are as follows:1. The liposome/water partition coefficient (Klip/w) values of ten nonsteroidal anti-inflammatory drugs (NSAIDs) were determined by the free liposome-water system, which provided essential data for the subsequent experiments. By exploring the effect of pH, ionic strength on the Klip/w and the octanol/water distribution coefficient (Doct/w), the mechanism of interaction between NSAIDs and phospholipid bilayers was elucidated. The other interaction besides hydrophobic interaction, such as ionic interaction plays an important role in the molecular interaction between NSAIDs and phospholipid bilayers.2. ILCC, which was prepared by physical adsorption method, was developed for studying the interactions between a set of NSAIDs and membrane and predicting the biological activity of NSAIDs. The normalized retention factor (KILCE) was the first time to be introduced into ILCE for evaluating drug-membrane interactions. The properties of ILCE for studying drug-membrane interactions were evaluated by comparing KILCE with the partition coefficient (Klip/w) in free liposome partitioning system and the normalized retention factor (KILC) determined by immobilized liposome chromatography (ILC). The strong relationships were observed between logKILCE values and logKlip/w values (R= 0.9855) or logKILC values (R= 0.9975). In order to further assess the capability of ILCE as an in vitro drug screening model at the early stage of drug discovery, the relationship between KILCE and IC50 for cyclooxygenase 2 (COX-2) determined on intact cells was investigated. IC50, an index of NSAIDs activity, is NSAIDs concentration required for 50%inhibition of COX-2. The logKLCE (phosphatidylcholine/cholesterol 80/20 mol%) values correlated well with the pIC50 (the minus logarithm of IC50) (R= 0.959). These results confirmed that ILCE, value as an evalutation index, can be effectively used for studying drug-membrane interactions and it has the potential to predict drug activity. Cholesterol-containing liposomes may be more suitable to mimic real cell membrane.3. A novel and economical covalent coupling approach was introduced to prepare permanent ILCCs. The coating conditions, such as the reaction time and temperature of liposome coupling, the content of dimyristoylphosphatidyl-ethanolamine (DMPE) in liposomes, the addition of Ca2+and the particle size of liposomes, were optimized. The final form of the coating was observed by atomic force microscopy. The performance of ILCCs was evaluated by investigating the effect of pH of BGE on EOF and by studying the repeatability, reproducibility and the lifetime. The intra-and inter-capillary variations in EOF are 4.02%RSD (n=30) and 6.72%RSD (n=4) respectively, and ILCC can be used to perform analysis at least for one month without any performance deterioration when stored at 4℃.4. ILCC, which was prepared by covalent coupling method, was applied in the study of interactions between a set of drugs with diverse structures and biological membrane. The influence of the components of liposome as well as the liposomal size on the KILCE values of drugs was investigated. The possibility of ILCE predicting the cell membrane permeability (Papp) of drugs, and the fraction dose absorbed in humans of drugs (Fa%) were also investigated. It was found that changing the DMPE content in liposomes had a strong influence on the retention of drugs; but changing the particle size of liposmes has little effect on the retention of drugs. Higher correlation was observed between logLILCE and Fa%with an increase in DMPE content of liposomes. A sigmodial relationship (R= 0.9017) between logKILCE and Fa%and a good linear relationship (R= 0.9568) between logKILCE and Papp were obtained when DMPE content was up to 20mol%. These results were demonstrated that ILCC can be used for in vitro prediction of Fa%and Papp of drugs that follow the transcellular passive transport rout.5. There are three preparation methods that are commonly used for coating capillaries with liposomes, namely physical adsorption, avidin-biotin binding and covalent coupling. Herein, ILCCs were coated with liposomes using the three different coating methods, and their performance was systematically studied and compared. The performance of ILCCs was evaluated by their EOF characterization, repeatability, reproducibility, lifetime and tolerance to salt. The amount of immobilized phospholipids and the electrophoretic behavior of neutral compounds for ILCCs prepared by these methods were also compared. Finally, the merit for each coating method was proposed. The amount of the immobilized phospholipids was almost independent on the preparation methods. The physical adsorption method was simple, fast and hence provided a good flexibility regarding the composition of the coating, but suffered poor repeatability, reproducibility, lifetime and tolerance. In contrast, the avidin-biotin binding and the covalent coupling methods provided higher repeatability and reproducibility, longer lifetime and better tolerance, while bearing several disadvantages. The preparation process of ILCC by these two methods was tedious. The extra interations generated through the prepration of ILCC might interfere with the interactions between analytes and phospholipid membranes.