Ceramide-1-phosphate, in contrast to ceramide, is not segregated into lateral lipid domains in phosphatidylcholine bilayers.

Sphingolipids are key lipid regulators of cell viability: ceramide is one of the key molecules in inducing programmed cell death (apoptosis), whereas other sphingolipids, such as ceramide 1-phosphate, are mitogenic. The thermotropic and structural behavior of binary systems of N-hexadecanoyl-D-erythro-ceramide (C(16)-ceramide) or N-hexadecanoyl-D-erythro-ceramide-1-phosphate (C(16)-ceramide-1-phosphate; C(16)-C1P) with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was studied with DSC and deuterium nuclear magnetic resonance ((2)H-NMR). Partial-phase diagrams (up to a mole fraction of sphingolipids X = 0.40) for both mixtures were constructed based on DSC and (2)H-NMR observations. For C(16)-ceramide-containing bilayers DSC heating scans showed already at X(cer) = 0.025 a complex structure of the main-phase transition peak suggestive of lateral-phase separation. The transition width increased significantly upon increasing X(cer), and the upper-phase boundary temperature of the mixture shifted to approximately 65 degrees C at X(cer) = 0.40. The temperature range over which (2)H-NMR spectra of C(16)-ceramide/DPPC-d(62) mixtures displayed coexistence of gel and liquid crystalline domains increased from approximately 10 degrees for X(cer) = 0.1 to approximately 21 degrees for X(cer) = 0.4. For C16-C1P/DPPC mixtures, DSC and (2)H-NMR observations indicated that two-phase coexistence was limited to significantly narrower temperature ranges for corresponding C1P concentrations. To complement these findings, C(16)-ceramide/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and C16-C1P/POPC mixtures were also studied by (2)H-NMR and fluorescence techniques. These observations indicate that DPPC and POPC bilayers are significantly less perturbed by C(16)-C1P than by C(16)-ceramide and that C(16)-C1P is miscible within DPPC bilayers at least up to X(C1P) = 0.30.

Antibiotic fusidic acid has strong interactions with negatively charged lipid membranes: an electrokinetic capillary chromatographic study.

Fusidic acid (FA), a narrow spectrum steroidal antibiotic, is useful for treatment of most skin, conjunctival, and corneal infections and also in infections caused by atypical microbes in the surface of the eye. Liposome electrokinetic capillary chromatography (LEKC) was used to study the interactions between FA and lipid membranes. Liposomes prepared by extrusion were composed of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleyl-sn-glysero-3-phosphor-L-serine (POPS), cholesterol, FA, and sphingomyelin (SM) in various molar ratios. 26 different liposome dispersions were studied as dispersed (pseudostationary) phase in LEKC. The hydrophobicities of the liposomes were evaluated by calculating the retention factors of model neutral steroids. The retention factors were calculated using the EOF and the effective electrophoretic mobilities of the analytes and the liposomes. The latter were separately determined by capillary electrophoresis with a polyacrylamide (PAA)-coated capillary. FA-lipid membrane interactions were studied by determining the retention factor of FA. In addition, liposomes prepared from lipids extracted from Escherichia coli bacterium were studied and used as dispersed phase in LEKC for interaction studies between FA and lipid membranes.

Quantitative determination of drug encapsulation in poly(lactic acid) nanoparticles by capillary electrophoresis.

Capillary electrophoretic (CE) methods were used for the quantitative determination of model drugs [salbutamol sulphate (SS), sodium cromoglycate (SCG) and beclomethasone dipropionate (BDP)] in poly(D,L-lactic acid) (PLA) nanoparticles, which were prepared by the nanoprecipitation method. Zeta potential and size distribution of the nanoparticles were determined by electrophoretic mobility determinations and photon correlation spectroscopy, respectively. Interactions between the drugs, the PLA nanoparticles and the fused-silica capillary were investigated by electrokinetic capillary chromatography (EKC). A quantitative CE method was developed for salbutamol sulphate and sodium cromoglycate, and the linearity and repeatability of migration times, peak areas and peak heights were determined. Microemulsion electrokinetic chromatography was used for the quantitative determination of beclomethasone dipropionate. According to this study, the applied electromigration techniques were suitable for the interaction, drug entrapment and dissolution studies of pharmaceutical nanoparticles. The results suggest that even quantitation of the drug located inside the nanoparticles was possible. Encapsulation of the more hydrophilic model drugs (SS, SCG) in the PLA nanoparticles was less efficient than in the case of BDP.

Novel, dynamic on-line analytical separation system for dissolution of drugs from poly(lactic acid) nanoparticles.

A novel method for investigating drug release in a dynamic manner from nanoparticles including, but not limited to, biodegradable poly(lactic acid) (PLA) is reported. The PLA nanoparticles were prepared by the nanoprecipitation method. Two poorly soluble drugs, beclomethasone dipropionate (BDP) and indomethacin, were encapsulated into PLA nanoparticles, and their dissolution from the nanoparticles were followed in a dynamic way. The on-line method comprised a short column (vessel) packed with the PLA nanoparticles, on-line connected to an analytical liquid chromatographic column via a multiport switching valve equipped with two loops. The system allowed monitoring of the drug release profiles in real time, and the conditions for the drug release could be precisely controlled and easily changed. The effects of solvent composition and temperature on the rate of dissolution of the drugs from the PLA nanoparticles were investigated. The system proved to be linear for the drugs tested over the concentration range 10-3000 ng (n=6, R(2)=0.999 and 0.997 for indomethacin and beclomethasone, respectively) and repeatable (RSD of peak areas <0.5%). The recoveries of the dissolution study were quantitative (120 and 103% for indomethacin and beclomethasone, respectively).

Interactions of fusidic acid and elongation factor G with lipid membranes.

Fusidic acid (FA) is a potent antibiotic and blocks the protein synthesis by binding to elongation factor G (EF-G) directly. Here we hypothesized that the antibiotic activity of FA would be potentiated by several orders of magnitude if both FA and EF-G would be residing in the lipid membranes and, hence, the probability of interaction would transform from three-dimensional to two-dimensional. Such detailed information could lead to more effective therapeutic interventions if they are understood on a molecular level. Interactions between FA and various lipid membranes composed of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and cholesterol (Chol) were studied by capillary electrochromatography (CEC). The influence of the lipid vesicle size--sonicated liposomes and liposomes extruded through 30-, 50-, and 100-nm filters--on the packing of vesicles on the silica capillary surface was investigated by CEC and dissipative quartz crystal microbalance. The CEC results evidenced that FA interacts with and resides in phospholipid membranes. Likewise, monolayer, asymmetrical flow field flow fractionation, and CEC studies confirmed that EF-G is hydrophobic and incorporated into POPC and POPC/Chol membranes. Including EF-G in phospholipid vesicles did not improve the binding of FA to the membranes.

Characterization of phosphatidylcholine/polyethylene glycol-lipid aggregates and their use as coatings and carriers in capillary electrophoresis.

PEG-stabilized lipid aggregates are a promising new class of model membranes in biotechnical and pharmaceutical applications. CE techniques, field-flow fractionation, light scattering, quartz crystal microbalance (QCM), and microscopic techniques were used to study aggregates composed of 1-palmitoyl-2-oleyl-sn-glycero-phosphatidylcholine (POPC) and PEG-lipid conjugates. The PEG-lipids, with PEG molar masses of 1000, 2000, and 3000, were 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(PEG)] derivatives with either dimyristoyl (DM, 14:0) or distearoyl (DS, 18:0) acyl groups. The 80/20 mol% POPC/PEG-lipid dispersions in HEPES at pH 7.4 were extruded through 100 nm size membranes. Asymmetrical flow field-flow fractionation (AsFlFFF), photon correlation spectroscopy (PCS), and dynamic light scattering (DLS) were used to determine the sizes of POPC and the PEGylated aggregates. All methods demonstrated that the DSPEG-lipid sterically stabilized aggregates were smaller in size than pure POPC vesicles. The zeta potentials of the aggregates were measured and showed an increase from -19 mV for pure POPC to -4 mV for the POPC/DSPEG3000 aggregates. Atomic force microscopy (AFM), electron cryo-microscopy (EM), and multifrequency QCM studies were made to achieve information about the PEGylated coatings on silica. Lipid aggregates with different POPC/DSPEG3000-lipid ratios were applied as capillary coating material, and the 80/20 mol% composition was found to give the most suppressed and stable EOFs. Mixtures of low-molar-mass drugs and FITC-labeled amino acids were separated with the PEGylated aggregates as carriers (EKC) or as coating material (CEC). Detection was made by UV and LIF.