Influence of vesicle size on complement-dependent immune damage to liposomes

Complement-dependent antibody-mediated damage to multilamellar lipid vesicles (MLVs) normally results in a maximum release of 50-60% of trapped aqueous marker. The most widely accepted explanation for this is that only the outermost lamellae of MLVs are attacked by complement. To test this hypothesis, complement damage to two different types of large unilamellar vesicles (LUVs), large unilamellar vesicles prepared by the reverse-phase evaporation procedure (REVs) and large unilamellar vesicles prepared by extrusion techniques (LUVETs), were determined. In the presence of excess antibody and complement the LUVs released a maximum of only approx. 25 to 40% of trapped aqueous marker, instead of close to 100% that would be expected. Since small unilamellar vesicles apparently differ from LUVs in that they can release 100% of trapped aqueous marker it appeared that the size of the vesicles was an important factor. Because of these observations the influence of MLV size on marker release was examined. Three populations of MLVs of different sizes were separated by a fluorescence activated cell sorter. Assays of the separated MLV populations showed that the degree of complement-dependent marker release was inversely related to MLV size. No detectable glucose was taken up by MLVs when glucose was present only outside the liposomes during complement lysis. Our results can all be explained by the closing, or loss, of complement channels. We conclude that complement channels are only transiently open in liposomes, and that loss of channel patency may be due to either channel closing or to loss of channels.     

Spontaneous transfer of ganglioside GM1 from its micelles to lipid vesicles of differing size.

The spontaneous incorporation of II3-N-acetylneuraminosylgangliotetraosylceramide (GM1) from its micelles into phospholipid bilayer vesicles has been investigated to determine whether curvature-induced changes in membrane lipid packing influence ganglioside uptake. Use of conventional liquid chromatography in conjunction with technically-improved molecular sieve gels permits ganglioside micelles to be separated from phospholipid vesicles of different average size including vesicles with diameters smaller than 40 nm and, thus, allows detailed study of native ganglioside GM1 incorporation into model membranes under conditions where complicating processes like fusion are readily detected if present. At 45 degrees C, the spontaneous transfer rate of GM1 from its micelles to small unilamellar vesicles (SUVs) comprised of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) is at least 3-fold faster than that to similar composition large unilamellar vesicles (LUVs) prepared by octyl glucoside dialysis. Careful analysis of ganglioside GM1 distribution among vesicle populations of differing average size reveals that GM1 preferentially incorporates into the smaller vesicles of certain populations. This behavior is observed in SUVs as well as in LUV-SUV mixtures and actually serves as a sensitive indicator for the presence of trace quantities of SUVs in various LUV preparations. Analysis of the results shows that both differences in the diffusional collision frequency between GM1 monomers and either SUVs or LUVs and curvature-induced changes in the interfacial lipid packing in either SUVs or LUVs can dramatically influence spontaneous ganglioside uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

CD Spectroscopy of Peptides and Proteins Bound to Large Unilamellar Vesicles

Circular dichroism (CD) spectroscopy is an essential tool for determining the conformation of proteins and peptides in membranes. It can be particularly useful for measuring the free energy of partitioning of peptides into lipid vesicles. The belief is broadly held that such CD measurements can only be made using sonicated small unilamellar vesicles (SUVs) because light scattering associated with extruded large unilamellar vesicles (LUVs) is unacceptably high. We have examined this issue using several experimental approaches in which a chiral object (i.e., peptide or protein) is placed both on the membrane and outside the membrane. We show that accurate CD spectra can be collected in the presence of LUVs. This is important because SUVs, unlike LUVs, are metastable and consequently unsuitable for equilibrium thermodynamic measurements. Our data reveal that undistorted CD spectra of peptides can be measured at wavelengths above 200 nm in the presence of up to 3 mM LUVs and above 215 nm in the presence of up to 7 mM LUVs. We introduce a simple way of characterizing the effect on CD spectra of light scattering and absorption arising from suspensions of vesicles of any diameter. Using melittin as an example, we show that CD spectroscopy can be used to determine the fractional helical content of peptides in LUVs and to measure their free energy of partitioning of into LUVs.     

Using giant unilamellar lipid vesicle micro-patterns as ultrasmall reaction containers to observe reversible ATP synthesis/hydrolysis of F0F1-ATPase directly

F(0)F(1)-ATPase within chromatophores, which was labeled with pH-sensitive quantum dots, was encapsulated in large unilamellar lipid vesicles (LUVs) through reverse-phase evaporation. Then a microarray of chromatophore-containing LUVs was created using a micro-contact printing (mu-CP) technique. Through controlled dehydration-rehydration of the lipid patterns, a microarray of single chromatophore-containing giant unilamellar lipid vesicles (GUVs) was formed with desired size and uniform shape. The reversible ATP synthesis/hydrolysis of F(0)F(1)-ATPase in GUVs was directly observed by fluorescence microscopy through the fluorescence intensity increase/decrease in the pH-sensitive quantum dots labeled on the outer surface of the chromatophore. To the best of our knowledge, this is the first direct observation of the reversible behavior of F(0)F(1)-ATPase at the bulk scale.     

GUVs Melt Like LUVs: The Large Heat Capacity of MLVs Is Not Due to Large Size or Small Curvature

The excess heat capacity functions (ΔC_p) associated with the main phase transition of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs) are very different. Two explanations are possible. First, the difference in vesicle size (curvature) results in different gel-fluid interactions in the membrane; those interactions have a large effect on the cooperativity of the phase transition. Second, there is communication between the bilayers in an MLV when they undergo the gel-fluid transition; this communication results in thermodynamic coupling of the phase transitions of the bilayers in the MLV and, consequently, in an apparent increase in the cooperativity of the transition. To test these hypotheses, differential scanning calorimetry was performed on giant unilamellar vesicles (GUVs) of pure dipalmitoylphosphatidylcholine. The ΔC_p curve of GUVs was found to resemble that of the much smaller LUVs. The transition in GUVs and LUVs is much broader (half-width ∼1.5°C) than in MLVs (∼0.1°C). This similarity in GUVs and LUVs indicates that their size has little effect on gel-fluid interactions in the phase transition. The result suggests that coupling between the transitions in the bilayers of an MLV is responsible for their apparent higher cooperativity in melting.     

Association of a protein with membrane vesicles at the collisional limit: studies with blood coagulation factor Va light chain also suggest major differences between small and large unilamellar vesicles

Vesicle size can be a very sensitive modulator of protein-membrane association. In addition, reactions at the collisional limit may be characteristic of many types of protein-membrane or protein-receptor interactions. To probe these effects quantitatively, we analyzed the association of blood clotting factor Va light chain (Va-LC) with phospholipid vesicles of 15-150-nm radius. The number of protein binding sites per vesicle was approximately proportional to vesicle surface area. Association rates approached the collisional limit, and the activation energy for the association reaction was 4.5 +/- 0.5 kcal/mol. In agreement with diffusional theory for this type of interaction at the collisional limit, the observed association rate constant for filling all sites was approximately proportional to the inverse of vesicle radius. This general property has important implications for many systems such as blood coagulation including possible slower association rates and higher Km values for reactions involving whole cells relative to those obtained for phospholipid vesicles. Dissociation rate constants for reactions that are near the collisional limit should also be proportional to the inverse of vesicle size if diffusional parameters are the only factors influencing dissociation. However, Va-LC bound to small unilamellar vesicles (SUVs, less than or equal to 15-nm radius) gave slower dissociation rates than Va-LC bound to large unilamellar vesicles (LUVs, greater than or equal to 35-nm radius). This indicated a change in KI, the intrinsic protein-phospholipid affinity constant for LUVs vs SUVs. The cumulative effect of association and dissociation rates resulted in higher affinity of Va-LC for SUVs than LUVs under equilibrium conditions. The latter was corroborated by competition binding studies. Furthermore, the temperature dependence of both rate constants indicated an entirely entropy-driven binding to LUVs but a largely enthalpy-driven binding to SUVs. Interactions which are largely entropic are thought to be ionic in nature. The differences observed between binding to LUVs and SUVs may reflect thermodynamic differences between these types of phospholipid structures.     

A membrane filtering method for the purification of giant unilamellar vesicles

The use of giant unilamellar vesicles (GUVs) for investigating the properties of biomembranes is advantageous compared to the use of small-sized vesicles such as large unilamellar vesicles (LUVs). Experimental methods using GUVs, such as the single GUV method, would benefit if there was a methodology for obtaining a large population of similar-sized GUVs composed of oil-free membranes. We here describe a new membrane filtering method for purifying GUVs prepared by the natural swelling method and demonstrate that, following purification of GUVs composed of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) membranes suspended in a buffer, similar-sized GUVs with diameters of 10–30 μm are obtained. Moreover, this method enabled GUVs to be separated from water-soluble fluorescent probes and LUVs. These results suggest that the membrane filtering method can be applied to GUVs prepared by other methods to purify larger-sized GUVs from smaller GUVs, LUVs, and various water-soluble substances such as proteins and fluorescent probes. This method can also be used for concentration of dilute GUV suspensions.     

Osmotic properties of large unilamellar vesicles prepared by extrusion.

We have examined the morphology and osmotic properties of large unilamellar vesicles (LUVs) prepared by extrusion. Contrary to expectations, we observe by cryo-electron microscopy that such vesicles, under isoosmotic conditions, are non-spherical. This morphology appears to be a consequence of vesicle passage through the filter pores during preparation. As a result when such LUVs are placed in a hypoosmotic medium they are able to compensate, at least partially, for the resulting influx of water by "rounding up" and thereby increasing their volume with no change in surface area. The increase in vesicle trapped volume associated with these morphological changes was determined using the slowly membrane-permeable solute [3H]-glucose. This allowed calculation of the actual osmotic gradient experienced by the vesicle membrane for a given applied differential. When LUVs were exposed to osmotic differentials of sufficient magnitude lysis occurred with the extent of solute release being dependent on the size of the osmotic gradient. Surprisingly, lysis was not an all-or-nothing event, but instead a residual osmotic differential remained after lysis. This differential value was comparable in magnitude to the minimum osmotic differential required to trigger lysis. Further, by comparing the release of solutes of differing molecular weights (glucose and dextran) a lower limit of about 12 nm diameter can be set for the bilayer defect created during lysis. Finally, the maximum residual osmotic differentials were compared for LUVs varying in mean diameter from 90 to 340 nm. This comparison confirmed that these systems obey Laplace's Law relating vesicle diameter and lysis pressure. This analysis also yielded a value for the membrane tension at lysis of 40 dyn cm-1 at 23 degrees C, which is in reasonable agreement with previously published values for giant unilamellar vesicles.     

Temperature-induced fusion of small unilamellar vesicles formed from saturated long-chain lecithins and diheptanoylphosphatidylcholine

Small unilamellar vesicles which form when gel-state long-chain phosphatidylcholines are mixed with micellar short-chain lecithins undergo an increase in size as the long-chain species melts to its liquid-crystalline form. Analysis of the vesicle population with quasi-elastic light scattering shows that the particle size increases from 90-A radius to greater than 5000-A radius. Resonance energy transfer experiments show total mixing of lipid probes with unlabeled vesicles only when the Tm of the long-chain phosphatidylcholine is exceeded. This implies that the large size change represents a fusion process. Aqueous compartments are also mixed during this transition. 31P NMR analysis of the vesicle mixtures above the phase transition shows a great degree of heterogeneity with large unilamellar particles coexisting with oligo- and multilamellar structures. Upon cooling the vesicles below the Tm, the original size distribution (e.g., small unilamellar vesicles) is obtained, as monitored by both quasi-elastic light scattering and 31P NMR spectroscopy. This temperature-induced fusion of unilamellar vesicles is concentration dependent and can be abolished at lower total phospholipid concentrations. It occurs over a wide range of long-chain to short-chain ratios and occurs with 1-palmitoyl-2-stearoylphosphatidylcholine and dimyristoylphosphatidylcholine as well. Characterization of this fusion event is used to understand the anomalous kinetics of water-soluble phospholipases toward these unusual vesicles.     

Stability of giant unilamellar vesicles and large unilamellar vesicles of liquid-ordered phase membranes in the presence of Triton X-100

We have investigated the stability of giant unilamellar vesicles (GUVs) and large unilamellar vesicles (LUVs) of lipid membranes in the liquid-ordered phase (lo phase) against a detergent, Triton X-100. We found that in the presence of high concentrations of Triton X-100, the structure of GUVs and LUVs of dipalmitoyl-PC (DPPC)/cholesterol (chol) and sphingomyelin (SM)/chol membranes in the lo phase was stable and no leakage of fluorescent probes from the vesicles occurred. We also found that ether-linked dihexadecylphosphatidylcholine (DHPC) membranes containing more than 20 mol% cholesterol were in the lo phase, and that DHPC/chol-GUV and DHPC/chol-LUV in the lo phase were stable and no leakage of internal contents occurred in the presence of Triton X-100. In contrast, octylglucoside solution could easily break these GUVs and LUVs of the lo phase membranes and induced internal contents leakage. These data indicate that GUVs and LUVs of the lo phase membranes are very valuable for practical use.     

Spectroscopic detection of endovesiculation by large unilamellar phosphatidylcholine vesicles: effects of chlorpromazine, dibucaine, and safingol.

Endovesiculation by large unilamellar vesicles (LUVs) induced by cationic amphiphiles is described in this work. A recent procedure to monitor phagocytosis of vesicles by macrophages by determining the amount of the simultaneously internalized water_soluble fluorescent dye HPTS with external quencher was adapted to LUVs (Daleke, D. L.; Hong, K.; Papahadjopoulos, D. Biochim. Biophys. Acta 1990, 1024, 352). Compared to dibucaine and safingol, the local anesthetic chlorpromazine (CPZ) was found to be the most efficient inducer of HPTS-internalization by LUVs. Control experiments using LUVs with entrapped HPTS indicated that the observed dye-internalization does not originate from transient lysis. A strong increase in activity above the critical micelle concentration of CPZ implies the importance of CPZ-micelles for endovesiculation. The significantly less efficient CPZ-induced HPTS-internalization by LUVs with 68 nm compared to 176 nm diameter further diminishes the likelihood of a micelle/bilayer fusion mechanism and supports the presence of 'zipper-type' endovesiculation by LUVs with diameters as small as 68 nm.     

New insight into the interaction of TRAF2 C-terminal domain with lipid raft microdomains

In this study we provide the first evidence of the interaction of a truncated-TRAF2 with lipid raft microdomains. We have analyzed this interaction by measuring the diffusion coefficient of the protein in large and giant unilamellar vesicles (LUVs and GUVs, respectively) obtained both from synthetic lipid mixtures and from natural extracts. Steady-state fluorescence measurements performed with synthetic vesicles indicate that this truncated form of TRAF2 displays a tighter binding to raft-like LUVs with respect to the control (POPC-containing LUVs), and that this process depends on the protein oligomeric state. Generalized Polarization measurements and spectral phasor analysis revealed that truncated-TRAF2 affects the membrane fluidity, especially when vesicles are heated up at physiological temperature. The addition of nanomolar concentration of TRAF2 in GUVs also seems to exert a mechanical action, as demonstrated by the formation of intraluminal vesicles, a process in which ganglioside GM1 plays a crucial role.     

Molecular properties of a stratum corneum model lipid system: large unilamellar vesicles.

Stratum corneum lipids are relatively complex, and there is little detailed understanding of their chemical and physical properties at the molecular level. Large unilamellar vesicles (LUVs) with lipid compositions similar to those of stratum corneum were prepared at pH 9 with commercially available lipids. This system was used as a model system for molecular studies of stratum corneum lipids. LUVs were chosen as the model system as they are comparatively more stable and can be characterized more quantitatively in terms of lipid concentration, surface area, and volume than model systems such as lipid mixture suspensions, lipid films, and small unilamellar vesicles. Results from freeze-fracture and cryo electron microscopy studies of our LUVs showed spherical vesicles. Quasi-elastic light scattering measurements revealed a narrow size distribution, centering around 119 nm. At room temperature, the LUVs were stable for several weeks at pH 9 and for more than 15 h but less than 24 h at pH 6. Differential scanning calorimetry measurements indicated broad endothermic transitions centered near 60-65 degrees C, closely matching the transition temperature reported for stratum corneum lipid extracts. Spin probes, 5-doxylstearic acid and 12-doxylstearic acid, were used for electron paramagnetic resonance (EPR) studies of the molecular dynamics of the lipids. EPR results indicated more restricted motion near the polar headgroup region than near the center of the alkyl chain region. Motional profiles of the spin labels near the polar headgroup and within the alkyl chain region in the LUVs were obtained as a function of temperature, ranging from 25 to 90 degrees C. We also found that the partitioning between the lipid and aqueous phases for each spin probe was temperature dependent and was generally correlated with phase transitions observed by differential scanning calorimetry and with alkyl chain mobility observed by EPR. Thus, this LUV system is well suited for additional molecular studies under different experimental conditions.     

Phospholipid vesicles promote human hemoglobin oxidation.

We have studied the heme oxidation kinetics of purified human hemoglobin (Hb) in the presence of lipid vesicles of dipalmitoyl phosphatidylcholine and bovine brain phosphatidylserine that exhibited minimal lipid peroxidation. We showed that the lipid vesicles enhanced Hb oxidation and that small unilamellar vesicles (SUVs) exerted a larger effect than large unilamellar vesicles (LUVs). We have determined pseudo first-order rate constants for the initial disappearance of oxygenated ferrous Hb (k0) and for the initial formation of several ferric Hb species (methemoglobin, hemichrome, and choleglobin) in the presence of SUVs and LUVs. k0 and other rate constants depended linearly on lipid-to-hemoglobin molar ratio (lipid/Hb), with k0SUV (h-1) = k0auto (h-1) + 3.7 x 10(-3) x lipid/Hb, and k0LUV (h-1) = k0auto (h-1) + 0.2 x 10(-3) x lipid/hb, where k0auto is the rate constant for Hb autoxidation in the absence of vesicles. Thus, in the absence of lipid peroxidation products, lipid vesicles themselves promote Hb oxidation by enhancing the rate of Hb oxidation. The enhanced oxidation was inhibited by catalase, but not by butylated hydroxytoluene. The rate constants were independent of Hb concentration, in the range of about 3.1 to 100 microM. We suggest that the lipid surface properties, including surface curvature, surface energy, and hydrophobicity, promote hemoglobin oxidation.     

Effect of dipalmitoylphosphatidylcholine vesicle curvature on the reaction with human apolipoprotein A-I

Large unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC) were prepared by sonication and were fractionated by gel filtration on Sepharose Cl-2B in the size range from 180- to 380-A Stokes radii. Negatively stained electron micrographs of these preparations indicated the presence of unilamellar, spheroidal structures of the expected size. Fluorescence polarization of diphenylhexatriene, dissolved in the vesicles, revealed progressively broader phase transitions, shifted to lower temperatures for vesicles of decreasing sizes. The fractionated unilamellar vesicles and multilamellar vesicles of DPPC were reacted with human apolipoprotein A-I at 41 degrees C for periods from 1 to 120 h. The reaction mixtures were then passed through a Bio-Gel A-5m column to separate unreacted lipid vesicles and protein from micellar complexes of DPPC with apolipoprotein A-I. Smaller vesicles were much more reactive than larger vesicles or multilamellar vesicles with the apolipoprotein. This difference in reactivity was explained by the increasing bilayer curvature of smaller vesicles which changes the packing of DPPC molecules in the bilayer and facilitates its penetration by the apolipoprotein.     

Preparation and Properties of Asymmetric Large Unilamellar Vesicles: Interleaflet Coupling in Asymmetric Vesicles Is Dependent on Temperature but Not Curvature

Asymmetry of inner and outer leaflet lipid composition is an important characteristic of eukaryotic plasma membranes. We previously described a technique in which methyl-β-cyclodextrin-induced lipid exchange is used to prepare biological membrane-like asymmetric small unilamellar vesicles (SUVs). Here, to mimic plasma membranes more closely, we used a lipid-exchange-based method to prepare asymmetric large unilamellar vesicles (LUVs), which have less membrane curvature than SUVs. Asymmetric LUVs in which sphingomyelin (SM) or SM + 1-palmitoyl-2-oleoyl-phosphatidylcholine was exchanged into the outer leaflet of vesicles composed of 1,2-dioleoyl-phosphatidylethanolamine (DOPE) and 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS) were prepared with or without cholesterol. Approximately 80–100% replacement of outer leaflet DOPE and POPS was achieved. At room temperature, SM exchange into the outer leaflet increased the inner leaflet lipid order, suggesting significant interleaflet interaction. However, the SM-rich outer leaflet formed an ordered state, melting with a midpoint at ∼37°C. This was about the same value observed in pure SM vesicles, and was significantly higher than that observed in symmetric vesicles with the same SM content, which melted at ∼20°C. In other words, ordered state formation by outer-leaflet SM in asymmetric vesicles was not destabilized by an inner leaflet composed of DOPE and POPS. These properties suggest that the coupling between the physical states of the outer and inner leaflets in these asymmetric LUVs becomes very weak as the temperature approaches 37°C. Overall, the properties of asymmetric LUVs were very similar to those previously observed in asymmetric SUVs, indicating that they do not arise from the high membrane curvature of asymmetric SUVs.     

Osmotically induced shape changes of large unilamellar vesicles measured by dynamic light scattering

Static and dynamic light scattering measurements have been used to characterize the size, size distribution, and shape of extruded vesicles under isotonic conditions. Dynamic light scattering was then used to characterize osmotically induced shape changes by monitoring changes in the hydrodynamic radius (R(h)) of large unilamellar vesicles (LUVs). These changes are compared to those predicted for several shapes that appear in trajectories through the phase diagram of the area difference elasticity (ADE) model (. Phys. Rev. E. 52:6623-6634). Measurements were performed on dioleoylphosphatidylcholine (DOPC) vesicles using two membrane-impermeant osmolytes (NaCl and sucrose) and a membrane-permeant osmolyte (urea). For all conditions, we were able to produce low-polydispersity, nearly spherical vesicles, which are essential for resolving well-defined volume changes and consequent shape changes. Hyper-osmotic dilutions of DOPC vesicles in urea produced no change in R(h), whereas similar dilutions in NaCl or sucrose caused reductions in vesicle volume resulting in observable changes to R(h). Under conditions similar to those of this study, the ADE model predicts an evolution from spherical to prolate then oblate shapes on increasing volume reduction of LUVs. However, we found that DOPC vesicles became oblate at all applied volume reductions.     

Antifungal property of dihydrodehydrodiconiferyl alcohol 9'-O-β-d-glucoside and its pore-forming action in plasma membrane of Candida albicans

The aims of this study were to investigate the antifungal activity as a bioactive property of dihydrodehydrodiconiferyl alcohol 9'-O-β-d-glucoside (DDDC9G) and the mode of action(s) involved in its effect. Antifungal susceptibility testing showed that DDDC9G possessed potent antifungal activities toward various fungal strains with almost no hemolytic effect. To understand the antifungal mechanism(s) of DDDC9G, we conducted the following experiments in this study using Candida albicans. Fluorescence experiments using the probes, 1, 6-diphenyl-1, 3, 5-hexatriene (DPH) and propidium iodide suggested that DDDC9G perturbed the fungal plasma membrane. Consecutively, the analysis of the transmembrane electrical potential (ΔΨ) with 3, 3'-dipropylthiadicarbocyanine iodide [DiSC(3)(5)] and bis-(1, 3-dibutylbarbituric acid) trimethine oxonol [DiBAC(4)(3)] indicated that DDDC9G induced membrane-depolarization. Furthermore, model membrane studies were performed with rhodamine-labeled giant unilamellar vesicles (GUVs), calcein encapsulating large unilamellar vesicles (LUVs), and FITC-dextran (FD) loaded LUVs. These results demonstrated that the antifungal effects of DDDC9G upon the fungal plasma membrane were through the formation of pores with the radii between 0.74nm and 1.4nm. Finally, in three dimensional (3D) flow cytometric contour plots, a reduced cell size was observed as a result of osmolarity changes from DDDC9G-induced structural and functional membrane damages. Therefore, the present study suggests that DDDC9G exerts its antifungal effect by damaging the membrane through pore formation in the fungal plasma membrane.     

siRNA carriers based on carbosilane dendrimers affect zeta potential and size of phospholipid vesicles

One of the major limitations in gene therapy is an inability of naked siRNA to passively diffuse through negatively charged cell membranes. Therefore, the siRNA transport into a cell requires efficient carriers. In this work we analyzed the charge-dependent interaction of the complexes of cationic carbosilane dendrimers (CBD) and anti-HIV siRNA (dendriplexes) with the model membranes - large unilamellar vesicles (LUV). We used the second generation of branched with CBD carbon-silicon bonds (CBD-CS) which are water-stable and that of oxygen-silicon bonds (CBD-OS) which are slowly hydrolyzed in aqueous solutions. The LUVs were composed of zwitterionic dimyristoylphosphatidylcholine (DMPC), negatively charged dipalmitoylphosphatidylglycerol (DPPG) and their mixture (DMPC/DPPG, molar ratio 7:3). The interaction of dendriplexes with LUVs affected both zeta potential and size of the vesicles. The changes of these values were larger for the negatively charged LUV. CBD-CS resulted in the decrease of zeta potential values to more negative ones, whereas an opposite effect took place for CBD-OS suggesting a different kind of interaction between LUVs and the dendriplexes. The results indicate that both CBD-CS and CBD-OS can be used for transport of siRNA into the cells. However, CBD-CS are preferred due to a better stability in water and improved bioavailability of siRNA on their surface.     

Vesicle fusion with bilayer lipid membrane controlled by electrostatic interaction

The fusion of proteoliposomes is a promising approach for incorporating membrane proteins in artificial lipid membranes. In this study, we employed an electrostatic interaction between vesicles and supported bilayer lipid membranes (s-BLMs) to control the fusion process. We combined large unilamellar vesicles (LUVs) containing anionic lipids, which we used instead of proteoliposomes, and s-BLMs containing cationic lipids to control electrostatic interaction. Anionic LUVs were never adsorbed or ruptured on the SiO₂ substrate with a slight negative charge, and selectively fused with cationic s-BLMs. The LUVs can be fused effectively to the target position. Furthermore, as the vesicle fusion proceeds and some of the positive charges are neutralized, the attractive interaction weakens and finally the vesicle fusion saturates. In other words, we can control the number of LUVs fused with s-BLMs by controlling the concentration of the cationic lipids in the s-BLMs. The fluidity of the s-BLMs after vesicle fusion was confirmed to be sufficiently high. This indicates that the LUVs attached to the s-BLMs were almost completely fused, and there were few intermediate state vesicles in the fusion process. We could control the position and amount of vesicle fusion with the s-BLMs by employing an electrostatic interaction.