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In a recent study, we have used phage display screening to identify peptide motifs with strong and selective affinity to bind to the surfaces of gypsum, an inorganic mineral of high abundance in geo(bio)chemical environments and large‐scale industrial use for construction purposes. The goal of the present work was to investigate the influence of such engineered peptides on the formation of gypsum crystals under different conditions. To establish a holistic picture, the previously discovered multi‐stage mineralization pathway of gypsum was scrutinized in detail by a combination of complementary characterization techniques, including potentiometric titration and cryogenic transmission electron microscopy. The results provide clear evidence that the added peptide interferes with practically all precursor and intermediate species on the way from dissolved ions to solid crystals. Key to this multifunctional behavior is the self‐assembly of the peptides to filamentous colloidal aggregates in the presence of calcium ions, which depends on peptide molecular weight and related changes in secondary structure formation. Overall, the findings made in this work highlight the potential of biotechnological selection techniques for the design of advanced concepts for crystallization control, including supramolecular peptide self‐assembly as a principle known from natural biomineralization and of utmost value for bioinspired material science. [ABSTRACT FROM AUTHOR]

Most nonconventional luminogens enjoy good water solubility and biocompatibility, showing unique application prospects in fields like biological imaging. Although clustering‐triggered emission (CTE) mechanisms have been proposed to explain such emissions, the have not been thoroughly elucidated, which limits their development and application. Here, the photoluminescence properties of carboxymethyl β‐cyclodextrin (CM‐β‐CD) aqueous solution are utilized to further investigate the effects of changes in concentration, in order to elucidate the emission mechanism through cryo‐transmission electron microscopy (cryo‐TEM), small‐angle X‐ray scattering (SAXS), molecular interaction analysis, and theoretical calculation. The results showed that the size distribution, morphology, and distance between water aggregates were successfully correlated with the cluster emission centers. The emission mechanism of nonconventional luminogen solutions was more clearly and intuitively elucidated, which has a promoting effect on the emission and application of this field. It is interesting that temperature‐dependent emission spectra show the blue‐shift phenomenon of PL with increasing excitation wavelengths. Moreover, due to its strong static quenching effect for Fe3+, CM‐β‐CD can efficiently detect Fe3+ in mixed‐ion aqueous solutions. It provides a strategy to clarify the CTE mechanism of nonconventional luminogen solutions more clearly and its application of mixed‐ion detection. [ABSTRACT FROM AUTHOR]

Oil-core nanocapsules (NCs, also known as nanoemulsions) are of great interest due to their application as efficient carriers of various lipophilic bioactives, such as drugs. Here, we reported for the first time the preparation and characterization of NCs consisting of chondroitin sulfate (CS)-based shells and liquid oil cores. For this purpose, two amphiphilic CS derivatives (AmCSs) were obtained by grafting the polysaccharide chain with octadecyl or oleyl groups. AmCS-based NCs were prepared by an ultrasound-assisted emulsification of an oil phase consisting of a mixture of triglyceride oil and vitamin E in a dispersion of AmCSs. Dynamic light scattering and cryo-transmission electron microscopy showed that the as-prepared core–shell NCs have typical diameters in the range of 30–250 nm and spherical morphology. Since CS is a strong polyanion, these particles have a very low surface potential, which promotes their stabilization. The cytotoxicity of the CS derivatives and CS-based NCs and their impact on cell proliferation were analyzed using human keratinocytes (HaCaTs) and primary human skin fibroblasts (HSFs). In vitro studies showed that AmCSs dispersed in an aqueous medium, exhibiting mild cytotoxicity against HaCaTs, while for HSFs, the harmful effect was observed only for the CS derivative with octadecyl side groups. However, the nanocapsules coated with AmCSs, especially those filled with vitamin E, show high biocompatibility with human skin cells. Due to their stability under physiological conditions, the high encapsulation efficiency of their hydrophobic compounds, and biocompatibility, AmCS-based NCs are promising carriers for the topical delivery of lipophilic bioactive compounds. [ABSTRACT FROM AUTHOR]

Solid polymer electrolytes (SPEs) make contact with highly reductive lithium (Li) metal anodes, forming the interphase that largely determines the battery performance. In this work, trace iodine doping in a poly(ethylene oxide) (PEO) electrolyte to achieve a stable interphase on Li metal surface for long battery cycling, is proposed. The triiodide ion (I3−) stemming from iodine additives can coordinate with the COC bond of PEO to enable the increased ionic conductivity of the SPE. The I‐doped interphase contains I3− and IO3−, which spontaneously react with the dead Li and Li2O at the initial interphase to smooth the Li metal surface, eventually leading to significant improvements in interfacial resistance and dendrite suppression. When matching with the LiFePO4 cathode, the full cell exhibits higher capacity (150 mAh g−1) and excellent cycling stability after 300 cycles (capacity retention of 96.5%) at 0.5 C and 50 °C. This work opens up a promising avenue for using halogen to design solid‐state Li metal batteries. [ABSTRACT FROM AUTHOR]

Most nonconventional luminogens enjoy good water solubility and biocompatibility, showing unique application prospects in fields like biological imaging. Although clustering-triggered emission (CTE) mechanisms have been proposed to explain such emissions, the have not been thoroughly elucidated, which limits their development and application. Here, the photoluminescence properties of carboxymethyl β-cyclodextrin (CM-β-CD) aqueous solution are utilized to further investigate the effects of changes in concentration, in order to elucidate the emission mechanism through cryo-transmission electron microscopy (cryo-TEM), small-angle X-ray scattering (SAXS), molecular interaction analysis, and theoretical calculation. The results showed that the size distribution, morphology, and distance between water aggregates were successfully correlated with the cluster emission centers. The emission mechanism of nonconventional luminogen solutions was more clearly and intuitively elucidated, which has a promoting effect on the emission and application of this field. It is interesting that temperature-dependent emission spectra show the blue-shift phenomenon of PL with increasing excitation wavelengths. Moreover, due to its strong static quenching effect for Fe 3+ , CM-β-CD can efficiently detect Fe 3+ in mixed-ion aqueous solutions. It provides a strategy to clarify the CTE mechanism of nonconventional luminogen solutions more clearly and its application of mixed-ion detection., (© 2024 John Wiley & Sons Ltd.)

We developed a series of highly crystalline double‐cable conjugated polymers for application in single‐component organic solar cells (SCOSCs). These polymers contain conjugated backbones as electron donor and pendant perylene bisimide units (PBIs) as electron acceptor. PBIs are connected to the backbone via alkyl units varying from hexyl (C6H12) to eicosyl (C20H40) as flexible linkers. For double‐cable polymers with short linkers, the PBIs tend to stack in a head‐to‐head fashion, resulting in large d‐spacings (e.g. 64 Å for the polymer P12 with C12H24 linker) along the lamellar stacking direction. When the length of the linker groups is longer than a certain length, the PBIs instead adopt a more ordered packing likely via H‐aggregation, resulting in short d‐spacings (e.g. 50 Å for the polymer P16 with C16H32 linker). This work highlights the importance of linker length on the molecular packing of the acceptor units and the influences on the photovoltaic performance of SCOSCs. [ABSTRACT FROM AUTHOR]

Abstract Background Microbial communities recurrently establish metabolic associations resulting in increased fitness and ability to perform complex tasks, such as xenobiotic degradation. In a previous study, we have described a sulfonamide-degrading consortium consisting of a novel low-abundant actinobacterium, named strain GP, and Achromobacter denitrificans PR1. However, we found that strain GP was unable to grow independently and could not be further purified. Results Previous studies suggested that strain GP might represent a new putative species within the Leucobacter genus (16S rRNA gene similarity

Environmental context: The molecular organisation of humic substances is key to understanding various natural and engineered processes, such as metal speciation and bioavailability. Two main models have been proposed: polymeric organisation and supramolecular organisation. A physicochemical approach, based on the interaction of humic acid with increasing amounts of cationic surfactants, leads to a sequence of molecular structures that provides support to the supramolecular view of humic organisation. The structural organisation of extracted humic matter, polymeric or supramolecular, has been a long-standing controversial issue. In this paper, we show that the interaction between a reference humic material, the Suwannee River Humic Acid (SRHA), and a homologous series of cationic surfactants (n -alkyl trimethyl ammonium chloride) provides key insights to resolve the controversy. By combining measurements of turbidity, electrophoretic mobility, surface tension and cryogenic transmission electron microscopy, we show that: (i) the binding of cationic surfactant to the anionic humic acid is initially triggered by electrostatic interactions; (ii) the contrasting evolution of turbidity–surfactant concentration curves implies that the surfactant alkyl chain interacts with the hydrophobic moieties of humic matter from very low concentrations of surfactant; (iii) a drastic restructuring of humic matter occurs on surfactant binding, which brings out supplementary anionic humic sites; (iv) in the presence of C12 cationic surfactant, SRHA spontaneously forms stable nanoscale unilamellar vesicles, which, in addition to the high surface activity of complexes, could be of interest in the remediation of oil-contaminated environments; (v) the sequence of molecular structures obtained from SRHA with increasing amounts of C12-cationic surfactant – humic-rich vesicles, aggregates, surfactant-rich vesicles – bears striking similarities with the phase behaviour of aqueous mixtures of anionic and cationic surfactants, thus supporting the supramolecular view of humic structure. Environmental context. The molecular organisation of humic substances is key to understanding various natural and engineered processes, such as metal speciation and bioavailability. Two main models have been proposed: polymeric organisation and supramolecular organisation. A physicochemical approach, based on the interaction of humic acid with increasing amounts of cationic surfactants, leads to a sequence of molecular structures that provides support to the supramolecular view of humic organisation. [ABSTRACT FROM AUTHOR]

Exosome selectivity mechanisms underlying exosome–target cell interactions and the specific traits affecting their capability to communicate still remain unclear. Moreover, the capacity of exosomes to efficiently deliver their molecular cargos intracellularly needs precise investigation towards establishing functional exosome‐based delivery platforms exploitable in the clinical practice. The current study focuses on: (a) exosome production from normal MRC‐5 and Vero cells growing in culture, (b) physicochemical characterization by dynamic light scattering (DLS) and cryo‐transmission electron microscopy; (c) cellular uptake studies of rhodamine‐labeled exosomes in normal and cancer cells, providing to exosomes either "autologous" or "heterologous" cellular delivery environments; and (d) loading exogenous Alexa Fluor 488‐labeled siRNA into exosomes for the assessment of their delivering capacity by immunofluorescence in a panel of recipient cells. The data obtained thus far indicate that MRC‐5 and Vero exosomes, indeed exhibit an interesting delivering profile, as promising "bio‐shuttles," being pharmacologically exploitable in the context of theranostic applications. [ABSTRACT FROM AUTHOR]

• Development of a baculovirus-free system for VLP production in Sf9 cells. • Study of the best DNA:PEI complexing conditions mediating transfection. • A 2.8-fold increase in VLP production is achieved by means of DoE and desirability functions. • High-throughput nanoparticle quantification using NTA and flow virometry. • Reproducibility proven for the production of eGFP, hSEAP and HIV-1 GageGFP VLPs. Insect cells have shown a high versatility to produce multiple recombinant products. The ease of culture, low contamination risk with human pathogens and high expression capacity makes an attractive platform to generate virus-like particles (VLPs). The baculovirus expression vector system (BEVS) has been frequently used to produce these complex nanoparticles. However, the BEVS entails several difficulties in the downstream phase as well as undesirable side-effects due to the expression of baculovirus-derived proteins. In this work, we developed a baculovirus-free system based on polyethylenimine (PEI)-mediated transient gene expression (TGE) of Sf9 cells. An exhaustive study of DNA:PEI polyplex formation was performed and the optimal TGE conditions were determined by the combination of Design of Experiments (DoE) and desirability functions. The TGE approach was successfully applied to produce three model recombinant products with different structural complexities, including eGFP, hSEAP and HIV-1 Gag VLPs. Cell membrane co-localization with the Gag polyprotein was detected by fluorescence microscopy, whereas nanoparticle tracking analysis and flow virometry were applied as high-throughput techniques to monitor the VLP production process. Analysis of VLP production revealed that 48 h after transfection were optimal for VLP harvesting since the ratio of VLPs to extracellular vesicles was the highest. In these conditions, a maximum of 1.9 ± 0.8·109 VLP/mL was achieved, representing a 2.8-fold increase compared to the initial transfection condition. In conclusion, the TGE approach proposed in this study provides a baculovirus-free platform to rapidly produce VLPs and potentially other recombinant products in insect cells. [ABSTRACT FROM AUTHOR]

Cells release membrane vesicles in their surrounding medium either constitutively or in response to activating signals. Two main types of extracellular vesicles (EVs) are commonly distinguished based on their mechanism of formation, membrane composition and size. According to the current model, EVs shed from the plasma membrane, often called microvesicles, expose phosphatidylserine (PS) and range in size from 100 nm to 1 µm, while EVs originating from endosomal multi-vesicular bodies, called exosomes, contain tetraspanin proteins, including CD63, and range in size from 50 to 100 nm. Heijnen et al. [1] have shown that activated platelets release EVs corresponding to these two types of vesicles, using negative staining electron microscopy (EM) and immuno-gold labeling. Here, we apply cryo-EM and immuno-gold labeling to provide a quantitative analysis of EVs released by platelets activated by thrombin, TRAP and CRP-XL, as well as EVs from serum. We show that EVs activated by these three agonists present a similar size distribution, the majority of them forming a broad peak extending from 50 nm to 1 µm, about 50% of them ranging from 50 to 400 nm. We show also that 60% of the EVs from TRAP or CRP-XL activation expose CD41, a majority of them exposing also PS. To explain the presence of large EVs CD41-negative or PS-negative, several alternative mechanisms of EV formation are proposed. We find also that the majority of EVs in activated platelet samples expose CD63, and distinguish two populations of CD63-positive EVs, namely large EVs with low labeling density and small EVs with high labeling density.

To elucidate the effect of fat crystals and liquid fat on the bubble interface in whipped cream, we observed structural changes in fat globules and bubble interfaces caused by whipping using cryo-transmission electron microscopy. The results suggested that fat crystals formed lamellae at the fat globule interface, together with liquid fat at the center of the fat globules, which were dissociated from the fat globule under whipping. As whipping progressed, fat crystals fragmented and were adsorbed on the bubble interface, such that the area occupied by the fat crystals in the bubbles gradually increased. This behavior was presumed to contribute to the stability of whipped cream bubbles. Furthermore, liquid fat released from the fat globules was inferred to occupy gaps between the fat globules. [ABSTRACT FROM AUTHOR]

• Cryogenic sample preparation by cryo-focused ion beam milling is used to study metal/electrolyte interfaces in NiCrMo alloys undergoing corrosion. • This approach successfully preserves oxides and hydroxides in their near-native hydrated states for cryo-TEM characterization. • Diffraction and dark-field imaging in cryo-TEM show that polycrystalline hydroxide has orientational relationships with the bulk metal and oxides. Hydroxide formation at the surface of corroded alloys is critical for understanding early-stage oxidation of many corrosion-resistant alloys. Many hydroxides are unstable in an ambient environment and are electron-beam sensitive, limiting the use of conventionally-prepared specimens for transmission electron microscopy characterization of these alloy-water interfaces. In order to avoid sample dehydration, NiCrMo alloys corroded in a Cl−-containing electrolyte solution were cryo-immobilized by plunge freezing. A cryo-focused ion beam microscope was used to thin the sample to electron transparency, while preserving the alloy-water interface, and the sample was then cryo-transferred to a transmission electron microscope for imaging and diffraction. The presence of rocksalt Ni 1-x Cr 2x/3 O and β-Ni 1-x Cr 2x/3 (OH) 2 phases and their orientational relationship to the underlying alloy were observed with electron diffraction, confirming the preservation of the surface structure through the fully-cryogenic sample preparation and analysis. [ABSTRACT FROM AUTHOR]

Image denoising is a very important step in cryo-transmission electron microscopy (cryo-TEM) and the energy filtering TEM images before the 3D tomography reconstruction, as it addresses the problem of high noise in these images, that leads to a loss of the contained information. High noise levels contribute in particular to difficulties in the alignment required for 3D tomography reconstruction. This paper investigates the denoising of TEM images that are acquired with a very low exposure time, with the primary objectives of enhancing the quality of these low-exposure time TEM images and improving the alignment process. We propose denoising structures to combine multiple noisy copies of the TEM images. The structures are based on Bayesian estimation in the transform domains instead of the spatial domain to build a novel feature preserving image denoising structures; namely: wavelet domain, the contourlet transform domain and the contourlet transform with sharp frequency localization. Numerical image denoising experiments demonstrate the performance of the Bayesian approach in the contourlet transform domain in terms of improving the signal to noise ratio (SNR) and recovering fine details that may be hidden in the data. The SNR and the visual quality of the denoised images are considerably enhanced using these denoising structures that combine multiple noisy copies. The proposed methods also enable a reduction in the exposure time.

The 3-D morphology of doxorubicin (DOX)–loaded liposomes with a size of circa 100 nm was characterized by atomic force microscopy in an aqueous environment. Prolate liposomes appear in accordance with linear expansion of DOX fiber bundles precipitated inside liposomes. Oblate and concave liposomes were simultaneously observed with increased DOX concentrations; however, their morphologies were not readily determined by 2-D cryo-TEM imaging. Precise data analysis of the 3-D parameters of each liposome allowed semiquantitative evaluation of the transformation of spherical liposomes into nonspherical—prolate, oblate, and concave liposomes. In addition, nonspherical liposomes became spherical on the replacement of the liposomal outer phase consisting of a sucrose solution, with water and subsequent water influx. All spherical liposomes transformed into oblate and concave liposomes with a return to hyperosmotic conditions, when transferred from water to sucrose solution. Furthermore, the concave liposomes did not appear under DOX incubation conditions (65°C), which could be due to the amorphous and supersaturated DOX inside the liposomes that restrained liposomal shrinkage. As atomic force microscopy has improved our ability to image 3-D morphologies of liposomes in various conditions, it is an alternative analytical tool to cryo-TEM and may have future applications in regulatory tests for quality control and assurance. [ABSTRACT FROM AUTHOR]

Cells release membrane vesicles in their surrounding medium either constitutively or in response to activating signals. Two main types of extracellular vesicles (EVs) are commonly distinguished based on their mechanism of formation, membrane composition and size. According to the current model, EVs shed from the plasma membrane, often called microvesicles, expose phosphatidylserine (PS) and range in size from 100 nm to 1 µm, while EVs originating from endosomal multi-vesicular bodies, called exosomes, contain tetraspanin proteins, including CD63, and range in size from 50 to 100 nm. Heijnen et al. [1] have shown that activated platelets release EVs corresponding to these two types of vesicles, using negative staining electron microscopy (EM) and immuno-gold labeling. Here, we apply cryo-EM and immuno-gold labeling to provide a quantitative analysis of EVs released by platelets activated by thrombin, TRAP and CRP-XL, as well as EVs from serum. We show that EVs activated by these three agonists present a similar size distribution, the majority of them forming a broad peak extending from 50 nm to 1 µm, about 50% of them ranging from 50 to 400 nm. We show also that 60% of the EVs from TRAP or CRP-XL activation expose CD41, a majority of them exposing also PS. To explain the presence of large EVs CD41-negative or PS-negative, several alternative mechanisms of EV formation are proposed. We find also that the majority of EVs in activated platelet samples expose CD63, and distinguish two populations of CD63-positive EVs, namely large EVs with low labeling density and small EVs with high labeling density. [ABSTRACT FROM AUTHOR]

The organization of Suwannee River Fulvic Acid (SRFA) is investigated through the interaction of SRFA with a cationic surfactant molecule (Dodecyl-trimethylammonium chloride (DTAC)). Turbidity measurements, Dynamic light scattering, electrophoretic mobility, surface tension, and cryo-transmission electron microscopy are combined to describe the SRFA/DTAC molecular structures thus obtained. Increasing DTAC concentration, fulvic acid-rich unilamellar vesicles, globules, aggregates of globules, disks, DTAC-rich unilamellar vesicles, and loosely aggregated spheroidal structures, are successively observed. Such sequence of molecular structures is typically found in phase diagrams of catanionic systems (mixture of oppositely charged surfactants). The strong surface activity of fulvic acid/DTAC complexes and the geometrical constraints associated with the formation of vesicles imply that a major component of SRFA is an individual amphiphile negatively charged of molecular size similar to that of DTAC. This soft matter approach supports a supramolecular organization for SRFA. [ABSTRACT FROM AUTHOR]

The size and size distribution of casein nanoparticles plays an important role in the functionality and stability of milk. Dynamic light scattering is an effective method of measuring the size and size distribution of casein nanoparticles. But as biomacromolecules, casein nanoparticles are quite sensitive to their microenvironment changes. And therefore, the physicochemical properties of casein nanoparticles could be easily altered in different treatments, resulting in the incorrect size information determined by dynamic light scattering. The object of the present research was to analyze the effect of pre-treatment methods on the size and size distribution of casein nanoparticles determined by dynamic light scattering. The results suggested that when water and calcium imidazole buffer were used as the diluents, the counting rate of the samples was less than 7 ×10N3 counts/s, indicating there were no particles that could influence the size determination of casein nanoparticles. However, it was also found when water and calcium imidazole buffer were used as the dilution of casein nanoparticles, a significant decrease in the counting rate could be observed. This indicated both water and calcium imidazole buffer could easily alter the microenvironments of casein micelle dispersions, resulting in the partially dissociation of nanoparticles. And thus, the water and calcium imidazole buffer were not suitable for the dilution of casein nanoparticles. When simulated milk ultrafiltrate and milk ultrafiltrate were used as the diluents, it was found that there were no significant changes in the counting rate of the casein micelle dispersions. This indicated the microenvironment of both simulated milk ultrafiltrate and milk ultrafiltrate (10 kDa) was quite similar to that of milk, and thus the casein nanoparticles could not dissociate in both simulated milk ultrafiltrate and milk ultrafiltrate. However, extra particles occurred in both simulated milk ultrafiltrate and milk ultrafiltrate 24 h after their preparation, and therefore they were not suitable for the size measurement of casein nanoparticles under such the situation. Furthermore, it could be observed that the temperature of the diluents could significantly affect the measurement accuracy: when the samples (25°C) were diluted with 4°C diluents, the counting rate could reduce by 16%; when the samples (4°C) were diluted with 25°C diluents, the counting rate could increase by 10%. Based on the conclusion obtained above, it was suggested that the best pre-treatment methods for the size and size distribution of casein nanoparticles by dynamic light scattering were: the temperatures of the samples diluted with simulated milk ultrafiltrate or milk ultrafiltrate (10 kDa) should be similar with the milk samples. Results indicated the size distribution of casein nanoparticles obtained by dynamic light scattering was quite similar with that observed by the cryo-transmission electron microscopy, indicating the pre-treatment methods used could not affect the structural properties of casein nanoparticles in their true microenvironments, and thus permitted us to obtain reliable information of size and size distribution. [ABSTRACT FROM AUTHOR]

Image denoising is a very important step in cryo-transmission electron microscopy (cryo-TEM) and the energy filtering TEM images before the 3D tomography reconstruction, as it addresses the problem of high noise in these images, that leads to a loss of the contained information. High noise levels contribute in particular to difficulties in the alignment required for 3D tomography reconstruction. This paper investigates the denoising of TEM images that are acquired with a very low exposure time, with the primary objectives of enhancing the quality of these low-exposure time TEM images and improving the alignment process. We propose denoising structures to combine multiple noisy copies of the TEM images. The structures are based on Bayesian estimation in the transform domains instead of the spatial domain to build a novel feature preserving image denoising structures; namely: wavelet domain, the contourlet transform domain and the contourlet transform with sharp frequency localization. Numerical image denoising experiments demonstrate the performance of the Bayesian approach in the contourlet transform domain in terms of improving the signal to noise ratio (SNR) and recovering fine details that may be hidden in the data. The SNR and the visual quality of the denoised images are considerably enhanced using these denoising structures that combine multiple noisy copies. The proposed methods also enable a reduction in the exposure time. [ABSTRACT FROM AUTHOR]

Halogenated dodecaborates, and especially dodecaiodododecaborate(2−), are found to trigger effectively the release of the contents of phospholipid liposomes, including liposomes containing distearoylphosphatidylcholine and cholesterol, which are used clinically in cancer therapy. The basis of the release is studied through differential scanning calorimetry, cryo-transmission electron microscopy, and atomic force microscopy. Upon administration at high concentrations, drastic morphological changes are induced by the dodecaborates. Their possible use in triggered release is suggested. [ABSTRACT FROM AUTHOR]

Unilamellar vesicles frequently serve as model membrane systems to study membrane-peptide and membrane-protein interactions. They are formed either from natural lipid extracts that contain complex lipid mixtures, or from synthetic one-, two-, or multi-component lipid mixtures mimicking the lipid composition of biological membranes. Unilamellar vesicles can be prepared by extrusion of aqueous multilamellar phospholipid suspensions and they can have limited stability due to aggregation and fusion processes. We used cryo-transmission electron microscopy (cryoTEM) to study the morphology of vesicles of phosphatidylethanolamines (PEs), phosphatidylglycerols (PGs), and cardiolipins (CLs) that represent the major lipid components of cytoplasmic membranes of Gram-negative bacteria. Our results show that single-component vesicles of PE and PG stored at RT are stable in shape and size for at least 1 wk. In contrast, two-component vesicles of PE/PG and PE/CL transform into lamellar sheets already several hours after preparation, which is probably due to a compensation of different spontaneous curvatures of PG lipids compared to PE and CL. With this investigation we intend to alert researchers working with model membranes to scrutinize the shape and stability of vesicles before using them in protein-vesicle interaction studies. Practical applications: Liposomes represent widely-used model membrane systems that are applied to mimic natural membranes. Usually, these liposomes are composed of one or two lipid components representing the major lipid constituents of the respective natural membrane. Their long-term shape, aggregation, and fusion stability is an important prerequisite for the study of peptide-liposome and protein-liposome interactions, where the binding of peptides or proteins can have serious impacts on the liposomal shape and size. Furthermore, shape and size stability is of outstanding importance for the application of liposomes as drug-delivery system. We showed that single-component liposomes composed of the major lipid components of cytoplasmic membranes of Gram-negative bacteria (PE and PG) are stable in shape and size for at least 1 wk, whereas the corresponding two-component liposomes become unstable within hours and transform into discs and sheets. This knowledge will be indispensable for future studies on interactions of liposomes with peptides and proteins. Cryo-transmission electron microscopy is the method of choice to study the size, morphology, and shape stability of liposomes being composed of the phospholipids PE, PG, and CL that represent the major lipid components of cytoplasmic membranes of Gram-negative bacteria. [ABSTRACT FROM AUTHOR]

Cryo-transmission electron microscopy (Cryo-TEM) and particularly single particle analysis is rapidly becoming the premier method for determining the three-dimensional structure of protein complexes, and viruses. In the last several years there have been dramatic technological improvements in Cryo-TEM, such as advancements in automation and use of improved detectors, as well as improved image processing techniques. While Cryo-TEM was once thought of as a low resolution structural technique, the method is currently capable of generating nearly atomic resolution structures on a routine basis. Moreover, the combination of Cryo-TEM and other methods such as X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular dynamics modeling are allowing researchers to address scientific questions previously thought intractable. Future technological developments are widely believed to further enhance the method and it is not inconceivable that Cryo-TEM could become as routine as X-ray crystallography for protein structure determination. [ABSTRACT FROM AUTHOR]

A series of novel functionalised dumbbell-shaped bifullerenes in which two [5.0] pentakis-adducts of C60 are covalently connected by cyclic bismalonates were synthesised. These dimeric compounds, carrying various combinations of hydrophilic and hydrophobic addends, self-assemble in aqueous solution towards supramolecular architectures of different structural complexity as observed by cryogenic transmission electron microscopy (cryo-TEM). The detailed analysis of the image data revealed an unprecedented hierarchical aggregation behaviour. Whereas completely hydrophilic substituted bifullerenes formed profoundly monodisperse populations of small oligomeric elementary micelles consisting of only three or four bifullerene molecules in a supposedly bent conformation, their amphiphilic equivalents underwent a hierarchical two-step assembly process towards larger spherical and even rod-like structures. The data suggest that the hierarchical assembly process is driven by hydrophobic interactions of preformed tetrameric elementary micelles. [ABSTRACT FROM AUTHOR]

Low cycling Coulombic efficiency (CE) and messy Li dendrite growth problems have greatly hindered the development of anode-free Li-metal batteries (AFLBs). Thus, functional electrolytes for uniform lithium deposition and lithium/electrolyte side reaction suppression are desired. Here, we report a locally fluorinated electrolyte (LFE) medium layer surrounding Cu foils to tailor the chemical compositions of the solid-electrolyte interphase (SEI) in AFLBs for inhibiting the immoderate Li dendrite growth and to suppress the interfacial reaction. This LFE consists of highly concentrated LiTFSI dissolved in a fluoroethylene carbonate and/or succinonitrile plastic mixture. The CE of Cu||LiNi 0.8 Co 0.1 Mn 0.1 O 2 (NCM811) AFLB increased to a high level of 99% as envisaged, and the cycling ability was also highly improved. These improvements are facilitated by the formation of a uniform, dense, and LiF-rich SEI. LiF possesses high interfacial energy at the LiF/Li interface, resulting in a more uniform Li deposition process as proved by density functional theory (DFT) calculation results. This work provides a simple yet utility tech for the enhancement of future high-energy-density AFLBs.

Abstract: The present study describes the production and characterization of monoolein aqueous dispersions (MAD) as drug delivery systems for curcumin (CR). MAD based on monoolein and different emulsifiers have been produced and characterized. Morphology and dimensional distribution have been investigated by Cryogenic Transmission Electron Microscopy (cryo-TEM), X-ray and Photon Correlation Spectroscopy (PCS). Monoolein in different mixtures with sodium cholate, sodium caseinate, bentonite and poloxamer resulted in heterogeneous dispersions constituted of unilamellar vesicles, cubosomes and sponge type phases, depending on the employed components, as found by cryo-TEM and X-ray studies. CR was encapsulated with entrapment efficiencies depending on the MAD composition, particularly the highest was reached in the case of monoolein/poloxamer/sodium cholate mixture. The same mixture was able to maintain CR stability also after 6months. CR release modalities were in vitro investigated in order to mimic a possible subcutaneous administration of MAD. It was found that MAD constituted of monoolein/poloxamer and monoolein/poloxamer/sodium cholate mixtures were able to sustain CR release. MAD viscous vehicles were produced by xanthan gum. CR percutaneous absorption has been studied in vitro using excised human skin membranes [stratum corneum epidermis (SCE)] mounted into Franz cells. It was found that fluxes (Fn) of CR incorporated in MAD are influenced by the presence of monoolein based nanosystems. In particular xanthan gum based MAD better control CR diffusion from MAD. [Copyright &y& Elsevier]

Abstract: A dedicated analytical scanning transmission electron microscope (STEM) with dual energy dispersive spectroscopy (EDS) detectors has been designed for complementary high performance imaging as well as high sensitivity elemental analysis and mapping of biological structures. The performance of this new design, based on a Hitachi HD-2300A model, was evaluated using a variety of biological specimens. With three imaging detectors, both the surface and internal structure of cells can be examined simultaneously. The whole-cell elemental mapping, especially of heavier metal species that have low cross-section for electron energy loss spectroscopy (EELS), can be faithfully obtained. Optimization of STEM imaging conditions is applied to thick sections as well as thin sections of biological cells under low-dose conditions at room and cryogenic temperatures. Such multimodal capabilities applied to soft/biological structures usher a new era for analytical studies in biological systems. [Copyright &y& Elsevier]

Sulfide electrolytes promise superior ion conduction in all-solid-state lithium (Li) metal batteries, while suffering harsh hurdles including interior dendrite growth and instability against Li and moist air. A prerequisite for solving such issues is to uncover the nature of the Li/sulfide interface. Herein, air-stable Li 4 SnS 4 (LSS) as a prototypical sulfide electrolyte is selected to visualize the dynamic evolution and failure of the Li/sulfide interface by cryo-electron microscopy. The interfacial parasitic reaction (2Li + 2Li 4 SnS 4 = 5Li 2 S + Sn 2 S 3 ) is validated by direct detection of randomly distributed Li 2 S and Sn 2 S 3 crystals. A bifunctional buffering layer is consequently introduced by self-diffusion of halide into LSS. Both the interface and the grain boundaries in LSS have been stabilized, eliminating the growing path of Li dendrites. The buffering layer enables the durability of Li symmetric cell (1500 h) and high-capacity retention of the LiFePO 4 full-cell (95%). This work provides new insights into the hierarchical design of sulfide electrolytes.

Abstract: The self-assembly behaviour of block copolymers in solution has been of significant interest over the past two decades for a number of applications — for example, as delivery vectors and micro-reactors. More recently, attention has turned to the formation of aggregates with complex internal structure, such as multi-compartment micelles and the so-called “Janus” particles (biphasic aggregates) for their promising application as vectors for the simultaneous inclusion of chemically-different encapsulates and their possible catalytic and templating properties. The challenge has been to observe these complex aggregates in such a way as to be able to characterise their internal morphology whilst preserving their intricate structure. To this end, cryogenic transmission electron microscopy (cryoTEM) has become a powerful and indeed a necessary tool for the elucidation and observation of self-assembled polymer systems. Through its use, a new class of complex micelles has been discovered: amphiphilic block copolymer nanospheres with internal bicontinuous structure. These structures have been observed from a variety of block copolymer amphiphiles, although rarely. Intriguingly, there is no seemingly obvious unifying blueprint for their formation. This review will present the importance of cryoTEM in the elucidation and characterisation of internally-structured polymeric aggregates in recent years and highlight its significance in the definition of bicontinuous dispersions. [Copyright &y& Elsevier]

Abstract: The presented work evaluates the use of bicellar systems as new delivery vectors for controlled release of compounds through the skin. Two different active principles were introduced into the bicellar systems: diclofenac diethylamine (DDEA) and flufenamic acid (Ffa). Bicellar systems are discoidal aggregates formed by long and short alkyl chain phospholipids. Characterization of the bicellar systems by dynamic light scattering (DLS) and cryogenic transmission electron microscopy (Cryo-TEM) showed that particle size decreased when DDEA was encapsulated and increased when Ffa was included in the bicellar systems. Percutaneous absorption studies demonstrated a lower penetration of DDEA and Ffa through the skin when the drugs were included in the bicellar systems than when the drugs were applied in an aqueous solution (DDEA) and in an ethanolic solution (Ffa); the reduction in penetration was more pronounced with Ffa. These bicellar systems may have retardant effects on percutaneous absorption, which result in a promising strategy for future drug or cosmetic delivery applications. [Copyright &y& Elsevier]

Abstract: This paper reports on the properties of bilayers composed of dioctadecyldimethylammonium bromide (DODAB) and oleic acid (OA) at various molar ratios. The mole fraction of OA, X OA, was varied in the range of 0–1 and the total lipid content was constant and equal to 10mM. The DODAB/OA dispersions were extruded at a temperature higher than that of the gel–liquid transition of DODAB. The morphology of bilayer structures formed in the dispersions was inspected using a cryogenic transmission electron microscopy (cryo-TEM) and a differential interference contrast microscopy (DIC). The observations revealed that the incorporation of OA into DODAB bilayer results in a decrease of the membrane curvature. Anisotropy measurements using 1,6-diphenylhexatriene (DPH) as a rotator probe demonstrated that the DODAB/OA membrane microviscosity decreased considerably for X OA >0.4. The thermal behavior of DODAB/OA membranes has been studied by differential scanning calorimetry (DSC). In the case of the systems in which X OA <0.8, the DODAB/OA membranes are in the gel phase at room temperature. Additionally, Langmuir monolayer experiments of the DODAB/OA mixtures showed that due to the electrostatic interactions between the oppositely charged head groups of DODAB and OA they get close to each other, which results in a decrease of the mean area per molecule. The results were next discussed based on the packing parameter concept. The reduction of the mean area per head group (a) in the DODAB/OA systems leads to subsequent increase in the so-called packing parameter (S), which governs the morphology of surfactant aggregates. [Copyright &y& Elsevier]

Abstract: Experimental evidence for the existence of ‘cubatic’ order in a colloidal dispersion of plate-like particles is presented. In a ‘cubatic’ phase, disk-like particles self-assemble with domains of a few parallel plates and the director tends to be orthogonal in adjacent domains. This phase has been predicted previously by computer simulation. The domains are approximately equiaxial and are predicted to exist only within a limited range of aspect ratios and volume fractions. This locally ordered structure cannot be identified readily using scattering techniques, since the patterns are expected to be similar to those of isotropic liquid phases. For this reason, we have used a real-space technique of cryo-transmission electron microscopy that directly probes such locally ordered structures to study dispersions of nickel hydroxide particles. Polydispersity of particle size is expected to require some local tilting in order to include larger particles in a dense structure and this is discussed with respect to the concentration range for which cubatic order is observed. This new structure offers the possibility of novel materials that could be prepared by self-assembly and have applications in a wide range of fields. [Copyright &y& Elsevier]

Abstract: Cryo-transmission electron microscopy was used in combination with turbidity and leakage measurements to explore and compare the membrane perturbing effects of melittin and alamethicin on POPC-based liposomes of varying composition. The results show that the two peptides, despite their differences in physico-chemical properties and proposed mode of action, induce similar structural effects on the liposomes. Importantly, whereas low peptide concentrations leave pure POPC liposomes intact and seemingly unperturbed, POPC liposomes supplemented with 40mol.% cholesterol change their shape, rupture and fuse in response to the addition of both melittin and alamethicin. In the case of alamethicin, but not melittin, fusion is effectively prevented by inclusion of 10mol.% POPG in the liposome membranes. By means of a competitive binding assay we furthermore show that alamethicin, in line with earlier findings for melittin, possess high affinity for positively curved lipid surfaces. Moreover, results from the present study show that magainin 2 has a similar preference for curved surfaces. [Copyright &y& Elsevier]

Abstract: Milk was processed with high hydrostatic pressure in order to modify the casein micelles. Images, that in details showed the casein micelle structure in untreated and pressure-treated skim milk, were obtained by using cryo-transmission electron microscopy (cryo-TEM). Sizes and shapes adopted by casein micelles in pressurised milk are concluded to be a result of an equilibrium distribution between self-assembling casein molecules in the serum phase and caseins adsorbed to surfaces of casein micelles and are governed by an initial pressure-dependent displacement of caseins into the serum phase. Pressurisation of milk at moderately high pressure, in the range 150–300MPa, favoured formation of a large number of small micelles that coexisted with a fraction of large micelles, and which appeared perfectly spherical with smooth and well-defined surfaces, features which are suggested to originate from secondary adsorption of caseins. Pressurisation of milk at 400MPa favoured formation of smaller casein assemblies, with sizes between 30nm and 100nm. Measurements of free calcium concentration [Ca2+] showed that calcium was rebound to casein micelles after pressurisation of milk. Furthermore, the electron microscopy images indicated that the substructures were similar for pressure-modified casein micelles and casein micelles in untreated milk. [Copyright &y& Elsevier]

Abstract: Detergent removal from mixed micelles was combined with preparative size exclusion chromatography (SEC) on Sephacryl S 500 HR to prepare unilamellar and spherical liposomes of defined sizes between 50 and 100nm with a very narrow size distribution (RSD of vesicle diameter between 13% and 25%). For neutral phosphatidylcholine and negatively charged phosphatidylcholine/phosphatidylglycerol liposome preparations, efficient sizing at the preparative scale was demonstrated by analyzing isolated SEC peak fractions with cryo-transmission electron microscopy and dynamic light scattering. The number-weighted average vesicle diameters obtained using both methods are in very good agreement for fractions of low polydispersity. [Copyright &y& Elsevier]

Abstract: Various β-cyclodextrin (β-CD) fatty esters with different chain lengths (C4–C14) were synthesised by transesterification of β-cyclodextrin by vinyl fatty ester using thermolysin in DMSO. For each cyclodextrin derivatives, two batches of synthesis were realized. The ability of these derivatives to form nano-organized systems was investigated through the solvent displacement technique. During the formulation step, the effects of the initial concentration of β-CD fatty esters in the organic phase and that of the final volume of the aqueous non-solvent phase were studied. Except for the β-CD C4 ester, the transesterified β-CD derivatives led to measurable nanoparticles. Cryo-electron microscopy images showed a significant morphological variability. Spherical, rod-like or more irregularly-shaped nano-objects were observed with either matricial or lamellar structures. A statistical analysis by a two-way ANOVA was computed for each class of β-cyclodextrin esters in order to determine the effects of batch and formulation on the final size of nanoparticles. [Copyright &y& Elsevier]

Synopsis The present investigation describes a comparative study for the design of innovative topical formulation for skin hydration. In particular, different colloidal forms based on lipidic components have been produced and characterized. Morphology and dimensional distribution have been investigated by means of electron microscopy and photon correlation spectroscopy. Nanoparticulate systems characterized by different morphology and dimensions depending on production procedures have been obtained, namely cubosomes, nanovesicles, solid lipid nanoparticles and liposomes. Hydration power has been studied by means of a corneometer, measuring the skin electrical capacitance before and after the application of opportunely viscosized nanoparticulate systems. It has been demonstrated that nanovesicle gel displayed a pronounced hydration power with respect to the other nanostructured forms, its hydration effect on skin was 3.5-fold higher, with respect to the untreated area, after 5 min from the application and 1.5-fold higher after 2 h. [ABSTRACT FROM AUTHOR]

Abstract: During the preparation of plant extracts by a press-slit technique, membranes of cell walls and cell organelles of the plant material form vesicles, which are colloidally dispersed. It was assumed that chlorophyll-containing green extracts enclose lipoidic structures. Vesicles in aqueous mistletoe extracts (extracts of Viscum album L.) were analyzed by cryo-transmission electron microscopy (cryo-TEM) without fixation. For the first time, it was possible to analyze unfixed vesicles in the mistletoe extract. Micrographs of cryo-TEM showed predominantly unilamellar vesicles of different sizes. The quantification of vesicles was established through the analysis of phospholipids, which are major components of membranes. The method was validated mainly according to ICH guidelines for the validation of analytical methods (Q2A and Q2B). For further characterization of the vesicle size, a method was developed which is based on the separation of the vesicles from low molecular weight substances by size exclusion chromatography. Fractions were collected and average sizes were determined by multi-angle laser light scattering (MALLS). Furthermore, the UV–vis absorbance and phospholipid concentration were analyzed. Phospholipid quantification was in agreement with photometrical data. Sizes determined by cryo-TEM and by light scattering showed consistent results. [Copyright &y& Elsevier]

Abstract: Idarubicin has been successfully encapsulated in cholesterol-free liposomes, however, little is known about how the rate of drug release from circulating liposomes influences therapeutic activity. The studies described herein assess the attributes of a liposome formulation required to significantly increase the plasma levels of idarubicin and further establish whether increases in the circulation longevity of the drug mediate improved antitumor activity. Pharmacokinetic assessments of 6 different 3[H]-labelled liposome formulations were compared to free idarubicin. The highest idarubicin plasma concentrations were observed with DSPC/DSPE-PEG2000 liposomes formulated with 2 mol% DSPE-PEG2000 and 150 mM (iso-osmotic) internal citrate concentration. It was shown that increased levels of PEG-lipid incorporation augmented IDA release and the optimal liposomal formulation needed to be prepared under iso-osmotic conditions. For efficacy studies in a murine leukemia model, groups of 12–14 mice were treated i.v. with saline or equivalent doses (1, 2, 3 mg/kg) of free or liposomal IDA. Liposomal treatment groups exhibited a higher % increase in life span (ILS) as compared to equivalent doses of free drug. Efficacy studies completed in two drug resistant models, P388/ADR and MDA435LCC6/MDR1, demonstrated that neither the free nor liposomal formulation of idarubicin was therapeutically active. Encapsulation of IDA in liposomes increased antitumor activity in an IDA sensitive model, however, the significant increase in plasma drug levels was not sufficient to overcome multidrug resistance. [Copyright &y& Elsevier]

Topotecan can be encapsulated in liposomes, however little is known about the role encapsulated counter ions play in drug loading efficiency and drug release. Using 1,2-distearoyl-sn-glycero-3 phosphatidylcholine and cholesterol liposomes (55:45 mole ratio), encapsulation was achieved using manganese ion gradients (MnSO4 or MnCl2), with the addition of A23187, a divalent cation/proton exchanger, to maintain a pH gradient. This methodology was compared to procedures where the pH gradient was generated by use of encapsulated (NH4)2SO4 or citrate (300 mM, pH 3.5). All methods facilitated topotecan encapsulation. Liposomes prepared in the presence of the citrate and MnCl2 (+A23187) exhibited reduced loading capacities. Liposomes prepared in the presence of (NH4)2SO4 and MnSO4 (+A23187) could be used to generate liposomes exhibiting a drug-to-lipid ratio of 0.3 (wt/wt) with an encapsulation efficiency of >90%. In vitro drug release data suggested that the (NH4)2SO4 and MnSO4 (+A23187) formulations released drug at a reduced rate. For these formulations, the drug release rates decreased as the drug-to-lipid ratio (wt/wt) increased from 0.1 to 0.2. Cryo-electron micrographs indicated that encapsulated topotecan precipitated as linear particles within liposomes. The stability of topotecan loaded liposomes appeared to be dependent on the presence of both a pH gradient and encapsulated sulfate. [Copyright &y& Elsevier]

Summary Cryo-transmission electron microscopy (cryo-TEM) is a rather artefact-free method, well suited to study the alveolar surfactant system. A pharmacy grade porcine lung surfactant extract (HL-10) was mixed with human SP-A and Ringer's solution (for calcium ions), and it was shown by cryo-TEM that the tubular myelin (TM) type of structure was reconstituted. These aggregates were associated to liposomal aggregates, and resulted in macroscopic phase-separation. This phase showed a weak birefringence in the polarising microscope, which is characteristic for a liquid-crystalline type of structure. TM from rabbit lung lavage was also examined, and showed the same periodic arrangement of bilayers as alveolar surface layer from freshly cut rabbit lungs deposited directly on the cryo-TEM grids. The distance between the bilayers of TM was 40–50 nm, and an electron dense material, assumed to be SP-A, was sometimes seen to occur periodically along the bilayers, oriented perpendicularly to the tubuli. The results are consistent with the surface-phase model of the alveolar lining. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the influence of pH changes on morphometric parameters of casein micelles and a general overview of their conformational structure through microscopy techniques, Raman spectroscopy and multivariate analysis. It was found that casein micelles morphology and protein secondary structure depend strongly upon pH. The changes of arithmetic average roughness (Ra), size, and shape of casein micelles at different pH are properly characterized by atomic force and cryo-transmission electron microscopy. Morphometric changes of casein micelles were correlated correctly with folding and unfolding of casein molecules as evaluated by Raman spectroscopy when the pH was varied. The novelty of this contribution consists in demonstrating that there is a close structure-functionality relationship between the morphometric parameters of proteins and their secondary structure. Knowledge about casein micelles can help improve their use of its diverse applications.