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.     

Structural Phase Behavior of High-Concentration,Alignable Biomimetic Bicelle Mixtures

The structures of bicelle mixtures composed of dimyristoyl and dihexanoyl phosphatidylcholines (DMPC and DHPC) with DMPC/DHPC molar ratios of 3.2 and 5 are characterized using polarized optical microscopy (POM) and small angle neutron scattering (SANS). Three phases, isotropic (I), chiral nematic (N^*) and smectic (S) are observed as temperature (T) varies from 10 to 70 °C. The structure of the magnetically alignable N^* phase, which was previously considered to be made up of discoidal micelles, is found to be composed of “ribbons”. Doping with the charged lipid, dimyristoyl phosphatidylglycerol (DMPG), which has the same 14:0 hydrocarbon chains as DMPC, results in a structural change of the aggregates where only the isotropic and smectic phases are observed. The smectic phase for the mixtures doped with DMPG is shear-alignable and follows one-dimensional swelling. However, at high-T zwitterionic DMPC/DHPC mixtures form multi-lamellar vesicles (MLV) with a relatively constant lamellar spacing of 66 Å, independent of water content.     

Salicylic acid-induced effects in the mixed-lipid (dipalmitoyl phosphatidylcholine-dipalmitoyl phosphatidylethanolamine) model membrane

The effect of the keratolytic drug salicylic acid (SA) on the thermotropic properties and fluidity of the mixed lipid membrane dipalmitoyl phosphatidylcholine (DPPC)-dipalmitoyl phosphatidylethanolamine (DPPE) had been studied using DSC, (1H and 31P) NMR, SAXS, and dynamic light scattering. The membrane was in multilamellar vesicular (MLV) and unilamellar vesicular (ULV) form with SA/(DPPC+DPPE) molar ratios, R(m), in the range from 0 to 0.5. It was found that the mechanism of interaction of SA with the lipid mixture exhibited similar patterns in both ULV and MLV. Both the NMR and DSC studies indicated that the drug molecules were probably localized in the lipid-water interfacial region neighboring the lipid headgroups or the glycerol moiety. The presence of the drug increased the fluidity of the membrane and the acyl chain order. However, studies on MLV showed that the presence of the drug in high concentration (R(m)0.2), caused destabilization of the DPPC-DPPE mixture, as indicated by the appearance of two endothermic transitions. DSC studies indicated that prolonged equilibration of the membrane led to reduced interaction between the lipid headgroups and the SA molecules. This reduced interaction could be due to the sequestering of the drug molecules into the lipid-water interfacial region, out of proximity to the polar headgroup or glycerol moiety. Effect of inclusion of cholesterol in the membrane systems was also studied.     

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.     

Spontaneously formed unilamellar vesicles with path-dependent size distribution

We observe the spontaneous formation of path-dependent monodisperse and polydisperse phospholipid unilamellar vesicles (ULV) from two different equilibrium morphologies specifically, disklike micelles and extended lamellae, respectively. On heating beyond a temperature Tc, low temperature disklike micelles, or so-called bicelles, transform into lamellae. Dilution of the lamellar phase, at a fixed temperature, results in a complete unbinding transition and the formation of polydisperse ULV, demonstrating the instability of the lamellar phase. On the other hand, heating of a dilute bicellar phase above Tc results in monodisperse ULV, which on cooling revert back to bicelles for lipid concentrations phi > or = 0.5 wt % and transform into oblate ellipsoids for phi = 0.1 wt %, a morphology not previously seen in "bicellar" lipid mixtures. Monodisperse ULV reform on heating of the oblate ellipsoids.     

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.     

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]).     

多菌灵与Pd（II）体系的共振瑞利散射光谱及分析应用

在pH=10.0的Britton-Robinson（BR）缓冲溶液中,多菌灵与Pd(Ⅱ)反应形成1∶1的六元螯合物,导致共振瑞利散射（RRS）、二级散射（SOS）和倍频散射（FDS）显著增强,并产生新的共振瑞利散射光谱,其最大RRS、SOS和FDS波长分别位于309、606和310 nm。 在一定范围内,3种散射增强（ΔIRRS、ΔISOS和ΔIFDS）均与多菌灵的浓度成正比,反应具有较高的灵敏度,对于多菌灵的检出限分别为7.1×10-9 g/mL（RRS）、7.4×10-9 g/mL（SOS）和10.7×10-9 g/mL（FDS）。 据此提出了测定多菌灵的光散射新方法。 以灵敏度最高的RRS法为例,测定了西芹和市售农药中多菌灵的含量,结果与标准方法一致。 文中还对反应机理和散射增强的原因进行了讨论。     

Reduced fluidity of dipalmitoyl phosphatidic acid membranes by salicylic acid

This paper presents DSC and NMR study of how the kerotolytic drug, salicylic acid (SA), affects the thermotropic and morphological behavior of a model membrane, dipalmitoyl phosphatidic acid (DPPA). The membrane-drug system has been studied in the multilamellar vesicular (MLV) and in the unilamellar vesicular (ULV) forms, for SA/DPPA molar ratios from 0 to 0.5. The mode of interaction of SA molecules with DPPA is similar in MLV and ULV. Chain-melting transition becomes sharper and shifts to higher temperatures in the presence of the drug, implying an enhanced co-operativity of the acyl chains. NMR and DSC data indicate that the drug molecules are located in the aqueous interfacial region neighboring the lipid headgroups. The membrane becomes more rigid in the presence of the drug molecules, due to a stronger interaction between the lipid headgroups leading to reduced permeability. ULVs are destroyed by even a short equilibration at room temperature, whereas prolonged equilibration of the MLV only leads to a slightly reduced interaction between the lipid headgroups due to sequestering of the drug molecules in the interfacial aqueous region.     

A study on the sizes and concentrations of gold nanoparticles by spectra of absorption, resonance Rayleigh scattering and resonance non-linear scattering

Liquid phase gold nanoparticles with different diameters and colors can be prepared using sodium citrate reduction method by controlling the amounts of sodium citrate. The mean diameters of gold nanoparticles are measured by transmission electron microscope (TEM). Gold nanoparticles with different sizes have specific absorption spectra. When the diameters of nanoparticles is between 12 and 41 nm, the maximum absorption peaks locate at 520-530 nm and there are red shifts gradually with the increase of diameters of gold nanoparticles. And when the size of gold nanoparticle is constant, the absorbance is proportional to the concentration of gold. Obvious resonance Rayleigh scattering (RRS) and the resonance non-linear scattering such as second-order scattering (SOS) and frequency-doubling scattering (FDS) appear at the same time as well, and the maximum scattering peaks are located at 286 nm (RRS), 480 nm (SOS) and 310 nm (FDS), respectively. When the concentration of gold is constant, absorbance and the intensities of RRS, SOS and FDS (I(RRS), I(SOS) and I(FDS)) have linear relationships with the diameters of gold nanoparticles. When the diameter of gold nanoparticle is constant, the absorbance and I(RRS), I(SOS), I(FDS) are directly proportional to the concentrations of gold nanoparticles. Therefore, it is very useful for studying the liquid phase gold nanoparticles by investigating the absorption, RRS, SOS and FDS spectra.     

Light scattering detection in aqueous high-performance gel permeation chromatography (HPGPC): Relative molecular mass distribution of eye lens proteins

Summary Light scattering techniques have been greatly improved since laser light sources, enabling dynamic light scattering and measurements at low angles have been available. Relative molecular mass determinations can be performed at low solute concentration, extrapolations to zero-angle are no longer required and, because of the low scattering volume, a flow-through cell construction has become possible where dust particles only interfere as spikes [1]. For HPLC detection the low-angle laser light scattering (LALLS) signal corresponds to the product of relative molecular mass and concentration; combination with a concentration detector (for example, differential refractometer, UV absorbance) enables calculation of relative molecular mass [2]. The sensitivities of LALLS and the usual concentration detectors are comparable for (bio)polymers with relative molecular masses near 100,000. The minimum relative molecular mass to be determined in this way is near 10,000 and, therefore, the only mode of HPLC using the LALLS detector to date is gel permeation (size exclusion) chromatography.     

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.     

Analytical Application of Resonance Rayleigh Scattering,Frequency Doubling Scattering,and Second-Order Scattering Spectra for the Sodium Alginate-CTAB System

Three new methods for the determination of trace amounts of sodium alginate (SA) based on the reaction of SA with cetyltrimethylammonium bromide (CTAB) by resonance Rayleigh scattering (RRS), frequency doubling scattering (FDS), and second-order scattering (SOS) have been investigated. The SA can react with CTAB in a pH 10.0 Britton–Robinson buffer to form a new product, which can lead to a significant enhancement of RRS, FDS, and SOS intensities and appearance of new spectra. The maximum scattering wavelengths, λex/λem, appear at 351 nm/351 nm for RRS, 240 nm/480 nm for SOS, and 870 nm/435 nm for FDS, respectively. The increments of the scattering intensities (ΔI) are proportional to the concentration of SA in a certain range. The detection limits (3σ) for SA are 3.69 ng mL^?1 for the RRS method, 6.91 ng mL^?1 for the FDS method, and 7.45 ng mL^?1 for the SOS method under optimum conditions. The proposed methods were applied to the determination of SA in real samples with satisfactory results.     

Volume transition and internal structures of small poly(N‐isopropylacrylamide) microgels

Monodispersed poly(N‐isopropylacrylamide) (PNIPAM) nanoparticles, with hydrodynamic radius less than 50 nm at room temperature, have been synthesized in the presence of a large amount of emulsifiers. These microgel particles undergo a swollen–collapsed volume transition in an aqueous solution when the temperature is raised to around 34 °C. The volume transition and structure changes of the microgel particles as a function of temperature are probed using laser light scattering and small angle neutron scattering (SANS) with the objective of determining the small particle internal structure and particle–particle interactions. Apart from random fluctuations in the crosslinker density below the transition temperature, we find that, within the resolution of the experiments, these particles have a uniform radial crosslinker density on either side of the transition temperature. This result is in contrast to previous reports on the heterogeneous structures of larger PNIPAM microgel particles, but in good agreement with recent reports based on computer simulations of smaller microgels. The particle interactions change across the transition temperature. At temperatures below the transition, the interactions are described by a repulsive interaction far larger than that expected for a hard sphere contact potential. Above the volume transition temperature, the potential is best described by a small, attractive interaction. Comparison of the osmotic second virial coefficient from static laser light scattering at low concentrations with similar values determined from SANS at 250‐time greater concentration suggests a strong concentration dependence of the interaction potential. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 849–860, 2005     

Study on Frequency Doubling Scattering and Second—Order Scattering Spectra of Heparin—Methylene Blue System

Binding of heparin with methylene blue(MB) in pH5.7 Britton-Robinson buffer can result in a significant enhancement of frequency doubling scattering (FDS) and second-order scattering (SOS).Their maximum scattering wavelengths(λmax) appear at 350nm for FDS and 700nm for SOS,respectively.The optimum conditions of the reaction,the influencing factors and the relationship between the two scattering intensities and the concentration of heparin have been investigated.The new methods for the determination of trace amounts of heparin bassed on the FDS and SOS methods have been developed,which exhibit high sensitivities.The detection limits of heparin are 4.36ng/mL for the FDS method and 3.55ng/mL for the SOS method,respectively.Both of the methods have fairly godd selectivity and ware applied to the determination of heparin in sodium heparinate injection samples with satisfactory results.Moreover,the relative mechamisms have also been discussed.     

Magnetically alignable phase of phospholipid "bicelle" mixtures is a chiral nematic made up of wormlike micelles

We have studied the phase behavior of binary mixtures of long- and short-chain lipids, namely, dimyristoyl phosphatidylcholine (DMPC) and dihexanoyl phosphatidylcholine (DHPC), using optical microscopy and small-angle neutron scattering. Samples with a total lipid content of 25 wt %, corresponding to ratios Q ([DMPC]/[DHPC]) of 5, 3.2, and 2, are found to exhibit an isotropic (I) --> chiral nematic (N) --> lamellar phase sequence on increasing temperature. The I-N transition coincides with the chain melting transition of DMPC at Q = 5 and 3.2, but the N phase forms at a higher temperature for Q = 2. All three samples form multilamellar vesicles in the lamellar phase. Our results show that disklike "bicellar" aggregates occur only in the lower temperature isotropic phase and not in the higher temperature magnetically alignable N phase, where they were previously believed to exist. The N phase is found to consist of long, flexible wormlike micelles, their entanglement resulting in the very high viscosity of this phase.     

Scattering functions of semidilute solutions of polymers in a good solvent

Expressions for analyzing small-angle scattering data from semidilute solutions of polymers in a good solvent over a broad range of scattering vectors are examined. Three different scattering function expressions are derived from Monte Carlo simulations. The expressions are similar to those of polymer reference interaction site models, with a scattering-vector-dependent direct correlation function. In the most advanced model, the screening of excluded-volume interactions beyond the overlap concentration is taken into account. Two simpler expressions, in which the screening of excluded-volume interactions is not included, are also applied. The three models are tested against small-angle neutron scattering (SANS) experiments on polystyrene in deuterated toluene for a broad range of molar masses and concentrations over a wide range of scattering vectors. For each model, simultaneous fits to all the measured scattering data are performed. The most advanced model excellently reproduces the SANS data over the full range of the parameters. The two simpler models fit the data almost equally well. On the basis of an extensive study, an optimal fitting strategy can be recommended for experimentalists, who want to analyze small-angle scattering data from polymers at any concentration. For data sets that do not contain data on the single-chain scattering function, the simpler model is recommended; it uses a direct correlation function equal to the form factor of an infinitely thin rod, which is independent of the concentration and molar mass. © 2004 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 42: 3081–3094, 2004     

Interaction of merocyanine 540 with charged membranes.

In this work, phospholipid liposomes were used to investigate the influence of lipid negative charge on the interaction of merocyanine 540 (MC540) with model membranes. Liposomes were prepared from a mixture of neutral dimyristoyl lecithin (DMPC) and negatively charged dimyristoyl phosphatidic acid (DMPA). A strong dependence between the presence of charges on the membrane and dye association was found. The affinity of the dye to liposomes was decreased with an increasing content of DMPA in liposomes. Changes in absorption spectra of MC540 suggest that the decrease in affinity of MC540 to charged membranes is accompanied by a hypsochromic solvatochromic shift and changes in monomer/dimer equilibrium of MC540 incorporated in the membrane.     

Salinity Controlled Vesicle Formation and Growth in Aqueous Mixture of Aerosol OT/Triton X‐100

Mixed vesicles can be formed spontaneously from aqueous mixture of the double‐tailed anionic surfactant sodium bis(2‐ethylhexyl) sulfosuccinate (AOT) and the nonionic surfactant octylphenoxypolyethoxyethanol (Triton X‐100) under the inducement of salt, the formation mechanism of which should be attributed to the compression of salt on the electric bilayers of the head groups. The stability and the polydispersity of the vesicles are superior to single‐component AOT vesicles, which can be proved by the TEM image and visual observation. The vesicle region was presented in a pseudo‐ternary diagram of AOT/TX‐100/brine. The size of the vesicle was measured using dynamic light scattering. It is found that the vesicle size increases with the salinity but decreases with the content of TX‐100 in the mixture at the same salinity. Especially, the vesicle size is independent of the surfactant concentration at fixed salinity.