Freeze-fracture Electron Microscopy Research Articles

Ionic liquid microemulsions of 1-butyl-3-methylimidazolium hexafluorophosphate, N,N-dimethylformamide, and water

The phase behavior of a ternary system consisting of the hydrophobic ionic liquid (IL) 1-butyl-3-methylimidazolium hexafluorophosphate (bmimPF6), N,N-dimethylformamide, and water was investigated. Single-phase microemulsion and multiphase regions were observed in the ternary phase diagram. To the best of our knowledge, this is the first report of aqueous IL microemulsions prepared in the absence of traditional surfactants, i.e., surfactant-free microemulsions (SFMEs) containing IL. The microstructures and structural transitions of the aqueous IL SFMEs were investigated using cyclic voltammetry, electrical conductivity, and dynamic laser light scattering techniques. The results showed that the aqueous IL SFMEs exhibit IL-in-water, bicontinuous, and water-in-IL microstructures, similar to the case of traditional surfactant-based microemulsions. The three kinds of microstructures were confirmed from freeze-fracture electron microscopy observations. Ultraviolet-visible and fluorescence spectroscopies were used to examine the polarities of microdomains in the microemulsions, using methyl orange and pyrene, respectively, as probes. It was found that these two techniques can be used to identify the microstructures and microstructural transitions of the aqueous IL microemulsions.

Nonaqueous ionic liquid microemulsions of 1-butyl-3-methylimidazolium tetrafluoroborate, toluene and ethanol

The phase behavior of the ternary system consisting of the hydrophilic ionic liquid (IL) 1-butyl-3-methylimidazolium tetrafluoroborate (bmimBF4), toluene and ethanol was investigated. Single-phase microemulsion and multiphase regions were observed in the ternary phase diagram. To the best of our knowledge, this is the first report on nonaqueous IL microemulsions prepared in the absence of traditional surfactants. The microstructures and structural transitions of the microemulsion system were investigated by cyclic voltammetry, pulse field gradient spin-echo nuclear magnetic resonance and electrical conductivity measurements. The microemulsions exhibit IL-in-toluene (IL/O), bicontinuous (BC) and toluene-in-IL (O/IL) microstructures, similarly to traditional surfactant-based microemulsions. The three kinds of microstructures were confirmed from freeze-fracture electron microscopy observations. A pearl necklace-like structure was observed in the BC microemulsion. The polarity of bmimBF4 domains in the IL/O microemulsion was investigated by UV-visible absorption spectroscopy using methyl orange as a probe. It was found that the polarity was lower than that of the bulk bmimBF4, and it increased with increasing bmimBF4 content at a constant toluene–ethanol ratio.

Visualization of STIM1 and Orai1 by Electron Microscopy

STIM1 and Orai1 constitute the core machinery of store-operated calcium entry. We used transmission and freeze-fracture electron microscopy to visualize these proteins at endoplasmic reticulum (ER)-plasma membrane (PM) junctions. In thin sections, ER formed frequent extensive cortical junctions in HEK-293 cells overexpressing STIM1 and Orai1 and exposed to thapsigargin (TG), but small and rare junctions in control cells. Extended molecules, most likely STIM1, decorated the ER and bridged the ER-PM gap. Freeze-fracture replicas of cells treated in the same manner showed elevated PM domains (“puncta”) packed with particles at a higher density than on the surface of control cells (2564±443/µm2 versus 1863±331/µm2). Puncta varied in size (∼0.01 to 0.45 µm2) and frequency. Clusters of particles in puncta typically exhibited a clear sharp border, but particle density sometimes declined more gradually to background. Since the puncta most likely correspond to sites of STIM1-Orai1 co-clustering at ER-PM junctions, targeting to puncta was used to guide the classification of particles potentially representing Orai1 channels based upon structural parameters. A high percentage of particles in puncta exhibited a common signature; “large and tall” particles were present at a significantly higher density within puncta than in untransfected cells (average 1300±309 versus 225±84/µm2). Three characteristics of these particles are similar to the tetrameric CaV1.1 channels of skeletal muscle: 1) diameter, only slightly smaller than that of CaV1.1; 2) unusual height indicated by the platinum free “shadow”; 3) square, slightly scalloped outline of the same shadow in some images. It is reasonable to postulate that the “large and tall” particles represent tetrameric Orai1 channels clustered in correspondence with cortical Stim-bearing ER junctions. These observations introduce a new approach to visualize individual unlabeled Stim and Orai molecules in situ. Funded by NIH RO1 HL-48093 (CFA) and NS-14609 (MDC).

Unique Aggregation Behavior of a Carboxylate Gemini Surfactant with a Long Rigid Spacer in Aqueous Solution

A new gemini surfactant with a long and rigid spacer, O,O'-bis(sodium 2-dodecylcarboxylate)-p-dibenzenediol (referred to as C(12)φ(2)C(12)), has been synthesized. Its aggregation in aqueous solution has been studied using static and dynamic light scattering measurements. The homologue O,O'-bis(sodium 2-dodecylcarboxylate)-p-benzenediol (C(12)φC(12)) whose spacer only contains a single phenyl group was also examined for comparison. Dynamic light scattering (DLS) revealed the unexpected existence of large aggregates in the solution of C(12)φ(2)C(12). However, C(12)φC(12) showed rather normal aggregation behavior. Both the results of intrinsic viscosity and light scattering demonstrated a loose structure for the large aggregates of C(12)φ(2)C(12). This behavior was attributed to an extending configuration of C(12)φ(2)C(12) with the two alkyl tails stretching toward the solution due to the rigidity of the long spacer. The large network-like aggregate formation was an inevitable outcome of spontaneously reducing the energy of the system. Freeze-fracture transmission electron microscopy (FF-TEM) images and (1)H NMR measurements supported this speculation. Due to the columnar-like molecular geometry, the large network-like aggregates were directly transformed into rodlike micelles with increasing surfactant concentration. Depending on further micellar growth, the wormlike micelles were finally formed as confirmed by rheological measurements.

Surfactant-Free Microemulsion Composed of Oleic Acid, n-Propanol, and H2O

Generally, a microemulsion consists of oil, water, surfactant, and sometimes cosurfactant. Herein, we report a surfactant-free microemulsion (denoted as SFME), consisting of oleic acid (oil phase), water, and n-propanol without the amphiphilic molecular structure of a traditional surfactant. The phase behavior of the ternary system was investigated, showing that there were a single-phase microemulsion region and a multiphase region in the ternary phase diagram. The electrical conductivity measurement was employed to investigate the microregions of the single-phase microemulsion region, and three different microregions, that is, water-in-oleic acid (W/O), a bicontinuous (B.C.) region, and oleic acid-in-water (O/W), were identified, which were further confirmed by freeze-fracture and cryogenic transmission electron microscopy (FF-TEM and Cryo-TEM) observations. The polarity and the salt solubility of water domains in the W/O SFME were investigated by UV-visible spectroscopy using methyl orange and potassium ferricyanide as probes, respectively. Experimental results showed that the water domains in the W/O microemulsion had a lower polarity than bulk water and a normal solubility for salt species, indicating that the SFMEs have much significance in the preparation of various nanomaterials.

Characterization of new DOPC/DHPC platform for dermal applications

Systems formed by mixtures of the phospholipids dioleoylphosphatidylcholine (DOPC) and dihexanoylphosphatidylcholine (DHPC) were characterized by use of differential scanning calorimetry, small angle X-ray scattering and two electron-microscopy techniques, freeze fracture electron microscopy and cryogenic transmission electron microscopy. These techniques allowed for the determination of the size, morphology, structural topology, self-assembly and thermotropic behavior of the nanostructures present in the mixtures. The interaction between the two phospholipids provides curvatures, irregularities and the increase of thickness and flexibility in the membrane. These effects led to the formation of different aggregates with a differential distribution of both phospholipids. The effect of these systems on the skin in vivo was evaluated by measurement of the biophysical skin parameters. Our results show that the DOPC/DHPC application induces a decrease in the permeability and in the hydration of the tissue. These effects in vivo are related to different microstructural changes promoted by these systems in the skin in vitro, published in a recent work. The fundamental biophysical analyses of DOPC/DHPC systems contribute to our understanding of the mechanisms that govern their interaction with the skin.

Microstructure transition of hydrophilic modified ibuprofen and Pluronic copolymer F127 complexes

The effect of the aggregation state of Pluronic copolymer (PEO100–PPO65–PEO100, F127) and the concentration of hydrophilic modified ibuprofen (Ibuprofen–PEG800, IP800) on the interaction between F127 and IP800 was systematically investigated by nuclear magnetic resonance, dynamic light scatter (DLS), surface tension, and freeze-fractured transmission electron microscopy. In the solution of F127 unimers (5 °C), F127 unimers tended to wrap around IP800 micelles, and the binding model of F127 unimers to IP800 micelles transferred from wrapping around to partly threading through with increasing IP800 concentration. The latter binding model was straightly confirmed by nuclear Overhauser enhancement spectroscopy. As the aggregation state of F127 is in the beginning of the micellization (20 °C), the addition of IP800 significantly promoted the micellization of F127 to form the F127/IP800 complex with F127 micelles as the skeleton called the F127–micelle complex. The sudden decrease of the size obtained from DLS stemmed from the disruption of the F127–micelle complex and accompanying rehydration of PPO which is weaker compared with refs. The amount of IP800 to disintegrate the F127–micelle complex increased in the F127–micelle-dominated solution (40 °C) compared to that at 20 °C.

A method for efficient observation of intracellular membranes of monolayer culture cells by quick-freeze and freeze-fracture electron microscopy

We previously developed a method of analyzing the two-dimensional distribution of membrane lipids by combining quick-freezing and freeze-fracture replica labeling (QF-FRL). In principle, this method can be applied to any membrane, but in practice it is not easy to observe cytoplasmic organelles efficiently without ice crystal damage. In this paper, we report a modification of our method that circumvents this problem. In the modified method, cells are cultured on a gold foil scratched with sandpaper and quick-frozen according to a high-pressure freezing method. This technique enables the efficient observation of intracellular structures such as the nucleus, endoplasmic reticulum, Golgi apparatus and mitochondria. Specific labeling of phosphoinositide 4,5-bisphosphate was confirmed in the obtained freeze-fracture replica. The present QF-FRL method is easy to use and should expedite the analysis of intracellular lipid distribution.

Unusual viscoelastic behavior of aqueous solutions of fluorocarbon–hydrocarbon hybrid surfactant and its morphological transformations

The mechanisms of the viscoelastic behavior and morphological transformations of aggregates of aqueous solutions of a sulfate-type hybrid surfactant (F8EH3OS), whose molecules have a hydrocarbon chain (propyl group) and a fluorocarbon chain (perfluorooctyl group), have been investigated. A 160mM aqueous solution of F8EH3OS is a transparent gel and exhibits high viscoelasticity. The zero-shear viscosity of aqueous F8EH3OS solutions is maximum at a concentration of 160mM. The frequency dependence of the dynamic viscoelasticity and freeze-fracture transmission electron microscopy observations of the 160mM F8EH3OS solution indicate the formation of wormlike micelles. The high viscoelasticity of the 160mM solution is attributed to the formation of wormlike micelles and these solutions are called micelle gels in this paper. F8EH3OS forms aggregates of various morphologies, such as spherical or spheroidal micelles, rod-like and wormlike micelles, vesicles, and lamellar phases, in aqueous solutions. A hydrocarbon surfactant (H8EH3OS), in which the fluorocarbon chain in F8EH3OS is replaced by a hydrocarbon chain, does not exhibit high viscoelasticity at any concentration. This unusual viscoelastic behavior is caused by the formation and disruption of micelle gels, and the fluorocarbon chain in F8EH3OS is essential for the formation of micelle gels. 1H NMR experiments suggest that the conformation of the hydrocarbon chain in F8EH3OS changed with surfactant concentration. This conformational change leads to transformation of the aggregates formed from F8EH3OS by changing the critical packing parameter. The hybrid nature of the hydrophobic part of F8EH3OS causes the unusual viscoelastic behavior and the formation of various morphologies.

Mitochondrial morphology, topology, and membrane interactions in skeletal muscle: a quantitative three-dimensional electron microscopy study

Dynamic remodeling of mitochondrial morphology through membrane dynamics are linked to changes in mitochondrial and cellular function. Although mitochondrial membrane fusion/fission events are frequent in cell culture models, whether mitochondrial membranes dynamically interact in postmitotic muscle fibers in vivo remains unclear. Furthermore, a quantitative assessment of mitochondrial morphology in intact muscle is lacking. Here, using electron microscopy (EM), we provide evidence of interacting membranes from adjacent mitochondria in intact mouse skeletal muscle. Electron-dense mitochondrial contact sites consistent with events of outer mitochondrial membrane tethering are also described. These data suggest that mitochondrial membranes interact in vivo among mitochondria, possibly to induce morphology transitions, for kiss-and-run behavior, or other processes involving contact between mitochondrial membranes. Furthermore, a combination of freeze-fracture scanning EM and transmission EM in orthogonal planes was used to characterize and quantify mitochondrial morphology. Two subpopulations of mitochondria were studied: subsarcolemmal (SS) and intermyofibrillar (IMF), which exhibited significant differences in morphological descriptors, including form factor (means ± SD for SS: 1.41 ± 0.45 vs. IMF: 2.89 ± 1.76, P < 0.01) and aspect ratio (1.97 ± 0.83 vs. 3.63 ± 2.13, P < 0.01) and circularity (0.75 ± 0.16 vs. 0.45 ± 0.22, P < 0.01) but not size (0.28 ± 0.31 vs. 0.27 ± 0.20 μm(2)). Frequency distributions for mitochondrial size and morphological parameters were highly skewed, suggesting the presence of mechanisms to influence mitochondrial size and shape. In addition, physical continuities between SS and IMF mitochondria indicated mixing of both subpopulations. These data provide evidence that mitochondrial membranes interact in vivo in mouse skeletal muscle and that factors may be involved in regulating skeletal muscle mitochondrial morphology.

Cochleates Derived from Vibrio cholerae O1 Proteoliposomes: The Impact of Structure Transformation on Mucosal Immunisation

Cochleates are phospholipid-calcium precipitates derived from the interaction of anionic lipid vesicles with divalent cations. Proteoliposomes from bacteria may also be used as a source of negatively charged components, to induce calcium-cochleate formation. In this study, proteoliposomes from V. cholerae O1 (PLc) (sized 160.7±1.6 nm) were transformed into larger (16.3±4.6 µm) cochleate-like structures (named Adjuvant Finlay Cochleate 2, AFCo2) and evaluated by electron microscopy (EM). Measurements from transmission EM (TEM) showed the structures had a similar size to that previously reported using light microscopy, while observations from scanning electron microscopy (SEM) indicated that the structures were multilayered and of cochleate-like formation. The edges of the AFCo2 structures appeared to have spaces that allowed penetration of negative stain or Ovalbumin labeled with Texas Red (OVA-TR) observed by epi-fluorescence microscopy. In addition, freeze fracture electron microscopy confirmed that the AFCo2 structures consisted of multiple overlapping layers, which corresponds to previous descriptions of cochleates. TEM also showed that small vesicles co-existed with the larger cochleate structures, and in vitro treatment with a calcium chelator caused the AFCo2 to unfold and reassemble into small proteoliposome-like structures. Using OVA as a model antigen, we demonstrated the potential loading capacity of a heterologous antigen and in vivo studies showed that with simple admixing and administration via intragastric and intranasal routes AFCo2 provided enhanced adjuvant properties compared with PLc.

Macrophages Sequester Clofazimine in an Intracellular Liquid Crystal-Like Supramolecular Organization

Clofazimine is a poorly-soluble but orally-bioavailable small molecule drug that massively accumulates in macrophages when administered over prolonged periods of time. To determine whether crystal-like drug inclusions (CLDIs) that form in subcellular spaces correspond to pure clofazimine crystals, macrophages of clofazimine-fed mice were elicited with an intraperitoneal thioglycollate injection. Inside these cells, CLDIs appeared uniform in size and shape, but were sensitive to illumination. Once removed from cells, CLDIs were unstable. Unlike pure clofazimine crystals, isolated CLDIs placed in distilled water burst into small birefringent globules, which aggregated into larger clusters. Also unlike pure clofazimine crystals, CLDIs fragmented when heated, and disintegrated in alkaline media. In contrast to all other organelles, CLDIs were relatively resistant to sonication and trypsin digestion, which facilitated their biochemical isolation. The powder x-ray diffraction pattern obtained from isolated CLDIs was consistent with the diffraction pattern of liquid crystals and inconsistent with the expected molecular diffraction pattern of solid, three dimensional crystals. Observed with the transmission electron microscope (TEM), CLDIs were bounded by an atypical double-layered membrane, approximately 20 nanometers thick. CLDIs were polymorphic, but generally exhibited an internal multilayered organization, comprised of stacks of membranes 5 to 15 nanometers thick. Deep-etch, freeze-fracture electron microscopy of unfixed snap-frozen tissue samples confirmed this supramolecular organization. These results suggest that clofazimine accumulates in macrophages by forming a membrane-bound, multilayered, liquid crystal-like, semi-synthetic cytoplasmic structure.

Thermotropic and structural effects of poly(malic acid) on fully hydrated multilamellar DPPC–water systems

The thermotropic and structural effects of low molecular weight poly(malic acid) (PMLA) on fully hydrated multilamellar dipalmitoylphosphatidylcholine (DPPC)–water systems were investigated using differential scanning calorimetry (DSC), small-angle X-ray scattering (SAXS), and freeze-fracture transmission electron microscopy (FFTEM). Systems of 20wt% DPPC concentration and 1 and 5wt% PMLA to lipid ratios were studied. The PMLA derivatives changed the thermal behavior of DPPC significantly and caused a drastic loss in correlation between lamellae in the three characteristic thermotropic states (i.e., in the gel, rippled gel and liquid crystalline phases). In the presence of PBS or NaCl, the perturbation was more moderate. The structural behavior on the atomic level was revealed by FTIR spectroscopy. The molecular interactions between DPPC and PMLA were simulated via modeling its measured infrared spectra, and their peculiar spectral features were interpreted. Through this interpretation, the poly(malic acid) is inferred to attach to the headgroups of the phospholipids through hydrogen bonds between the free hydroxil groups of PMLA and the phosphodiester groups of DPPC.

GRecon: A Method for the Lipid Reconstitution of Membrane Proteins

Membrane proteins account for about 20–30 % of the protein-encoding genes in the genomes of all living organisms.2 Their fundamental importance in human health and disease is underlined by the fact that many drugs in current use act on them.3 Functional and structural studies of membrane proteins are therefore of increasing importance but more difficult and demanding than studies with soluble proteins, because membrane proteins function in the lipid bilayer of cell membranes. For in vitro studies, the proteins are first extracted from the membrane by detergent solubilization and then purified in detergent solution. Sensitive membrane proteins are often unstable in detergent solution, but quite stable once they are reconstituted into a lipid bilayer. Moreover, many membrane proteins require a lipid environment for activity. The reconstitution process, in which detergent is replaced by lipid, must be carefully controlled, as otherwise the proteins tend to denature and aggregate.4

GRecon: A Method for the Lipid Reconstitution of Membrane Proteins

GRecon: A Method for the Lipid Reconstitution of Membrane Proteins

Study on the Morphology Feature of 1-Butyl-3-methylimidazolium Tetrafluoroborate Based Ionic Liquid Reverse Microemulsions

The micromorphology of 1-butyl-3-methylimidazolium tetrafluoroborate (bmimBF4)-in-cyclohexane and bmimBF4-in-triethylamine ionic liquid microemulsions was investigated by two-dimensional NMR and freeze-fracture transmission electron microscopy. The reverse micelles of Triton X-100/cyclohexane were destroyed by adding bmimBF4 and reverse microemulsions were induced to form by successively adding bmimBF4. However, no micelles appeared in triethylamine. But if adding bmimBF4 to a certain extent, a reverse microemulsion was also formed. The driving force of such aggregations could be attributed to the presence of different types of interactions between Triton X-100 and bmimBF4. A staggered arrangement of surfactant led to the irregular droplet structure and large aggregate size.

Influence of Counterions on Micellization of Tetramethylammonium Perfluorononanoic Carboxylate in 1-Butyl-3-methylimidazolium Ionic Liquid

The influence of counterions on micellization of perfluorononanoic carboxylate ammonium salts in water and 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim]BF(4)) solutions was investigated by surface tension and (19)F NMR measurements and freeze-fracture transmission electron microscopy (FF-TEM) observations. Changes in the counterions of the fluorocarbon surfactants have different effects on the two solvents. With the increase of counterion volume, the critical micelle concentration (cmc) value of relevant fluorinated surfactant decreases in aqueous solutions. This is because the counterions with larger size, such as (+)N(CH(3))(4), can be little hydrated, which can screen the electrostatic repulsion of the headgroups of the fluorocarbon surfactant and thus facilitate micelle formation. However, the fluorocarbon surfactants can dissolve and form micelles in [bmim]BF(4) only when they provide with largest counterion such as (+)N(CH(3))(4). This is because the counterion, (+)N(CH(3))(4), disperses the charge of the cations, which could weaken the electrostatic interaction between the ion pair of the surfactant, leading to a higher degree of counterion binding. The thermodynamic parameters estimated from the temperature dependence of the cmc values tell us that the micelle formation for tetramethylammonium perfluorononanoic carboxylate (C(8)F(17)COON(CH(3))(4), PFNT) in ionic liquids (ILs) is an entropy-driven process at low temperature but an enthalpy-driven process at high temperature. The driving force of the micellization for fluorocarbon surfactants in [bmim]BF(4) is the solvophobic effect, due to the hydrophobic and oleophobic properties of fluorocarbon chains.

Protein-Containing PEGylated Cubosomic Particles: Freeze-Fracture Electron Microscopy and Synchrotron Radiation Circular Dichroism Study

The purpose of this work is to investigate the entrapment of protein molecules in cubosomic nanocarriers that are sterically stabilized by an amphiphilic poly(ethylene glycol) (PEG) derivative. Toward that aim, the mechanism of fragmentation of a self-assembled, PEGylated cubic lipid phase into nanoparticles (NPs) is investigated in excess aqueous medium. The molar ratio between the cubic-phase-forming lipid monoolein (MO) and its PEGylated derivative (MO-PEG(2000)) is selected as to favor the formation of inverted-type liquid-crystalline (LC) structures (permitting one to reveal the stages of the fragmentation and bicontinuous membrane NP assembly process) rather than a phase transformation to lamellar or micellar phases. The PEGylated amphiphile considerably affects the interfacial curvature of the cubic lipid membrane and, under agitation, contributes to the fragmentation of the bicontinuous cubic lattice into NPs. Freeze-fracture electron microscopy (FF-EM), quasi-elastic light scattering (QELS), and confocal laser scanning fluorescence microscopy (CLSFM) are applied for determination of the NPs' sizes, inner organization, and stability with regard to a thermal stimulus. Entrapped protein molecules can essentially stabilize the cubosomic particles (proteocubosomes), which display well-defined inner organization of nanochannels in their freeze-fracture planes. The protein α-chymotrypsinogen A is studied in proteocubosome dispersions by means of far-UV synchrotron radiation circular dichroism (SRCD) spectroscopy. It is suggested that the protein molecules are entrapped in the interior of the PEGylated cubosomes via a "nanopockets" mechanism. The LC PEGylated proteocubosomes offer new possibilities for investigation of protein loading in sterically stabilized ("Stealth") nanostructured lipid carriers, which differ from Poloxamer-stabilized isasomes.

Ultrastructural freeze-fracture immunolabeling identifies plasma membrane-localized syndapin II as a crucial factor in shaping caveolae

Membrane topology control is thought to involve peripheral membrane proteins of the F-BAR domain family including syndapins. These proteins are predestined to shape membranes by partial insertion and by imposing their curved shape onto the lipid bilayer. Direct observation of such functions on cellular membranes, however, was precluded by the difficulty to combine high-resolution imaging with visualization of membrane topology. Here, we report the ultrastructural visualization of endogenous syndapin II at the plasma membrane of NIH 3T3 cells using a combination of freeze-fracturing, immunogold labeling and transmission electron microscopy. Surprisingly, syndapin II was detected at flat and curved membrane areas. Ultrastructural colocalization with caveolin 1 identified syndapin II-positive invaginations as caveolae. Consistent with the syndapin II F-BAR domain interacting with caveolin 1, F-BAR overexpression affected caveolin 1 localization. Syndapin II knockdown did not alter caveolin 1 expression or plasma membrane recruitment. Instead, syndapin II knockdown reduced the density of caveolae and strongly increased the number of caveolin 1 molecules at flat membrane areas. Comparative immunoelectron microscopy and tilt series revealed that syndapin II was asymmetrically localized at the neck of caveolae. Double-immunogold labeling showed that the caveolae-shaping molecule PTRF/cavin 1 behaved similarly and that syndapin II and PTRF/cavin 1 colocalized. Visualization of a transiently membrane-binding F-BAR protein in direct relation to membrane topology of mammalian cells thereby revealed that syndapin II binds to both flat and curved membranes in vivo and that it plays an important role in caveolar shaping, a role that it shares with PTRF/cavin 1.

Reformulation of etoposide with solubility-enhancing rubusoside

Etoposide (ETO), a widely used anti-cancer drug, is constrained by its low aqueous solubility and by side effects from both the drug and its solubilizing excipients. In this study, a recently discovered natural solubilizer rubusoside (RUB) was used to achieve the solubilization of ETO. Dynamic light scattering and freeze-fracture transmission electron microscopy studies showed that ETO and RUB formed ETO–RUB nanoparticles (∼6nm in diameter). The powder of ETO–RUB nanoparticles was completely reconstitutable in water and remained stable in this solution at 25 and 37°C for at least 24h. Under other physiologic conditions, ETO solution was clear and free of precipitation at 25°C, but underwent various structural transformations. In PBS and simulated intestinal fluid, RUB-solubilized ETO underwent epimerization and equilibrated to cis-ETO. In simulated gastric fluid, RUB-solubilized ETO degraded to 4′-demethylepipodophyllotoxin-beta-d-glucoside and 4′-demethylepipodophyllotoxin. Higher temperatures favored epimerization or degradation. Furthermore, a side-by-side comparison with DMSO-solubilized ETO confirmed that the RUB-solubilized ETO showed no significant differences in cytotoxicity in colon, breast and prostate cancer cell lines. RUB effectively solubilized and stabilized etoposide, which sets the stage for further toxicology, bioavailability, and efficacy investigations.