Dynamic assessment of bilayer thickness by varying phospholipid and hydraphile synthetic channel chain lengths

A library of "hydraphile" synthetic ion channel analogues that differ in overall length from approximately 28-58 A has been prepared. A new and convenient ion-selective electrode (ISE) method was used to assay Na(+) release. Liposomes were formed from three different phospholipids: 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC). The acyl chains of the lipids comprise cis-unsaturated 14:1, 18:1, or 22:1 residues, respectively. Sodium release was measured for each liposome system with each of the synthetic channels. Peak activity was observed for shorter channels in liposomes formed from DMPC and for longer channels in DEPC. A separate study was then conducted in DMPC liposomes in the presence of the putative membrane-thickening agents cholesterol and decane. Peak activity was clearly shifted to longer channel lengths upon addition of 20 or 40 mol % cholesterol or n-decane to the liposome preparation.     

Investigation of Factors Affecting Controlled Release from Photosensitive DMPC and DSPC Liposomes

An investigation of liposomes comprised of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) lipids with cholesterol and zinc phthalocyanine (ZnPC) revealed that several fundamental liposome properties are influenced by composition and by lipid-specific features. DMPC and DSPC liposomes were synthesized, and their compositional changes, encapsulation capacities, morphologies, and release properties were evaluated. In this research, liposome degradation, lysis, and content release were initiated by photolysis, i.e., rupture induced by exposure to light. A controlled release mechanism was created through the introduction of photosensitizers (i.e., ZnPC) embedded within the cholesterol-stabilized liposome membrane. The light wavelength and light exposure time accelerated photodegradation properties of DMPC liposomes compared to DSPC liposomes, which exhibited a slower release rate. Morphological changes in the liposomes were strongly influenced by light wavelength and light exposure time. For both the DMPC and DSPC liposomes, visible light with wavelengths in the red end of the spectrum and broad spectrum ambient lighting (400?C700?nm) were more effective for lysis than UV-A light (365?nm). Heating liposomes to 100?°C decreased the stability of liposomes compared to liposomes kept at room temperatures. In addition, the optimal lipid-to-cholesterol-to-photoactivator ratio that produced the most stable liposomes was determined.     

Liposome electrokinetic capillary chromatography in the study of analyte-phospholipid membrane interactions. Application to pesticides and related compounds

Interactions between low-molar mass analytes and phospholipid membranes were studied by liposome electrokinetic capillary chromatography (LEKC). The analytes were pesticides, some degradation products, and compounds associated with the manufacture of pesticides. Negatively charged liposome dispersions with different zwitterionic lipids (PC) were applied to the determination of retention factors (k) of 15 charged and uncharged compounds. The liposome dispersions consisted of 80:20 mol% of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/POPS, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/POPS. Retention factors were calculated from the effective electrophoretic mobilities of the analytes under LEKC and CZE conditions and from the effective electrophoretic mobilities of the liposomes, determined by CZE with a polyacrylamide-coated capillary. Determining the liposome mobilities in this way proved to be a good alternative to the conventional method employing a liposome marker compound. The log k values of the analytes for the different liposome dispersed phases were correlated with one another. In addition, correlation curves were determined between log k and calculated octanol-water partition coefficients. The results showed that the zwitterionic phospholipid in the liposome has a major impact on the interactions between the tested compounds and the lipid membranes.     

Isomerization dynamics of photochromic spiropyran molecular switches in phospholipid bilayers

Transient absorption spectroscopy was used to investigate the dynamics of the photochromic indolinobenzospiropyran reaction in toluene solution and in phosphatidylcholine bilayers (1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)). After excitation with UV light, colorless (R/S)-2-(3',3'-dimethyl-6-nitro-3'H-spiro[chromene-2,2'-indol]-1'-yl)ethanol derivatives are converted to colored merocyanine products in high yield; Phi = 0.45 in DMPC liposomes. We find that the reaction occurs in the bilayer aliphatic region in the gel (P(beta)(')) and liquid (L(alpha)) phases. The Arrhenius activation energy for the isomerization in DMPC bilayers was approximately 3.5 times larger in the liquid phase (L(alpha), E(a) = 26.0 +/- 1.0 kJ mol(-1)) than that in the gel phase (P(beta)('), E(a) = 7.3 +/- 1.6 kJ mol(-1)). Analysis of the isomerization rate constant temperature dependence allows an estimation of the bilayer viscosity and free volume properties in the L(alpha) phase.     

Langmuir balance investigation of superoxide dismutase interactions with mixed-lipid monolayers

Higher than theoretical encapsulation efficiencies in liposomes of the cytoplasmic protein, superoxide dismutase (SOD), were previously observed. The high encapsulation of SOD led to the consideration of lipid-protein interactions and the embedding of SOD in the lipid bilayer. Difficulty in other methods such as dynamic scanning calorimetry due to cholesterol obscuring the measurements brought about the interest for a modified Langmuir monolayer relaxation study. A novel method was devised to distinguish between different lipid compositions that formed either a favorable or an unfavorable environment for SOD. Normalized monolayer relaxations with SOD were compared between mixed-lipid compositions containing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and cholesterol (Chol). Lipid-monolayer relaxation with and without SOD in the subphase was plotted over 30 min to determine if the protein was altering the lipid-monolayer relaxation. The monolayer relaxation with SOD was normalized to the monolayer relaxation without SOD over the 30 min period. The results indicated that lipid length and mole percent of cholesterol were important parameters that must be adjusted in order to support a favorable environment for SOD interaction with the lipid. It was determined that hydrophobic interactions were dominant over electrostatic forces; thus, SOD was embedding into the lipid monolayer. Additionally, this study was correlated to a previous liposome study and proved that lipid-protein interactions were the reason for the higher encapsulation efficiencies. The significance of this method is that it (1) provides a connection between lipid-protein interactions observed in monolayers and bilayers and (2) establishes a simple and effective manner to test lipid compositions for lipid-protein interaction that will aid in optimization of liposome encapsulation efficiency.     

Surface thermodynamic properties of monolayers versus reconstitution of a membrane protein in solid-supported bilayers

Atomic force microscopy (AFM) was used to study the influence of a membrane protein, lactose permease of Escherichia coli (LacY), on the surface spreading behavior and the features of self-assembled phospholipids bilayers on mica. The miscibility of phospholipids used, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), was investigated by surface pressure area isotherm measurements at the air-water interface. A composition with an equimolar proportion of POPC and DMPC was used to form the liposomes. Surface layers formed with DMPC:POPC (0.5:0.5, mol/mol) or LacY reconstituted in proteoliposomes with the same phospholipid composition were imaged by using AFM. When lactose permease was reconstituted in DMPC:POPC (0.5:0.5, mol/mol), self-assembled structures that remained firmly adsorbed onto the mica surface were observed. These sheets had an irregular shape and their upper layer was more corrugated than that obtained for the phospholipid matrix.     

Micellar aggregates formed following the addition of hexafluoroisopropanol to phospholipid membranes

The addition of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) to aqueous phospholipid membranes leads to perturbation of the bilayer. In the case of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), calorimetric and small-angle X-ray scattering analyses indicate that effects are already apparent at bound molar HFIP/lipid ratios of less than 1:150, with a pronounced decrease in the temperature of the main (gel to liquid crystalline) phase transition and a decrease in the intensity of the first- and second-order scattering reflections. As the HFIP concentration is raised further, at bound molar HFIP/lipid ratios >2:1, uniform isotropic particulate structures are formed with higher intrinsic curvature than the parent liposomes. These observations are supported by the results of thin-film experiments and are consistent with the formation of DMPC/HFIP adducts that are detergent-like in nature. In the case of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) the effects are much less marked, with no blebbing observed over a comparable range of HFIP concentrations. Although HFIP interacts strongly with DOPC membranes, it appears that membrane rupture is not promoted as readily with this lipid. Data from electron microscopy, laser correlation spectroscopy, and marker release experiments suggest that some of the immediate (nonequilibrium) effects of HFIP on membranes are the consequence of microinhomogeneity in water/HFIP mixtures. On the basis of our observations, we propose a model for the interaction of HFIP with phospholipid membranes.     

Kinetics of liposome adhesion on a mercury electrode

The adhesion of liposomes on a mercury electrode leads to capacitive signals due to the formation of islands of lecithin monolayers. Integration of the current-time transients gives charge-time transients that can be fitted by the empirical equation Q(t) = Q(0) + Q(1)(1 - exp(-t/tau(1))) + Q(2)(1 - exp(-t/tau(2))), where the first term on the right side is caused by the docking of the liposome on the mercury surface, the second term is caused by the opening of the liposome, and the third term is caused by the spreading of the lecithin island on the mercury surface. The temperature dependence of the two time constants tau(1) and tau(2) and the temperature dependence of the overall adhesion rate allow determination of the activation energies of the opening, the spreading, and the overall adhesion process both for gel-phase 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and for liquid-crystalline-phase DMPC liposomes. In all cases, the spreading is the rate-determining process. Negative apparent activation energies for the spreading and overall adhesion process of liquid-crystalline-phase DMPC liposomes can be explained by taking into account the weak adsorption equilibria of the intact liposomes and the opened but not yet spread liposomes. A formal kinetic analysis of the reaction scheme supports the empirical equation used for fitting the charge-time transients. The developed kinetic model of liposome adhesion on mercury is similar to kinetic models published earlier to describe the fusion of liposomes. The new approach can be used to probe the stability of liposome membranes.     

Peroxidase-like catalytic activity of anion-exchange resins modified with metal-porphyrins in oxidative reaction of hetrocyclic amines with hydrogen peroxide

To elucidate the peroxidase (POD)-like catalytic activity of anion-exchange resins modified with metal-tetrakis(sulfophenyl)porphine (M-TSPP(r)s), an oxidative reaction of seven mutagenic heterocyclic amines (HCAs) with hydrogen peroxide, which reaction is catalyzed by horse radish POD, was investigated in the presence of M-TSPP(r)s. Among six M-TSPP(r)s tested, Mn- and Fe-TSPP(r)s were found to have a relatively strong POD-like activity for HCAs, in particular for a typical HCA, 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ). The optimal condition for the POD-like activity was selected using Fe- and Mn-TSPP(r)s. For evaluation of an oxidation product of IQ produced in the presence of Fe-TSPP(r), the absorption, NMR and FAB-mass spectra thereof were compared with those of an oxidation product of IQ produced by horse radish POD or a chemical oxidizing agent, sodium hypochlorite. When Fe-TSPP(r) was present as a catalysts, IQ was converted into the dimmer (hydorazone type) which has no mutagenic activity in umu-test. It was revealed that Fe- and Mn-TSPP(r)s exhibit a POD-like catalytic activity in oxidative reaction of HCAs with hydrogen peroxide.     

Effect of the Phospholipid Chain Length and Head Group on Beta‐Phase Formation of Poly(9,9‐dioctylfluorene) Enclosed in Liposomes

We have studied the effect of head group and alkyl chain length on β‐phase formation in poly(9,9‐dioctylfluorene) (PFO) solubilized in phospholipid liposomes. Systems studied have three different alkyl chain lengths (1,2‐dimyristoyl‐sn‐glycero‐3‐phosphatidylcholine [DMPC], 1,2‐didodecanoyl‐sn‐glycero‐3‐phosphatidylcholine [DLPC], 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphatidylcholine [DPPC]) and head groups (1,2‐dimyristoyl‐sn‐glycero‐3‐phosphate monosodium salt [DMPA], 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphoethanolamine [DMPE] and 1,2‐dimyristoyl‐sn‐glycero‐3‐phospho‐l ‐serine sodium salt [DMPS]). Changes in liposome size upon addition of PFO are followed by dynamic light scattering. All the phospholipids induce the formation of PFO β‐phase, which is followed by the emission intensity and deconvolution of the absorption spectra. Both the head group and alkyl chain length affect the yield of β‐phase. The photophysics of PFO incorporated in liposomes is characterized by stationary and time‐resolved fluorescence, whereas the polymer‐phospholipid interactions have been studied by the effect of the PFO concentration on the phospholipid phase transitions (differential scanning calorimetry [DSC]).     

Role of trehalose in prevention of giant vesicle adsorption and encapsulated solute leakage in anhydrobiotic preservation

Anhydrobiotic preservation has the potential to allow the processing and storage of mammalian cells in a state of suspended animation at ambient conditions in trehalose glasses; however, stresses--particularly to the lipid bilayer--during desiccation and rehydration have thus far prevented the full realization of the promise of this technique. Giant gel-phase 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and liquid-crystalline-phase 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) vesicles provide a model cell system with which to elucidate the role of trehalose in surface-lipid bilayer interactions, as well as the part played by lipid phase. In the absence of trehalose, DSPC liposomes adsorbed to polystyrene, producing irreversible structural changes and apparent leakage of all intravesicular solute upon drying and rehydration. Addition of trehalose significantly reduced vesicle adsorption with only transient intravesicular solute leakage for the rehydrated vesicles; however, at very low moisture contents, the vesicles underwent permanent structural changes. In contrast to the results with DSPC vesicles, DLPC vesicles largely avoided adsorption and exhibited high intravesicular solute retention when dried and rehydrated even in the absence of trehalose, despite significant internal structural changes.     

Resistive-pulse detection of multilamellar liposomes

The resistive-pulse method was used to monitor the pressure-driven translocation of multilamellar liposomes with radii between 190 and 450 nm through a single conical nanopore embedded in a glass membrane. Liposomes (0% and 5% 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (sodium salt) in 1,2-dilauroyl-sn-glycero-3-phosphocholine or 0%, 5%, and 9% 1,2-dipalmitoyl-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine) were prepared by extrusion through a polycarbonate membrane. Liposome translocation through a glass nanopore was studied as a function of nanopore size and the temperature relative to the lipid bilayer transition temperature, T(c). All translocation events through pores larger than the liposome, regardless of temperature, show translocation times between 30 and 300 μs and current pulse heights between 0.2% and 15% from the open pore baseline. However, liposomes at temperatures below the T(c) were captured at the pore orifice when translocation was attempted through pores of smaller dimensions, but squeezed through the same pores when the temperature was raised above T(c). The results provide insights into the deformation and translocation of individual liposomes through a porous material.     

Membrane-mediated capillary electrophoresis: interaction of cationic peptides with bicelles

Electrokinetic capillary chromatography is applied to determine the membrane affinity of peptides using both 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micelles and DHPC/1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bicelles under controlled conditions. The effect of temperature and the bicelle q value in surface association with cationic peptides is studied. The cationic peptides selected have a well-defined membrane structure (indolicidin), induced secondary structure (melittin, magainin 2), or do not possess classical secondary structure (atrial natriuretic peptide (ANP) 1-28, 4-28, 5-27). Electrokinetic capillary chromatography facilitated by DMPC and DHPC additives provides a rapid means of estimating lipophilicity and screening for peptides that have membrane affinity.     

Directed growth of pure phosphatidylcholine nanotubes in microfluidic channels

The morphology of self-assembled phospholipid membranes (e.g., micelles, vesicles, rods, tubes, etc.) depends on the method of formation, secondary manipulation, temperature, and storage conditions. In this contribution, microfluidic systems are used to create pure phosphatidylcholine (PC) micro- and nanotubes with unprecedented lengths. Tubes up to several centimeters in length and aligned with the long axis of the microchannel were created from spots of dry films of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). These high aspect ratio structures, which, to our knowledge, represent the first examples of extended tubes formed from pure PC lipids, were examined by fluorescence microscopy, electron and optical microscopy, and optical manipulation tools (i.e., a laser trap and laser scalpel) to characterize structure and stability. In particular, the tubular structure was confirmed by observation of fluorescent dyes that were sequestered within the aqueous cavity or within the phospholipid tube. Compared to other phospholipid tubes, the tubes formed from PC lipids in microfluidic channels show high mechanical stability and rigidity that depend on tube size, age, and storage conditions.     

Peroxidase-like catalytic activity of Mn- and Fe-tetrakis(4-carboxyphenyl)porphines bound to aminopropyl-glass bead in oxidative reaction of heterocyclic amines

Fe- or Mn-tetrakis(4-carboxyphenyl)porphine (Fe- and Mn-TCPP) bound to aminopropyl-glass bead (Fe- and Mn-TCPP_gs) was examined for the peroxidase (POD)-like function in order to develop a solid catalyst which can exhibit POD-like activity without adsorbing heterocyclic amines (HCAs). Mn-TCPP in aqueous solution had only a slight POD-like catalytic activity on HCAs (IQ and MeIQ). As for Fe-TCPP, it was impossible to examine the POD-like activity since it reacted with hydrogen peroxide in a liquid reaction system. However, both Fe- and Mn-TCPP when immobilized on aminopropyl-glass bead via peptide bond (Fe- and Mn-TCPP_gs), catalyzed the oxidative reaction of mutagenic HCAs with hydrogen peroxide. The catalytic activity of Fe- and Mn-TCPP_gs was investigated in more detail using as a substrate IQ and MeIQ which were oxidized more rapidly among the tested HCAs. Consequently, the optimal conditions for the oxidative reaction catalyzed by Fe- and Mn-TCPP_gs were determined. In addition, ESI-mass and absorption spectra of oxidation products of IQ and MeIQ showed that they are dimers. Thus, it was demonstrated that a solid catalyst with POD-like activity can be obtained by immobilizing Fe- and Mn-TCPPs on aminopropyl-glass beads.     

Interaction and lipid-induced conformation of two cecropin-melittin hybrid peptides depend on peptide and membrane composition

The interaction of two hybrid peptides of cecropin A and melittin [CA(1-8)M(1-18) and CA(1-7)M(2-9)] with liposomes was studied by differential scanning calorimetry (DSC), circular dichroism (CD), and quasi-elastic light scattering (QELS). The study was carried out with large unilamellar vesicles (LUVs) of three different lipid compositions: 1,2-dimyristoil-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DMPG) and a binary mixture of DMPC/DMPG, in a wide range of peptide-to-lipid (P:L) molar ratios (0 to 1:7). DSC results indicate that, for both peptides, the interaction depends on membrane composition, with very different behavior for zwitterionic and anionic membranes. CD data show that, although the two peptides have different secondary structures in buffer (random coil for CA(1-7)M(2-9) and predominantly beta-sheet for CA(1-8)M(1-18)), they both adopt an alpha-helical structure in the presence of the membranes. Overall, results are compatible with a model involving a strong electrostatic surface interaction between the peptides and the negatively charged liposomes, which gives place to aggregation in the gel phase and precipitation after a threshold peptide concentration. In the case of zwitterionic membranes, a progressive surface coverage with peptide molecules destabilizes the membrane, eventually leading to membrane disruption. Moreover, delicate modulations in behavior were observed depending on the peptide.     

Fluorescence study of the fluidity and cooperativity of the phase transitions of zwitterionic and anionic liposomes confined in sol-gel glasses

The current work makes use of different fluorescent reporter molecules and fluorescent spectroscopic techniques to characterize the thermotropic, physical, and dynamical properties of large unilamellar liposomes formed from either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-glycerol] (DMPG) encapsulated in sol-gel matrixes. In particular, cooperativity of the phase transition is analyzed from steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH), the interfacial properties are studied by measuring the spectral shift of Laurdan, and the structural organization (heterogeneity) of the lipid bilayer is determined from the fluorescence lifetime of trans-parinaric acid (t-PnA). In addition, information regarding order and dynamical properties in the bulk hydrophobic core is obtained from time-resolved fluorescence anisotropy of t-PnA and 3-(4-(6-phenyl)-1,3,5-hexatrienyl)-phenylpropionic acid (PA-DPH). The spectroscopic study reveals that upon encapsulation, the basic thermodynamic properties as well as the fluidity of the lipid bilayer practically remain intact for DMPG liposomes but not for DMPC liposomes, whose lipid bilayer exhibits large gel-fluid heterogeneity. On the basis of these experimental results, electrostatic interactions between phospholipid polar heads and the porous surface of the host matrix seem to play a capital role for the preservation of the structural integrity of encapsulated bilayer.     

Sequestration of amitriptyline by liposomes

We study the uptake of amitriptyline, which is a common cause of overdose-related fatalities, in aqueous solutions by 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes and liposomes composed of a mixture of DMPC and 1,2-dioleoyl-sn-glycero-3-[phospho-rac(1-glycerol)] (DOPG) lipids. The effect of drug concentration, liposomal charge, pH, salt, and protein presence on the drug uptake is investigated using two different methodologies, a precipitation and a centrifugation method. Furthermore, the time scale of the drug uptake is studied through qualitative observations at high pH and through conductivity measurements at neutral pH and found to be <5 s. The results of the quantitative studies show that the fractional drug uptake decreases with increasing drug concentration, and for a given concentration it increases with the pH and decreases in the presence of salt. We find that a larger amount of drug is sequestered by negatively charged liposomes (those containing DOPG) than liposomes with no net charge (DMPC). We speculate that the mechanism of drug uptake is due to both electrostatic interactions as well as hydrophobic effects. The fractional uptake by DMPC:DOPG in a 70:30 ratio is as high as 95% in water and about 90% in physiological buffer. The fractional uptake is also measured in presence of 2% (w/w) bovine serum albumin (BSA), which is approximately the protein concentration in the intercellular fluid. In presence of protein the fractional uptakes by 70:30 DMPC:DOPG liposomes and 50:50 DMPC:DOPG liposomes are 82 and 90%, respectively, at 125 muM drug amitriptyline. In the absence of liposomes, 67% of the drug is taken up by the protein in a 2% (w/w) BSA, 125 muM amitriptyline solution. Thus, addition of 50:50 DMPC:DOPG liposomes reduces the free drug concentration by a factor of about 3.5, making them attractive candidates for drug detoxification.     

Effects of incorporation of various amphiphiles into recipient liposome membranes on inter-membrane protein transfer.

To obtain information about the factors governing spontaneous inter-membrane protein transfer, we examined the effects of incorporation of various amphiphilic compounds in dimyristoylphosphatidylcholine (DMPC) liposomes on protein transfer from influenza virus-infected cells to the liposomes, and analyzed the physical properties of these liposome membranes. The incorporation of amphiphilic compounds, negatively charged dicetylphosphate (DCP), dipalmitoylphosphatidylserine (DPPS) or positively charged dimethyldipalmitoylammonium (DMDPA), into DMPC liposomal membranes enhanced protein transfer. The liposomes containing DCP, DPPS or DMDPA were unaffected by osmotic shock caused by external addition of glucose, suggesting a decrease in lipid packing in the liposomal membranes. Furthermore, calorimetric study of these liposomes showed that a phase separation occurred partially in the liposomal membranes. Accordingly, the membranes of DMPC liposomes containing DCP, DPPS and DMDPA should be distorted due to the coexistence of two phases, gel and liquid crystalline, in the membranes. Consequently, the membrane distortion could be responsible for the enhancement effects of the amphiphiles on the inter-membrane protein transfer from influenza virus-infected cells to the liposomes.     

Immobilization of liposomes on hydrophobically modified polymer gel particles in batch mode interaction

Immobilization of liposomal phospholipids onto Sephacryl S-1000 gels that were chemically conjugated with hydrophobic alkyl moieties, octyl, dodecyl and hexadecyl, was examined in batch mode interaction. Compared with the octyl gel, the dodecyl and the hexadecyl gels were found to immobilize the three to four times more phospholipids with the less hydrophobic moieties. The encapsulation of a water-soluble marker, with other evidences, suggests that the majority of the immobilized phospholipids maintained liposomal morphology. As the lipid of the interacting liposomes, egg yolk phosphatidylcholine (eggPC), 1,2-dimyristoylphosphatidylcholine (DMPC) and a mixture of DMPC and 1,2-dimyristamido-1,2-deoxyphosphatidylcholine were examined. At 22 °C, DMPC liposomes showed higher extent of immobilization than at 37 °C but not eggPC liposomes, suggesting that the phase of liposomal membrane could have influence on the immobilization. Exchange between the immobilized liposomes and free ones was found to be small, less than 3%. The gel that had been first interacted with liposomes to apparent saturation could further immobilize the newly added liposomes. The rate of this second immobilization was similar to that of the slow adsorption process; the both could be based on the same mechanism, possibly involving rearrangement of the immobilized liposomes on the gel as proposed by Lundahl. As had been observed in the flow mode, the immobilization had preference for smaller liposomes. In application of the system in batch mode, the size distributions of the immobilized liposomes and of those left in the supernatant may differ from that of the originally added liposomes.