Chemically-Induced Lipoprotein Breakdown for Improved Extracellular Vesicle Purification.

Extracellular vesicles (EVs) are nanosized biomolecular packages involved in intercellular communication. EVs are released by all cells, making them broadly applicable as therapeutic, diagnostic, and mechanistic components in (patho)physiology. Sample purity is critical for correctly attributing observed effects to EVs and for maximizing therapeutic and diagnostic performance. Lipoprotein contaminants represent a major challenge for sample purity. Lipoproteins are approximately six orders of magnitude more abundant in the blood circulation and overlap in size, shape, and density with EVs. This study represents the first example of an EV purification method based on the chemically-induced breakdown of lipoproteins. Specifically, a styrene-maleic acid (SMA) copolymer is used to selectively breakdown lipoproteins, enabling subsequent size-based separation of the breakdown products from plasma EVs. The use of the polymer followed by tangential flow filtration or size-exclusion chromatography results in improved EV yield, preservation of EV morphology, increased EV markers, and reduced contaminant markers. SMA-based EV purification enables improved fluorescent labeling, reduces interactions with macrophages, and enhances accuracy, sensitivity, and specificity to detect EV biomarkers, indicating benefits for various downstream applications. In conclusion, SMA is a simple and effective method to improve the purity and yield of plasma-derived EVs, which favorably impacts downstream applications.

Selective labeling of phosphatidylserine for cryo-TEM by a two-step immunogold method.

Immunogold labeling in transmission electron microscopy (TEM) utilizes the high electron density of gold nanoparticles conjugated to proteins to identify specific antigens in biological samples. In this work we applied the concept of immunogold labeling for the labeling of negatively charged phospholipids, namely phosphatidylserine, by a simple protocol, performed entirely in the liquid-phase, from which cryo-TEM specimens can be directly prepared. Labeling included a two-step process using biotinylated annexin-V and gold-conjugated streptavidin. We initially applied it on liposomal systems, demonstrating its specificity and selectivity, differentiating between 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) membranes. We also observed specific labeling on extracellular vesicle samples isolated from THP1 cells and from MDA-468 cells, which underwent stimulations. Finally, we compared the levels of annexin-V labeling on the cells vs. on their isolated EVs by flow cytometry and found a good correlation with the cryo-TEM results. This simple, yet effective labeling technique makes it possible to differentiate between negatively charged and non-negatively charged membranes, thus shillucidating their possible EV shedding mechanism.

Extracellular Vesicle MicroRNA That Are Involved in ß-Thalassemia Complications.

Beta thalassemia major (ßT) is a hereditary anemia characterized by transfusion-dependency, lifelong requirement of chelation, and organ dysfunction. MicroRNA (miRNA) can be packed into extracellular vesicles (EVs) that carry them to target cells. We explored EV-miRNA in ßT and their pathophysiologic role. Circulating EVs were isolated from 35 ßT-patients and 15 controls. EV miRNA was evaluated by nano-string technology and real-time quantitative polymerase chain reaction (RT-qPCR). We explored effects of EVs on cell culture proliferation, apoptosis, and signal transduction. Higher amounts of small EV (exosomes) were found in patients than in controls. The expression of 21 miRNA was > two-fold higher, and of 17 miRNA < three-fold lower in ßT-EVs than control-EVs. RT-qPCR confirmed differential expression of six miRNAs in ßT, particularly miR-144-3p, a regulator of erythropoiesis. Exposure of endothelial, liver Huh7, and pancreatic 1.1B4 cells to ßT-EVs significantly reduced cell viability and increased cell apoptosis. ßT-EV-induced endothelial cell apoptosis involved the MAPK/JNK signal-transduction pathway. In contrast, splenectomized ßT-EVs induced proliferation of bone marrow mesenchymal stem cells (BM-MSC). In summary, the miR-144-3p was strongly increased; ßT-EVs induced apoptosis and decreased endothelial, pancreatic, and liver cell survival while supporting BM-MSC proliferation. These mechanisms may contribute to ßT organ dysfunction and complications.

Synergistic Effect of Two Organogelators for the Creation of Bio-Based, Shape-Stable Phase-Change Materials.

Two organogelators of different chemistry (a fatty acid derivative and a bis-urea derivative), as well as their blends, were used to impart shape stability to a bio-based phase-change material (PCM) bearing a near-ambient phase-transition temperature. Characterization of the individual gelators and their blends revealed their ability to immobilize the PCM by forming a continuous fibrillar network. The fibrils formed by the fatty acid derivative were helical, while the bis-urea derivative formed smooth fibrils. Also, the bis-urea derivative formed a continuous network at a lower critical concentration than the fatty acid derivative. At each fixed concentration, the bis-urea derivative yielded gels with higher thermal stability than the fatty acid derivative. The two gelators blended in certain ratios demonstrated a strong synergistic effect, providing gels with a significantly higher modulus (∼20-fold) and yield stress (∼1.5-fold) than each gelator individually. PCM gelation did not significantly affect its thermal behavior, however, affected its crystalline morphology. The gelled PCM displayed stacked structures, consisting of alternating pure PCM layers separated by layers formed by gelator fibrils. The phase diagram of the triple system comprising both gelators and PCM demonstrated either single or double gelation behavior depending on the composition. These findings may provide guidelines for the development of novel, shape-stable PCMs, which could be of potential use in various thermal energy storage applications.

A direct-imaging cryo-EM study of shedding extracellular vesicles from leukemic monocytes.

The human leukemia monocytic cell line (THP-1) is known to shed extracellular vesicles (EVs) under various stimulations. We studied the effects of two types of common stimulation types, lipopolysaccharide (LPS) and starvation conditions by high resolution cryogenic electron microscopy, namely, cryo-SEM and cryo-TEM. Cryo-SEM data of cells undergoing EV blebbing and shedding is presented here for the first time. The high-resolution images show good agreement with models describing the membrane processes of shedding. Cells that underwent a 48-h starvation treatment exhibited differing morphological features, including shrunken nucleus and elongated membrane protrusions. LPS treated cells, however, showed extensive blebbing originating from the cell membrane, in good agreement with the sizes of EVs imaged by cryo-TEM. EVs isolated from both types of stimulations were measured by nanoparticle tracking analysis (NanoSight), by which LPS-EVs samples exhibited higher concentration and smaller mean diameter, as compared to starvation-EVs. Our results suggest a difference in the effects of the two stimulation types on the shedding process and possibly on the type of EVs shed. Our unique methodologies provide an important and innovative outlook of the shedding process and on its products, paving the way to further discoveries in this developing field of research, in which much is still unknown.

Biomineralization pathways in a foraminifer revealed using a novel correlative cryo-fluorescence-SEM-EDS technique.

Foraminifera are marine protozoans that are widespread in oceans throughout the world. Understanding biomineralization pathways in foraminifera is particularly important because their calcitic shells are major components of global calcium carbonate production. We introduce here a novel correlative approach combining cryo-SEM, cryo-fluorescence imaging and cryo-EDS. This approach is applied to the study of ion transport processes in the benthic foraminifer genus Amphistegina. We confirm the presence of large sea water vacuoles previously identified in intact and partially decalcified Amphistegina lobifera specimens. We observed relatively small vesicles that were labelled strongly with calcein, and also identified magnesium (Mg)-rich mineral particles in the cytoplasm, as well as in the large sea water vacuoles. The combination of cryo-microscopy with elemental microanalysis and fluorescence imaging reveals new aspects of the biomineralization pathway in foraminifera which are, to date, unique in the world of biomineralization. This approach is equally applicable to the study of biomineralization pathways in other organisms.

Hyaluronic acid: An overlooked extracellular vesicle contaminant.

The variable presence of contaminants in extracellular vesicle (EV) samples is one of the major contributors to a lack of inter-study reproducibility in the field. Well-known contaminants include protein aggregates, RNA-protein complexes and lipoproteins, which resemble EVs in shape, size and/or density. On the contrary, polysaccharides, such as hyaluronic acid (HA), have been overlooked as EV contaminants. Here, it is shown that low and medium molecular weight HA polymers are unexpectedly retained to some extent in EV fractions using two common isolation methods known for high purity: size-exclusion chromatography and tangential flow filtration. Although these isolation techniques are capable of efficient removal of non-EV-associated proteins, this is not the case for HA polymers, which are partially retained in a molecular weight-dependent manner, especially with size-exclusion chromatography. The supramolecular structure and hydrodynamic size of HA are likely to contribute to isolation in EV fractions of filtration-based approaches. Conversely, HA polymers were not retained with ultracentrifugation and polymer-based precipitation methods, which are known for co-isolating other types of contaminants. HA has a broad range of immunomodulatory effects, similar to those ascribed to various sources of EVs. Therefore, HA contaminants should be considered in future studies to avoid potential inaccurate attributions of functional effects to EVs.

Altered ubiquitin signaling induces Alzheimer's disease-like hallmarks in a three-dimensional human neural cell culture model.

Alzheimer's disease (AD) is characterized by toxic protein accumulation in the brain. Ubiquitination is essential for protein clearance in cells, making altered ubiquitin signaling crucial in AD development. A defective variant, ubiquitin B + 1 (UBB+1), created by a non-hereditary RNA frameshift mutation, is found in all AD patient brains post-mortem. We now detect UBB+1 in human brains during early AD stages. Our study employs a 3D neural culture platform derived from human neural progenitors, demonstrating that UBB+1 alone induces extracellular amyloid-ß (Aß) deposits and insoluble hyperphosphorylated tau aggregates. UBB+1 competes with ubiquitin for binding to the deubiquitinating enzyme UCHL1, leading to elevated levels of amyloid precursor protein (APP), secreted Aß peptides, and Aß build-up. Crucially, silencing UBB+1 expression impedes the emergence of AD hallmarks in this model system. Our findings highlight the significance of ubiquitin signalling as a variable contributing to AD pathology and present a nonclinical platform for testing potential therapeutics.

Encapsulation of Thymol and Eugenol Essential Oils Using Unmodified Cellulose: Preparation and Characterization.

Essential oils (EOs) are volatile natural organic compounds, which possess pesticidal properties. However, they are vulnerable to heat and light, limiting their range of applications. Encapsulation of EOs is a useful approach to overcome some of these limitations. In this study, a novel emulsification technique is utilized for encapsulation of thymol (TY) and eugenol (EU) (EOs) within microcapsules with an unmodified cellulose shell. Use of low cost materials and processes can be beneficial in agricultural applications. In the encapsulation process, unmodified cellulose was dissolved in 7% aqueous NaOH at low temperature, regenerated to form a dispersion of cellulose hydrogels, which was rigorously mixed with the EOs by mechanical mixing followed by high-pressure homogenization (HPH). Cellulose:EO ratios of 1:1 and 1:8 utilizing homogenization pressures of 5000, 10,000 and 20,000 psi applied in a microfluidizer were studied. Light microscopy, high-resolution cryogenic scanning electron microscopy (cryo-SEM) and Fourier transform infrared spectroscopy (FTIR) revealed successful fabrication of EO-loaded capsules in size range of 1 to ~8 µm. Stability analyses showed highly stabilized oil in water (O/W) emulsions with instability index close to 0. The emulsions exhibited anti-mold activity in post-harvest alfalfa plants, with potency affected by the cellulose:EO ratio as well as the EO type; TY showed the highest anti-mold activity. Taken together, this study showed potential for anti-fungal activity of cellulose-encapsulated EOs in post-harvest hay.

Phase Change Material with Gelation Imparting Shape Stability.

Blending two gelators with different chemistries (12-hydroxystearic acid and a bis-urea derivative, Millithix MT-800) was used to impart shape stability to CrodaTherm 29, a bio-based phase change material (PCM), melting/crystallizing at near-ambient temperature. The gelators immobilized the PCM by forming an interpenetrating fibrillar network. 15 wt % concentration of the gelators was found to be effective in preventing liquid PCM leakage. In order to improve the mechanical properties and thermal conductivity (TC) of the PCM, gelation of suspensions of multiwalled carbon nanotubes (MWCNTs) and graphene nanoplatelets (GnPs) in a molten material was done at concentrations exceeding their percolation thresholds. Compared to pristine PCM, the gelled PCM containing 3.0 wt % of GnPs demonstrated a shorter crystallization time, ∼1.5-fold increase in strength, improved stability, and ∼65% increase in TC. At the same time, PCM filled with up to 0.6 wt % of MWCNTs had diminished strength and increased leakage with a slight TC improvement. Gelation of PCM did not significantly alter its thermal behavior, but it did change its crystalline morphology. The developed shape-stable PCMs may have a wide range of applications in ambient temperature solar-thermal installations, for example, temperature-controlled greenhouses, net zero-energy buildings, and water heaters.

Sucrose-based cryoprotective storage of extracellular vesicles.

Advancements in extracellular vesicle (EV) studies necessitate the development of optimized storage conditions to ensure preservation of physical and biochemical characteristics. In this study, the most common buffer for EV storage (phosphate-buffered saline/PBS) was compared to a cryoprotective 5% sucrose solution. The size distribution and concentration of EVs from two different sources changed to a greater extent after -80 °C storage in PBS compared to the sucrose solution. Additionally, molecular surface protrusions and transmembrane proteins were more prevalent in EVs stored in the sucrose solution compared to those stored in PBS. This study demonstrates, for the first time, that distinct ring-like molecular complexes and cristae-like folded membranous structures are visible upon EV degradation. Taken together, the size, concentration, molecular surface extensions, and transmembrane proteins of EVs varied substantially based on the buffer used for -80 °C storage, suggesting that biocompatible cryoprotectants, such as sucrose, should be considered for EV studies.

Cryogenic Electron Microscopy Methodologies as Analytical Tools for the Study of Self-Assembled Pharmaceutics.

Many pharmaceutics are aqueous dispersions of small or large molecules, often self-assembled in complexes from a few to hundreds of molecules. In many cases, the dispersing liquid is non-aqueous. Many pharmaceutical preparations are very viscous. The efficacy of those dispersions is in many cases a function of the nanostructure of those complexes or aggregates. To study the nanostructure of those systems, one needs electron microscopy, the only way to obtain nanostructural information by recording direct images whose interpretation is not model-dependent. However, these methodologies are complicated by the need to make liquid systems compatible with high vacuum in electron microscopes. There are also issues related to the interaction of the electron beam with the specimen such as micrograph contrast, electron beam radiation damage, and artifacts associated with specimen preparation. In this article, which is focused on the state of the art of imaging self-assembled complexes, we briefly describe cryogenic temperature transmission electron microscopy (cryo-TEM) and cryogenic temperature scanning electron microcopy (cryo-SEM). We present the principles of these methodologies, give examples of their applications as analytical tools for pharmaceutics, and list their limitations and ways to avoid pitfalls in their application.

Molecular self-assembly under nanoconfinement: indigo carmine scroll structures entrapped within polymeric capsules.

Molecular self-assembly forms structures of well-defined organization that allow control over material properties, affording many advanced technological applications. Although the self-assembly of molecules is seemingly spontaneous, the structure into which they assemble can be altered by carefully modulating the driving forces. Here we study the self-assembly within the constraints of nanoconfined closed spherical volumes of polymeric nanocapsules, whereby a mixture of polyester-polyether block copolymer and methacrylic acid methyl methacrylate copolymer forms the entrapping capsule shell of nanometric dimensions. We follow the organization of the organic dye indigo carmine that serves as a model building unit due to its tendency to self-assemble into flat lamellar molecular sheets. Analysis of the structures formed inside the nanoconfined space using cryogenic-transmission electron microscopy (cryo-TEM) and cryogenic-electron tomography (cryo-ET) reveal that confinement drives the self-assembly to produce tubular scroll-like structures of the dye. Combined continuum theory and molecular modeling allow us to estimate the material properties of the confined nanosheets, including their elasticity and brittleness. Finally, we comment on the formation mechanism and forces that govern self-assembly under nanoconfinement.

Structure elucidation of silica-based core-shell microencapsulated drugs for topical applications by cryogenic scanning electron microscopy.

We present here a technology to microencapsulate drugs by the sol-gel process, and cryo-SEM methodology that allows the nanostructural characterization of the formed capsules in their native state without any artifacts, related to their drying prior to imaging. The methodology utilizes three signals generated by the electron beam scanning the specimen: Secondary electrons, backscattered electrons, and x-rays. The first gives topographical information of the fracture-surface of the thermally-fixed specimen, the second gives contrast between elements of different atomic numbers, and the third allows the identification of those elements. Combined, the three signals provide full microstructural characterization of the studied specimen. Using this methodology, we were able to demonstrate that the sol-gel technology does indeed enable the encapsulation of two hydrophobic active molecules with a silica shell. This technology allows the active ingredient in the drug product to slowly migrate from the microcapsule onto the skin, thus obtaining the desired effect with minimal side-effects, as was exhibited in several clinical studies. The successful application of the cryo-SEM methodology in this case, demonstrates that it can be used to characterize a wide range of liquid-phase suspensions, in their native state, with minimal specimen preparation or imaging artifacts.

Flow induced stability of pluronic hydrogels: Injectable and unencapsulated nucleus pulposus replacement.

Poloxamers, or pluronics, have been proposed as biomimetic substitutes for physiological gels. Concern regarding their ability to resist swelling under fluid flows has impeded their implementation. Using a combination of techniques including cryo-TEM and rapid X-ray imaging, we found that rapid flow rates stabilized the gels against dissolution. Energy balance calculations confirmed that disentanglement of individual micelles was not possible at time scales faster than the reptation time when the system response was that of a solid which dissipated the hydrodynamic force field via cooperative deformation. In-vivo tests were performed where the hydrogel was injected as a substitute for the nucleus pulposus following discectomy in dogs. The results indicated that the gel was still present after 3 months, and radiographs indicated that compression of the disc space was prevented despite the gel being exposed to constant perfusion. STATEMENT OF SIGNIFICANCE: This paper demonstrates a highly unexpected result and counter intuitive result, namely the inverse dependence of the dissociation rate of a physical hydrogel on the flow velocity of the liquid medium. Using cryo-electron microscopy we demonstrate that the gel responds like deformable solid in high flow rates, with minimal dissociation. Since these gels are thermoreversible, they were injected into dogs, where we show that they were a viable alternative to the nucleus pulposus, without dissolution in physiological fluid flows for at least three months.

Extracellular Vesicle Characteristics in ß-thalassemia as Potential Biomarkers for Spleen Functional Status and Ineffective Erythropoiesis.

ß-thalassemia major (ß-TM) is a therapeutically challenging chronic disease in which ineffective erythropoiesis is a main pathophysiological factor. Extracellular vesicles (EVs) are membrane-enclosed vesicles released by cells into biological fluids; they are involved in intercellular communication and in multiple physiological and pathological processes. The chaperone heat-shock protein 70 (HSP70), which is released from cells via EVs, aggravates ineffective erythropoiesis in ß-TM. We propose that ß-TM EVs may show specific signatures, reflecting disease mechanisms, stages and severity. Our study aims were to define EV profiles in ß-TM patients, investigate the influence of hypersplenism and splenectomy on EV features, and explore the association of circulating EVs with ineffective erythropoiesis and iron-overload parameters. We characterized circulating EVs in 35 transfusion-dependent ß-thalassemia patients and 35 controls using several techniques. Nanoparticle-tracking analysis revealed increased EV concentration in patients vs. controls (P = 0.0036), with smaller EV counts and sizes in patients with hypersplenism. Flow cytometry analysis showed lower levels of RBC and monocyte EVs in patients vs. controls. RBC-EV levels correlated with patient hematocrit, reflecting degree of anemia. The procoagulant potential of the EVs evaluated by flow cytometry revealed lower levels of endothelial protein C receptor-labeled EVs in patients vs. controls, and increased tissue factor-to-tissue factor pathway inhibitor-labeled EV ratio in splenectomized patients, suggesting a hypercoagulable state. Protein content, evaluated in EV pellets, showed increased levels of HSP70 in patients (P = 0.0018), inversely correlated with transfusion requirement and hemoglobin levels, and positively correlated with reticulocyte, erythropoietin and lactate dehydrogenase levels. This first description of EVs in patients with hypersplenism reveals the spleen's importance in EV physiology and clearance. Circulating EV-HSP70 levels were associated with markers of ineffective erythropoiesis, hemolysis and hematological disease severity. EV analysis in ß-TM-reflecting spleen status, hypercoagulability state and ineffective erythropoiesis-may serve as a biomarker of disease dynamics, supporting both anticipation of the risk of complications and optimizing treatment.

Cryo-Imaging of Hydrogels Supermolecular Structure.

Gelatin, derived from collagen, has both the mechanical properties required for tissue growth, as well the functional domains required for cell binding. In its natural state, gelatin derives its properties from a network of structured, intertwined, triple helical chains, which is stabilized by hydrogen bonds at temperatures below 37 °C. The mechanical properties of such a structure can be further controlled by additional enzymatic cross-linking. But, in contrast to simple polymer systems, the response to an imposed deformation is here determined by two competing factors: the establishment of the cross-linked mesh vs. the self-assembly of the fibrils into larger and stronger hierarchical structures. Therefore, properties deduced from the response to measurements such as rheology or swelling, are a combination of these two very different factors, hence a modeling is impossible unless more precise knowledge regarding the internal structure is available. The cryogenic-temperature scanning electron microscopy (cryo-SEM) was adopted to image the fully hydrated gelatin network in which distinct chain folding was observed at low densities, while cross-linked networks were observed at higher densities. Based on these images, a theoretical model which results in good agreement between the mesh sizes of both networks and their mechanical properties was developed.

Transport of membrane-bound mineral particles in blood vessels during chicken embryonic bone development.

During bone formation in embryos, large amounts of calcium and phosphate are taken up and transported to the site where solid mineral is first deposited. The initial mineral forms in vesicles inside osteoblasts and is deposited as a highly disordered calcium phosphate phase. The mineral is then translocated to the extracellular space where it penetrates the collagen matrix and crystallizes. To date little is known about the transport mechanisms of calcium and phosphate in the vascular system, especially when high transport rates are needed and the concentrations of these ions in the blood serum may exceed the solubility product of the mineral phase. Here we used a rapidly growing biological model, the chick embryo, to study the bone mineralization pathway taking advantage of the fact that large amounts of bone mineral constituents are transported. Cryo scanning electron microscopy together with cryo energy dispersive X-ray spectroscopy and focused-ion beam imaging in the serial surface view mode surprisingly reveal the presence of abundant vesicles containing small mineral particles in the lumen of the blood vessels. Morphologically similar vesicles are also found in the cells associated with bone formation. This observation directly implicates the vascular system in solid mineral distribution, as opposed to the transport of ions in solution. Mineral particle transport inside vesicles implies that far larger amounts of the bone mineral constituents can be transported through the vasculature, without the danger of ectopic precipitation. This introduces a new stage into the bone mineral formation pathway, with the first mineral being formed far from the bone itself.

Nanostructure formation in the lecithin/isooctane/water system.

We present here for the first time a study of the self-assembled nanostructures in the lecithin/isooctane/water system by direct-imaging techniques, namely, cryogenic transmission electron microscopy (cryo-TEM) and cryogenic scanning electron microscopy (cryo-SEM). Along the dilution line [water]/[lecithin] = 5, we identified a nanostructural development with the increase of lecithin concentration. The system changes from a single reverse micellar phase, through a reverse micellar phase coexisting with a lamellar phase, and finally to a reverse liquid crystalline cubic phase and a lamellar phase. We compared the nanostructures formed when phosphatidylcholine rather than naturally occurring lecithin is used and found that both phase behavior and nanostructure are significantly different. The use of the two complementary cryo-EM techniques proved very efficient in the nanostructural characterization of the system. We also performed small-angle X-ray scattering to confirm our findings. Since the system is very sensitive to changes in composition, the cryo-EM specimens were prepared in a Controlled Environment Vitrification System (CEVS) that has been modified for our specimen preparation needs. We were able to overcome the challenges involved in directly imaging this nonaqueous (oil-rich), concentrated complex liquid systems, thus extending the usefulness of those characterization techniques.