Freeze-fracture Electron Microscopy Research Articles

The distal C terminus of the dihydropyridine receptor β1a subunit is essential for tetrad formation in skeletal muscle

The skeletal muscle dihydropyridine receptor (DHPR) β1a subunit is indispensable for full trafficking of DHPRs into triadic junctions (i.e., the close apposition of transverse tubules and sarcoplasmic reticulum [SR]), facilitation of DHPRα1S voltage sensing, and arrangement of DHPRs into tetrads as a consequence of their interaction with ryanodine receptor (RyR1) homotetramers. These three features are obligatory for skeletal muscle excitation–contraction (EC) coupling. Previously, we showed that all four vertebrate β isoforms (β1–β4) facilitate α1S triad targeting and, except for β3, fully enable DHPRα1S voltage sensing [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488–7493 (2013)]. Consequently, β3 failed to restore EC coupling despite the fact that both β3 and β1a restore tetrads. Thus, all β-subunits are able to restore triad targeting, but only β1a restores both tetrads and proper DHPR–RyR1 coupling [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488–7493 (2013)]. To investigate the molecular region(s) of β1a responsible for the tetradic arrangement of DHPRs and thus DHPR–RyR1 coupling, we expressed loss- and gain-of-function chimeras between β1a and β4, with systematically swapped domains in zebrafish strain relaxed (β1-null) for patch clamp, cytoplasmic Ca2+ transients, motility, and freeze-fracture electron microscopy. β1a/β4 chimeras with either N terminus, SH3, HOOK, or GK domain derived from β4 showed complete restoration of SR Ca2+ release. However, chimera β1a/β4(C) with β4 C terminus produced significantly reduced cytoplasmic Ca2+ transients. Conversely, gain-of-function chimera β4/β1a(C) with β1a C terminus completely restored cytoplasmic Ca2+ transients, DHPR tetrads, and motility. Furthermore, we found that the nonconserved, distal C terminus of β1a plays a pivotal role in reconstitution of DHPR tetrads and thus allosteric DHPR–RyR1 interaction, essential for skeletal muscle EC coupling.

Stabilization of all-aqueous droplets by interfacial self-assembly of fatty acids bilayers

All-aqueous microdroplets produced by liquid–liquid phase separation have emerged as promising models of artificial cells, and offer new approaches for the solvent-free encapsulation of fragile solutes. Yet, the lack of a membrane on such droplets makes them intrinsically unstable against coarsening, and precludes a fine control over chemical localization, as solutes can freely diffuse through the interface. Herein, we report the construction of stable and impermeable water-in-water emulsions via the interfacial self-assembly of mixed sodium oleate/1-decanol bilayers on dextran-rich droplets produced by segregative liquid–liquid phase separation with poly(ethylene glycol). Lipids spontaneously self-assemble as multilamellar structures at the surface of the droplets as revealed by freeze-fracture transmission electron microscopy and small-angle X-ray scattering. We further demonstrate that the lipid-based membrane is impermeable to oligonucleotides and proteins, but also to a low molecular weight dye, so that a strict chemical encapsulation can be achieved by spontaneous partitioning within the droplets before membrane self-assembly. Taken together, our results highlight the ease of production of fatty acid-stabilized all-aqueous emulsions droplets able to encapsulate a range of solutes without the need of oil or organic solvents, paving the way to the construction of robust membrane-bounded, polymer-rich artificial cells.

Brij 30 Induced Transition of Rodlike Micelles to Wormlike Micelles and Gels in the Imidazole Ionic Liquid Surfactants: The Alkyl Chain Length Effect.

The effect of the hydrocarbon chain length of ionic liquid surfactants 1-hexadecyl-3-alkyl imidazolium bromide [C16imCn]Br (n = 1-16) on their aggregation behavior with polyoxyethylene lauryl ether (Brij 30) in aqueous solution was inspected. The rheological behavior, thermal properties, and microstructures of the different samples were studied using freeze-fractured electron microscopy. The interactions between [C16imCn]Br and Brij 30 were studied using nuclear magnetic resonance spectroscopy and theoretical simulation. With the addition of Brij 30, the rodlike micelles of [C16imCn]Br (n = 1, 2, 4, and 6) transition into wormlike micelles. The effects of the molar ratio of Brij 30 and [C16imCn]Br and the hydrocarbon chain length of [C16imCn]Br on the Brij 30/[C16imCn]Br (n = 1, 2, 4, and 6) wormlike micelles were studied. When Brij 30 was mixed with the rodlike micelles of [C16imC8]Br, the Brij 30/[C16imC8]Br mixtures form wormlike micelles at low Brij 30 concentration and gels at high Brij 30 concentration. The [C16imCn]Br (n = 10, 12, 14, and 16) rodlike micelles were induced by Brij 30 to turn into the Brij 30/[C16imCn]Br gels. The effect of the [C16imCn]Br hydrocarbon chain length on their rodlike micelles with the addition of Brij 30 is also theoretically discussed.

Elastic and electro-optical properties of flexible fluorinated dimers with negative dielectric anisotropy

ABSTRACT We report the synthesis and the temperature dependencies of optical, dielectric, and elastic properties of four homologous dimeric mesogens in the uniaxial nematic (N) phase. The studied dimers are derived from 2ʹ,3ʹ-difluoroterphenyl units connected via flexible alkyl chains. The compounds differ in the length of either the flexible alkyl chain or the terminal groups. These achiral bimesogens exhibit the twist-bend nematic (NTB) phase with a nanoscale pitch of the modulated nematic director, as established by the freeze-fracture transmission electron microscopy. All four homologs have negative dielectric anisotropy owing to the permanent dipole oriented normal to the long molecular axes. A longer flexible bridge length yields an expanded temperature range of the N phase, stronger dielectric anisotropy, larger birefringence and bend elastic constant . Longer alkyl terminal chains increase birefringence but decrease the absolute value of dielectric anisotropy and . All four bimesogens show a dramatic decrease of down to ~0.5 pN on approaching the N-NTB transition. A longer bridge yields a longer period of twist-bend modulation in the NTB phase.

Potential of quercetin in combination with antioxidants of different polarity incorporated in oil-in-water nanoemulsions to control enzymatic browning of apples

Quercetin in combination with other antioxidants from natural sources and different polarity, including quercetin-3-rhamnoside, ascorbic acid, and α-tocopherol, were incorporated into oil-in-water (O/W) nanoemulsions to evaluate their effects on the activity of polyphenol oxidase (PPO) and browning of Granny Smith apple fruits. Additionally, molecular modeling methods were used to determine the ability and possible mechanisms of action of quercetin and quercetin-3-rhamnoside to inhibit PPO and to reduce the o-quinones derived from (–)-epicatechin, chlorogenic acid, catechol and methylcatechol (PPO substrates). Nanoemulsions were prepared using a low-energy emulsification method. In all nanoemulsions, Dynamic Light Scattering (DLS) analysis showed a size distribution of droplets between 100 and 500 nm diameter, whereas microscopical observation by freeze-fracture transmission electron microscopy (FF-TEM) corroborated the presence of the droplets and their size. Also, occasional arrangements consisting of stacked layers, with vesicle-like structures between them, were seen. The nanoemulsion 1, that contained the four antioxidants was the one that showed the best effect on PPO, inhibiting it by 41%, and inhibiting the browning of the apple flesh between 81 and 74% in a period of 6 h. Calculations with molecular modeling methods suggest that quercetin may be a more effective reducing agent than ascorbic acid and quercetin-3-rhamnoside, and could also act as a non-competitive inhibitor of PPO. While quercetin-3-rhamnoside represents a selective and competitive inhibitor of PPO. The most important function of α-tocopherol in the nanoemulsions could be the regeneration of quercetin to prolong its antioxidant activity.

Dissection of Barrier Dysfunction in Organoid-Derived Human Intestinal Epithelia Induced by Giardia duodenalis

The protozoa Giardia duodenalis is a major cause of gastrointestinal illness worldwide, but underlying pathophysiological mechanisms remain obscure, partly due to the absence of adequate cellular models. We aimed at overcoming these limitations and recapitulating the authentic series of pathogenic events in the primary human duodenal tissue by using the human organoid system. We established a compartmentalized cellular transwell system with electrophysiological and barrier properties akin to duodenal mucosa and dissected the events leading to G. duodenalis-induced barrier breakdown by functional analysis of transcriptional, electrophysiological, and tight junction components. Organoid-derived cell layers of different donors showed a time- and parasite load-dependent leak flux indicated by collapse of the epithelial barrier upon G. duodenalis infection. Gene set enrichment analysis suggested major expression changes, including gene sets contributing to ion transport and tight junction structure. Solute carrier family 12 member 2 and cystic fibrosis transmembrane conductance regulator-dependent chloride secretion was reduced early after infection, while changes in the tight junction composition, localization, and structural organization occurred later as revealed by immunofluorescence analysis and freeze fracture electron microscopy. Functionally, barrier loss was linked to the adenosine 3',5'-cyclic monophosphate (cAMP)/protein kinase A-cAMP response element-binding protein signaling pathway. Data suggest a previously unknown sequence of events culminating in intestinal barrier dysfunction upon G. duodenalis infection during which alterations of cellular ion transport were followed by breakdown of the tight junctional complex and loss of epithelial integrity, events involving a cAMP/protein kinase A-cAMP response element-binding protein mechanism. These findings and the newly established organoid-derived model to study G. duodenalis infection may help to explore new options for intervening with disease and infection, in particular relevant for chronic cases of giardiasis.

Rhodopsins build up the birefringent bodies of the dinoflagellate Oxyrrhis marina

The ultrastructure of the birefringent bodies of the dinoflagellate Oxyrrhis marina was investigated by transmission electron microscopy. Ultrathin sectioning revealed that the bodies consist of highly ordered and densely packed lamellae, which show a regular striation along their longitudinal axis. A lattice distance of 6.1 nm was measured for the densely packed lamellae by FFT (Fast Fourier Transformation) analysis. In addition, a rather faint and oblique running striation was registered. Lamellae sectioned rather oblique or almost close to the surface show a honeycombed structure with a periodicity of 7.2–7.8 nm. Freeze-fracture transmission electron microscopy revealed that the lamellae are composed of highly ordered, crystalline arrays of particles. Here, FFT analysis resulted in lattice distances of 7.0–7.6 nm. Freeze-fracture transmission electron microscopy further revealed that the bodies remained intact after cell rupture followed by ascending flotation of the membrane fractions on discontinuous sucrose gradients. The birefringent bodies most likely are formed by evaginations of membranes, which separate the cytoplasm from the food vacuoles. Distinct, slightly reddish-colored areas, which resembled the birefringent bodies with respect to size and morphology, were registered by bright field light microscopy within Oxyrrhis marina cells. An absorbance maximum at 540 nm was registered for these areas, indicating that they are composed of rhodopsins. This was finally proven by immuno-transmission electron microscopy, as antisera directed against the C-terminal amino acid sequences of the rhodopsins AEA49880 and ADY17806 intensely immunolabeled the birefringent bodies of Oxyrrhis marina.

Lipid Polymorphism of the Subchloroplast-Granum and Stroma Thylakoid Membrane-Particles. II. Structure and Functions.

In Part I, by using 31P-NMR spectroscopy, we have shown that isolated granum and stroma thylakoid membranes (TMs), in addition to the bilayer, display two isotropic phases and an inverted hexagonal (HII) phase; saturation transfer experiments and selective effects of lipase and thermal treatments have shown that these phases arise from distinct, yet interconnectable structural entities. To obtain information on the functional roles and origin of the different lipid phases, here we performed spectroscopic measurements and inspected the ultrastructure of these TM fragments. Circular dichroism, 77 K fluorescence emission spectroscopy, and variable chlorophyll-a fluorescence measurements revealed only minor lipase- or thermally induced changes in the photosynthetic machinery. Electrochromic absorbance transients showed that the TM fragments were re-sealed, and the vesicles largely retained their impermeabilities after lipase treatments—in line with the low susceptibility of the bilayer against the same treatment, as reflected by our 31P-NMR spectroscopy. Signatures of HII-phase could not be discerned with small-angle X-ray scattering—but traces of HII structures, without long-range order, were found by freeze-fracture electron microscopy (FF-EM) and cryo-electron tomography (CET). EM and CET images also revealed the presence of small vesicles and fusion of membrane particles, which might account for one of the isotropic phases. Interaction of VDE (violaxanthin de-epoxidase, detected by Western blot technique in both membrane fragments) with TM lipids might account for the other isotropic phase. In general, non-bilayer lipids are proposed to play role in the self-assembly of the highly organized yet dynamic TM network in chloroplasts.

The Molecular Basis of Glucose Galactose Malabsorption in a Large Swedish Pedigree.

Glucose-galactose malabsorption (GGM) is due to mutations in the gene coding for the intestinal sodium glucose cotransporter SGLT1 (SLC5A1). Here we identify the rare variant Gln457Arg (Q457R) in a large pedigree of patients in the Västerbotten County in Northern Sweden with the clinical phenotype of GGM. The functional effect of the Q457R mutation was determined in protein expressed in Xenopus laevis oocytes using biophysical and biochemical methods. The mutant failed to transport the specific SGLT1 sugar analog α-methyl-D-glucopyranoside (αMDG). Q457R SGLT1 was synthesized in amounts comparable to the wild-type (WT) transporter. SGLT1 charge measurements and freeze-fracture electron microscopy demonstrated that the mutant protein was inserted into the plasma membrane. Electrophysiological experiments, both steady-state and presteady-state, demonstrated that the mutant bound sugar with an affinity lower than the WT transporter. Together with our previous studies on Q457C and Q457E mutants, we established that the positive charge on Q457R prevented the translocation of sugar from the outward-facing to inward-facing conformation. This is contrary to other GGM cases where missense mutations caused defects in trafficking SGLT1 to the plasma membrane. Thirteen GGM patients are now added to the pedigree traced back to the late 17th century. The frequency of the Q457R variant in Västerbotten County genomes, 0.0067, is higher than in the general Swedish population, 0.0015, and higher than the general European population, 0.000067. This explains the high number of GGM cases in this region of Sweden.

Polymer-mediated drug supersaturation – A spotlight on the interplay between phase-separated amorphous drug colloids and dissolved molecules

HypothesisColloidal aggregation phenomena have been found responsible for the supersaturation of poorly water-soluble drugs, potentially leading to bioavailability enhancements. Unlike coarse precipitates, phase separation in the form of colloids, is expected to enhance drug supersaturation performance. Therefore, a high proportion of these colloids should correlate with the extent and the kinetics of supersaturation. The prime objective of the current study is to provide a mechanistic understanding on supersaturation for the model drug albendazole (ALB) in combination with twelve polymers. ExperimentsSpecies separated after a pH-shift were characterized by dynamic light scattering (DLS), freeze-fracture electron microscopy (FF-EM) and transmission X-ray diffraction (XRD). Laser diffraction (LD) in a liquid cell was introduced for a relative quantification of the colloidally separated species, described as colloid fraction. The pH-dependent supersaturation was assessed online using a miniaturized dissolution assay. FindingsHere, a measure of the extent of amorphous colloidal phase separation was established, and its impact on supersaturation was evaluated. As a result, a correlation was found between the extent of supersaturation and the colloid fraction. This confirmed the dependence of polymer-mediated enabling and preservation of supersaturation on the ability of polymers to stabilize colloid fractions. Furthermore, a fixed ratio was suggested between the dissolved drug and colloidally separated drug as the kinetic profiles of both species showed similar trajectories. In conclusion, colloid fractions were identified to be responsible for dissolved and potentially bioavailable drug molecules.

Abstract P-42: Structure of Hydrogels of an Anionic Polysaccharide Studied by Freeze-Fracture Transmission Electron Microscopy

Background: Polysaccharide hydrogels draw attention due to the ability to form mechanically tough gels at low concentrations (typically 1 wt% or lower), combined with biocompatibility and biodegradability. Biopolymer hydrogels can be used as a matrix for cell growth, in order to obtain materials for the replacement of damaged tissues. “Physical” gels with macromolecules cross-linked by dynamic reversible cross-links are of great interest due to their self-healing ability. However, investigation of the native un-perturbed structure of such hydrogels presents a challenge, since they collapse upon drying, and present a difficulty for preparing a thin specimen for cryo-TEM experiments due to very high viscosity. The aim of this work is to study the native structure of hydrogels of an anionic polysaccharide – carboxymethyl hydroxypropyl guar (CMHPG) – cross-linked by borax. Methods: Freeze-fracture transmission electron microscopy (FF-TEM) was conducted on a Phillips EM-301 microscope. A small volume of the sample (100 μl) was put into the copper cell and cooled down by liquid nitrogen, put under vacuum (10−5 torr) at continuous cooling with liquid nitrogen, and fractured. The surface was etched for 10–20 min at 10−5 torr and then replicated by spraying platinum and carbon. Results: The gels have a microphase-separated microstructure – a rather thick (several nm) polymer backbone is seen, which is presumably formed by multiple aggregated macromolecules, and meshes between the backbone do not contain polymer and are filled with solvent. Mesh size determined from the micrographs qualitatively coincides with the value determined from the elastic modulus of the gels. Upon increasing the concentrations of cross-linker, the network becomes denser: the mesh size becomes lower, and the thickness of the backbone increases. Thus, the addition of cross-linker favors the aggregation of polymer chains forming the backbone. Conclusion: It was shown by FF-TEM that cross-linked CMHPG gels have a microphase-separated structure with a dense backbone formed by polymer chains and rather large meshes between them.

(Invited) Optical Control of the Membrane Potential of Living Cells with Donor-Acceptor-Linked Molecules

In living animal cells, there exists a difference in the electrical charge on the two extreme sides of the plasma membrane, that is, a slight excess of positive and negative ions outside and inside a cell, respectively. Such a charge difference produces a membrane potential (V m) across the plasma membrane. The V m (< 0) is controlled by various ion channels, and, especially, in neuronal cells, it is drastically changed for neurotransmission. In response to some input, the ion channels of neuronal cells get quickly activated such that the abovementioned difference is cancelled (∆V m > 0, depolarization). This electrical signal propagates along the neuronal axons like a wave through coordinated activation and deactivation of the ion channels. Therefore, the control of membrane potential and ion transport across the plasma membrane using a light source is an attractive strategy that can allow targeted, fast control of precisely defined events in the plasma membrane. In this symposium, we have demonstrated a novel general strategy for the control of membrane potential and ion transport by using donor-acceptor-linked molecules (D-A molecules) capable of intramolecular long-lived charge separation, nanocarriers for solubilization, membrane targeting of D-A molecules, and light (Figure 1).For the preparation of the D-A molecule, porphyrin (H2P) derivatives with ferrocene (Fc) and fullerene (C60) were synthesized to create a triadic arrangement (Fc-H2P-C60). We had previously reported that, under light irradiation, Fc-H2P-C60 yields the ferrocenium cation (Fc+)-H2P-C60 radical anion (C60 •-) with a long lifetime of ~0.01 ms [1]. For its solubilization in the physiological media and safer plasma membrane delivery, we focused on a lipoprotein in our body considering its amphiphilic property. High-density lipoprotein (HDL) can be reconstituted with recombinant lipid-binding proteins and phospholipids. We had previously reported that HDL genetically and chemically engineered with cytophilic peptides and fusogenic lipids is capable of high plasma membrane binding without significant cytotoxicity [2]. For this purpose, Fc-H2P-C60 was solubilized with one of our engineered HDL nanoparticles (cpHDL) and added to a cell culture medium of rat pheochromocytoma cells (PC12). The cell membrane delivery of Fc-H2P-C60 was suggested by confocal analysis and freeze-fracture electron microscopy. Subsequent light irradiation of PC12 cells increased the membrane potential (depolarization), which was not observed for the reference molecule (H2P) that was incapable of charge separation [3]. In addition, the inhibition of potassium ion flow across the membrane was found to be responsible for this depolarization (Figure 1). Our following study with 3 D-A molecules, including Fc-H2P-C60, revealed that the degree of the depolarization was positively correlated with the charge separation yield [4]. In this symposium, I will discuss more about the potential of applying our D-A molecules/carriers system in the biomedical fields.Figure 1. Schematic illustration of optical control of membrane potential and cation flow using a D-A molecule (Fc-H2P-C60)[1] Imahori, H. et al., J. Am. Chem. Soc. 2001, 123, 2607-2617.[2] Murakami, T., Biotechncol. J. 2012, 7, 762-767.; Kim, H. et al. Biochim. Biophys. Acta Biomembr. 2019, 1861, 183008.[3] Numata, T. et al., J. Am. Chem. Soc. 2012, 134, 6092-6095.[4] Takano, Y. et al., Chem. Sci. 2016, 7, 3331-3337. Figure 1

The distribution of phosphatidylinositol 4,5-bisphosphate in the budding yeast plasma membrane.

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is generated through phosphorylation of phosphatidylinositol 4-phosphate (PtdIns(4)P) by Mss4p, the only PtdIns phosphate 5-kinase in yeast cells. PtdIns(4,5)P2 is involved in various kinds of yeast functions. PtdIns(4)P is not only the immediate precursor of PtdIns(4,5)P2, but also an essential signaling molecule in the plasma membrane, Golgi, and endosomal system. To analyze the distribution of PtdIns(4,5)P2 and PtdIns(4)P in the yeast plasma membrane at a nanoscale level, we employed a freeze-fracture electron microscopy (EM) method that physically immobilizes lipid molecules in situ. It has been reported that the plasma membrane of budding yeast can be divided into three distinct areas: furrowed, hexagonal, and undifferentiated flat. Previously, using the freeze-fracture EM method, we determined that PtdIns(4)P is localized in the undifferentiated flat area, avoiding the furrowed and hexagonal areas of the plasma membrane. In the present study, we found that PtdIns(4,5)P2 was localized in the cytoplasmic leaflet of the plasma membrane, and concentrated in the furrowed area. There are three types of PtdIns 4-kinases which are encoded by stt4, pik1, and lsb6. The labeling density of PtdIns(4)P in the plasma membrane significantly decreased in both pik1ts and stt4ts mutants. However, the labeling densities of PtdIns(4,5)P2 in the plasma membrane of both the pik1ts and stt4ts mutants were comparable to that of the wild type yeast. These results suggest that PtdIns(4)P produced by either Pik1p or Stt4p is immediately phosphorylated by Mss4p and converted to PtdIns(4,5)P2 at the plasma membrane.

Experimental evidence of a transition from a sponge-like to a foam-like nanostructure in water-rich L3 phases

HypothesisThe micrometer-sized gas bubbles of a liquid foam with a dispersed gas phase of > 74 vol% are polyhedral and surrounded by a continuous aqueous phase. The structure of a water-rich microemulsion with a water phase of > 74 vol% normally consists of oil droplets in water or is bicontinuous. We hypothesize that at these high water contents polyhedral water droplets in oil can also exist. ExperimentsWe (a) carried out phase studies on the water-rich side of the phase diagram of the quaternary system water/NaCl – hexyl methacrylate – AOT, because AOT is known for its propensity to form water-in-oil structures and hexyl methacrylate can be polymerized, (b) measured the electrical conductivities and viscosities, and (c) visualized the nanostructure with freeze-fracture electron microscopy (FFEM). FindingsWe found narrow 1-phase regions emanating from the L3 phase of the oil-free water/NaCl – AOT system by adding small amounts of oil. In these regions the conductivities become extremely low and the viscosities are extremely high. In addition, FFEM images clearly show the foam-like nanostructure.

Transmembrane phospholipid translocation mediated by Atg9 is involved in autophagosome formation

The mechanism of isolation membrane formation in autophagy is receiving intensive study. We recently found that Atg9 translocates phospholipids across liposomal membranes and proposed that this functionality plays an essential role in the expansion of isolation membranes. The distribution of phosphatidylinositol 3-phosphate in both leaflets of yeast autophagosomal membranes supports this proposal, but if Atg9-mediated lipid transport is crucial, symmetrical distribution in autophagosomes should be found broadly for other phospholipids. To test this idea, we analyzed the distributions of phosphatidylcholine, phosphatidylserine, and phosphatidylinositol 4-phosphate by freeze-fracture electron microscopy. We found that all these phospholipids are distributed with comparable densities in the two leaflets of autophagosomes and autophagic bodies. Moreover, de novo-synthesized phosphatidylcholine is incorporated into autophagosomes preferentially and shows symmetrical distribution in autophagosomes within 30 min after synthesis, whereas this symmetrical distribution is compromised in yeast expressing an Atg9 mutant. These results indicate that transbilayer phospholipid movement that is mediated by Atg9 is involved in the biogenesis of autophagosomes.

From water-rich to oil-rich gelled non-toxic microemulsions.

Gelled non-toxic microemulsions have great potential in transdermal drug delivery: the microemulsion provides an optimum solubilizing capacity for drugs and promotes drug permeation through the skin barrier, while the gel network provides mechanical stability. We have formulated such a gelled non-toxic microemulsion consisting of H2O - isopropyl myristate (IPM) - Plantacare 1200 UP (technical-grade alkyl polyglucoside with an average composition of C12G1.4) - 1,2-octanediol in the presence of the low molecular weight gelator (LMWG) 1,3:2,4-dibenzylidene-d-sorbitol (DBS) at an oil-to-water ratio of φ = 0.50. The study at hand aimed to develop gelled non-toxic microemulsions that can contain both oil- and water-soluble drugs and are either water- or oil-based, depending on the application. To accomplish this, we varied the oil-to-water ratio from being water-rich to oil-rich, i.e. 0.2 ≤ φ ≤ 0.8. Phase studies were carried out along the middle phase trajectory, and a suitable LMWG was identified for all φ-ratios. Electrical conductivity measurements showed that the structure can be tuned from water- to oil-continuous by adjusting the amount of 1,2-octanediol and φ-ratios. The existence of the gel network was visualized by freeze-fracture electron microscopy (FFEM) at three different φ-ratios. We found that all systems from φ = 0.35 to φ = 0.80 form strong gels with nearly the same rheological behavior, while the system with φ = 0.20 is a much weaker gel. We attribute this behavior on the one hand to the microemulsion microstructure and on the other hand to the solvent-dependent gelation properties of DBS, which can be described by the Hansen solubility parameters (HSPs).

Applications of Chemically Modified Clay Minerals and Clays to Water Purification and Slow Release Formulations of Herbicides

This review deals with modification of montmorillonite and other clay-minerals and clays by interacting them with organic cations, for producing slow release formulations of herbicides, and efficient removal of pollutants from water by filtration. Elaboration is on incorporating initially the organic cations in micelles and liposomes, then producing complexes denoted micelle- or liposome-clay nano-particles. The material characteristics (XRD, Freeze-fracture electron microscopy, adsorption) of the micelle– or liposome–clay complexes are different from those of a complex of the same composition (organo-clay), which is formed by interaction of monomers of the surfactant with the clay-mineral, or clay. The resulting complexes have a large surface area per weight; they include large hydrophobic parts and (in many cases) have excess of a positive charge. The organo-clays formed by preadsorbing organic cations with long alkyl chains were also addressed for adsorption and slow release of herbicides. Another examined approach includes “adsorptive” clays modified by small quaternary cations, in which the adsorbed organic cation may open the clay layers, and consequently yield a high exposure of the siloxane surface for adsorption of organic compounds. Small scale and field experiments demonstrated that slow release formulations of herbicides prepared by the new complexes enabled reduced contamination of ground water due to leaching, and exhibited enhanced herbicidal activity. Pollutants removed efficiently from water by the new complexes include (i) hydrophobic and anionic organic molecules, such as herbicides, dissolved organic matter; pharmaceuticals, such as antibiotics and non-steroidal drugs; (ii) inorganic anions, e.g., perchlorate and (iii) microorganisms, such as bacteria, including cyanobacteria (and their toxins). Model calculations of adsorption and kinetics of filtration, and estimation of capacities accompany the survey of results and their discussion.

Formulation of Gelled Non-toxic Bicontinuous Microemulsions Stabilized by Highly Efficient Alkanoyl Methylglucamides

Gelled non-toxic bicontinuous microemulsions have a great potential for transdermal drug delivery as the microemulsion facilitates the solubilization of both hydrophilic and hydrophobic drugs, while the gel network provides mechanical stability and thus an easy application on the skin. In our previous study, we formulated a gelled non-toxic bicontinuous microemulsion: we gelled the system H2O-isopropyl myristate (IPM)-Plantacare 1200 UP (C12G1.4)-1,2-octanediol with the low molecular weight organogelator 1,3:2,4-dibenzylidene-d-sorbitol (DBS). However, a large amount of Plantacare 1200 UP (12 wt %) is needed to form a bicontinuous microemulsion. To solve this problem, we studied a new class of surfactants, namely, alkanoyl methylglucamides (MEGA), which have been rarely used for the formulation of microemulsions. The phase behavior of microemulsions stabilized by MEGA-8/10, MEGA-12/14-PC, and MEGA-12/14-HC was compared with that of systems stabilized by alkyl polyglucosides. We found that even with 2 wt % MEGA-12/14-HC, a bicontinuous microemulsion can be formed, which is 1/6 of the amount of Plantacare 1200 UP. The bicontinuous microstructure of the non-toxic microemulsion H2O-IPM-MEGA-12/14-HC-1,2-octanediol was confirmed by small-angle neutron scattering. Furthermore, the phase boundaries remained unchanged when gelled by DBS. The rheological properties of the gel were studied by oscillatory shear rheometry. Finally, freeze-fracture electron microscopy images show the coexistence of gel fibers and bicontinuous oil and water domains. These results suggest that the new gelled non-toxic bicontinuous microemulsion is an orthogonal self-assembled system.

Supramolecular assembly of ionic liquid induced by UO2 2+: a strategy for selective extraction-precipitation

Abstract In this work, a novel task specific ionic liquid (TSIL) [tributyl(hexyl)phosphonium]2[diglycolic acetate] ([P6,4,4,4]2[DGA]) was prepared and used to construct a vesicle system. The addition of UO2 2+, La3+ or Th4+ exhibited different effects on the system. It was found that small amount of UO2 2+ could induce large-sized aggregation of vesicles and make the precipitation happen, while La3+ and Th4+ did not have such capacity. The whole process was characterized by dynamic light scattering and freeze-fracture transmission electron microscopy. An extraction-precipitation strategy was then developed for the selective recovery of UO2 2+. Different factors were further studied to optimize the separation efficiency of the extraction-precipitation process.

Microfluidisation trends in the development of nanodelivery systems: characterisation and release study of resveratrol‐loaded nanostructured lipid carrier prepared with high‐pressure microfluidisation

The release behaviour and kinetic evaluation of resveratrol (Res) from nanostructured lipid carrier (NLC) have been conducted after characterisation for Res-NLC in this study. High-pressure microfluidisation technique was successfully applied to manufacturing Res-NLC, and the morphology of nanoparticle was observed via freeze-fracture transmission electron microscopy. Photon correlation spectroscopy was used to characterise the NLC system for particle size (PS) and polydispersity index (PDI). The in vitro release research was implemented by the dialysis method with active ingredient determined by high-performance liquid chromatography. Fitting of release data to different kinetic models was carried out. The optimised Res-NLC was physically stable with the PS below 200 nm, PDI below 0.3 and zeta potential (ZP) above 50 mV. The encapsulation efficiency was above 80%. Moreover, an in vitro release analysis demonstrated the sustained release profile of contents from NLC formulation over 12 h, with about 90% of the active ingredient released. Res-NLC presented the best fit with the Weibull model accompanied by better correlation value. In conclusion, all the obtained results demonstrated that the release study of Res-loaded NLC exhibited the promising application of the NLC formulation system in active ingredient sustained release carrier.