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Bicellar Mixtures Containing Pluronic F68: Morphology and Lateral Diffusion from Combined SANS and PFG NMR Studies
Abstract
Small angle neutron scattering (SANS) and pulsed field gradient (PFG) nuclear magnetic resonance (NMR) diffusion measurements were applied to examine morphology and diffusion in dimyristoyl- plus dihexanoyl-phosphatidylcholine bicellar mixtures, either neutral or negatively charged, incorporating a Pluronic triblock copolymer (F68). Negatively charged bicellar mixtures, doped with dimyristoylphosphatidylglycerol (DMPG), exhibited SANS profiles consistent with a perforated lamellar morphology for the magnetically alignable phase. Correspondingly, F68 diffusion in this magnetically aligned phase was normal Gaussian, in that the mean square displacements increased linearly with the experimental diffusion time, with a lateral diffusion coefficient of 1.9 x 10(-11) m(2) s(-1) consistent with a lipid bilayer inserted configuration. Neutral bicellar mixtures, that is, lacking DMPG, in contrast, displayed SANS profiles characteristic of ribbons arranged in such a fashion as to produce extended lamellae. Within the lamellae, the ribbons exhibited an in-plane periodicity (interribbon) of between 120 and 140 A. Correspondingly, F68 diffusion was non-Gaussian, exhibiting a square root diffusion time dependence of the mean square displacement indicative of one-dimensional curvilinear diffusion. The presence or absence of DMPG, rather than of F68, dictated the ribbon versus lamellar morphology, with F68 reflecting this difference via its lateral diffusion behavior. Although ribbons have been reported previously, this is the first study to show that they aggregate, most likely into extended lamellar sheets, and eventually fold into multilamellar vesicles.
... 36 In the case of concentrated samples, where strong Bragg diffraction peaks, as a result of positionally correlated bilayers, are present, a phenomenological model, as expressed in eq 1, was used to fit the SANS data in order to determine the interlamellar spacing, or d spacing. 37 The model assumes that Bragg peaks sit on top of the background signal, which, depending on the q range, contains two distinct slopes (i.e., −m and −n) in the low-and high-q regimes, respectively, as described in the first term on the r.h.s. of eq 1 ...
... The SANS patterns and corresponding morphologies of DMPC/DHPC as a function of temperature have been reported previously. 26,37 They have been interpreted as follows: at temperatures below the gel to liquid-crystalline phase transition temperature of DMPC (T M ≈ 296 K), discoidal bicelles (bilayered micelles) are formed. At 25 wt % and with increasing temperature, the bicelles progressively transform into ribbons, then ribbon-meshed lamellae, and eventually multilamellar vesicles (MLVs) (i.e., regularly spaced, stacked onionlike structures that cannot swell once the temperature exceeds ∼320 K 22,26,37 ). ...
... 26,37 They have been interpreted as follows: at temperatures below the gel to liquid-crystalline phase transition temperature of DMPC (T M ≈ 296 K), discoidal bicelles (bilayered micelles) are formed. At 25 wt % and with increasing temperature, the bicelles progressively transform into ribbons, then ribbon-meshed lamellae, and eventually multilamellar vesicles (MLVs) (i.e., regularly spaced, stacked onionlike structures that cannot swell once the temperature exceeds ∼320 K 22,26,37 ). Importantly, the SANS intensity profile from each morphology is distinct. ...
Spontaneously forming structures of a system composed of dimyristoyl phosphatidylcholine (DMPC) and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) were studied by small angle neutron scattering (SANS), (31)P NMR and stimulated echo (STE) pulsed field gradient (PFG) (1)H NMR diffusion measurements. The charged lipid dimyristoyl phosphatidylglycerol (DMPG) was used to induce different surface charge densities. The structures adopted were investigated as a function of temperature and lipid concentration for samples with a constant molar ratio of long-chain to short-chain lipid (=3). In the absence of DMPG, zwitterionic bicellar mixtures exhibited a phase transition from discoidal bicelles, or ribbons, to multi-lamellar vesicles, either upon dilution or with increased temperature. CHAPSO-containing mixtures showed a higher thermal stability in morphology than DHPC-containing mixtures at the corresponding lipid concentrations. In the presence of DMPG, discoidal bicelles (or ribbons) were also found at low temperature and lower lipid concentration mixtures. At high temperature perforated lamellae were observed in high concentration mixtures (≥ 7.5 wt%), while uniform unilamellar vesicles and bicelles formed in low-concentration mixtures (≤ 2.5 wt%), respectively, when the mixtures were moderately and highly charged. From the results, spontaneous structural diagrams of the zwitterionic and charged systems were constructed.
... It has been previously reported that the DMPC/DHPC/ DMPG mixtures form bicelles and perforated lamellae at low and high temperatures, respectively. [39][40][41] The low-T bicelles are not alignable, while the high-T lamellae are alignable under a shear flow with their bilayer normal perpendicular to the confined surfaces. 33 More interestingly, the aligned structure remains even several hours after the flow ceases. ...
... 42 The bicelle-to-lamellae transition has also been reported elsewhere. 39 To evaluate the degree of alignment of the sample under the shear flow, "rocking curve" measurement was performed by fixing the detector angle at the 1st order Bragg peak position (i.e., 2θ B ) and collecting the scattering intensity as a function of sample angle, θ. The attainable θ range was between −40 • and 40 • lest that the supporting posts might block the incident beam as descried in "Instrumental Design" section. ...
We have designed and constructed a temperature-controllable shear flow cell for in-situ study on flow alignable systems. The device has been tested in the neutron diffraction and has the potential to be applied in the small angle neutron scattering configuration to characterize the nanostructures of the materials under flow. The required sample amount is as small as 1 ml. The shear rate on the sample is controlled by the flow rate produced by an external pump and can potentially vary from 0.11 to 3.8 × 10(5) s(-1). Both unidirectional and oscillational flows are achievable by the setting of the pump. The instrument is validated by using a lipid bicellar mixture, which yields non-alignable nanodisc-like bicelles at low T and shear-alignable membranes at high T. Using the shear cell, the bicellar membranes can be aligned at 31 °C under the flow with a shear rate of 11.11 s(-1). Multiple high-order Bragg peaks are observed and the full width at half maximum of the "rocking curve" around the Bragg's condition is found to be 3.5°-4.1°. It is noteworthy that a portion of the membranes remains aligned even after the flow stops. Detailed and comprehensive intensity correction for the rocking curve has been derived based on the finite rectangular sample geometry and the absorption of the neutrons as a function of sample angle [See supplementary material at http://dx.doi.org/10.1063/1.4908165 for the detailed derivation of the absorption correction]. The device offers a new capability to study the conformational or orientational anisotropy of the solvated macromolecules or aggregates induced by the hydrodynamic interaction in a flow field.
... Since their discovery, bicelles have gained significant interest in biochemistry and biophysical chemistry, because they mimic biological membranes and are often orientable in magnetic fields, making them attractive vessels for NMR studies of membrane proteins [1,4,[6][7][8][9][10]. Various different morphologies are found in systems containing disc-like bicelles, such as perforated or unperforated uni-and multilamellar vesicles, 'infinite' lamellae and perforated lamellae, branched and unbranched worm-like micelles, and ribbons [3,4,7,8,11,12]. Apart from worm-like micelles, all these structures are related as the curvature for the long-chain lipid approaches zero, the difference being the degree of intramicellar segregation of the two lipids. ...
Motivation:
Surfactants like C8E8CH2COOH have such bulky headgroups that they cannot show the common sphere-to-cylinder transition, while surfactants like C18:1E2CH2COOH are mimicking lipids and form only bilayers. Mixing these two types of surfactants allows one to investigate the competition between intramicellar segregation leading to disc-like bicelles and the temperature dependent curvature constraints imposed by the mismatch between heads and tails.
Experiments:
We establish phase diagrams as a function of temperature, surfactant mole ratio, and active matter content. We locate the isotropic liquid-isotropic liquid phase separation common to all nonionic surfactant systems, as well as nematic and lamellar phases. The stability and rheology of the nematic phase is investigated. Texture determination by polarizing microscopy allows us to distinguish between the different phases. Finally, SANS and SAXS give intermicellar distances as well as micellar sizes and shapes present for different compositions in the phase diagrams.
Findings:
In a defined mole ratio between the two components, intramicellar segregation wins and a viscoelastic discotic nematic phase is present at low temperature. Partial intramicellar mixing upon heating leads to disc growth and eventually to a pseudo-lamellar phase. Further heating leads to complete random mixing and an isotropic phase, showing the common liquid–liquid miscibility gap. This uncommon phase sequence, bicelles, lamellar phase, micelles, and water-poor packed micelles, is due to temperature induced mixing combined with dehydration of the headgroups. This general molecular mechanism explains also why a metastable water-poor lamellar phase quenched by cooling can be easily and reproducibly transformed into a nematic phase by gentle hand shaking at room temperature, as well as the entrapment of air bubbles of any size without encapsulation by bilayers or polymers.
... The temperature and the concentration conditions in our experiments are marked by a purple rectangle on the diagram presented in Figure S1. Previously, the reorganization of bicelles into elongated ribbon-and wormlike aggregates was detected by different methods (SAS, electron microscopy, NMR, polarized optical microscopy) 24,38,75,85,86 . Since the DMPC tends to form a bilayer, these elongated "worm-like" objects should have a flattened cross-section (ribbon-like) with a thickness close to the thickness of the DMPC bilayer. ...
Despite remarkable progress, mainly due to the development of LCP and ‘bicelle’ crystallization, lack of structural information remains a bottleneck in membrane protein (MP) research. A major reason is the absence of complete understanding of the mechanism of crystallization. Here we present small-angle scattering studies of the evolution of the “bicelle” crystallization matrix in the course of MP crystal growth. Initially, the matrix corresponds to liquid-like bicelle state. However, after adding the precipitant, the crystallization matrix transforms to jelly-like state. The data suggest that this final phase is composed of interconnected ribbon-like bilayers, where crystals grow. A small amount of multilamellar phase appears, and its volume increases concomitantly with the volume of growing crystals. We suggest that the lamellar phase surrounds the crystals and is critical for crystal growth, which is also common for LCP crystallization. The study discloses mechanisms of “bicelle” MP crystallization and will support rational design of crystallization.
... The effects of relaxation are ignored during the echo delay periods. The mean square displacement ( 〈z 2 〉 ) along the direction of the applied magnetic field gradient for a specific diffusion interval can be computed from [60]. ...
We studied the thermogelation of a triblock copolymer Pluronic F127 (poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide)) in an aqueous solvent in the presence of gold nanoparticles, using pulsed-field gradient diffusion, NMR temperature experiments, relaxation measurements, and 2D heteronuclear NMR experiments. Pulsed field gradient diffusion NMR is a powerful technique to study the transition between diffusive regimes in a polymer mesh which are modulated by phase transitions in the polymeric network. In the isotropic phase, the triblock copolymer diffusion is a classical Fickian process. As the onset of gelation occurs, diffusion in the system becomes anomalous and the mean square displacement in the direction of the applied magnetic field gradient shows a power law dependence. Our experiments show that the introduction of gold nanoparticles leads to a disruption of gelation and the shifting of the formation of the ordered phase of the triblock copolymer to a higher temperature.
... Both have been commonly observed in other bicellar mixtures with saturated long-chain lipid [75][76][77]. The observation of ribbons [ Fig. 1(b)] is consistent with another known intermediate morphology in the absence of charged lipid, ribbon which is sandwiched between low and high T for the high-C lp DMPC/DHPC bicellar mixtures [56,86]. Since the experimental temperature was not lower than T M of the POPC in all cases, the discoidal bicelles was not expected in this study. ...
The spontaneously formed structures of physiologically relevant lipid model membranes made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)) and 1,2-hexanoyl-sn-glycero-3-phosphocholine have been evaluated in depth using small angle neutron scattering. Although a common molar ratio of long- to short- chain phospholipids (~4) as reported in many bicellar mixtures was used, discoidal bicelles were not found as the major phase throughout the range of lipid concentration and temperature studied, indicating that the required condition for the formation of bicelle is the immiscibility between the long- and short- chain lipids, which were in the gel and Lα phases, respectively, in previous reports. In this study, all lipids are in Lα phase. The characterization outcome suggests that the spontaneous structures tie strongly with the physical parameters of the system such as melting transition temperature of the long-chain lipid, total lipid concentration and charge density of the system. Multilamellar vesicles, unilamellar vesicles, ribbons and perforated lamellae can be obtained based on the analysis of the small angle neutron scattering result, leading to the construction of structural diagrams. This report provides the important map to choose suitable lipid systems for the structural study of membrane-associated proteins, design of theranostic nanocarriers or other related research fields.
... Nanodiscs containg reasonable amounts of charged lipids are known to be very stable [11]. However, as their charge density decreases, they transform into bilayered ribbons, and subsequently into vesicles due to the depletion of the short-chain lipids from the ribbon high curvature regions [33]. It is understood that strong Coulombic interactions can inhibit nanodiscs from coalescing with each other. ...
Uniform nanodisc structures can be self assembled from mixtures of different phospholipids. This study focuses on the theoretical and experimental growth kinetics of phospholipid based nanodiscs. Motivation for this project comes from the nanodisc’s small size and their potential use as a carrier for drug delivery. It was observed that at high total lipid concentration the nanodiscs are stable at approximately 10 nm in radius and can remain stable for a long period of time. However, growth of these nanodiscs are observed at relatively low total lipid concentration. The observed growth mechanism is not well understood. In this thesis dynamic light scattering is used to monitor the size and growth rate of nanodiscs at different solution conditions. It is determined that disc-disc interactions play an important role in the stability of the disc structures. More importantly surface charge on the nanodisc prevent growth due to the electric repulsive force. The growth at low concentrations is caused by the transfer of charged lipids from the discs to the solution, therefore reducing the Columbic interaction between two interacting discs. The effect of size and different surface potentials can be modeled by the Smoluchowski transport equation along with the transport limited boundary conditions. Chapter 1 to 5 include the experimental method, results and mathematical model for this research. The last part of this thesis, chapter 6, presents the relevant microrheology experiments. The aim is to obtain viscoelastic properties of complex fluids in the frequency range between 103 to 105 Hz, gives insight into the frequency dependent particle-particle or disc-disc interactions in suspensions.
... The apparent concentration of Lamotrigine in optimized formulation as a function of temperature is shown in fig.(3).The solubility of Lamotrigine in water at 25 0 is 0.17 mg/ml. 22 The solubility of Lamotrigine in Pluronic solution significantly increases with an increase in temperature. In general, with an increase in temperature, the dehydration of the PEO shell occurs and PPO chains become more hydrophobic, the drug might be expected to be solubilized in micellar core which is confirmed by UV spectroscopy due to the micellar environment is different than that of water. ...
Objective: The objective of the study was to investigate the solubilization of poorly water-soluble drug Lamotrigine in pure & mixed Pluronic polymeric micelles. Method: Two different Pluronic (Pluronic F68, Pluronic L81) were chosen and micelle formulations were prepared by using various drug:polymer ratios and model drug Lamotrigine. Formulations were characterized by critical micellization concentration (CMC) values, cloud point of copolymers, micelle size and size distribution, zeta potential, loading efficiency,drug release and stability. Result: Mixed micelles(hydrophilic and hydrophobic) also helped to overcome the limitations of monosystem of Pluronic L81 and Pluronic F68. The solubilized drug and salt decreased the cloud point of copolymers. Results show that the solubilization of Lamotrigine enhances with the rise in concentration of block copolymers, negative Gs0 and temperature, but no significant increase was observed with added salt and at a lower pH the drug show highest solubility. Conclusion: Mixed micelles showed fairly high entrapment efficiency, loading capacity and sustained release profile for Lamotrigine, a model hydrophobe than that of plain Pluronic micelles. © 2014, Association of Pharmaceutical Teachers of India. All rights reserved.
... 3 However, as their charge density decreases, they transform into bilayered ribbons, and subsequently into vesicles due to the depletion of the short-chain lipids from the ribbon high curvature regions. 7 It is understood that strong Coulombic interactions can inhibit nanodiscs from coalescing with each other. Recently, studies of coalescing nanodiscs made from egg yolk lecithin and bile salt were conducted as a function of salinity. ...
Lipid-based nanodiscs composed of long- and short- chain lipids [namely,
dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol
(DMPG) and dihexanoyl phosphatidylcholine (DHPC)] constantly form at
high lipid concentrations and at low temperatures (i.e., below the
melting transition temperature of DMPC, TM). The initial size
of these nanodiscs (at high total lipid concentration, CL>
20 wt.%) is relatively uniform and of similar dimension (according to
dynamic light scattering and small angle neutron scattering
experiments), seemingly independent of thermal history. Upon dilution,
the nanodiscs slowly coalesce and grow in size with time irreversibly.
Our preliminary result shows that the growth rate strongly depends on
several parameters such as charge density, CL and
temperature. We have also found that the nanodisc coalescence is a
reaction limit instead of diffusion limit process through a
time-resolved study.
... All the interfacial tensions reported in Table 2 Following the analysis of van der Graaf et al [33], these times correspond to a region where the rate of increase in interfacial area will be low, but there may still be a slight increase in interfacial tension over the equilibrium value and, therefore, increased drop size. The effective diffusion coefficient of Pluronic F68 is reported as 1.9×10 -11 m 2 s -1 [34] while for Tween 20 Luschtinetz and Dosche reported a value of 7.7×10 -11 m 2 s -1 [35]. Since the loading of the interface with surfactant is directly proportional to the effective diffusion coefficient [33], it will take longer for Pluronic F68 molecules to stabilise the forming oil droplet which may also explain the larger diameters and reaches its greatest value at the transitional radius. ...
StIrred cell membrane emulsification was used to create double water-in-oil-in-water emulsions at the dispersed phase flux of up to 3200 m-2 h-1. The oil phase was unrefined pumpkin oil or sunflower oil containing 30% by volume of internal water phase and the external water phase 2% Tween 20 (polyoxyethylene sorbitan monolaurate) or 2% Pluronic F-68 (polyoxyethylene-polyoxipropylen copolymer). Using microengineered nickel membranes with pore sizes between 15 and 40 micrometer, median drop sizes of double emulsion were in the range between 100 and 430 micrometer, depending mainly on the shear at the membrane surface and dispersed phase injection rate. In most cases the drops were very uniform, with span (i. e. 90% drop size minus 10% drop size devided by median size) values of around 0.5. This data is similar to what was obtained previously for simple O/W emulsions of the same materials. Hence, the internal water phase, and internal surfactant, 5% PGRP (polyglycerol polyricinoleate), did not adversely influence the emulsification process. A marker material, copper sulfate, was added to the internal water phase and the release was monitoring with respect to time. For both lipid systems, at the larger droplet size, there was a significant period of no copper release, followed by almost linear release with time. This initial period was absent when the drop size was close to 100 micrometer. The initial entrapment efficiency of the copper, in all experiments, was higher than 94%.
... The ribbons transformed into MLVs with increasing temperature, while the charged perforated lamellae remained unaltered. In the case of high total lipid concentration (≥20 wt.%) systems, the lamellar phase is comprised of entangled ribbons, as was recently confirmed using a combination of SANS and pulsed field gradient NMR studies ( Soong et al., 2010). It should be pointed out that bilayered disks were identified by SANS to exist at low temperature ( Harroun et al., 2005), where the NMR signal was found to be isotropic. ...
Scattering techniques, in particular electron, neutron and X-ray scattering have played a major role in elucidating the static and dynamic structure of biologically relevant membranes. Importantly, neutron and X-ray scattering have evolved to address new sample preparations that better mimic biological membranes. In this review, we will report on some of the latest model membrane results, and the neutron and X-ray techniques that were used to obtain them.
Bicellar systems have become popularized as their rich morphology can be applied in biochemistry, physical chemistry, and drug delivery technology. To the biochemical field, bicelles are powerful model membranes for the study of transmembrane protein behavior, membrane transport, and environmental interactions with the cell. Their morphological responses to environmental changes reveal a profound fundamental understanding of physical chemistry related to the principle of self-assembly. Recently, they have also drawn significant attention as theranostic nanocarriers in biopharmaceutical and diagnostic research due to their superior cellular uptake compared to liposomes. It is evident that applications are becoming broader, demanding to understand how the bicelle will form and behave in various environments. To consolidate current works on the bicelle's modern applications, this review will discuss various effects of composition and environmental conditions on the morphology, phase behavior, and stability. Furthermore, various applications such as payload entrapment and polymerization templating are presented to demonstrate their versatility and chemical nature.
We report a generalized platform for synthesizing a polymer nanoweb with a high specific surface area via a bicellar template, composed of 1,2-dipalmitoyl phosphocholine (DPPC), 1,2-dihexanoyl phosphocholine (DHPC), and 1,2-dipalmitoyl phosphoglycerol (DPPG). The pristine bicelle (in the absence of monomer or polymer) yields a variety of well-defined structures, including disc, vesicle, and perforated lamella. The addition of styrene monomers in the mixture causes bicelles to transform into lamellae. Monomers are miscible with DPPC and DPPG initially, while polymerization drives polymers to the DHPC-rich domain, resulting in a polymer nanoweb supported by the outcomes of small angle neutron scattering, differential scanning calorimetry, and transmission electron microscopy.
Bicellar mixtures have been used as alignable membrane substrates under a magnetic field applicable for the structural characterization of membrane-associated proteins. Recently, it has shown that bicelles can serve as nanocarriers to effectively deliver hydrophobic therapeutic molecules to cancer cells with a three- to ten-fold enhancement compared to that of liposomes of a chemically identical composition. In this chapter, detailed preparation protocol, common structural characterization methods, the structural stability, the cellular uptake and a few unique functions of bicellar nanodiscs are discussed.
Bicellar mixtures have been used as alignable membrane substrates for the structural characterization of membrane-associated proteins. Most recently, it has been shown that bicelles can serve as nanocarriers to effectively deliver hydrophobic molecules to cancer cells with a 3- to 10-fold enhancement compared to that of chemically identical liposomes. In this chapter, a detailed preparation protocol, common structural characterization methods, the structural stability and the cellular uptake of bicellar nanodisks are discussed.
This mini-review describes diffusion NMR studies of polymers in bicelles using the pulsed field gradient (PFG) NMR technique. Bicelles are first introduced and their morphology and applications as membrane mimetics and confinement media are briefly covered. The PFG NMR diffusion method is then introduced, emphasizing its specific advantages and limitations when applied to diffusion measurements in macroscopically-oriented lamellar systems such as magnetically-aligned bicelles. The utility of PFG NMR diffusion measurements in bicellar model membrane systems for examining lateral diffusion of membrane-bound, surface-grafted poly(ethylene glycol) (PEG) molecular species is demonstrated, focusing on those features of lateral diffusion and / or bicelle morphology that such studies illuminate. Further, PFG NMR diffusion studies of various molecular weight soluble PEG species confined within the parallel-plate geometry of magnetically aligned bicelles are reviewed, again focusing on revelations concerning bicelle morphology. The discussion concludes with an outline of future prospects for diffusion NMR studies in bicelles.
Lipid-based carriers (liposomes) are available for delivering therapeutics. Naturally, micron-size multilamellar vesicles (MLVs) are found in zwitterionic long-chain lipids in aqueous solutions. The lamellar spacing of these MLVs does not swell upon dilution. In order to produce uniform, nano-size unilamellar vesicles (ULVs), which have higher loading capacity for hydrophilic drugs, multi-stage extrusion is commonly applied to MLV solutions. The mass production of ULVs through this method is labor intensive and sometimes membrane fouling and rupture can be problematic. It has also been investigated that doping short-chain and charged lipids into the zwitterionic long-chain lipids results in uniform nano-sized ULVs at increased temperature (i.e., T > TM, the melting transition temperature of the long-chain lipid). The ULVs are found to be thermodynamically stable.
In this report, we will demonstrate that kinetically trapped ULVs are attainable in a lipid mixture, where MLVs are thermodynamically stable. The lipid solutions initially form uniform nano-discs, which undergo a slow self-folding process into ULVs, presumably related to the rigidity of the bilayered membrane induced by charged lipids.
The influence of adding salt on the self-assembly in sodium octyl sulfate (SOS)-rich mixtures of the anionic surfactant SOS and the cationic surfactant hexadecyltrimethylammonium bromide (CTAB) have been investigated with the two complementary techniques, small-angle neutron scattering (SANS) and cryo-transmission electron microscopy. We are able to conclude that addition of a substantial amount of inert salt, NaBr, mainly has three effects on the structural behaviors: (i) the micelles become much larger at the transition from micelles to bilayers, (ii) the fraction of bilayer disks increases at the expense of vesicles, and (iii) bilayer aggregates perforated with holes are formed in the most diluted samples. A novel form factor valid for perforated bilayer vesicles and disks is introduced for the first time and, as a result, we are able to directly observe the presence of perforated bilayers by means of fitting SANS data with an appropriate model. Moreover, we are able to conclude that the morphology of bilayer aggregates changes according to the following sequence of different bilayer topologies, vesicles → disks → perforated bilayers, as the electrolyte concentration is increased and surfactant mole fraction in the bilayer aggregates approaches equimolarity. We are able to rationalize this sequence of transitions as a result of a monotonous increase of the bilayer saddle-splay constant (k̅cbi) with decreasing influence from electrostatics, in agreement with theoretical predictions as deduced from the Poisson–Boltzmann theory.
Lipid-based nanodiscs (bicelles) are able to form in mixtures of long- and short-chain lipids. Initially, they are of uniform size but grow upon dilution. Previously, nanodisc growth kinetics have been studied using time-resolved small angle neutron scattering (SANS), a technique which is not well suited for probing their change in size immediately after dilution. To address this, we have used dynamic light scattering (DLS), a technique which permits the collection of useful data in a short span of time after dilution of the system. The DLS data indicate that the negatively charged lipids in nanodiscs play a significant role in disc stability and growth. Specifically, the charged lipids are most likely drawn out from the nanodiscs into solution, thereby reducing interparticle repulsion and enabling the discs to grow. We describe a population balance model, which takes into account Coulombic interactions and adequately predicts the initial growth of nanodiscs with a single parameter - i.e., surface potential. The results presented here strongly support the notion that the disc coalescence rate strongly depends on nanoparticle charge density. The present system containing low-polydispersity lipid nanodiscs serves as a good model for understanding how charged discoidal micelles coalesce.
The self-assembling morphologies of low-concentration (mostly 1 and 10mg/mL) bicellar mixtures composed of zwitterionic dipalmitoyl (di-C16) phosphatidylcholine (DPPC), dihexanoyl (di-C6) phosphatidylcholine (DHPC), and negatively charged dipalmitoyl (di-C16) phosphatidylglycerol (DPPG) were investigated using small angle neutron scattering, dynamic light scattering and transmission electron microscopy. A polyethylene glycol conjugated (PEGylated) lipid, distearoyl phosphoethanolamine-[methoxy (polyethyleneglycol)-2000] (PEG2000-DSPE), was incorporated in the system at 5 mole% of the total lipid composition. The effects of several parameters on the spontaneous structures are studied, including temperature, lipid concentration, salinity, and PEG2000-DSPE. In general, nanodiscs (bicelles) were observed at low temperatures (below the melting temperature, TM of DPPC) depending on the salinity of the solutions. Nanodisc-to-vesicle transition is found upon the elevation of temperature (above TM) in the cases of low lipid concentration in absence of PEG2000-DSPE or high salinity. Both addition of PEG2000-DSPE and high lipid concentration stabilize the nanodiscs, preventing the formation of multilamellar vesicles, while high salinity promotes vesiculation and the formation of aggregation. This study suggests that the stability of such nanodiscs is presumably controlled by the electrostatic interactions, the steric effect induced by PEG2000-DSPE and the amount of DHPC located at the disc rim.
This minireview focuses on diffusion NMR studies in bicelles. Following a discourse on diffusion fundamentals, and a comparative overview of fluorescence and NMR-based techniques for measuring diffusion, the pulsed field gradient (PFG) NMR diffusion method is introduced, emphasizing its specific advantages and limitations when applied to diffusion measurements in macroscopically oriented lamellar systems such as magnetically aligned bicelles. The utility of PFG NMR diffusion measurements in bicellar model membrane systems for examining lateral diffusion of membrane-bound molecular species is demonstrated, along with certain features of lateral diffusion that such studies illuminate. Further, those aspects of bicelle morphology that have been resolved using PFG NMR diffusion studies of various molecular weight soluble polymeric species are reviewed. The discussion concludes with an outline of future prospects for diffusion NMR studies in bicelles.
Bicellar dispersions of chain perdeuterated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC-d54) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) were prepared with the molar fraction of DHPC held fixed at 20% and varying amounts of DMPC replaced by the anionic lipid 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG). 2H NMR spectra were examined to assess the effect of added DMPG on mixture phase behavior and morphology. Quadrupole echo decay and quadrupole-Carr-Purcell-Mieboom-Gill echo train measurements provided information about slow motions contributing to echo decay in the high temperature phases. The spectra and quadrupole echo decay properties of DMPC-d54/DHPC (4:1) and DMPC-d54/DMPG/DHPC (3:1:1) were qualitatively similar. With increasing DMPG concentration, the transition between the magnetically orientable phase and the higher temperature phase became increasingly distinct, and the spectral shape and echo decay characteristics of the high temperature bicellar phase became increasingly similar to those of DMPC-d54 in the liquid crystalline phase. The observation that DMPG changes spectra in the orientable phase incrementally while increasing the distinction between the orientable and high temperature bicellar phases provides new insights into how DMPG influences bicellar mixture morphology.
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.
Bicellar model membranes composed of 1,2-dimyristoylphosphatidylcholine (DMPC) and 1,2-dihexanoylphosphatidylcholine (DHPC), with a DMPC/DHPC molar ratio of 5, and doped with the negatively charged lipid 1,2-dimyristoylphosphatidylglycerol (DMPG), at DMPG/DMPC molar ratios of 0.02 or 0.1, were examined using small angle neutron scattering (SANS), (31)P NMR, and (1)H pulsed field gradient (PFG) diffusion NMR with the goal of understanding temperature effects on the DHPC-dependent perforations in these self-assembled membrane mimetics. Over the temperature range studied via SANS (300-330 K), these bicellar lipid mixtures exhibited a well-ordered lamellar phase. The interlamellar spacing d increased with increasing temperature, in direct contrast to the decrease in d observed upon increasing temperature with otherwise identical lipid mixtures lacking DHPC. (31)P NMR measurements on magnetically aligned bicellar mixtures of identical composition indicated a progressive migration of DHPC from regions of high curvature into planar regions with increasing temperature, and in accord with the "mixed bicelle model" (Triba, M. N.; Warschawski, D. E.; Devaux, P. E. Biophys. J.2005, 88, 1887-1901). Parallel PFG diffusion NMR measurements of transbilayer water diffusion, where the observed diffusion is dependent on the fractional surface area of lamellar perforations, showed that transbilayer water diffusion decreased with increasing temperature. A model is proposed consistent with the SANS, (31)P NMR, and PFG diffusion NMR data, wherein increasing temperature drives the progressive migration of DHPC out of high-curvature regions, consequently decreasing the fractional volume of lamellar perforations, so that water occupying these perforations redistributes into the interlamellar volume, thereby increasing the interlamellar spacing.
Mixtures of long- and short-tail phosphatidylcholine lipids are known to self-assemble into a variety of aggregates combining flat bilayerlike and curved micellelike features, commonly called bicelles. Atomistic simulations of bilayer ribbons and perforated bilayers containing dimyristoylphosphatidylcholine (DMPC, di-C(14) tails) and dihexanoylphosphatidylcholine (DHPC, di-C(6) tails) have been carried out to investigate the partitioning of these components between flat and curved microenvironments and the stabilization of the bilayer edge by DHPC. To approach equilibrium partitioning of lipids on an achievable simulation timescale, configuration-bias Monte Carlo mutation moves were used to allow individual lipids to change tail length within a semigrand-canonical ensemble. Since acceptance probabilities for direct transitions between DMPC and DHPC were negligible, a third component with intermediate tail length (didecanoylphosphatidylcholine, di-C(10) tails) was included at a low concentration to serve as an intermediate for transitions between DMPC and DHPC. Strong enrichment of DHPC is seen at ribbon and pore edges, with an excess linear density of approximately 3 nm(-1). The simulation model yields estimates for the onset of edge stability with increasing bilayer DHPC content between 5% and 15% DHPC at 300 K and between 7% and 17% DHPC at 323 K, higher than experimental estimates. Local structure and composition at points of close contact between pores suggest a possible mechanism for effective attractions between pores, providing a rationalization for the tendency of bicelle mixtures to aggregate into perforated vesicles and perforated sheets.
Variable-pressure deuterium nuclear magnetic resonance ((2)H NMR) has been used to study the pressure-temperature phase diagram of bicellar mixtures containing 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC). Spectra were obtained for DMPC-d(54)/DHPC (3:1), DMPC-d(54)/DHPC (4.4:1), DMPC/DHPC-d(22) (3:1), and DMPC/DHPC-d(22) (4.4:1) in the range 10-68 degrees C at ambient pressure, 66 MPa, 102 MPa, and 135 MPa. Isotropic-to-nematic and nematic-to-lamellar transition temperatures were found to rise with pressure at approximately 0.15 and approximately 0.14 degrees C/MPa, respectively, for DMPC-d(54)/DHPC (3:1) and at at approximately 0.19 and approximately 0.18 degrees C/MPa, respectively, for DMPC-d(54)/DHPC (4.4:1). Pressure had little effect on the range of DMPC-d(54) chain orientational order through the nematic phase temperature range, but the behavior of chain orientational order at the nematic-to-lamellar transition was found to vary slightly with pressure. Comparison of differential scanning calorimetry (DSC) observations with ambient-pressure (2)H NMR observations of DMPC-d(54) in the bicellar mixtures suggests that absorption of heat persists for a few degrees above the onset of axially symmetric DMPC-d(54) reorientation.
We present the first experimental measurements of surfactant curvilinear diffusion in giant wormlike micelles. The surfactant diffusion was monitored by pulsed field gradient NMR for various observation times t ranging from 0.020 to 1.5 s. The surfactant mean-square displacement was found to scale as t1/2. A model of lateral diffusion along wormlike micellar aggregates with Gaussian statistics is found to describe the echo attenuation well. This type of diffusion is analogous to polymer segment diffusion in the tube-reptation model.
Small angle neutron scattering (SANS) instruments typically cover a q (scattering vector) range from 0.001 to 0.6 Å <sup>-1</sup> . This range in q is achieved through a combination of cold neutrons (λ≫4 Å ) and a highly collimated beam. However, as a direct result of the unavailability of a cold source at the Canadian Neutron Beam Centre (CNBC), we have resorted to adapting a triple-axis spectrometer to perform SANS measurements. This is achieved through the use of multiple converging incident beams which enhance the neutron flux on the sample by a factor of 20, compared to a single beam of the same spot size. Furthermore, smearing effects due to vertical divergence from the slit geometry are reduced through the use of horizontal Soller collimators. As a result, this modified triple-axis spectrometer enables SANS measurements to a minimum q value (q<sub> min </sub>) of ∼0.006 Å <sup>-1</sup> . Data obtained from the modified triple-axis spectrometer are in good agreement with those data from the 30 m NG3-SANS instrument located at the National Institute of Standards and Technology (Gaithersburg, MD, USA).
The spin-echo attenuation in NMR field-gradient diffusometry experiments is treated for the tube model in a time scale longer than the entanglement time taue. The theory comprises the Doi-Edwards [M. Doi and S. F. Edwards, The Theory of Polymer Dynamics (Clarendon, Oxford, 1986)] limits of the (anomalous) segment displacement as well as the (ordinary) center-of-mass diffusion. This formalism is to be distinguished from formalisms for anomalous diffusion on fractal networks: The reptation mechanism implies an intrinsically different character of the displacement probability density. It is shown that the expressions usually applied in NMR diffusometry are inadequate for the reptation problem and can cause misinterpretations. Applications of the formalism to polymer chains in bulk and confined in porous media are discussed.
Over the past decade "bicellar" lipid mixtures composed of the long-chain dimyristoyl phosphatidylcholine (DMPC) and the short-chain dihexanoyl PC (DHPC) molecules have emerged as a powerful medium for studying membrane associated, biologically relevant macromolecules and assemblies. Depending on temperature, lipid concentration and composition these lipid mixtures can assume a variety of morphologies, some of them alignable in the presence of a magnetic field. This article will examine the biophysical studies that have elucidated the various morphologies assumed by these lipid mixtures, and their use in the biochemical studies of biomolecules.
The diffusion of water in randomly oriented lamellae of Aerosol OT (AOT)/water solutions has been measured by using the pulsed field gradient nuclear magnetic resonance (PFG NMR) method. Three concentration regimes are identified and the transitions between them correspond with transitions in both the X-ray repeat distances and solution conductivity. By varying the diffusion observation interval we observe the displacement of water within a single domain or two and more domains. Diffusive barriers are indicated within each domain and a model is proposed which is consistent with both the pulsed field gradient NMR results and the X-ray and conductivity data.
Self-diffusion coefficients of tritiated water in normal (H2O) and heavy water (D2O) have been measured over the temperature range 1-45°. The diaphragm-cell technique was used and the results are considered to be probably accurate to ±0.2%. The data of Longsworth1,2 for HDO diffusion in both H2O and D2O in conjunction with the tritiated water values measured here have been used to calculate the self-diffusion coefficients of pure normal water (H2O-H2O) and pure heavy water (D2O-D2O). These coefficients have been tabulated and compared with molecular dynamics and nmr data.
The stimulated echo in a three‐rf‐pulse experiment is shown to be useful in extending the range of measurement of diffusion coefficients to more viscous substances or the measurement of barrier separations to wider spacings in systems where the diffusing substance has T1 > T2. The spin‐echo attenuation due to self‐diffusion is derived for the general case of a time‐dependent field gradient, and the result is found experimentally to be correct for the special case of a field gradient applied in two equal, square pulses.
Micellization of poly(oxyethylene-b-oxypropylene-b-oxyethylene) triblock copolymers (Pluronic polymers F68, P85, and F108) in aqueous solutions was studied, and critical micellization concentrations (cmc) were determined using surface tension measurements and fluorescent probes (pyrene, 1,6-diphenyl-1,3,5-hexatriene), The dependence of cmc on temperature was observed, and critical micellization temperatures characterizing temperature-dependent transitions of Pluronic unimers to multimolecular micelles were measured. The molecular characteristics of P85 and F108 micelles including their dimensions, molecular masses and surfactant aggregation numbers were determined using lightscattering and ultracentrifugation techniques. Depending on the type of Pluronic, the micelles had an average hydrodynamic diameter ranging from about 15 to about 35 nm, a molecular mass of about 200 kDa and aggregation numbers ranging from one to several dozens. The partitioning of fluorescent probes between aqueous and micellar phases was analyzed within the frame of a pseudophase model, and the partitioning coefficients were determined using the fluorescence data. The results are compared with previous reports and are discussed in relationship to the application of block copolymer micelles as microcontainers for drug delivery.
An X-ray study of highly oriented films of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) in the L(c') phase is presented. The data describe the physical characteristics of the hydrophilic phosphorylcholine headgroups and hydrocarbon chains in the L(c') phase and show that the L(c') --> L(beta F) phase transition is characterized by a change in hydrocarbon chain packing, tilt angle and direction. Also, 1D electron density profiles indicate that L(c') and dry L(beta F) bilayers differ significantly in the bilayer interface region from those in the L(beta 1) (molecular tilt toward nearest neighbors) and hydrated L(beta F) (molecular tilt between nearest neighbors) phases.
We report on the diffusion of a surfactant confined in a branched cylindrical “micellar” network, formed by lecithin and small amounts of water in the solvent isooctane. By means of the pulsed field gradient 1H NMR technique, the measured surfactant mean square displacement, z2, allows for a detailed investigation on the microstructure of the micellar network. Our results show that the structure depends weakly on the micellar volume fraction, Φ, and strongly on the water-to-lecithin molar ratio, W0. We have studied the lecithin diffusion along two different oil dilution lines, corresponding to different water-to-lecithin molar ratios, 2 and 3. The time window in the diffusion experiments was varied in the range from 50 ms to 1 s. At W0 = 3, a Gaussian diffusion, characterized by a mean square displacement varying linearly with time, was observed for all concentrations and all observation times investigated. Furthermore, the self-diffusion coefficient was found to be independent of the concentration in the micellar volume fraction range studied from Φ = 0.1 to Φ = 0.38. The value of the diffusion coefficient is approximately 1/3 of the value of the lateral diffusion coefficient, Dc. At the second dilution line, W0 = 2, the situation is markedly different. At lower concentrations (Φ < 0.11), we found at shorter times a mean square displacement z2 scaling as t1/2 consistent with curvilinear diffusion. For longer times, there was a crossover to a Gaussian diffusion with z2 t. The observation time where there is a crossover from curvilinear to a Gaussian diffusion shifts to shorter times with increasing Φ. At higher concentrations, only a Gaussian diffusion was observed within the experimental time window. The diffusion coefficient evaluated from the Gaussian regime increases linearly with Φ, the value varying from Dc/100 to Dc/20. The high diffusion coefficients evaluated at W0 = 3 clearly indicate that the structure is a branched micellar network where the curvilinear distance along the cylindrical micelles between two branch points is smaller than the persistence length. At W0 = 2, the data can also be interpreted in terms of a branched network, however with a much smaller density of branch points. The branching density increases with increasing Φ. Finally, the measured water diffusion along the two oil dilution lines was found to be Gaussian with a time-independent, single diffusion coefficient. The dominating mechanism for the water diffusion was found to be the motion inside the giant wormlike reverse micelles mediated by an interaggregate exchange with a characteristic time of the order of microseconds.
Static and dynamic light-scattering studies on the temperature-induced micellization behavior in aqueous solutions of poly (oxyethylene-oxypropylene-oxyethylene) block copolymer, poloxamer 188, are presented. A wide temperature range from 21.5 to 78°C was covered in this work. Three temperature regions exist, namely, unimer, transition, and micelle. At room temperatures, i.e., below the critical micellization temperature, molecularly dispersed particles of an average radius of ∼2.3 nm with a broad polydispersity have been detected. In the micelle region, the micellar weights measured are of the order of 105 g mol−1 and increase linearly with increasing temperature, while the hydrodynamic radius of micelles remains nearly constant (∼8.0 nm). Such a dual effect of temperature could be interpreted in terms of enhanced dehydration with temperature. The polydispersity of polymolecular micelles becomes much narrower than that of unimers. In the intermediate transition region, the equilibrium unimers ⇌ micelles shifts toward micelles with rising temperature and increasing concentration. Thus a reasonably complete picture about the association process in the aqueous poloxamer system has been given in accordance with the closed association model.
The phase behavior of lipid bilayered micelles (`bicelles') (dimyristoyl-phosphatidylcholine, DMPC/dihexanoyl-phosphatidyl-choline, DHPC 2.6/1) has been studied by small angle X-ray scattering and 31P NMR. Below 3% w/v the bilayers are arranged in tightly packed stacks. At intermediate concentrations single units are observed, whereas at 24% w/v and higher, weak stacking occurs again. The DMPC/DHPC ratio in the bicelles strongly increases at low concentration, which is correlated with an increase in the bicelle size and stacking. The increase of the order parameter in a magnetic field is related to the stack formation. Below 297 K there is no stacking at any concentration and no magnetic alignment.
A procedure based on a phase-cycling and pulse-swapping technique is described which eliminates unwanted echoes and FIDs in three-pulse electron spin-echo experiments and allows the measurement of the envelope of the stimulated echo as a function of the time τ′ for 0 < τ′ ⩽ τ. The performance of the method is demonstrated experimentally.
Abstract: We have carried out neutron diffraction experiments on a lipid mixture in a macroscopically confined sample geometry (1 mm gap) and have observed the transformation of bilayered micelles into two distinct orientationally aligned domains of perforated lamellae seemingly littered with defects. Depending on the separation of the confining surfaces, the defects can lie on a two-dimensional centered rectangular lattice where the angle, , between lamellar domains can vary. The particular lamellar phase is shown to be metastable and eventually relaxes, either in time or by perturbation of the sample by centrifugation, into aligned multibilayer stacks of single orientation.
Abstract: The structural phases of magnetically alignable lipid mixtures were investigated as a function of temperature and lipid concentration using small-angle neutron scattering (SANS). Two systems were examined: (a) an aqueous mixture of DMPC (dimyristoyl phosphatidylcholine) and DHPC (dihexanoyl phosphatidylcholine) lipids doped with Tm3+ ions resulting in the positive alignment of the system with the applied magnetic field and (b) the above aqueous Tm3+ doped lipid mixture containing a negatively charged lipid, DMPG (dimyristoylphosphatidylglycerol). For both systems, three different scattering patterns were observed corresponding to distinct structural phases at specific temperatures and lipid concentrations. At 45 C and a lipid concentration of >0.05 g/mL, the high-viscosity liquid crystalline phase was found to be a perforated and possibly undulating lamellar phase consistent with NMR results. Upon dilution (<0.05 g/mL) at the same temperature (45 C), the perforated lamellar phase transformed into a unilamellar vesicular phase, in which the bilayers may also be perforated. Below about 25 C, the viscosity decreases considerably and the scattering data suggest that the lamellae present at higher temperatures break up into smaller entities characterized by the bicellar morphology proposed previously for the nondoped system. The structural dimensions of the vesicular and bicellar phases have been determined as a function of lipid concentrations from the SANS data. In the lamellar phase, the influence of Tm3+ ions and DMPG on bilayer structure (e.g., lamellar repeat spacing, bilayer rigidity, and magnetic alignment) were also investigated.
Advances in colloid and interface science have stimulated a renewed interest in the study of lipid-water systems. In recent years, much progress has been achieved in the domains of sample preparation and sample environments, offering the unique possibility of studying these systems under physiologically relevant conditions. In the case of neutron reflectometry, new experimental protocols allow for the unique structural determination of one-dimensional membrane profiles, while the advantages offered by synchrotron radiation (e.g., high flux and spatial resolution) make X-rays an excellent tool for addressing questions pertaining to membrane interactions. Most recently, holographic techniques are evolving so that one day they may be able to resolve, to atomic resolution, the structure of poorly crystallized membrane associated proteins.
To test a prediction of the mixed bicelle model, stimulated echo (STE) pulsed field gradient (PFG) (1)H nuclear magnetic resonance (NMR) measurements of water diffusion between and across bicellar lamellae were performed in positively and negatively magnetically aligned bicelles, composed of mixtures of DHPC (1,2-dihexanoyl-sn-glycero-3-phosphocholine) and DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), as a function of temperature and of the proportion of added short-chain lipid DHPC. (31)P NMR spectra obtained for each situation confirmed that the DHPC undergoes fast exchange between curved and planar regions as per the mixed bicelle model and permitted an estimate of the proportion of the two DHPC populations. Water diffusion across the bicellar lamellae was shown to scale directly with q*, the fraction of edge versus planar phospholipid, rather than simply the ratio q, the global fraction of long-chain to short-chain phospholipid. Geometric modeling of the dependence of water diffusion on q* suggested an upper limit of 400 A for the size of DHPC-rich toroidal perforations within the bicelle lamellae. These findings constitute an independent confirmation of the mixed bicelle model in which DHPC is not confined to edge regions but enjoys, instead, a finite miscibility with DMPC.
Mixtures of long-chain and short-chain phosphatidylcholine (PC) were characterized by multinuclear (13C, 31P, 2H) solid-state nuclear magnetic resonance. This work complements and extends previous characterization of such mixtures by focusing on concentrated mixtures at temperatures above the gel to liquid crystalline phase transition temperature (Tm) of the long-chain PC component. Above Tm it was observed that highly oriented, bilayer-like assemblies could be formed of mixtures of dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC) in molar ratios ranging from approximately 1:3.5 to 1:2 (DHPC:DMPC) over a considerable range of lipid concentrations (at least 3-40% w/v total lipid, for a 1:2.5 sample). Orientation was observed to occur only in an L alpha-like phase. The NMR data can be accounted for by a general model of the DHPC-DMPC aggregates in which DHPC can be found in two distinct populations (one highly ordered, one not). The averaged conformations of the glycerol backbone/headgroup regions of the long- and short-chain PC composing the assemblies were judged by solid-state 13C NMR to be similar to each other. The information gleaned about these mixtures and the quality of the oriented NMR spectra obtained suggest that DHPC-DMPC mixtures may prove to be useful as model membrane media in solid-state NMR studies of biomembranes.
Small-angle x-ray diffraction studies were performed on gel phase-oriented bilayers of dipalmitoylphosphatidylcholine (DPPC) and DPPC containing 40 mol% of either palmitic acid (PA) or palmitic acid brominated at the 2-position (BPA). Oriented samples were prepared using a method developed by us, which is as simple as powder sample preparations while offering all the advantages of oriented samples made by traditional methods. Phases were determined using swelling experiments with structure factors plotted in reciprocal space, creating a relatively smooth curve as the amount of water between the bilayers was changed. Continuous Fourier transforms were also calculated to further test the consistency of the phase assignments. The diffraction data were used to calculate absolute electron density profiles for different bilayers to a resolution of 5-6 A. Analysis indicates the following: (a) The electron density profiles for the three preparations are virtually identical in the hydrocarbon chain region. (b) There is a decrease in the electron density of the glycerol backbone-headgroup region and d-space in DPPC-PA compared to DPPC. (c) The bromine of fatty-acid brominated at the 2-position is in the vicinity of the glycerol backbone. (d) The bilayer thickness of DPPC containing either brominated or unbrominated fatty acid remains relatively constant with increased levels of hydration, unlike DPPC bilayers.
Using a combination of X-ray diffraction data from oriented films and multilamellar liposomes of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) in the subgel phase, we have established the presence of a 2D molecular lattice containing two lipid molecules. The proposed 2D lattice is consistent with all the X-ray diffraction data on the subgel phase of DPPC available in the literature. In this phase, the DPPC molecules are ordered in the plane of the bilayer and are also found to be positionally correlated across a single bilayer but not with those in adjacent bilayers. We also present the possible molecular arrangements for the proposed lattice.
Solid-state phosphorus (31P) and deuterium (2H) nuclear magnetic resonance (NMR) spectroscopy over the temperature range of 25-50 degreesC were used to investigate bilayered micelles (bicelles) composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1, 2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) in the presence of either the anionic lipid 1,2-dimyristoyl-sn-3-phosphoglycerol (DMPG) or the cationic lipid 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP). The 31P-NMR spectra demonstrate that bicellar structures form with DMPG/DMPC ratios ranging from 0 to 50/50 and with DMTAP/DMPC ratios from 0 to 40/60, while the overall concentration of DHPC remains constant. The formation of bicelles containing charged amphiphiles is contingent upon the presence of NaCl, with 50 mM NaCl being sufficient for bicelle formation at all concentrations of charged amphiphile investigated, while 150 mM NaCl affords better resolution of the various 31P-NMR resonance signals. The 2H-NMR spectra demonstrate that the quadrupolar splittings (Deltanu) of head group-deuterated DMPC change inversely as a function of the amount of negative versus positive charge present, and that the changes for deuterons on the alpha-carbon are opposite in sense to those for deuterons on the beta-carbon. This indicates that head group-deuterated phosphatidylcholine functions as a molecular voltmeter in bicelles in much the same fashion as it does in spherical vesicles.
Considering one-dimensional diffusion as a sequence of exchange processes between occupied and vacant sites, the long-time limit of the mean-square displacement of the diffusants is shown to be easily calculable as the net effect of the random walk of the vacancies.
With the aim of establishing acidic bicellar solutions as a useful membrane model system, we have used deuterium NMR spectroscopy to investigate the properties of dimyristoyl/dihexanoylphosphatidylcholine (DMPC/DHPC) bicelles containing 25% (w/w in H(2)O) of either dimyristoylphosphatidylserine (DMPS) or dimyristoylphosphatidylglycerol (DMPG). The addition of the acidic lipid component to this lyotropic liquid crystalline system reduces its range of stability because of poor miscibility of the two dimyristoylated phospholipids. Compared to the neutral bicelles, which are stable at pH 4 to pH 7, acidic bicelles are stable only from pH 5.5 to pH 7. Solid-state deuterium NMR analysis of d(54)-DMPC showed similar ordering in neutral and acidic bicelles. Fully deuterated DMPS or DMPG is ordered in a way similar to that of DMPC. Study of the binding of the myristoylated N-terminal 14-residue peptide mu-GSSKSKPKDPSQRR from pp60(nu-src) to both neutral and acidic bicelles shows the utility of these novel membrane mimetics.
Aligned samples of lipid bilayers have been fully hydrated from water vapor in a different type of x-ray chamber. Our use of aligned samples resolves issues concerning the ripple phase that were ambiguous from previous powder studies. In particular, our x-ray diffraction data conclusively demonstrate that, on cooling from the L alpha to the P beta' phase, both chiral and racemic samples of dipalmitoyl phosphatidylcholine (DPPC) exhibit phase coexistence of long and short ripples with a ripple wavelength ratio lambda L/lambda S approximately 1.8. Moreover, the long ripple always forms an orthorhombic unit cell (gamma L = 90 degrees), strongly supporting the possibility that these ripples are symmetric. In contrast, gamma S for short ripples was consistently different from 90 degrees, implying asymmetric ripples. We continue to find no evidence that chirality affects the structure of rippled bilayers. The relative thermodynamic stability of the two types of ripples was investigated and a qualitative free energy diagram is given in which the long ripple phase is metastable. Finally, we suggest a kinetic mechanism, involving loss of water, that promotes formation of the metastable long ripple phase for special thermal protocols.
The structural phase behavior of phospholipid mixtures consisting of short-chain (dihexanoyl phosphatidylcholine) and long-chain lipids (dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol), with and without lanthanide ions was investigated by small-angle neutron scattering (SANS). SANS profiles were obtained from 10 degrees C to 55 degrees C using lipid concentrations ranging from 0.0025 g/ml to 0.25 g/ml. The results reveal a wealth of distinct morphologies, including lamellae, multi-lamellar vesicles, unilamellar vesicles, and bicellar disks.
Pluronic block copolymers have been used extensively in a variety of pharmaceutical formulations including delivery of low molecular mass drugs and polypeptides. This review describes novel applications of Pluronic block copolymers in the treatment of drug-resistant tumors. It has been discovered that Pluronic block copolymers interact with multidrug-resistant cancer (MDR) tumors resulting in drastic sensitization of these tumors with respect to various anticancer agents, particularly, anthracycline antibiotics. Furthermore, Pluronic affects several distinct drug resistance mechanisms including inhibition of drug efflux transporters, abolishing drug sequestration in acidic vesicles as well as inhibiting the glutathione/glutathione S-transferase detoxification system. All these mechanisms of drug resistance are energy-dependent and therefore ATP depletion induced by Pluronic block copolymers in MDR cells is considered as one potential reason for chemosensitization of these cells. Following validation using in vitro and in vivo models, a formulation containing doxorubicin and Pluronic mixture (L61 and F127), SP1049C, has been evaluated in phase I clinical trials. Further mechanistic studies and clinical evaluations of these systems are in progress.
Drug efflux transporters can influence the absorption, tissue distribution and elimination of many therapeutic agents. Modulation of drug efflux transporter activity is being explored as a means for improving the pharmacokinetic and pharmacodynamic properties of various drugs. In this regard, several polymer formulations have been shown to inhibit drug efflux transporters such as P-glycoprotein (P-gp). The current review will focus on Pluronic block copolymers in particular, the mechanisms involved in the effects of Pluronic on drug efflux transporters, and the optimal polymer compositions required for inhibition of drug efflux transporters. Special emphasis will be placed on the potential applications of Pluronic in enhancing the blood-brain barrier (BBB) penetration of drugs.
The relationship between molecular architecture and the nature of interactions with lipid bilayers has been studied for a series of poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers using small-angle X-ray scattering (SAXS) and thermal analysis (differential scanning calorimetry, DSC). The number of molecular repeat units in the hydrophobic poly(propylene oxide), PPO, block has been found to be a critical determinant of the nature of triblock copolymer-lipid bilayer association. For dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-based biomembrane structures, polymers possessing a PPO chain length commensurate with the acyl chain dimensions of the lipid bilayer yield highly ordered, swollen lamellar structures consistent with well-integrated (into the lipid bilayer) PPO blocks. Triblock copolymers of lesser PPO chain length yield materials with structural characteristics similar to a simple dispersion of DMPC in water. Increasing the concentration (from 4 to 12 mol %) of well-integrated triblock copolymers enhances the structural ordering of the lamellar phase, while concentrations exceeding 16 mol % result in the formation of a hexagonal phase. Examination of temperature-induced changes in the structure of these mesophases (complex fluids) reveals that if the temperature is reduced sufficiently, all compositions exclude polymer and thus exhibit the characteristic SAXS pattern for hydrated DMPC bilayers. Increasing the temperature promotes better insertion of the polymers possessing PPO chain lengths sufficient for membrane insertion. No temperature-induced structural changes are observed in compositions prepared with PEO-PPO-PEO polymers that feature PPO length insufficient to permit full incorporation into the lipid bilayer.
The effects caused by poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO; Pluronic) copolymers on the structure and stability of dioleoylphosphatidylethanolamine (DOPE) liposomes were studied by means of turbidity, leakage, and cryo-transmission electron microscopy investigations. The results show that by inclusion of Pluronics in the DOPE dispersion it is possible to stabilize the lamellar Lalpha phase and to produce liposomes that are stable and nonleaky at low pH (pH 5). The stabilizing capacity was observed to depend critically on the molecular composition of the Pluronics. Block copolymers with comparably long PPO and PEO segment lengths, such as F127 and F108, most effectively protected DOPE liposomes prepared at high pH from aggregation and subsequent structural rearrangements induced by acidification. A sufficiently long PPO block was found to be the most decisive parameter in order to obtain adequate coverage of the liposome surface at low Pluronic concentrations. Upon increasing the copolymer concentration, however, Pluronics with comparably short PPO and PEO segment lengths, such as F87 and P85, could also be used to stabilize the DOPE liposomes. Essentially the same trends were observed when the Pluronics were added to preformed DOPE liposomes instead of being included in the preparation mixture. In this case the least effective copolymers failed, however, to completely prevent the DOPE liposomes from releasing encapsulated hydrophilic markers.
We have used cryo-transmission electron microscopy (cryo-TEM) for inspection of aggregates formed by dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC) in aqueous solution at total phospholipid concentrations cL < or = 5% and DMPC/DHPC ratios q < or = 4.0. In combination with ocular inspections, we are able to sketch out this part of phase-diagram at T = 14-80 degrees C. The temperature and the ratio q are the dominating variables for changing sample morphology, while cL to a lesser extent affects the aggregate structure. At q = 0.5, small, possibly disc-shaped, aggregates with a diameter of approximately 6 nm are formed. At higher q-values, distorted discoidal micelles that tend to short cylindrical micelles are observed. The more well-shaped discs have a diameter of around 20 nm. Upon increasing q or the temperature, long slightly flattened cylindrical micelles that eventually branch are formed. A holey lamellar phase finally appears upon further elevation of q or temperature. The implications for biological NMR work are two. First, discs prepared as membrane mimics are frequently much smaller than predicted by current "ideal bicelle" models. Second, the q approximately 3 preparations used for aligning water-soluble biomolecules in magnetic fields consist of perforated lamellar sheets. Furthermore, the discovered sequence of morphological transitions may have important implications for the development of bicelle-based membrane protein crystallization methods.
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.
Lateral diffusion measurements of PEG-lipid incorporated into magnetically aligned bicelles are demonstrated using stimulated echo (STE) pulsed field gradient (PFG) proton (1H) nuclear magnetic resonance (NMR) spectroscopy. Bicelles were composed of dimyristoyl phosphatidylcholine (DMPC) plus dihexanoyl phosphatidylcholine (DHPC) (q = DMPC/DHPC molar ratio = 4.5) plus 1 mol % (relative to DMPC) dimyristoyl phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG 2000) at 25 wt % lipid. 1H NMR STE spectra of perpendicular aligned bicelles contained only resonances assigned to residual HDO and to overlapping contributions from a DMPE-PEG 2000 ethoxy headgroup plus DHPC choline methyl protons. Decay of the latter's STE intensity in the STE PFG 1H NMR experiment (g(z) = 244 G cm(-1)) yielded a DMPE-PEG 2000 (1 mol %, 35 degrees C) lateral diffusion coefficient D = 1.35 x 10(-11) m2 s(-1). Hence, below the "mushroom-to-brush" transition, DMPE-PEG 2000 lateral diffusion is dictated by its DMPE hydrophobic anchor. D was independent of the diffusion time, indicating unrestricted lateral diffusion over root mean-square diffusion distances of microns, supporting the "perforated lamellae" model of bicelle structure under these conditions. Overall, the results demonstrate the feasibility of lateral diffusion measurements in magnetically aligned bicelles using the STE PFG NMR technique.
Nonionic amphiphiles and particularly block copolymers of ethylene oxide and propylene oxide (Pluronics) cause pronounced chemosensitization of tumor cells that exhibit multiple resistance to antineoplastic drugs. This effect is due to inhibition of P-glycoprotein (P-gp) responsible for drug efflux. It was suggested that the inhibition of P-gp might be due to changes in its lipid surrounding. Indeed, high dependence of P-gp activity on the membrane microviscosity was demonstrated [Regev et al. (1999) Eur. J. Biochem. 259, 18-24], suggesting that the ability of Pluronics to affect the P-gp activity is mediated by their effect on the membrane structure. We have found recently that adsorption of Pluronics on lipid bilayers induced considerable disturbance of the lipid packing [Krylova et al. (2003) Chemistry 9, 3930-3936]. In the present paper, we studied 19 amphiphilic copolymers, including newly synthesized hyperbranched polyglycerols, Pluronic and Brij surfactants, for their ability to accelerate flip-flop and permeation of antitumor drug doxorubicin (DOX) in liposomes. It was found that not only bulk hydrophobicity but also the chemical microstructure of the copolymer determines its membrane disturbing ability. Copolymers containing polypropylene oxide caused higher acceleration of flip-flop and DOX permeation than polysurfactants containing aliphatic chains. The effects of copolymers containing hyperbranched polyglycerol "corona" were more pronounced, as compared to the copolymers with linear poly(ethylene oxide) chains, indicating that a bulky hydrophilic block induces additional disturbances in the lipid bilayer. A good correlation between the copolymer flippase activity and a linear combination of copolymer bulk hydrophobicity and the van der Waals volume of its hydrophobic block was found. The relationship between the structure of a copolymer and its ability to disturb lipid membranes presented in this paper may be useful for the design of novel amphiphilic copolymers capable of affecting the activity of membrane transporters in living cells.
The morphology and stability of small unilamellar egg yolk phosphatidylcholine (EggPC) liposomes modified with the Pluronic copolymer (poly (oxyethylene)-poly (oxypropylene)-poly (oxyethylene) (PEO-PPO-PEO)) with different compositions on mica surface have been investigated using atomic force microscopy. Morphology studies reveal significant morphological changes of liposomes upon incorporating the Pluronic copolymer. Bilayers are observed for Pluronic with small hydrophilic (PEO) chain lengths such as L81 [(PEO)2(PPO)40(PEO)2] and L121 [(PEO)4(PPO)60(PEO)4]; bilayer and vesicle coexistence is observed for P85 [(PEO)26(PPO)39.5(PEO)26] and F87 [(PEO)61.1(PPO)39.7(PEO)61.1]; and stable vesicles are observed for F88 [(PEO)103.5(PPO)39.2(PEO)103.5], F127 [(PEO)100(PPO)65(PEO)100], and F108 [(PEO)132.6(PPO)50.3(PEO)132.6]. The micromechanical properties of Pluronic-modified EggPC vesicles were studied by analyzing AFM approaching force curve. The bending modulus (k(c)) of the Pluronic-modified EggPC vesicles increased several-fold compared with that of the pure EggPC vesicles. The significant difference is due to the enhanced rigidity of the EggPC vesicles as a result of the incorporation of PPO molecules and PEO chains. Based on the analysis of onset point by AFM and diameters of vesicles by light scattering, it was concluded that the favorable model to describe the polymer-bilayer interaction is the membrane-spanning model.
Mixtures of long- and short-chain phospholipids, specifically 14:0 and 6:0 phosphatidylcholines (DMPC and DHPC), have been used successfully in NMR studies as magnetically alignable substrates for membrane-associated proteins. However, recent publications have shown that the phase behavior of these mixtures is much more complex than originally thought. Using polarized light microscopy and small-angle neutron scattering, phase diagrams of DMPC/DHPC mixtures at molar ratios of 2, 3.2, and 5 have been determined. Generally, at temperatures below the main-chain melting transition of DMPC (T(M) = 23 degrees C), an isotropic phase of disk-like micelles is found. At high temperatures (T > 50 degrees C), a lamellar phase consisting of either multilamellar vesicles (MLV) or extended lamellae is formed, which at low lipid concentrations (e.g., MLV) coexists with an excess of water. At intermediate temperatures and lipid concentrations, a chiral nematic phase made up of worm-like micelles was observed.
Lateral diffusion measurements of polyethylene glycol(PEG)-lipid incorporated into magnetically aligned lipid bilayers, composed of dimyristoyl phosphatidylcholine (DMPC) plus dihexanoyl phosphatidylcholine (DHPC) plus 1 mol % (relative to DMPC) dimyristoyl phosphatidylethanolamine-n-[methoxy(polyethylene glycol)-2000] (DMPE-PEG 2000), were performed using stimulated-echo pulsed-field-gradient proton ((1)H) nuclear magnetic resonance spectroscopy. The DMPE-PEG 2000 (1 mol %, 35 degrees C) lateral diffusion coefficient D varied directly with the mole fraction of DMPC, X(DMPC) = q/(1+q) where q = DMPC/DHPC molar ratio, decreasing progressively from D = 1.65 x 10(-11) m(2) s(-1) at q approximately 4.7 to D = 0.65 x 10(-11) m(2) s(-1) at q approximately 2.5. Possible sources of this dependence, including orientational disorder, obstruction, and PEG-lipid sequestration, were simulated using, respectively, a diffusion-in-a-cone model, percolation theory, and a two-phase PEG distribution model. Orientational disorder alone was not capable of reproducing the observations, but in combination with either obstruction or PEG-lipid two-phase distribution models did so satisfactorily. A combination of all three models yielded the most reasonable fit to the observed dependence of lateral diffusion on q. These same effects would be expected to influence lateral diffusion of any bilayer-associating species in such systems.
The morphology of DMPC/DHPC mixtures at total lipid concentration cL = 5% (w/w) and DMPC/DHPC ratio q approximately 3, doped with small amounts of DMPG or CTAB, was investigated. 31P NMR was used to identify the magnetically aligning phase, and cryo-transmission electron microscopy (cryo-TEM) was employed for structural characterization. Magnetic alignment was found to occur between approximately 30 and approximately 45 degrees C, and cryo-TEM showed that the magnetically aligning phase consisted of extended sheets with a lacelike structure. The aggregates are best described as intermediates between two-dimensional networks of flattened, highly branched, cylindrical micelles and lamellar sheets perforated by large irregular holes. DHPC most likely covers the edges of the holes, while DMPC makes up the bilayer bulk of the aggregates. However, 20-43% of the DHPC takes part in the bilayer, corresponding to 6-12% of the bilayer being made up of DHPC. This fraction increases with increasing temperature. At temperatures above 45 degrees C, the aligning phase collapses.
This Account describes the ability of amphiphilic polymers (e.g., EO/PO/EO block copolymers) and polycations [e.g., quaternized poly(4-vinylpyridine)] to accelerate translocation from the inside leaflet to the outside leaflet ("flip-flop") within vesicle bilayer membranes. Driving forces and mechanisms of flip-flop catalyzed by the nonionic and cationic polymers are different. The nonionics are bound to the biological membrane via incorporation of their hydrophobic blocks into the inner part of the lipid bilayer occupied by the hydrocarbon chains. The resulting scrambling of lipid molecules is favored by the overall hydrophobicity of the copolymer and the volume of its hydrophobic block. External binding of the cationic polymers, on the other hand, is driven by electrostatic interactions between the positively charged polymer units and the negatively charged lipid headgroups within the outside leaflet. Electrostatic binding favors both the flip-flop of anionic lipid from the inner to outer leaflet and the formation of anionic domains in the outer leaflet. When it is considered that less than 1% of the liposome surface is occupied by certain bound polymers, their effect upon membrane dynamics, as will be described herein, is considerable. A distinct correlation has been found between the "flippase" activity of the polymers and their ability to mediate drug permeation through biomembranes.
The diffusion of various molecular weight poly(ethyleneglycol)s (PEG) confined between the lamellae of magnetically aligned bicelles has been measured using stimulated echo (STE) pulsed field gradient (PFG) 1H nuclear magnetic resonance (NMR) spectroscopy. Bicelles were formulated to contain dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), and dihexanoylphosphatidylcholine (DHPC) in the proportion DMPG/DMPC = 0.05 and q = (DMPC + DMPG)/DHPC = 4.5. PEG diffusion within the interlamellar spaces between such bicelles was found to be unrestricted over diffusion distances of tens of microns. Two confinement regimes could be differentiated according to the dependence of the reduced PEG diffusivity D/D0, where D0 is the unconfined PEG diffusion coefficient, on the relative confinement Rh/H, where Rh is the unperturbed hydration radius of the particular PEG and H approximately 60 A is the separation between apposing lamellae of the magnetically aligned bicelles. In the regime Rh/H < 0.4, the reduced PEG diffusivity was altered only in proportion to the viscosity increase associated with the bicelle dispersion relative to bulk solution. In the regime Rh/H > 0.4, the reduced PEG diffusivity scaled as (Rh/H)-2/3, in agreement with scaling theories for confined polymers.
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