Curvature effect on the structure of phospholipid bilayers

High-resolution small-angle X-ray scattering (SAXS), complemented by small-angle neutron scattering (SANS) and dynamic light scattering (DLS) experiments, was used to study the effect of curvature on the bilayer structure of dioleoyl-phosphatidylcholine (DOPC) and dioleoyl-phosphatidylserine (DOPS) unilamellar vesicles (ULVs). Bilayer curvature, as a result of finite vesicle size, was varied as a function of vesicle radius and determined by DLS and SANS measurements. Unilamellarity of large DOPC ULVs was achieved by the addition of small amounts (up to 4 mol %) of the charged lipid, DOPS. A comparison of SANS data over the range of 0.02 < q <0.2 A-1 indicated no change in the overall bilayer thickness as a function of ULV diameter (620 to 1840 A). SANS data were corroborated by high-resolution (0.06 < q <0.6 A-1) SAXS data for the same diameter ULVs and data obtained from planar samples of aligned bilayers. Both the inner and outer leaflets of the bilayer were found to be indistinguishable. This observation agrees well with simple geometric models describing the effect of vesicle curvature. However, 1220-A-diameter pure DOPS ULVs form asymmetric bilayers whose structure can most likely be rationalized in terms of geometrical constraints coupled with electrostatic interactions, rather than curvature alone.     

Formation of kinetically trapped nanoscopic unilamellar vesicles from metastable nanodiscs

Zwitterionic long-chain lipids (e.g., dimyristoyl phosphatidylcholine, DMPC) spontaneously form onion-like, thermodynamically stable structures in aqueous solutions (commonly known as multilamellar vesicles, or MLVs). It has also been reported that the addition of zwitterionic short-chain (i.e., dihexanoyl phosphatidylcholine, DHPC) and charged long-chain (i.e., dimyristoyl phosphatidylglycerol, DMPG) lipids to zwitterionic long-chain lipid solutions results in the formation of unilamellar vesicles (ULVs). Here, we report a kinetic study on lipid mixtures composed of DMPC, DHPC, and DMPG. Two membrane charge densities (i.e., [DMPG]/[DMPC] = 0.01 and 0.001) and two solution salinities (i.e., [NaCl] = 0 and 0.2 M) are investigated. Upon dilution of the high-concentration samples at 50 °C, thermodynamically stable MLVs are formed, in the case of both weakly charged and high salinity solution mixtures, implying that the electrostatic interactions between bilayers are insufficient to cause MLVs to unbind. Importantly, in the case of these samples small angle neutron scattering (SANS) data show that, initially, nanodiscs (also known as bicelles) or bilayered ribbons form at low temperatures (i.e., 10 °C), but transform into uniform size, nanoscopic ULVs after incubation at 10 °C for 20 h, indicating that the nanodisc is a metastable structure. The instability of nanodiscs may be attributed to low membrane rigidity due to a reduced charge density and high salinity. Moreover, the uniform-sized ULVs persist even after being heated to 50 °C, where thermodynamically stable MLVs are observed. This result clearly demonstrates that these ULVs are kinetically trapped, and that the mechanical properties (e.g., bending rigidity) of 10 °C nanodiscs favor the formation of nanoscopic ULVs over that of MLVs. From a practical point of view, this method of forming uniform-sized ULVs may lend itself to their mass production, thus making them economically feasible for medical applications that depend on monodisperse lipid-based systems for therapeutic and diagnostic purposes.     

Highly stable phospholipid unilamellar vesicles from spontaneous vesiculation: A DLS and SANS study

Spontaneously formed unilamellar vesicles (ULV) composed of short- and long-chain phospholipids, dihexanoyl phosphorylcholine (DHPC) and dimyristoyl phosphorylcholine (DMPC), respectively, were doped with a negatively charged lipid, dimyristoyl phosphorylglycerol (DMPG), and studied with small-angle neutron scattering (SANS) and dynamic light scattering (DLS). Upon dilution, the spontaneous formation of vesicles was found to take place from bilayered micelles, or so-called "bicelles". SANS and DLS data show that ULV with narrow size distributions are highly stable at low lipid (C(lp) < 0.50 wt %) and NaCl salt (C(s)) concentrations. ULV size was found to be independent of both C(lp) and C(s) when they were below 0.33 and 0.5 wt %, respectively. Surface charge and salinity were found to be important factors in preparing ULV of a certain size. This observation is not in complete agreement with previous experimental results and cannot be completely explained with current theoretical predictions based on equilibrium calculations for catanionic surfactant mixtures. ULV size is found to be invariant over a wide range of temperatures, both below and above the phase-transition temperature, T(M), of DMPC, and was stable for periods of weeks and months, even after sonication.     

Solubilization of phosphatidylcholine vesicles by hydrophobically modified poly(acrylamide)-co-(acrylic acid): effects of acrylic acid fraction and polymer concentration

The interaction of hydrophobically modified copolymers of acrylamide and acrylic acid, designated as PAM-C12-AA (X%) (X% indicates the percentage of acrylic acid unit and X = 5, 10, 20), with dimyristoylphosphatidylcholine (DMPC) vesicles has been studied. Complementary techniques including isothermal titration microcalorimetry (ITC), differential scanning calorimetry (DSC), turbidity measurement, calcein leakage measurement, dynamic light scattering (DLS), and transmission electron microscopy (TEM) were used to get comprehensive information. The results show that PAM-C12-AA leads to solubilization of DMPC vesicles. There is a critical concentration (C(s)) for PAM-C12-AA to induce obvious vesicle disruption. This concentration is very close to the critical aggregation concentration (CAC) for the polymer self-aggregation. The Cs values are found to be similar for the three polymers. However, the disruption of DMPC vesicles induced by the polymers increases to a greater degree at higher AA fraction, owing to the increasing strength of interaction between the polymer and the lipid bilayer.     

Nanoscopic Characterization of Stearic Acid Release from Bovine Serum Albumin Hydrogels

The release behavior of 16‐doxyl stearic acid (16‐DSA) from hydrogels made from bovine serum albumin (BSA) is characterized. 16‐DSA serves as a model tracer molecule for amphiphilic drugs. Various hydrogel preparation procedures are tested and the fatty acid release from the different gels is compared in detail. These comparisons reach from the macroscopic level, the viscoelastic behavior via rheological characterization to changes on the nanoscopic level concerning the secondary structure of the protein during gelation through infrared (ATR‐IR) spectroscopy. 16‐DSA‐BSA interaction via continuous wave electron paramagnetic resonance (CW EPR) spectroscopy in addition gives a nanoscopic view of small molecule–hydrogel interaction. The combined effects of fatty acid concentration, hydrogel incubation time, and gelation procedures on release behavior are studied via CW EPR spectroscopy and dynamic light scattering (DLS) measurements, which provide deep insight on the interaction of 16‐DSA with BSA hydrogels and the nature and size of the released components, respectively. It is found that the release rate of the fatty acid from BSA hydrogels depends on and can thus be tuned through its loading percentage, duration of hydrogel formation and the type of gelation methods. All of the results confirm the potential of these gels as delivery hosts in pharmaceutical applications allowing the sustained release of drug.     

Colloidal dispersions of monodisperse magnetite nanoparticles modified with poly(ethylene glycol)

Monodisperse magnetite nanoparticles modified with poly(ethylene glycol) (PEG) were synthesized using a silane functionalized PEG obtained by reacting 3-aminopropyl triethoxysilane with carboxylic acid-methoxy PEG (mPEG-COOH) using amide reactions. Transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential measurements show the particles are monodisperse (sigma(gv) approximately 0.2) and stable in water for pH of 3-9 and ionic strengths, up to 0.3 M NaCl. Thermogravimetric analysis coupled with TEM and DLS indicates formation of a dense graft layer on the particle surface. An analysis of the interparticle interaction energy indicates that the particles are stabilized by strong steric repulsions between PEG chains on their surface.     

Lipid bilayer disruption by polycationic polymers: the roles of size and chemical functional group

Polycationic polymers are used extensively in biology to disrupt cell membranes and thus enhance the transport of materials into the cell. The highly polydisperse nature of many of these materials makes obtaining a mechanistic understanding of the disruption processes difficult. To design an effective mechanistic study, a monodisperse class of polycationic polymers, poly(amidoamine) (PAMAM) dendrimers, has been studied in the context of supported dimyristoylphosphatidylcholine (DMPC) lipid bilayers using atomic force microscopy (AFM). Aqueous solutions of amine-terminated generation 7 (G7) PAMAM dendrimers caused the formation of 15-40-nm-diameter holes in lipid bilayers. This effect was significantly reduced for smaller G5 dendrimers. For G3, no hole formation was observed. In addition to dendrimer size, surface chemistry had a strong influence on dendrimer-lipid bilayer interactions. In particular, acetamide-terminated G5 did not cause hole formation in bilayers. In all instances, the edges of bilayer defects proved to be points of highest dendrimer activity. A proposed mechanism for the removal of lipids by dendrimers involves the formation of dendrimer-filled lipid vesicles. By considering the thermodynamics, interaction free energy, and geometry of these self-assembled vesicles, a model that explains the influence of polymer particle size and surface chemistry on the interactions with lipid membranes was developed. These results are of general significance for understanding the physical and chemical properties of polycationic polymer interactions with membranes that lead to the transport of materials across cell membranes.     

Spontaneously forming ellipsoidal phospholipid unilamellar vesicles and their interactions with helical domains of saposin C

We have observed a bimodal distribution of ellipsoidal unilamellar vesicles (ULVs) in a phospholipid mixture composed of dioleoyl phosphatidylserine (DOPS) and dipalmitoyl and dihexanoyl phosphatidylcholine, DPPC and DHPC, respectively. Dynamic light scattering and transmission electron microscopy data indicate a bimodal size distribution of these nanoparticles with hydrodynamic radii of approximately 200 and >500 nm, while small-angle neutron scattering data were fit using a model of coexisting monodisperse morphologies, namely, oblate and triaxial ellipsoidal vesicles. Unlike DOPS ULV formed by sonication, which can fuse days after being formed, these ULVs are stable over a period of 12 months at 4 degrees C. We also report on the structure of these ULVs associated with the two helical peptide domains (H1 and H2) of a glucosylprotein, namely, Saposin C, to gain some insight into protein-membrane interactions.     

Bicellar systems as modifiers of skin lipid structure

The characterization of different bicellar aggregates and the effects of these systems on the stratum corneum (SC) microstructure have been studied. Dynamic light scattering (DLS) and freeze fracture electron microscopy (FFEM) techniques showed that both of the systems studied, dimyristoyl-phosphatidylcholine/dihexanoyl-phosphocholine (DMPC/DHPC) and dipalmitoyl-phosphocholine (DPPC)/DHPC, were formed by small discoidal aggregates at room temperature (20°C). Treating skin with DMPC/DHPC bicelles does not affect the SC lipid microstructure, whereas bicellar systems formed by DPPC and DHPC can promote the formation of new structures in the SC lipid domains. This indicates the passage of lipids from bicelles through the SC layers and also a possible interaction of these lipids with the SC lipids. Given the absence of surfactant in the bicellar composition and the small size of these structures, the use of these smart nano-systems offers great advantages over other lipid systems for dermatological purposes. Bicelles could be promising applications as drug carriers through the skin. This contribution, based on the new biological use of bicelles, may be useful to scientists engaged in colloid science and offers a new tool for different applications in skin and cosmetic research.     

Interaction of sodium N-lauroylsarcosinate with N-alkylpyridinium chloride surfactants: spontaneous formation of pH-responsive, stable vesicles in aqueous mixtures

The interaction of sodium N-lauroylsarcosinate (SLS) with N-cetylpyridinium chloride (CPC) and N-dodecylpyridinium chloride (DPC) was investigated in aqueous mixtures. A strong interaction between the anionic and cationic surfactants was observed. The interaction parameter, β was determined for a wide composition range and was found to be negative. The mixed systems were found to have much lower critical micelle concentration (cmc) and surface tension at cmc. The surfactant mixtures exhibit synergism in the range of molar fractions investigated. The self-assembly formation in the mixtures of different compositions and total concentrations were studied using a number of techniques, including surface tension, fluorescence spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), confocal fluorescence microscopy (CFM). Thermodynamically stable unilamellar vesicles were observed to form upon mixing of the anionic and cationic surfactants in a wide range of composition and concentrations in buffered aqueous media. TEM as well as DLS measurements were performed to obtain shape and size of the vesicular structures, respectively. These unilamellar vesicles are stable for periods as long as 3 months and appear to be the equilibrium form of aggregation. Effect of pH, and temperature on the stability was investigated. The vesicular structures were observed to be stable at pH as low as 2.0 and at biological temperature (37°C). In presence of 10 mol% of cholesterol the mixed surfactant vesicles exhibited leakage of the encapsulated calcein dye, showing potential application in pH-triggered drug release.     

Creation of highly stable selenium nanoparticles capped with hyperbranched polysaccharide in water

Water-dispersible selenium nanoparticles (SeNPs) were created by using natural hyperbranched polysaccharide (HBP) as the stabilizer and capping agent under extremely safe conditions. The structure, morphology, size, and stability of the nanocomposites were investigated by transmission electron microscopy (TEM), atomic force microscopy (AFM), and static and dynamic light scattering (DLS) measurements. The results revealed that the spherical selenium nanoparticles (mean particle size of about 24 nm) were ligated with HBP to form nanocomposites (Se-HBP) in aqueous solution and were stable for over one month. In our findings, supported by the results of FTIR, TEM, AFM, and DLS, SeNPs were capped with the HBP macromolecules, as a result of strong physical adsorption of OH groups on Se surfaces, leading to a highly stable structure of Se nanoparticles in water. This work provided reaction sites for the complexation between HBP and Se to fabricate well-dispersed Se nanoparticles in aqueous system with potential bioapplications.     

Spontaneously Forming Unilamellar Phospholipid Vesicles

Unilamellar vesicles (ULV) consisting of a single lipid bilayer are of special interest as drug delivery vehicles. Here, we report on a spontaneously forming ULV system composed of the short- and long-chain phospholipids, dihexanoyl (DHPC) and dimyristoyl (DMPC) phosphorylcholine, respectively, doped with the negatively charged lipid, dimyristoyl phosphorylglycerol (DMPG). Small-angle neutron scattering (SANS) and dynamic light scattering (DLS) were employed to systematically investigate the effects of lipid concentration, salinity, and time on vesicle stability. It is found that ULV size is practically constant over a range of lipid concentration and temperature. The spontaneously formed ULV are stable for periods of four months, or greater, without the use of stabilizers.     

Subgel studies of dimyristoylphosphatidylcholine bilayers

It is known that when bilayers of some saturated phosphatidylcholines are stored for 3 or more days at approximately 0 degrees C, a lamellar subgel (Lc) phase is detected at temperatures below the pretransition by differential scanning calorimetry (DSC). However, the subgel (Lc) phase and the corresponding subtransition (Lc--> Lbeta') for dimyristoylphosphatidylcholine (DMPC) has not been clearly characterized. In this study, using the temperature jump protocol first developed by Tristram-Nagle et al. for the dipalmitoylphosphatidylcholine (DPPC) system, new and accurate data characterizing the subgel formation and subtransition of DMPC were obtained through DSC and fluorescence spectroscopy with 1,6-diphenyl-1,3,5-hexatriene (DPH). It was discovered that the formation of the DMPC subgel phase requires incubation at temperatures of -5 degrees C or lower for 2 h or more. Kinetics of the subgel formation indicate that it is a very complex process and demonstrates that the planar gel phase is merely metastable below the subtransition, and not the thermodynamically stable phase. The subgel growth of DMPC is proven to be the dehydration of the headgroup region, and the subtransition is a process in which poorly hydrated DMPC becomes hydrated.     

Characterizing assembly morphology changes during solubilization process of dimyristoyl phosphocholine vesicles by n-dodecyl triethylammonium bromide

In the present work, the assembly morphology changes during the solubilization process of the sonicated unilamellar vesicles from dimyristoyl phosphocholine (DMPC) by a cationic surfactant, n-dodecyl triethylammonium bromide (DTEAB) were well characterized with DSC, FF-TEM and DLS and fluorescence probes technique. Based on an analysis on the above results, a primary multi-stage model was brought forward to sketch the assembly morphology changes during the DMPC vesicle solubilization by DTEAB. In comparison with classical models, vesicles division, tubule-like structure formation and fission to vesicle were found in the middle stages of this model. Additionally, it is the first time that the transversally-cut profiles of tubule-like structures were observed during vesicle solubilization process.     

Solubilization and phase equilibria of water-in-oil microemulsions : II. Effects of alcohols, oils, and salinity on single-chain surfactant systems

The solubilization and phase equilibria of w/o microemulsions have been shown to be dependent on two phenomenological parameters, namely the spontaneous curvature and elasticity of the interfacial film, when interfacial tension is very low. The spontaneous curvature of an interface is basically determined by the geometric packing of surfactant and cosurfactant molecules at the interface, whereas the interfacial elasticity is related to the energy required to bend the interface. The droplet size and solubilization of microemulsions is mainly determined by the radius of spontaneous curvature, and is further influenced by interfacial elasticity and interdroplet interactions. A w/o microemulsion with a highly curved and relatively rigid interfacial film can exist in equilibrium with excess water at the solubilization limit due to the interfacial bending stress. Increasing the natural radius and fluidity of the interface can increase the droplet size and hence the solubilization in the microemulsion. On the other hand, a w/o microemulsion with a highly fluid interfacial film can exist in equilibrium with an excess oil phase containing a low density of microemulsion droplets due to attractive interdroplet interaction. Increasing the interfacial rigidity and decreasing the natural radius in this case can increase water solubilization in the microemulsion by retarding the phase separation process. Thus, a maximum water solubilization in a w/o microemulsion can be obtained by minimizing both the interfacial bending stress of rigid interfaces and the attractive interdroplet interaction of fluid interfaces at an optimal interfacial curvature and elasticity. The study of phase equilibria of microemulsions can serve as a simple method to evaluate the property of the interface and provide phenomenological guidance for the formulation of microemulsions with maximum solubilization capacity.     

Light scattering and luminescence studies on self-aggregation behavior of amphiphilic copolymer micelles

The self-aggregation behavior of three amphiphilic graft copolymers, oligo(9,9-dihexyl)fluorence-graft-poly(ethylene oxide) (OHF-g-PEO), with different architectures was studied by dynamic and static light scattering (DLS and SLS) in combination with fluorescence spectroscopy and transmission electron microscopy (TEM). The formation of self-assembled polymeric micelles was confirmed by SLS and TEM. DLS and SLS analyses showed that the architecture of graft copolymers has a dramatic effect on critical aggregation concentration (CAC), micelle size distribution, apparent aggregation number (Nagg app), and apparent molecular weight of polymer aggregates (Mw,agg app). An architecture-dependent excimer emission, resulting from the pi-pi stacking of the oligofluorene backbones, was also observed from the photoluminescence spectra of the micelle aqueous solutions, which indicated a strong intermolecular interaction among the polymeric molecules. The excimer emission was further investigated by time-resolved fluorescence spectroscopy.     

Charge-induced unilamellar vesicle formation and phase separation in solutions of Di-n-decylmethylamine oxide

A double-tail amine oxide surfactant, di-n-decylmethylamine oxide (2C10MAO), was prepared, and the effects of protonation on aggregate structure were examined by small-angle neutron scattering (SANS), cryo-transmission electron microscopy (cryo-TEM), turbidity, electric conductivity, and solubilization of an oil-soluble dye at various degrees of neutralization, X, defined as the mole ratio of HCl/2C10MAO. The surfactant makes an L(2) phase in the nonprotonated state (X = 0) in water. The L(2) phase is in equilibrium with an aqueous L(1) phase. On protonation, unilamellar vesicles (ULVs) are formed over a wide range of compositions (0.05 < X< 0.4-0.5 at C = 10 mM) as observed by cryo-TEM. At X = 0.2, the ULV is stable over a wide concentration range (3 mM < or = C < 0.1 M), but an L(alpha) phase replaces the vesicle phase at C > 0.1 M. SANS results show that the mean radius of the ULV is about 25 nm and the bilayer thickness is about 2 nm, consistent with the extended configuration of the alkyl chains of the surfactant. An important contribution to the enhanced stability of the bilayer structures over the L(2) phase is suggested to be the translational entropy of the counterions. The enhanced stability of the bilayers diminishes as the counterion concentration increases either by an increase of X or by the addition of a salt. When the counterion concentration exceeds a critical value, the ULV solutions transform into the L(2) phase (or L(2)/L(1) two-phase system at low surfactant concentrations). The critical composition X is about 0.4-0.5 in water, but it is below 0.4 in D(2)O. The critical NaCl concentration is below 5 mM at X = 0.2. The stability of ULVs against multilamellar vesicles is ascribed partly to undulation forces and partly to the adjustable nature of the spontaneous curvature of amine oxide monolayers. The characteristics of the ULV of the surfactant remain the same within a temperature range 25-50 degrees C at X = 0.2. An iridescent lamellar phase and possibly an L(3) phase were observed in a very narrow X range (0 < X < 0.02) prior to the vesicle phase.     

Interaction of the glycoalkaloid tomatine with DMPC and sterol monolayers studied by surface pressure measurements and Brewster angle microscopy

The interaction of the glycoalkaloid tomatine with monolayers of dimyristoylphosphatidylcholine (DMPC) and cholesterol, as well as other selected sterols, has been investigated using surface pressure measurements at constant area and Brewster angle microscopy (BAM). The interaction of tomatine with sterol monolayers is found to vary with the structure of the sterol. The interaction of tomatine with cholesterol-containing monolayers results in a surface pressure increase accompanied by the appearance of a mottled texture. Morphological changes are observed that suggest the formation of tomatine-cholesterol complexes that aggregate at the water-air interface. No morphology change observable by BAM is observed for monolayers containing epicholesterol, suggesting that the stereochemistry of hydrogen bonding between the sterol and the sugar units on tomatine is of particular significance. Strong interactions are observed with cholestanol- and coprostanol-containing monolayers, and BAM reveals formation of spiked aggregates upon interaction with 7:3 mole ratio DMPC/coprostanol mixed monolayers. More modest surface pressure changes are observed for cholestenone- and epicoprostanol-containing monolayers. A much smaller surface pressure increase is observed when tomatine is injected beneath a pure DMPC monolayer.     

可聚合囊泡体系(烯丙基-二甲基-十二烷基溴化胺和十二烷基硫酸钠)在溶液中的盐效应

高稳定的囊泡广泛用于制作生物模型、药物输送以及合成纳米材料的模板。获得高稳定囊泡结构的重要方法之一是用聚合反应固定囊泡结构。作为可聚合囊泡制备的前期基础工作,研究了一种可聚合的囊泡体系：1-丙烯基－2,2,二甲基－十二烷基溴化胺（ADDB）和ADDB与十二烷基磺酸钠（SDS）的等摩尔比混合体系。该囊泡体系即使在高浓度盐水中也能够自发地形成均相的囊泡溶液。在聚合之前,采用动态激光光散射（DLS）、冷冻蚀刻透射电镜（FF-TEM）技术研究了可聚合囊泡的盐效应。DLS测试发现没有盐存在时,囊泡大小为83 nm,盐的浓度增加到250 mmol/L时,囊泡尺寸增大到250 nm。然而继续增大盐浓度到1000 mmol/L, 囊泡尺寸减小到180nm. FF-TEM结果发现盐浓度小于150 mM时, 单个囊泡为70 nm左右,然而明显存在囊泡的絮凝与融合;当盐浓度增加到400 mM时,单个囊泡尺寸减小到20 nm. 因此DLS 观测到囊泡尺寸增大的原因是由于囊泡的絮凝与融合;而尺寸减小的原因是由于在高盐浓度下,盐屏蔽了带电颗粒之间的静电相互作用,在熵增的驱使下,大囊泡变成小囊泡。