Mechanism Of Vesicle Formation Research Articles

Polymersomes for Therapeutic Protein and Peptide Delivery: Towards Better Loading Properties.

Therapeutics based on proteins and peptides have profoundly transformed the landscape of treatment for diseases, from diabetes mellitus to cancers, yet the short half-life and low bioavailability of therapeutic proteins and peptides hinder their wide applications. To break through this bottleneck, biomolecules-loaded polymersomes with strong adjustability and versatility have attracted more and more attentions recently. Loading proteins or peptides into polymersomes is the first but extremely important step towards developing high-quality formulation products. However, increasing protein and peptide loading content is quite challenging due to the inherent nature of self-assembled vesicle formation mechanism and physiochemical characteristics of biomacromolecules. This review highlights the potential of polymersomes as the next-generation therapeutic proteins and peptides carrier and emphatically introduces novel approaches and recent progress to achieve satisfactory encapsulation capability of polymersomes for proteins and peptides. On the one hand, with the help of intermolecular interactions, such as electrostatic, lipid-protein, and hydrophobic interactions, the drug loading could be significantly improved. On the other hand, loading improvement could be attained through innovation of preparation methods, ranging from modified traditional film hydration techniques to the novel phase-guided assembly method.

Energetic and Entropic Motifs in Vesicle Morphogenesis in Amphiphilic Diblock Copolymer Solutions

Coarse-grained molecular dynamic simulations are employed to investigate the spatiotemporal evolution of vesicles (polymersomes) via self-assembly of randomly distributed amphiphilic diblock copolymers PB-PEO (Poly(Butadiene)-b-Poly(Ethylene Oxide)) in water. The vesiculation pathway consists of several intermediate structures, such as spherical/rodlike aggregates, wormlike micelles, lamellae, and cavities. The lamella-to-vesicle transition occurs at a constant aggregation number and is accompanied by a reduction in the solvent-accessible surface area. Simulation predictions are in qualitative agreement with the mechanism of vesicle formation in which the unfavorable hydrophobic interactions between water molecules and polymer segments, along the edge of the lamella, are eliminated at the expense of gaining curvature energy. However, rod–lamella–vesicle transition is accompanied by an increase in copolymer packing density. Hence, the change in the surface area accompanying vesiculation predicted by the simulations is significantly lower than theoretical estimates. Changes in information entropy, quantified by the expectation of the logarithm of the probability distribution function of the segmental stretch parameter s, defined as the difference between the maximum and instantaneous segmental extension, are statistically insignificant along the vesiculation pathway. For rods, lamellae, and polymersomes, s follows a log normal distribution. This is explained based on the configurational dynamics of a single diblock chain in water.

In vitro reconstitution of COPII vesicles from Arabidopsis thaliana suspension-cultured cells.

Transport vesicles mediate protein traffic between endomembrane organelles in a highly selective and efficient manner. In vitro reconstitution systems have been widely used for studying mechanisms of vesicle formation, polar trafficking, and cargo specificity in mammals and yeast. However, this technique has not yet been applied to plants because of the large lytic vacuoles and rigid cell walls. Here, we describe an Arabidopsis-derived in vitro vesicle formation system to reconstitute, purify and characterize plant-derived coat protein complex II (COPII) vesicles. In this protocol, we provide a detailed method for the isolation of microsomes and cytosol from Arabidopsis thaliana suspension-cultured cells (7-8 h), in vitro COPII vesicle reconstitution and purification (4-5 h) and biochemical and microscopic analysis using specific antibodies against COPII cargo molecules for reconstitution efficiency evaluation (2 h). We also include detailed sample-preparation steps for analyzing vesicle morphology by cryogenic electron microscopy (1 h) and vesicle cargoes by quantitative proteomics (4 h). Routinely, the whole procedure takes ~18-20 h of operation time and enables plant researchers without specific expertise to achieve organelle purification or vesicle reconstitution for further characterization.

Do Extracellular Vesicles Derived from Mesenchymal Stem Cells Contain Functional Mitochondria?

Extracellular vesicles (EV) derived from stem cells have become an effective complement to the use in cell therapy of stem cells themselves, which has led to an explosion of research into the mechanisms of vesicle formation and their action. There is evidence demonstrating the presence of mitochondrial components in EV, but a definitive conclusion about whether EV contains fully functional mitochondria has not yet been made. In this study, two EV fractions derived from mesenchymal stromal stem cells (MSC) and separated by their size were examined. Flow cytometry revealed the presence of mitochondrial lipid components capable of interacting with mitochondrial dyes MitoTracker Green and 10-nonylacridine orange; however, the EV response to the probe for mitochondrial membrane potential was negative. Detailed analysis revealed components from all mitochondria compartments, including house-keeping mitochondria proteins and DNA as well as energy-related proteins such as membrane-localized proteins of complexes I, IV, and V, and soluble proteins from the Krebs cycle. When assessing the functional activity of mitochondria, high variability in oxygen consumption was noted, which was only partially attributed to mitochondrial respiratory activity. Our findings demonstrate that the EV contain all parts of mitochondria; however, their independent functionality inside EV has not been confirmed, which may be due either to the absence of necessary cofactors and/or the EV formation process and, probably the methodology of obtaining EV.

Imaging vesicle formation dynamics supports the flexible model of clathrin-mediated endocytosis

Clathrin polymerization and changes in plasma membrane architecture are necessary steps in forming vesicles to internalize cargo during clathrin-mediated endocytosis (CME). Simultaneous analysis of clathrin dynamics and membrane structure is challenging due to the limited axial resolution of fluorescence microscopes and the heterogeneity of CME. This has fueled conflicting models of vesicle assembly and obscured the roles of flat clathrin assemblies. Here, using Simultaneous Two-wavelength Axial Ratiometry (STAR) microscopy, we bridge this critical knowledge gap by quantifying the nanoscale dynamics of clathrin-coat shape change during vesicle assembly. We find that de novo clathrin accumulations generate both flat and curved structures. High-throughput analysis reveals that the initiation of vesicle curvature does not directly correlate with clathrin accumulation. We show clathrin accumulation is preferentially simultaneous with curvature formation at shorter-lived clathrin-coated vesicles (CCVs), but favors a flat-to-curved transition at longer-lived CCVs. The broad spectrum of curvature initiation dynamics revealed by STAR microscopy supports multiple productive mechanisms of vesicle formation and advocates for the flexible model of CME.

Regulation of anionic structure and CO2 on the vesicle formation of imidazolium-based ionic liquids in water

The understanding of vesicle formation mechanism of ionic liquids (ILs) in aqueous solution is of great significance for the innovation and application of IL vesicles. In this work, a series of single-tailed amphiphilic ionic liquids (ILs) consisted of 1-tetradecyl-3-methylimidazolium [C14mim]+ and five kinds of anions have been designed to examine the regulation of anionic structures on the vesicle formation of ILs in water without any additives by surface tension, conductivity, cryogenic transmission electron microscopy, dynamic light scattering, small-angle X-ray scattering, and NMR spectroscopy. It is found that synergistic effect of hydrophobic and π-π interaction plays a vital role in the vesicle formation of 1-tetradecyl-3-methylimidazolium 1, 2, 3-benztriazole ([C14mim][1, 2, 3-Ben]). Interestingly, reversible structural transition between vesicle and micelle of this IL is observed by alternating CO2 and N2 bubbling due to the reversible formation of carbamate arising from the reaction between CO2 and the anion of the IL.

Revealing the mechanisms of vesicle formation with multiple spectral methods.

The investigation of the self-assembly of amphiphilic molecules and the formation of micelles/vesicles has attracted significant attention. However, in situ and real-time methods for such studies are rare. Here, a surface-sensitive second harmonic generation (SHG) technique was applied to study the formation of vesicles in solutions of an anti-cancer drug, doxorubicin (DOX), and a generally used surfactant (sodium bis (2-ethylhexyl) sulfosuccinate, AOT). With the aid of two-photon fluorescence (TPF), Rayleigh scattering and TEM, we revealed the structural evolution of the aggregated micelles/vesicles. It was found that AOT and DOX molecules rapidly aggregated and formed micelles in the solution. The residual DOX then acted as a "glue" that induced the aggregating/growing of the micelles and the transformation from aggregates to vesicles. The existence of lipid films, which was considered as the necessary intermediate state for vesicle formation, was excluded via the SHG observations, indicating that hollow shells may be directly transformed from solid aggregated micelles in the self-assembly formation of complex vesicles. The combined spectroscopic methods were also used to investigate the formation of vesicles from a commonly used lipid (i.e., 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, DOPG) from its stacked bilayers. The swelling, curving and sealing of the DOPG bilayers for vesicle formation was monitored and clear dynamics were revealed. This work shows that the vesicle formation mechanism varies with the initial state of the surfactant/lipid molecules. It not only demonstrates the capability of the combined spectroscopic methods in investigating the aggregated systems but also provides new insight for understanding the formation of vesicles.

Abstract P419: Revealing The Dynamics Of Vesicle Formation During Clathrin-mediated Endocytosis In Living Endothelial Cells

Clathrin-mediated endocytosis (CME) is an essential cellular process for internalizing nutrients and therapeutics at endothelial cell barriers. Studying the formation of cargo containing endocytic vesicles in living cells is challenging due to the limited resolution of fluorescence microscopy and the highly dynamic nature of CME. Moreover, it is currently unknown how the physiological conditions present in vasculature affect CME in endothelial cells. To address this challenge we used a novel microscopy approach, Simultaneous Two-wavelength Axial Ratiometry (STAR), to image vesicle formation dynamics with nanometer axial resolution in living cells. High-throughput analysis revealed that 80% of de novo clathrin accumulations contributed to endocytosis while 20% remained flat, consistent in both human umbilical vein endothelial cells (HUVECs) and our test-bed model green monkey kidney fibroblast-like (Cos-7) cells. We next investigated the interplay between coat curvature and clathrin accumulation in vesicle initiation to identify the mechanism of vesicle formation. Our results support the flexible model of vesicle formation with curvature and clathrin accumulation initiating together at shorter-lived vesicles (<20s) and through a flat-to-curved transition of clathrin lattices at longer-lived vesicles (>20s). Finally, we addressed if physiological conditions present in vasculature alter the dynamics of vesicle formation. We show that increasing osmotic pressure decreased the total number of internalizations but had no impact on the number of flat clathrin accumulations or the mechanism of vesicle formation in Cos-7 cells. In future research, we will test the hypothesis that HUVECs have distinct mechanisms to retain vesicle formation under osmotic pressure or shear stress conditions similar to their native environment. Additionally targeted drug delivery to vascular endothelial cells, for example nanocarriers binding to flat lattices or CCVs leading to different therapeutic outcomes or bioavailability, can potentially be informed by identifying clathrin morphology and dynamics and the mechanisms of endocytosis using STAR microscopy.

Filopodium-derived vesicles produced by MIM enhance the migration of recipient cells.

Extracellular vesicles (EVs) are classified as large EVs (l-EVs, or microvesicles) and small EVs (s-EVs, or exosomes). S-EVs are thought to be generated from endosomes through a process that mainly depends on the ESCRT protein complex, including ALG-2 interacting protein X (ALIX). However, the mechanisms of l-EV generation from the plasma membrane have not been identified. Membrane curvatures are generated by the bin-amphiphysin-rvs (BAR) family proteins, among which the inverse BAR (I-BAR) proteins are involved in filopodial protrusions. Here, we show that the I-BAR proteins, including missing in metastasis (MIM), generate l-EVs by scission of filopodia. Interestingly, MIM-containing l-EV production was promoted by invivo equivalent external forces and by the suppression of ALIX, suggesting an alternative mechanism of vesicle formation to s-EVs. The MIM-dependent l-EVs contained lysophospholipids and proteins, including IRS4 and Rac1, which stimulated the migration of recipient cells through lamellipodia formation. Thus, these filopodia-dependent l-EVs, which we named as filopodia-derived vesicles (FDVs), modify cellular behavior.

A REVIEW ON PRONIOSOMES: A PRO-COLLOIDAL PARTICULATE DRUG CARRIER

Colloidal particulate carrier systems are the systems which carry particulates in a nanometre size. These systems are substantially effective for transportation and distribution of variety of loaded drugs to desired site and increase efficacy and decrease toxicity, to provide therapeutic activity in a controlled manner for a prolonged period of time. One such new emerging colloidal systems is proniosomes which has capacity to improve the bioavailability and also permeation of drugs across the stratum corneum to provide a controlled release action and reduce toxic effects associated with drugs. These are the dry formulations of water-soluble non-ionic surfactant coated carrier systems which immediately on hydration form niosomes. They have the capacity to overcome the various problems associated with niosomes and liposomes, like instability, transportation, distribution, storage and dosing. They offer versatile drug delivery concept for both hydrophilic and hydrophobic drugs. They have the capacity to deliver drugs effectively through different routes at specific site to achieve controlled release action. This review concentrates on preparation, characterization, components, structure, types, evaluation parameters, proniosomes in drug delivery and targeting, toxicities associated with proniosomes, proniosomes versus niosomes, clinical applications, mechanism of vesicle formation in proniosomes and future trends associated with it. Also, drug delivery via different routes, such as oral, parenteral, topical and transdermal, ocular, vaginal, mucosal, pulmonary and intranasal were discussed.

Quantification of outer membrane vesicles: a potential tool to compare response in Pseudomonas putida KT2440 to stress caused by alkanols.

The bacterial release of outer membrane vesicles (OMVs) is an important physiological mechanism of Gram-negative bacteria playing numerous key roles. One function of the release of OMVs is related to an increase in surface hydrophobicity. This phenomenon initiates biofilm formation, making bacteria more tolerant to environmental stressors. Recently, it was qualitatively shown for Pseudomonas putida that vesicle formation plays a crucial role in multiple stress responses. Yet, no quantification of OMVs for certain stress scenarios has been conducted. In this study, it is shown that the quantification of OMVs can serve as a simple and feasible tool, which allows a comparison of vesicle yields for different experimental setups, cell densities, and environmental stressors. Moreover, the obtained results provide insight to the underlying mechanism of vesicle formation as it was observed that n-alkanols, with a chain length of C7 and longer, caused a distinct and steep increase in vesiculation (12-19-fold), compared to shorter chain n-alkanols (2-4-fold increase).

A novel stimulus-responsive temozolomide supramolecular vesicle based on host–guest recognition

Temozolomide is a potent chemotherapeutic agent for glioblastoma multiforme treatment. However, its low aqueous solubility and short half-life (only about 1.8 h) in plasm limit its clinical therapeutics. Herein, a supramolecular vesicle based on hydroxypropyl-β-cyclodextrin and temozolomide was firstly constructed by elaborate design and preparation, which can load temozolomide into membranous layer of vesicle effectively. The morphologies and diameters of this temozolomide-loaded vesicle were characterized through transmission electron microscope, scanning electron microscope, and dynamic light scattering. The possible vesicle formation mechanism was further studied by X-ray diffraction, Fourier transform infrared spectrum, ultraviolet-visible spectroscopy, 1H nuclear magnetic resonance, and 2D nuclear magnetic resonance (ROSEY). Finally, the stimulus responsiveness of this vesicle was studied. Temozolomide can be released from the membrane of the vesicle once copper ions were dropped into the vesicle solution.

Size-Defined Cracked Vesicle Formation via Self-Assembly of Gold Nanoparticles Covered with Carboxylic Acid-Terminated Surface Ligands

The self-assembly of gold nanoparticles (GNPs) into a defined structure, particularly hollow capsule structures, provides great potential for applications in materials science and medicine. However, the complexity of the parameters for the preparation of those structures through self-assembly has limited access to critical mechanistic questions. With this in mind, we have studied GNP vesicle (GNV) formation through self-assembly by the surface modification of GNPs with low-molecular-weight ligands. Here, we successfully prepared GNVs composed of GNPs with a diameter of 30 nm by surface modification with carboxylic acid-terminated fluorinated oligo(ethylene glycol) ligands (CFLs). As the carboxylic acid has two states (protonated and deprotonated), the balance of the attraction and repulsion between GNPs covered with CFLs is tunable. Sodium carboxylate-terminated fluorinated oligo(ethylene glycol) ligands (SCFLs) provided smaller GNVs than did CFLs at 0.8 × 1011 NPs/mL. Time-course study revealed that CFL-covered GNPs quickly form small aggregates and gradually grow to larger GNVs (ca. 200 nm), but no gradual growth was observed for SCFL-covered GNPs. This result indicated that the electrostatic repulsion inhibits fusion of the small GNVs. The size of the GNVs formed with the aid of CFLs was independent of the initial GNP concentration, but the extinction spectra were concentration-dependent. Electron microscopy imaging and simulations supported the defect formation in the assemblies. These results provided new insights into the vesicle formation mechanism.

Phase evolution, structural characteristics and mechanism of vesicle formation from a synthetic amphiphile: Controlled morphology by tuning solution phase parameters

We report herein, the aggregation behavior of 3, 4-di(dodecyloxy)benzoic acid-4-hydroxy phenyl ester (DDBE), a synthetic amphiphile and a true non-ionic surfactant system as per the geometrical considerations. The true surfactant nature of the system stems from its hydrophilic-lipophilic-balance (HLB = 4.7), comparable to that of Span-60, also a true non-ionic surfactant. This compound undergoes spontaneous vesicle formation in THF:water binary solvent mixtures which further underwent fission at lower DDBE concentrations and fusion at higher concentrations, leading to giant vesicles of the order of 3000 nm. These vesicles are sensitive to the polarity of their environment. The predominant mode of interaction as observed from the molecular dynamics simulations were found to be π-π stacking with the phenyl rings of the molecule. Further, the system, upon complete extraction into water, formed spherical aggregates of size 50 nm based on the good solvent-poor solvent combination as the necessary condition for the vesicle formation.GRAPHICAL ABSTRACT

Thermal structural evolutions of DMPC-water biomimetic systems investigated by Raman Spectroscopy

Many cell membranes of living organisms can be represented as phospholipid bilayers immersed into a water environment. The physical-chemical interactions at the membranes/water interface are responsible for the stabilization of the membranes. In addition, the drug efficiency, the pharmaceutical mechanism and the improvement of the drug design can be addressed to the interactions between the membranes-water interface with the drug and to the membrane-drug interface. In this framework, it is important to find membranes models able to simulate and simultaneously simplify the biological systems to better understand both physical and chemical interactions at the interface level. Dimyristoylphosphatidylcholine (DMPC) is a synthetic phospholipid used in order to make Multilamellar Vesicle (MLV), Large Unilamellar Vesicle (LUV) and Giant Unilamellar Vesicle (GUV). In order to understand the mechanisms of vesicle formation, we have analyzed mixtures of DMPC and water by micro-Raman spectroscopy at different temperatures in the range between 10 and 35 °C. Particularly, we analyzed the temperature dependence of the CN vibrational frequency, which appears well correlated to the order degree of the various phases. These investigations, beyond the determination of phospholipid hydrocarbon chains order, provide information about the conformation of the lipid membranes.We have identified the mixture of DMPC/water that is best suited for Raman studies and can be used as an in-vitro model for biological systems.A peculiar frequency shift across the transition gel-ripple-liquid crystalline phases has been proposed as a useful diagnostic marker to detect the “order degree” and subsequently the phases of biomimetic membranes made by DMPC.

Polymersome formation mechanism and formation rate in stirred‐tank reactors

ABSTRACTUniform polymersomes (polymer vesicles) made of poly(2‐methyloxazoline)15‐b‐poly(dimethylsiloxane)68‐b‐poly(2‐methyloxazoline)15 (PMOXA15–PDMS68–PMOXA15) can be formed in miniaturized‐stirred tank reactors by the aid of a recently published process. In this study, the occurring self‐assembly mechanism was elucidated by using transmission electron microscopy. Subsequent to the initial formation of small spherical micelles and the following fusion to worm‐like micelles, two simultaneously occurring pathways, describing the transformation of further intermediate structures to the desired vesicles, were found. The resulting particle increase was followed by dynamic light scattering. Thus, the vesicle formation rate was judged by the linear increase of the particle diameter over time. While temperature showed no influence, higher initial polymer concentrations and lower final solvent concentrations accelerated the polymersome formation. Besides, the process was crucially dependent on the agitation speed. While spherical micelles did not transform into polymersomes when no stirring or too slow stirring is applied, the self‐assembly process was accelerated by increasing the agitation speed. Uniform polymeric vesicles can be formed under vigorous stirring in stirred‐tank reactors in short process times. In this study, the underlying mechanisms of vesicle formation were elucidated, showing that the polymer forms small micellar structures before undergoing two separate pathways to form the desired vesicular structures. The formation rate of the polymer vesicles was mainly dependent on the agitation speed but also on the polymer and solvent concentrations, highlighting the need for controlled formation conditions. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018, 135, 46077.

Analysis of exosome release as a cellular response to MAPK pathway inhibition.

Exosome size distributions and numbers of exosomes released per cell are measured by asymmetric flow-field flow fractionation/multi-angle light scattering (A4F/MALS) for three thyroid cancer cell lines as a function of a treatment that inhibits MAPK signaling pathways in the cells. We show that these cell lines release exosomes with well-defined morphological features and size distributions that reflect a common biological process for their formation and release into the extracellular environment. We find that those cell lines with constitutive activation of the MAPK signaling pathway display MEK-dependent exosome release characterized by increased numbers of exosomes released per cell. Analysis of the measured exosome size distributions based on a generalized extreme value distribution model for exosome formation in intracellular multivesicular bodies highlights the importance of this experimental observable for delineating different mechanisms of vesicle formation and predicting how changes in exosome release can be modified by pathway inhibitors in a cell context-dependent manner.

A multistimuli-responsive supramolecular vesicle constructed by cyclodextrins and tyrosine

Stimuli-responsive vesicle is a significant self-assembly. The supramolecular amphiphile strategy provides a simple way to construct the vesicles. Here, we report a β-cyclodextrin/tyrosine supramolecular vesicle based on host-guest recognition. Tyrosine, which is a nontoxic amino acid, is used as the vesicles building blocks. The β-cyclodextrin/tyrosine vesicles were identified by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and dynamic light scattering (DLS). X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), ultraviolet-visible (UV-vis) spectrum, hydrogen-1 nuclear magnetic resonance (1H NMR), and two-dimensional nuclear magnetic resonance spectroscopy rotating frame nuclear Overhauser effect spectroscopy (2D NMR ROESY) were further employed to elucidate the vesicle formation mechanism. Furthermore, different host molecules, including α-cyclodextrin and γ-cyclodextrin, can all form vesicles with tyrosine. Finally, the vesicles can respond to multiple external stimuli. Competitive guest molecules and copper ions can all disrupt the vesicles’ architectures.

The emerging role of extracellular vesicles as biomarkers for urogenital cancers.

The knowledge gained from comprehensive profiling projects that aim to define the complex genomic alterations present within cancers will undoubtedly improve our ability to detect and treat those diseases, but the influence of these resources on our understanding of basic cancer biology is still to be demonstrated. Extracellular vesicles have gained considerable attention in past years, both as mediators of intercellular signalling and as potential sources for the discovery of novel cancer biomarkers. In general, research on extracellular vesicles investigates either the basic mechanism of vesicle formation and cargo incorporation, or the isolation of vesicles from available body fluids for biomarker discovery. A deeper understanding of the cargo molecules present in extracellular vesicles obtained from patients with urogenital cancers, through high-throughput proteomics or genomics approaches, will aid in the identification of novel diagnostic and prognostic biomarkers, and can potentially lead to the discovery of new therapeutic targets.

Dynamin-related proteins in plant post-Golgi traffic.

Membrane traffic between two organelles begins with the formation of transport vesicles from the donor organelle. Dynamin-related proteins (DRPs), which are large multidomain GTPases, play crucial roles in vesicle formation in post-Golgi traffic. Numerous in vivo and in vitro studies indicate that animal dynamins, which are members of DRP family, assemble into ring- or helix-shaped structures at the neck of a bud site on the donor membrane, where they constrict and sever the neck membrane in a GTP hydrolysis-dependent manner. While much is known about DRP-mediated trafficking in animal cells, little is known about it in plant cells. So far, two structurally distinct subfamilies of plant DRPs (DRP1 and DRP2) have been found to participate in various pathways of post-Golgi traffic. This review summarizes the structural and functional differences between these two DRP subfamilies, focusing on their molecular, cellular and developmental properties. We also discuss the molecular networks underlying the functional machinery centering on these two DRP subfamilies. Furthermore, we hope that this review will provide direction for future studies on the mechanisms of vesicle formation that are not only unique to plants but also common to eukaryotes.