Drug partition chromatography on immobilized porcine intestinal brush border membranes

We immobilized porcine intestinal brush border membrane vesicles (BBMVs) for chromatographic analyses of drug partitioning into the membranes determined as Ks, the drug retention per phospholipid amount. For positive and neutral drugs Ks decreased day by day, whereas Ks for negative drugs increased marginally. Similar results on vesicle-lipid liposomes indicated a gradual loss of negative charge from the columns. The Ks values for positive drugs were higher than those for negative drugs with the same octanol/water partitioning or the same Ks on egg yolk phospholipid bilayers. Electrostatic interactions seem to be important for the partitioning of charged drugs into brush border membranes.     

Immobilized liposome chromatography to study drug-membrane interactions. Correlation with drug absorption in humans

For rapid screening of drug-membrane interactions and predicting drug absorption in vivo, unilamellar liposomes were stably immobilized in the pores of gel beads by avidin-biotin binding. Interactions of a diverse set of well-described drugs with the immobilized liposomal membranes were reflected by their elution profiles. The membrane partitioning coefficients (KLM) of the drugs were determined from the retention volumes. The drug retentions on egg phosphatidylcholine (EPC)-phosphatidylserine (PS)-cholesterol (chol) and EPC-PS-phosphatidylethanolamine (PE)-chol columns intended to mimic small intestine membranes were similar, although the positively-charged drugs were more strongly retarded on the negatively-charged liposomes than the negatively-charged drugs. The relationship between log KLM with the drug fraction absorbed in humans showed that the log KLM values obtained with unilamellar liposomes can be used to predict drug passive transcellular absorption, similarly to that previously shown for entrapped multilamellar liposomes. The immobilized liposome chromatography method should be useful for screening compounds at an early stage of the drug discovery process. The avidin-biotin immobilization of the liposomes prolongs the lifetime of the columns.     

Steady-state oxidation of cholesterol catalyzed by cholesterol oxidase in lipid bilayer membranes on platinum electrodes

Cholesterol oxidase is immobilized in electrode-supported lipid bilayer membranes. Platinum electrodes are initially modified with a self-assembled monolayer of thiolipid. A vesicle fusion method is used to deposit an outer leaflet of phospholipids onto the thiolipid monolayer forming a thiolipid/lipid bilayer membrane on the electrode surface. Cholesterol oxidase spontaneously inserts into the electrode-supported lipid bilayer membrane from solution and is consequently immobilized to the electrode surface. Cholesterol partitions into the membrane from buffer solutions containing cyclodextrin. Cholesterol oxidase catalyzes the oxidation of cholesterol by molecular oxygen, forming hydrogen peroxide as a product. Amperometric detection of hydrogen peroxide for continuous solution flow experiments are presented, where flow was alternated between cholesterol solution and buffer containing no cholesterol. Steady-state anodic currents were observed during exposures of cholesterol solutions ranging in concentration from 10 to 1000 μM. These data are consistent with the Michaelis-Menten kinetic model for oxidation of cholesterol as catalyzed by cholesterol oxidase immobilized in the lipid bilayer membrane. The cholesterol detection limit is below 1 μM for cholesterol solution prepared in buffered cyclodextrin. The response of the electrodes to low density lipoprotein solutions is increased upon addition of cyclodextrin. Evidence for adsorption of low density lipoprotein to the electrode surface is presented.     

Purification of the membrane protein enzyme lipoamidase by affinity chromatography

Lipoamidase, a membrane glycoprotein enzyme, was purified from brain membrane by means of various affinity columns. A column with immobilized Arg-Phe-NH2 was found to be the most effective. After loading the crude material of the membrane, and extensive washing of the column with sodium chloride (0.3 M) solution, the enzyme activity was eluted by a solution containing 1% of nonionic detergent (Nonidet P-40). The fractions containing the lipoamidase activity were analyzed by SDS-PAGE, and a single protein band detected in this fraction. On the other hand, lipoyl-affinity columns with various resins were not effective in enzyme purification. Single step chromatography on the Arg-Phe-NH2 column enriched the membrane enzyme lipoamidase by 40-fold. The mechanism by which this affinity resin effectively enriches the enzyme remains to be elucidated.     

Immobilized lipodisks as model membranes in high-throughput HPLC-MS analysis

Lipodisks, also referred to as polyethylene glycol (PEG)-stabilized bilayer disks, have previously been demonstrated to hold great potential as model membranes in drug partition studies. In this study, an HPLC-MS system with stably immobilized lipodisks is presented. Functionalized lipodisks were immobilized on two different HPLC support materials either covalently by reductive amination or by streptavidin–biotin binding. An analytical HPLC column with immobilized lipodisks was evaluated by analysis of mixtures containing 15 different drug compounds. The efficiency, reproducibility, and stability of the system were found to be excellent. In situ incorporation of cyclooxygenase-1 (COX-1) in immobilized lipodisks on a column was also achieved. Specific binding of COX-1 to the immobilized lipodisks was validated by interaction studies with QCM-D. These results, taken together, open up the possibility of studying ligand interactions with membrane proteins by weak affinity chromatography.     

Self-assembly of solid-supported membranes using a triggered fusion of phospholipid-enriched proteoliposomes prepared from the inner mitochondrial membrane

A general procedure for the formation ofsolid-supported artificial membranes containing transmembrane proteins is reported. The main objective was to directly use the pool of proteins of the native biomembrane (here the inner membrane from mitochondria of human carcinogenic hepatic cells) and to avoid purification steps with detergent. Proteoliposomes of phospholipid-enriched inner membranes from mitochondria were tethered and fused onto a tailored surface via a streptavidin link. The failure of some preliminary experiments on membrane formation was attributed to strong nonspecific interactions between the solid surface and the protuberant hydrophilic parts of the transmembrane complexes. The correct loading of uniform membranes was performed after optimization of a tailored surface, covered with a grafted short-chain poly(ethylene glycol), so that nonspecific interactions are reduced. Step-by-step assembly of the structure and triggered fusion of the immobilized proteoliposomes were monitored by surface plasmon resonance and fluorescence photobleaching recovery, respectively. The long-range lateral diffusion coefficient (at 22 degrees C) for a fluorescent lipid varies from 2.5 x 10(-8) cm2 s(-1) for a tethered lipid bilayer without protein to 10(-9) cm2 s(-1) for a tethered membrane containing the transmembrane proteins of the respiratory chain at a protein area fraction of about 15%. The decrease in the diffusion coefficient in the tethered membrane with increase in protein area fraction was too pronounced to be fully explained by the theoretical models of obstructed lateral diffusion. Covalent tethering links with the solid are certainly involved in the decrease of the overall lateral mobility of the components in the supported membrane at the highest protein-to-lipid ratios.     

Characterization of interaction kinetics between chiral solutes and human serum albumin by using high-performance affinity chromatography and peak profiling

Peak profiling and high-performance columns containing immobilized human serum albumin (HSA) were used to study the interaction kinetics of chiral solutes with this protein. This approach was tested using the phenytoin metabolites 5-(3-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) as model analytes. HSA columns provided some resolution of the enantiomers for each phenytoin metabolite, which made it possible to simultaneously conduct kinetic studies on each chiral form. The dissociation rate constants for these interactions were determined by using both the single flow rate and multiple flow rate peak profiling methods. Corrections for non-specific interactions with the support were also considered. The final estimates obtained at pH 7.4 and 37°C for the dissociation rate constants of these interactions were 8.2-9.6 s(-1) for the two enantiomers of m-HPPH and 3.2-4.1 s(-1) for the enantiomers of p-HPPH. These rate constants agreed with previous values that have been reported for other drugs and solutes that have similar affinities and binding regions on HSA. The approach used in this report was not limited to phenytoin metabolites or HSA but could be applied to a variety of other chiral solutes and proteins. This method could also be adopted for use in the rapid screening of drug-protein interactions.     

Micellar liquid chromatography for lipophilicity determination of new biologically active 1,3‐purinodiones

A series of newly synthesized 1,3‐purinodiones with potential anticonvulsant activity, exhibiting affinity to adenosine A₁ and/or A_2A receptors, were subjected to micellar LC (MLC) with SDS as micelle‐forming agent and n‐propanol as organic modifier. Two C18 silica‐based columns were employed in MLC: a particle one and a monolithic. In parallel, those derivatives were also analyzed in RP‐LC on four silica‐based columns and on an immobilized artificial membrane column. The correlations between the relevant logarithms of the retention factors of analytes obtained in MLC, immobilized artificial membrane and RP‐LC systems on the one hand, and the calculated log P (clog P) and log D values (clog D) on the other, were examined. The level of the correlations of retention data from MLC and RP‐LC systems with clog P and clog D obtained is similar but it could be stressed that MLC allows increasing the speed of analysis and using only one mobile phase. Moreover, there is no need of applying an extrapolation procedure in lipophilicity determination. Therefore, the MLC systems, providing chromatographic data in a fast and efficient manner, were demonstrated as promising alternatives to the classical RP‐LC systems to estimate the lipophilicity of drugs and drug candidates.     

Electrokinetic migration across artificial liquid membranes. New concept for rapid sample preparation of biological fluids

Basic drug substances were transported across a thin artificial organic liquid membrane by the application of 300 V d.c. From a 300 microl aqueous donor compartment (containing 10 mM HCl), the drugs migrated through a 200 microm artificial liquid membrane of 2-nitrophenyl octyl ether immobilized in the pores of a polypropylene hollow fiber, and into a 30 microl aqueous acceptor solution of 10 mM HCl inside the lumen of the hollow fiber. The transport was forced by an electrical potential difference sustained over the liquid membrane, resulting in electrokinetic migration of drug substances from the donor compartment to the acceptor solution. Within 5 min of operation at 300 V, pethidine, nortriptyline, methadone, haloperidol, and loperamide were extracted with recoveries in the range 70-79%, which corresponded to enrichments in the range 7.0-7.9. The chemical composition of the organic liquid membrane strongly affected the permeability, and may serve as an efficient tool for controlling the transport selectivity. Water samples, human plasma, and human urine were successfully processed, and in light of the present report, electrokinetic migration across thin artificial liquid membranes may be an interesting tool for future isolation within chemical analysis.     

Preparation,evaluation, and comparison of wide bore (320 μm) and narrow bore (50 μm) cyanosilicone-coated capillary columns for gas chromatography

The preparation of wide bore (320 μm) and narrow bore (50 μm) fused silica capillary columns is described for immobilized cyanopropyl substituted silicones containing 60 and 88% substitution. The effect of high temperature deactivation with cyanopropylcyclosiloxanes was studied with a special test mixture. Curing was achieved with dicumyl peroxide or azo-tert-butane. The columns were evaluated and compared in terms of efficiency, activity, polarity, and temperature stability. Different coating methods were compared for the narrow bore columns. The activity of the 60% cyanopropyl columns that had been immobilized with dicumyl peroxide was significantly larger than for azo-tert-butane immobilized columns. The polarity of polar columns appeared to depend greatly on column temperature and is completely different for wide and narrow bore columns.     

Development of immobilized membrane-based affinity columns for use in the online characterization of membrane bound proteins and for targeted affinity isolations

Membranes obtained from cell lines that express or do not express a target membrane bound protein have been immobilized on a silica-based liquid chromatographic support or on the surface of an activated glass capillary. The resulting chromatographic columns have been placed in liquid chromatographic systems and used to characterize the target proteins and to identify small molecules that bind to the target. Membranes containing ligand gated ion channels, G-protein coupled receptors and drug transporters have been prepared and characterized. If a marker ligand has been identified for the target protein, frontal or zonal displacement chromatographic techniques can be used to determine binding affinities (K_d values) and non-linear chromatography can be used to assess the association (k_on) and dissociation (k_off) rate constants and the thermodynamics of the binding process. Membrane-based affinity columns have been created using membranes from a cell line that does not express the target protein (control) and the same cell line that expresses the target protein (experimental) after genomic transfection. The resulting columns can be placed in a parallel chromatography system and the differential retention between the control and experimental columns can be used to identify small molecules and protein that bind to the target protein. These applications will be illustrated using columns created using cellular membranes containing nicotinic acetylcholine receptors and the drug transporter P-glycoprotein.     

On‐chip generation of monodisperse giant unilamellar lipid vesicles containing quantum dots

Monodispersed lipid vesicles have been used as a drug delivery vehicle and a biochemical reactor. To generate monodispersed lipid vesicles in the nano‐ to micrometer size range, an extrusion step should be included in conventional hand‐shaking method of lipid vesicle synthesis. In addition, lipid vesicles as a drug carrier still need to be improved to effectively encapsulate concentrated biomolecules such as cells, proteins, and target drugs. To overcome these limitations, this paper reports a new microfluidic platform for continuous synthesis of small‐sized (～10 μm) giant unilamellar vesicles (GUVs) containing quantum dots (QDs) as a nanosized model drug. To generate GUVs, we introduced an additional cross‐flow to break vesicles into small size. 1,2 ‐ dimyristoyl‐sn‐glycero ‐ 3 ‐ phosphocholine (DMPC) in an octanol–chloroform mixture was used in the construction of self‐assembled membrane. Consequently, we have successfully demonstrated the fabrication of monodispersed GUVs with 7?12 μm diameter containing QDs. The proposed synthesis method of cell‐sized GUVs would be highly desirable for applications such as multipurpose drug encapsulation and delivery.     

Phospholipid-Modified ODS Monolithic Column for Affinity Prediction of Hydrophobic Basic Drugs to Phospholipids

A phospholipid-modified octadecyl silica (ODS) monolithic column was prepared and its interaction with basic hydrophobic drugs was studied. These drugs are of interest in pharmaceutical research because of their strong interaction with biomembranes. The amount of dimyristoylphosphatidylcholine trapped on the ODS surface was reproducible, and the results of the trinitrobenzenesulfonic acid assay suggested the formation of a monolayer on the surface. Both hydrophobic and electrostatic interactions acted between the model drugs and the phosphatidylcholine. The column was stable for 10 days at least. The column was applied to the affinity screening of basic drugs to phospholipid. Good correlation was obtained between log k and log P for the basic drugs lidocaine, quinidine, propranolol, imipramine, and chlorpromazine. The monolithic silica column allowed highly hydrophobic basic drugs such as imipramine and chlorpromazine to be assayed; these are difficult to analyze by using a conventional particle-packed column. These drugs were clearly separated from acidic drugs naproxen and warfarin on the log k versus log P plots. The thermodynamic studies revealed that the retention of the drug was an enthalpy-driven process, and that the decrease in enthalpy for the phospholipid-modified ODS monolithic column was larger than those for immobilized artificial membrane columns. Our results suggest that the phospholipid-modified ODS monolithic column is applicable to affinity screening of drugs to phospholipids.     

Functional immobilization of biomembrane fragments on planar waveguides for the investigation of side-directed ligand binding by surface-confined fluorescence

A method for the functional immobilization of Na,K-ATPase-rich membrane fragments on planar metal oxide waveguides has been developed. A novel optical technique based on the highly sensitive detection of surface-confined fluorescence in the evanescent field of the waveguide allowed us to investigate the interactions of the immobilized protein with cations and ligands. For specific binding studies, a FITC-Na,K-ATPase was used, which had been labelled covalently within the ATP-binding domain of the protein. Fluorophore labels of the surface-bound enzyme can be selectively excited in the evanescent field. A preserved functional activity of the immobilized enzyme was only found when a phospholipid monolayer was preassembled onto the hydrophobic chip surface to form a gentle, biocompatible interface. In situ atomic force microscopy (AFM) was used to examine and optimize the conditions for the lipid and membrane fragment assembly and the quality of the formed layers. The enzyme's functional activity was tested by selective K+ cation binding, interaction with anti-fluorescein antibody 4-4-20, phosphorylation of the protein and binding of inhibitory ligand ouabain. The comparison with corresponding fluorescence intensity changes found in bulk solution provides information about the side-directed surface binding of the Na,K-ATPase membrane fragments. The affinity constants of K+ ions to the Na,K-ATPase was the same for the immobilized and the non-immobilized enzyme, providing evidence for the highly native environment on the surface. The method for the functional immobilization of membrane fragments on waveguide surfaces will be the basis for future applications in pharmaceutical research where advanced methods for exploring the molecular mechanisms of membrane receptor targets and drug screening are required.     

Effect of increase in orientational order of lipid chains and head group spacing on non steroidal anti-inflammatory drug induced membrane fusion

Membrane fusion is a key event in many biological processes. The fusion process, both in vivo and in vitro, is induced by different agents which include mainly proteins and peptides. For protein- and peptide-mediated membrane fusion, conformational reorganization serves as a driving force. Small drug molecules do not share this advantage; hence, drug induced membrane fusion occurring in absence of any other fusogenic agent and at physiologically relevant concentration of the drugs is a very rare event. To date, only three drugs, namely, meloxicam (Mx), piroxicam (Px), and tenoxicam (Tx), belonging to the oxicam group of non steroidal anti-inflammatory drugs (NSAIDs), have been shown by us to induce fusion at very low drug to lipid ratio without the aid of any other fusogenic agent. In our continued effort to understand the interplay of different physical and chemical parameters of both the participating drugs and the membrane on the mechanism of this drug induced membrane fusion, we present here the effect of increase in orientational order of the lipid chains and increase in head group spacing. This is achieved by studying the effect of low concentration cholesterol (<10 mol %) at temperatures above the chain-melting transition. Low concentration cholesterol (<10 mol %), above the gel to fluid transition temperature, is mainly known to increase orientational order of the lipid chains and increase head group spacing. To isolate the effect of these parameters, small unilameller vesicles (SUVs) formed by dimyristoylphosphatidylcholine (DMPC) with an average diameter of 50-60 nm were used as simple model membranes. Fluorescence assays were used to probe the time dependence of lipid mixing, content mixing, and leakage and also used to determine the partitioning of the drugs in the membrane bilayer. Differential scanning calorimetry (DSC) was used to study the effect of drugs in the presence of cholesterol on the chain-melting temperature which reflects the fluidization effect of the hydrophobic tail region of the bilayer. Our results show contradictory effect of low concentration cholesterol on the fusion induced by the three drugs, which has been explained by parsing the effect of orientational order and increase in head group spacing on the fusion process.     

Molecular dynamics simulation of nonsteroidal antiinflammatory drugs,naproxen and relafen,in a lipid bilayer membrane

Naproxen and relafen, as nonsteroidal antiinflammatory drugs, were simulated in neutral and charged forms and their effects on a lipid bilayer membrane were investigated by molecular dynamics simulation using Groningen machine for chemical simulations software (GROMACS). Simulation of 10 systems was performed, which included different dosages of the drug molecules, naproxen and Relafen, in charged and neutral forms, and a mixture of naproxen and Relafen in neutral forms. The effects of the mixture and the individual drugs' dosages on membrane properties, such as electrostatic potential, order parameter, diffusion coefficients, and hydrogen bond formation, were analyzed. Hydration of the drugs in the membrane system was investigated using radial distribution function analysis. Using fully hydrated dimyristoylphosphatidylcholine (DMPC) as a reference system, 128 lipid molecules and water molecules were simulated exclusively, and the same simulation technique was performed on 10 other systems, including drug mixtures and a DMPC membrane. Angular distributions of lipid chains of the membrane were calculated, and the effects of the drug insertion and chain orientation in the membrane were evaluated. © 2013 Wiley Periodicals, Inc.     

Evaluation and application of liquid chromatographic columns coated with "intelligent" ligands. (III). Immobilized phospholipid column.

Immobilized enzyme columns have been developed for use as high-performance liquid chromatographic enzyme reactors. Enzyme reactors were prepared by immobilizing trypsin or cytochrome-c on phospholipid columns. Dynamic coating was employed to prepare the reactors by recycling a buffer solution containing trypsin or cytochrome-c through a phospholipid-coated column, on which the enzymes were immobilized by hydrophobic binding. The immobilized trypsin column displayed hydrolytic activity which catalyzed the hydrolysis of L-amino acid esters to amino acid. The immobilized cytochrome-c column exhibited oxidation activity which catalyzed N-demethylation of N,N-dimethylaniline, codeine, and dihydrocodeine in the presence of hydrogen peroxide as an oxygenating agent. The enzyme reaction proceeded rapidly in the column; both product and substrate could be separated and detected simultaneously. The immobilized enzyme columns could be readily regenerated using the original phospholipid column by repeating the dynamic coating. These immobilized enzyme columns could be utilized as enzyme reactors in the high-performance liquid chromatographic mode. Complete hydrolysis of amino acid ester was observed with the trypsin column. Demethylation of codeine and of dihydrocodeine were observed with the cytochrome-c column.     

Immobilization of active facilitated glucose transporters (GLUT-1) in supported biological membranes

Membrane fragments or membrane proteins within a lipid mixture were immobilized over metal electrodes. This procedure has been developed to study biological membranes without interferences from cell machinery. To obtain a smooth hydrophilic biomembrane support and a mode of binding of the membrane, either a crosslinked gel or an aromatic polyamine-polymer doped with avidin was deposited at the metal electrode by electropolymerization. This layer (less than 10 nm thick) also served as a submembrane compartment. The facilitated glucose transporter (GLUT-1) purified from human erythrocytes was integrated into a lipid membrane containing artificial biotinylated lipids and reacted with the activated surface of the glucose sensitive electrode. It was demonstrated that the lipid layer was attached to the polymer-containing avidin and could only be removed by detergent extraction. The presence of an active membrane transporter was demonstrated by electrochemical detection of glucose in the submembrane compartment, and by inhibition of glucose transport with the specific inhibitor Cytochalasin-B.     

Immobilization and electrochemical behavior of gold nanoparticles modified leukemia K562 cells and application in drug sensitivity test

A new strategy for immobilization of tumor cells on electrode surface and accelerating electron transfer between electrode and the immobilized cells was proposed to study the electrochemical behavior of cells and the effect of antitumor drug on cell viability. The leukemia K562 cells immobilized in a microporous cellulose membrane were firstly modified with colloidal gold nanoparticles to retain efficiently the activity of immobilized living tumor cells and promote electron transfer between electroactive centers of the cells and the electrode, exhibiting a well-defined anodic peak of guanine at +0.830 V at 50 mV s⁻¹. The electrochemical response could be used to describe cell growth and evaluate the effectiveness of antitumor drug methotrexate on tumor cells. The proposed method offered potential advantages for drug sensitivity test with little usage of cells. It could be developed as a convenient means for the study of the tumor cells growth and the cytotoxicity of antitumor drugs.     

Spatial control of protein binding on lipid bimembrane using photoeliminative linker

Protein adsorption and dissociation on cell membrane surfaces is a topic of important study to reveal biological processes including signal transduction and protein trafficking. We demonstrated here the establishment of a mimic model system for the spatial control of protein adsorption/elimination on a lipid bimembrane using a photochemical technique. The novel photoeliminative linker that we synthesized here consists of three distinct components: a substrate (biotin), a photoeliminative group (4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butanoic acid), and a lipid bimembrane-adsorbent group (farnesyl). The photoeliminative linker was inserted on the entire surface of the lipid bimembrane and two-dimensionally eliminated by spatial UV irradiation onto the membrane to create a biotin pattern. A target protein, streptavidin was selectively immobilized on the patterned biotin, although it was almost not attached on the nonirradiated region. The streptavidin array was selectively dissociated by UV irradiation onto the entire membrane.