Mats Almgren

Cryo transmission electron microscopy of liposomes and related structures

Abstract Cryo-transmission electron microscopy, c-TEM, has during the last 10 years contributed significantly to the understanding of the numerous, and often complex, structures formed by amphiphilic molecules in dilute aqueous solutions. In particular, the method has evolved as an important tool for the investigation of liposomes. In this review, we discuss and show examples of how the technique has been utilised to gain new information on the form and structure of liposomes, as well as on the morphological changes taking place upon encapsulation of drugs or interactions with surfactants, DNA and other polyelectrolytes. Several examples where c-TEM has been successfully employed to visualise related amphiphilic structures, such as thread-like micelles and particles of reversed phases, are also presented. In addition, we discuss recent developments concerning sample preparation and interpretation of images, as well as possible artefacts and their origin.

Complexes between cationic liposomes and DNA visualized by cryo-TEM

The association structures formed by cationic liposomes and DNA-plasmids have been successfully employed as gene carriers in transfection assays. In the present study such complexes was studied by cryo-TEM (cryo-transmission electron microscopy). Cationic liposomes made up by DOPE (dioleoylphosphatidylethanolamine) and various amounts of three different cationic surfactants were investigated. The cryo-TEM analysis suggests that an excess of lipid in terms of charge, leads to entrapment of the DNA molecules between the lamellas in clusters of aggregated multilamellar structures. With increasing amounts of DNA free or loosely bound plasmids were found in the vicinity of the complexes. The importance of the choice of surfactant, as reported from many transfection assays, was not reflected in changes of the type of DNA-vesicle association. A tendency towards polymorphism of the lipid mixtures is reported and its possible implications are discussed.

Self-aggregation and phase behavior of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymers in aqueous solution

The phase behavior and aggregation properties of block copolymers of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (Pluronics, poloxamers) in aqueous solution have recently attracted much attention. Both experimental and theoretical studies are reviewed, not comprehensively, but with the focus on studies, partly cooperative, partly independent, performed by groups in Uppsala (light scattering and fluorescence), Roskilde (rheology and calorimetry), Risø (SANS), Graz (x-ray and speed of sound), and Lund (theoretical model calculations).The phase behavior of these copolymers is similar in many respects to that of conventional nonionic surfactants, with the appearance of hexagonal, cubic, and lamellar liquid crystalline phases at high concentrations. In the isotropic solution phase the critical concentration for micelle formation is strongly temperature dependent, and at a given concentration the monomer to micelle transition occurs gradually over a broad temperature range, partly due to the broad size polydispersity of both the PO- and EO-blocks. For some Pluronic copolymers a transition from globular to long rod-like micelles occurs above a transition temperature, resulting in a strong and sudden increase of viscosity and viscoelasticity of the solution.Size and aggregation numbers have been determined for the globular micelles in some cases, and also the rod-like micelles have been characterized. NMR and fluorescence measurements have provided further information on the properties of the micellar core and mantle. In combination, results from different measurements on the same Pluronics material indicate that the aggregation number of the micelles increases with the temperature, whereas the hydrodynmic radius varies much less. The PEO-mantle of the micelles seems to contract with increasing temperature. The core appears to contain appreciable amounts of PEO in addition to PPO (and also some water). The segregation between core and mantle is not as distinct as in normal micelles, a conclusion which is in line with the predictions from the model calculations.

Mixed micelles and other structures in the solubilization of bilayer lipid membranes by surfactants

The solubilization of lipid bilayers by surfactants is accompanied by morphological changes of the bilayer and the emergence of mixed micelles. From a phase equilibrium perspective, the lipid/surfactant/water system is in a two-phase area during the solubilization: a phase containing mixed micelles is in equilibrium with bilayer structures of the lamellar phase. In some cases three phases are present, the single micelle phase replaced by a concentrated and a dilute solution phase. In the case of non-ionic surfactants, the lipid bilayers reach saturation when mixed micelles, often flexible rod-like or thread-like, start to form in the aqueous solution, at a constant chemical potential of the surfactant. The composition of the bilayers also remains fixed during the dissolution. The phase behavior encountered with many charged surfactants is different. The lamellar phase becomes destabilized at a certain content of surfactant in the membrane, and then disintegrates, forming mixed micelles, or a hexagonal phase, or an intermediate phase. Defective bilayer intermediates, such as perforated vesicles, have been found in several systems, mainly with charged surfactants. The perforated membranes, in some systems, go over into thread-like micelles via lace-like structures, often without a clear two-phase region. Intermediates in the form of disks, either micelles or bilayer fragments, have been observed in several cases. Most noteworthy are the planar and circular disks found in systems containing a large fraction of cholesterol in the bilayer. Bile salts are a special class of surfactants that seem to break down the bilayer at low additions. Originally, disk-like mixed micelles were conjectured, with polar membrane lipids building the disk, and the bile salts covering the hydrophobic rim. Later work has shown that flexible cylinders are the dominant intermediates also in these systems, even if the disk-like structures have been re-established as transients in the transformation from mixed micelles to vesicles.

Size of sodium dodecyl sulfate micelles in the presence of additives i. alcohols and other polar compounds

The steady-state fluorescence quenching method has been used to examine how 12 polar additives affects the size of SDS micelles. The picture that emerges is that the controlling factor for micelle size is the surface area available per charged headgroup, or its inverse the surface charge density. In first approximation, the surface charge density is the same (linear in a wide range) function of the mole fraction additive in the micelle for all the alcohols from C4 and up. Depending on the volume of the hydrophobic part of the additive the micelle either grows directly or first passes a minimum size on increasing amounts of additives. The fluorescence quenching methods primarily give the number of micelles in the solution, from which number average aggregation number and micelle size are calculated.

Effect of inorganic salts on the micellar behaviour of ethylene oxide-propylene oxide block copolymers in aqueous solution

The aggregation behaviour of two ethylene oxide-propylene oxide block copolymers (PEO-PPO-PEO) in aqueous solution has been investigated in the presence of added salts (KCNS, KI, KBr, KCl and KF) by viscosity, cloud point, light scattering, pulse gradient spin echo NMR, and solubilization measurements. The salts have a strong effect on the cloud points of the pluronics. Both P-85 and L-64 form micelles which increase in size and change into elongated shapes when the cloud point is approached. The changes of size and shape of the micelles, revealed by the intrinsic viscosity and rheological properties, seem to occur at the same temperature relative to the cloud point, independent of the nature of the salt. The onset of micelle formation is also shifted in the same direction as the cloud point by the salts, but by a much smaller amount.

Solubilization of lecithin vesicles by C12E8 : structural transitions and temperature effects

Abstract The structural transitions induced by addition of the nonionic surfactant C 12 E 8 (octaethylene glycol n -dodecyl monoether) to small unilamellar lecithin vesicles has been studied by means of static and dynamic light scattering as well as with cryo-transmission electron microscopy. At low surfactant concentration a slight swelling of the vesicles, due to the incorporation of surfactant monomers into the vesicle membrane, can be seen. Large unilamellar vesicles begin to form as the surfactant concentration exceeds 30 mol%. The vesicle size increases with C 12 E 8 concentration until about 40 mol% has been added. At this point the intensity of light scattered at 90° starts to decrease and dynamic light scattering indicates bimodal size distributions. The cryo-TEM micrographs show cylindrical micelles in coexistence with bilayer sheets. At C 12 E 8 concentrations above 70 mol%, the lipid vesicles are completely solubilized into mixed micelles that gradually become spherical and decrease in size with surfactant concentration. Depending on the C 12 E 8 concentration the formation of the lipid/surfactant mixed aggregates may take from less than a second to more than an hour. Increasing the temperature shifts the lipid/surfactant molar ratios at which the different amphiphilic structures start to appear toward lower values.

Static and dynamic properties of a (PEOPPOPEO) block copolymer in aqueous solution

Abstract Aqueous solutions of a triblock copolymer of the type polyethyleneoxide-block-polypropyleneoxide-block-polyoxyleneoxide (Pluronic L-64, average composition 26 EO-units and 30 PO-units), were examined with static and dynamic light scattering (DLS), pulsed-gradient spin-echo (PGSE) NMR and fluorescence spectroscopy over a range of concentrations (0.2–25 wt%) and temperatures (15–60°C). Relaxation time distributions from DLS show L-64 to be molecularly dissolved at 21°C, and to form micelles at higher temperatures, which remain at high concentrations (25%) without formation of gel or liquid crystalline phases. The temperature where micelle formation starts is strongly concentration dependent, in contrast to the cloudpoint with remains fairly constant at 60°C. The hydrodynamic radii of the micelles, as obtained from DLS and PGSE NMR, are in reasonable agreement (60–80 A), and also agree with an aggregation number reported from fluorescence-quenching studies, whereas static light scattering, evaluated according to normal practice, indicates much smaller aggregates. This is due to the presence in the solution of a mixture of monomers, micelles, and at low temperatures, also some strongly scattering larger aggregates, possibly emanating from a small percentage of a diblock impurity in the preparation. The diblock impurity aggregates are dissolved by the proper micelles at higher temperatures. Their presence is indicated also by the anomalous excimer formation at low temperatures, caused by pyrene becoming concentrated in the premicellar aggregates.

Self-Assembly in Monoelaidin Aqueous Dispersions: Direct Vesicles to Cubosomes Transition

Background In the present study, synchrotron small-angle X-ray scattering (SAXS) and Cryo-TEM were used to characterize the temperature-induced structural transitions of monoelaidin (ME) aqueous dispersion in the presence of the polymeric stabilizer F127. We prove that the direct transition from vesicles to cubosomes by heating this dispersion is possible. The obtained results were compared with the fully hydrated bulk ME phase. Methodology/Principal Findings Our results indicate the formation of ME dispersion, which is less stable than that based on the congener monoolein (MO). In addition, the temperature-dependence behavior significantly differs from the fully hydrated bulk phase. SAXS findings indicate a direct Lα-V2 internal transition in the dispersion. While the transition temperature is conserved in the dispersion, the formed cubosomes with internal Im3m symmetry clearly contain more water and this ordered interior is retained over a wider temperature range as compared to its fully hydrated bulk system. At 25°C, Cryo-TEM observations reveal the formation of most likely closely packed onion-like vesicles. Above the lamellar to non-lamellar phase transition at 65°C, flattened cubosomes with an internal nanostructure are observed. However, they have only arbitrary shapes and thus, their morphology is significantly different from that of the well-shaped analogous MO cubosome and hexosome particles. Conclusions/Significance Our study reveals a direct liposomes-cubosomes transition in ME dispersion. The obtained results suggest that the polymeric stabilizer F127 especially plays a significant role in the membrane fusion processes. F127 incorporates in considerable amount into the internal nanostructure and leads to the formation of a highly swollen Im3m phase.

Diffusion-influenced deactivation processes in the study of surfactant aggregates

Abstract This review follows the evolution of the theories for excited state deactivation by diffusion-influenced quenching in microheterogeneous media, and the application of methods on this basis for the study of surfactant aggregates. From the starting point of the simple model for fluorescence quenching in monodisperse small micelles, the theoretical treatment has taken up polydisperse systems, quenching in long rods and lamellae, various possibilities for exchange of reactants between aggregates, and deactivation in clusters of small micelles. The information that can be obtained by time-resolved excited state deactivation studies is different in different types of systems, and is illustrated with examples from experimental studies. Limitations and possibilities are discussed.