Preparation Of Vesicles Research Articles

Preparation of outer membrane vesicles from rabbit Bordetella bronchiseptica and their protein composition analysis

Preparation of outer membrane vesicles from rabbit Bordetella bronchiseptica and their protein composition analysis

Analysis of peptides and small proteins in preparations of horse milk exosomes, purified on anti-CD81-Sepharose

Exosomes are nanovesicles (40–100 nm) involved in intracellular communication. Milk is a unique source of exosomes. In our previous papers, our group was the first to describe horse milk exosomes. Here we show how crude vesicle preparations were obtained from 18 horse milk samples by standard centrifugation, ultracentrifugation, and gel-filtration methods. Transmission electron microscopy and flow cytometry confirmed that exosomes contain tetraspanins on their surface. Affinity chromatography of vesicle preparations on anti-CD81-Sepharose produced extra-purified exosomes with more than 20 peptides and small proteins of 0.8–10 kDa as detected by MALDI-TOF mass spectrometry. The peptides are natural intrinsic components of horse milk exosomes and are not formed either as a result of or during sample preparation. We assume some of the hundreds and thousands of exosome proteins previously described by other authors in milk exosomes by highly sensitive liquid chromatography-mass spectrometry to have been misidentified as full-length proteins.

Extracellular vesicle-associated repetitive element DNAs as candidate\xa0osteosarcoma biomarkers

Osteosarcoma (OS) is the most common malignant bone tumor in children and young adults. Despite that high-risk factors have been identified, no test for early detection is available. This study aimed to identify circulating nucleic acid sequences associated with serum extracellular vesicle (EV) preparations at the time of OS diagnosis, as a step towards an OS early detection assay. Sequencing of small nucleic acids extracted from serum EV preparations revealed increased representation of diverse repetitive element sequences in OS patient versus control sera. Analysis of a validation cohort using qPCR of PEG-precipitated EV preparations revealed the over-representation of HSATI, HSATII, LINE1-P1, and Charlie 3 at the DNA but not RNA level, with receiver operating characteristic (ROC) area under the curve (AUC) ≥ 0.90. HSATI and HSATII DNAs co-purified with EVs prepared by precipitation and size exclusion chromatography but not by exosome immunocapture, indicative of packaging in a non-exosomal complex. The consistent over-representation of EV-associated repetitive element DNA sequences suggests their potential utility as biomarkers for OS and perhaps other cancers.

Preparation and utility of asymmetric lipid vesicles for studies of perfringolysin O-lipid interactions.

Studying the interaction of pore-forming toxins, including perfringolysin O (PFO), with lipid is crucial to understanding how they insert into membranes, assemble, and associate with membrane domains. In almost all past studies, symmetric lipid bilayers, i.e., bilayers having the same lipid composition in each monolayer (leaflet), have been used to study this process. However, practical methods to make asymmetric lipid vesicles have now been developed. These involve a cyclodextrin-catalyzed lipid exchange process in which the outer leaflet lipids are switched between two lipid vesicle populations with different lipid compositions. By use of alpha class cyclodextrins, it is practical to include a wide range of sterol concentrations in asymmetric vesicles. In this article, protocols for preparing asymmetric lipid vesicles are described, and to illustrate how they may be applied to studies of pore-forming toxin behavior, we summarize what has been learned about PFO conformation and its lipid interaction in symmetric and in asymmetric artificial lipid vesicles.

Mesenchymal stromal cell extracellular vesicles rescue mitochondrial dysfunction and improve barrier integrity in clinically relevant models of ARDS.

Alveolar epithelial–capillary barrier disruption is a hallmark of acute respiratory distress syndrome (ARDS). Contribution of mitochondrial dysfunction to the compromised alveolar-capillary barrier in ARDS remains unclear. Mesenchymal stromal cells-derived extracellular vesicles (MSC-EVs) are considered as a cell-free therapy for ARDS. Mitochondrial transfer was shown to be important for the therapeutic effects of MSCs and MSC-EVs. Here we investigated the contribution of mitochondrial dysfunction to the injury of alveolar epithelial and endothelial barriers in ARDS and the ability of MSC-EVs to modulate alveolar–capillary barrier integrity through mitochondrial transfer.Primary human small airway epithelial and pulmonary microvascular endothelial cells and human precision cut lung slices (PCLSs) were stimulated with endotoxin or plasma samples from patients with ARDS and treated with MSC-EVs, barrier properties and mitochondrial functions were evaluated. Lipopolysaccharide (LPS)-injured mice were treated with MSC-EVs and degree of lung injury and mitochondrial respiration of the lung tissue were assessed.Inflammatory stimulation resulted in increased permeability coupled with pronounced mitochondrial dysfunction in both types of primary cells and PCLSs. Extracellular vesicles derived from normal MSCs restored barrier integrity and normal levels of oxidative phosphorylation while an extracellular vesicles preparation which did not contain mitochondria was not effective. In vivo, presence of mitochondria was critical for extracellular vesicles ability to reduce lung injury and restore mitochondrial respiration in the lung tissue.In the ARDS environment, MSC-EVs improve alveolar–capillary barrier properties through restoration of mitochondrial functions at least partially via mitochondrial transfer.

Proteomics of extracellular vesicles produced by Granulicatella adiacens, which causes infective endocarditis.

When oral bacteria accidentally enter the bloodstream due to transient tissue damage during dental procedures, they have the potential to attach to the endocardium or an equivalent surface of an indwelling prosthesis and cause infection. Many bacterial species produce extracellular vesicles (EVs) as part of normal physiology, but also use it as a virulence strategy. In this study, it was hypothesized that Granulicatella adiacens produce EVs that possibly help it in virulence. Therefore, the objectives were to isolate and characterize EVs produced by G. adiacens and to investigate its immune-stimulatory effects. The reference strain G. adiacens CCUG 27809 was cultured on chocolate blood agar for 2 days. From subsequent broth culture, the EVs were isolated using differential centrifugation and filtration protocol and then observed using scanning electron microscopy. Proteins in the vesicle preparation were identified by nano LC-ESI-MS/MS. The EVs proteome was analyzed and characterized using different bioinformatics tools. The immune-stimulatory effect of the EVs was studied via ELISA quantification of IL-8, IL-1β and CCL5, major proinflammatory cytokines, produced from stimulated human PBMCs. It was revealed that G. adiacens produced EVs, ranging in diameter from 30 to 250 nm. Overall, G. adiacens EVs contained 112 proteins. The proteome consists of several ribosomal proteins, DNA associated proteins, binding proteins, and metabolic enzymes. It was also shown that these EVs carry putative virulence factors including moonlighting proteins. These EVs were able to induce the production of IL-8, IL-1β and CCL5 from human PBMCs. Further functional characterization of the G. adiacens EVs may provide new insights into virulence mechanisms of this important but less studied oral bacterial species.

Preparation and characterization of extracellular vesicles.

Extracellular vesicles (EVs) are heterogeneous membranous vesicles secreted by every cell type and offer significant potential in therapy and diagnostics. Differential ultracentrifugation is the gold standard for EV isolation, although other techniques including, polyethylene glycol (PEG) precipitation, immunoprecipitation, size exclusion chromatography, and immuno-isolation approaches are common. Purified EVs can be characterized based on their physical characteristics, biochemical composition, or cell of origin. For size and concentration measurement, nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and electron microscopy are commonly employed methods. Biochemical analyses of EVs are typically performed using flow cytometry, immunoblotting, or proteomic investigation. Based on tissue of origin, EVs have specific markers that can be used to isolate and purify specific cell-associated EVs using an affinity selection approach. Despite existence of several methods for isolation and characterization, major limitations associated with each method hinder the progress of the field. Evolving concepts in EV biology possess great promise for better isolation and characterization leading to a better insight of biological function and have immense clinical implications. In this review, we discuss recent advancements in EV isolation and characterization approaches.

Electromagnetic Piezoelectric Acoustic Sensor Detection of Extracellular Vesicles through Interaction with Detached Vesicle Proteins.

An electromagnetic piezoelectric acoustic sensor (EMPAS) was used to study the non-specific adsorption of human red blood cell-derived extracellular vesicle preparations. Vesicle storage history (temperature and duration) highly affected the obtained results: The signal change, namely the frequency decrease of the crystal measured at 20 °C, was negligibly small (<1 s−2) when the vesicle solutions had previously been stored at 4 °C, and was in the order of 10 s−2 when the vesicle solutions had been stored at −30 °C. Moreover, the rate of frequency decrease increased exponentially with the storage time at −30 °C. Upon a 4 °C storage period following the −30 °C storage period of the same sample, the measured frequency decrease dropped, suggesting a partial relaxation of the system. The results are explained by the disintegration of the vesicles triggered by the freeze–thaw cycle, likely due to the detachment of proteins from the vesicle surface as was proved by size-exclusion chromatography. Surface modification of the sensor crystal provided the possibility of signal enhancement, as the maximum rate of the frequency change for the same vesicle concentrations was higher on hydrophobic, octadecyl trichlorosilane–modified quartz than on hydrophilic, bare quartz. The EMPAS signal has been associated with the amount of detached proteins, which in turn is proportional to the originating vesicle concentration.

Characterization of exosome release and extracellular vesicle-associated miRNAs for human bronchial epithelial cells irradiated with high charge and energy ions

Exosomes are extracellular vesicles that mediate transport of nucleic acids, proteins, and other molecules. Prior work has implicated exosomes in the transmission of radiation nontargeted effects. Here we investigate the ability of energetic heavy ions, representative of species found in galactic cosmic rays, to stimulate exosome release from human bronchial epithelial cells in vitro. Immortalized human bronchial epithelial cells (HBEC3-KT F25F) were irradiated with 1.0Gy of high linear energy transfer (LET) 48Ti, 28Si, or 16O ions, or with 10Gy of low-LET reference γ-rays, and extracellular vesicles were collected from conditioned media. Preparations were characterized by single particle tracking analysis, transmission electron microscopy, and immunoblotting for the exosomal marker, TSG101. Based on TSG101 levels, irradiation with high-LET ions, but not γ-rays, stimulated exosome release by about 4-fold, relative to mock-irradiated controls. The exosome-enriched vesicle preparations contained pro-inflammatory damage-associated molecular patterns, including HSP70 and calreticulin. Additionally, miRNA profiling was performed for vesicular RNAs using NanoString technology. The miRNA profile was skewed toward a small number of species that have previously been shown to be involved in cancer initiation and progression, including miR-1246, miR-1290, miR-23a, and miR-205. Additionally, a set of 24 miRNAs was defined as modestly over-represented in preparations from HZE ion-irradiated versus other cells. Gene set enrichment analysis based on the over-represented miRNAs showed highly significant association with nonsmall cell lung and other cancers.

Enhanced antimicrobial photodynamic activity of photosensitizer encapsulated copper based metallocatanionic vesicles against E.coli using visible light

In this research, we demonstrated preparation of metallo-catanionic vesicles by proper mixing of desired stoichiometric molar ratios of a cationic metallosurfactant, hexadecyltrimethyl ammonium copper trichloride and anionic conventional surfactant, sodium dodecyl sulphate. The characterization of synthesized metallo-catanionic vesicles was done by using various analytical tools. Further, these synthesized metallo-catanionic vesicles were used for encapsulation of a cationic dye (Toluidine blue (TB)), as well as an anionic dye (Rose Bengal (RB)). The dye encapsulated metallo-catanionic vesicles were tested for singlet oxygen generation and were employed for antimicrobial Photodynamic Therapy (aPDT) against Escherichia coli (E.coli). Also, a comparison is made between TB and RB encapsulated metallo-catanionic vesicles. These vesicles exhibited enhanced singlet oxygen quantum yield thereby showing photodynamic activity against Escherichia coli that was investigated using colony forming unit count method. The bacterial growth was tremendously decreased from 5.84× 106 CFU/mL to 0.06 × 106 CFU/ mL using TB encapsulated metallo-catanionic vesicles SDS:CuC I (30:70) under irradiation and to 0.34 × 106 CFU/mL using TB encapsulated 70:30 fraction. With variable charge and the presence of metal, the metallo-catanionic vesicles work effectively for PDT against E.coli. Interestingly, the RB encapsulated metallocatanionic vesicles exhibited much enhanced effect, as the bacterial growth was diminshed and almost zero colony formation was observed. Herein developed metallo-catanionic vesicles of variable charge perform efficiently and effectively in PDT against E. coli and can be scalable for affordable industrial applications.

Nanoliposome Precursors for Shape Modulation: Use of Heuristic Algorithm and QBD Principles for Encapsulating Phytochemicals.

Screening of multiple methods is worthless for formulators due to material losses, wastage of time, and expenditures. It is imperative to make a quick decision. The present investigation describes the systematic approach to select the best suitable method for the development of nanoliposomes (NL), the precursor of nanocochleates encapsulating curcumin using Analytic Hierarchy Process (AHP). Pair-wise comparison matrices were used to achieve the overall priority weight and ranking for the selection of appropriate technique. Furthermore, Plackett-Burman screening Design (PBD) was exploited to investigate specific effects of associated formulation and process variables on particle size (Y1), drug content (Y2), and entrapment efficiency (Y3), while fabricating NL. Results revealed the reliability of the pair-wise comparison matrices and selected the ethanol injection method with the highest priority weight (0.337). Bland-Altman plot and control chart validated the results of AHP. The preparation of vesicles with the preferred diameter and size distribution was essentially fulfilled. Stirring speed (X5), amount of phospholipid (X4), and cholesterol (X8) showed significant influence (p<0.05;) on Y1 and Y3, PBD revealed. These factors can be further optimized using the design of experiments. AHP being an effective tool, has assisted in selecting the best alternative for fabricating NL, whilst PBD enabled a clear understanding of the effects of diverse formulation variables on responses studied. Results ensure that NL is a riveting candidate for modulating effectively into tailormade diverse shaped nanoformulations for further in vitro; and in vivo; studies.

Engineering Stem Cell Derived Biomimetic Vesicles for Versatility and Effective Targeted Delivery

AbstractTo date, only few a types of nanomedicines have been made available for clinical use even in the most common liposomal delivery systems, due to significant constraints, including poor targeting of specific cells, hindered penetration, and accelerated clearance. Various intricate designs have been generated and show improvements, while also bringing new problems. Biological cells, possessing advantages like low immunogenicity, long circulation time, receptors integration, and innate targeting capability. Herein, liposomes are innovatively functionalized with a stem cell membrane through a facile approach. The biomimetic vesicles achieve controlled release, exhibit better stability, longer circulation time, and targeted delivery. Loading with curcumin leads to enhanced survival rate of ischemic stroke mice with a single injection (from ≈30% to over 90%). The versatility of the method is verified by differently charged liposomes modification and erythrocyte membrane derived vesicles preparation. Overall, the cell membrane derived biomimetic vesicles achieve targeted delivery and versatility prepared by a facile approach, as well as effortless preparation.

Biophysical Analysis of Lipid Domains by Fluorescence Microscopy.

The study of the structure and dynamics of membrane domains in vivo is a challenging task. However, major advances could be achieved through the application of microscopic and spectroscopic techniques coupled with the use of model membranes, where the relations between lipid composition and the type, amount and properties of the domains present can be quantitatively studied.This chapter provides protocols to study membrane organization and visualize membrane domains by fluorescence microscopy both in artificial membrane and living cell models of Gaucher Disease (GD ). We describe a bottom-up multiprobe methodology, which enables understanding how the specific lipid interactions established by glucosylceramide, the lipid that accumulates in GD , affect the biophysical properties of model and cell membranes, focusing on its ability to influence the formation, properties and organization of lipid raft domains. In this context, we address the preparation of (1) raft-mimicking giant unilamellar vesicles labeled with a combination of fluorophores that allow for the visualization and comprehensive characterization of those membrane domains and (2) human fibroblasts exhibiting GD phenotype to assess the biophysical properties of biological membrane in living cells using fluorescence microscopy.

A novel method of vesicle preparation by simple dilution of bicelle solution

We prepared phospholipid vesicles in continuous dilution from small phospholipid bilayer bicelles. As vesicles are useful as delivery carriers, it is necessary to develop a method of continuous encapsulation. Bicelles are composed of long-chain 1,2-dimyristoyl-sn-glycero-3-phosphocholine in bilayers and short-chain 1,2-dihexanoyl-sn-glycero-3-phosphocholine as a detergent. Bicelles were diluted in a batch system and a flow system and their membrane properties were compared; sizes were analyzed by dynamic light scattering and membrane properties by fluorescence spectroscopy. In the flow system, the initial bicelles (10−20 nm) coalesced by dilution and hydration of the detergent into large vesicles (>30 nm). Uptake of methylene blue solution revealed the internal aqueous phase. Transmission electron microscopy confirmed the formation of vesicles by dilution. The membrane fluidity of the vesicles showed an ordered gel phase. The results suggest that bicelles can form vesicles by continuous dilution. This method is suitable for encapsulating materials.

Racemic Phospholipids for Origin of Life Studies

Although prebiotic condensations of glycerol, phosphate and fatty acids produce phospholipid esters with a racemic backbone, most experimental studies on vesicles intended as protocell models have been carried out by employing commercial enantiopure phospholipids. Current experimental research on realistic protocell models urgently requires racemic phospholipids and efficient synthetic routes for their production. Here we propose three synthetic pathways starting from glycerol or from racemic solketal (α,β-isopropylidene-dl-glycerol) for the gram-scale production (up to 4 g) of racemic phospholipid ester precursors. We describe and compare these synthetic pathways with literature data. Racemic phosphatidylcholines and phosphatidylethanolamines were obtained in good yields and high purity from 1,2-diacylglycerols. Racemic POPC (rac-POPC, (R,S)-1-palmitoyl-2-oleoyl-3-phosphocholine), was used as a model compound for the preparation of giant vesicles (GVs). Confocal laser scanning fluorescence microscopy was used to compare GVs prepared from enantiopure (R)-POPC), racemic POPC (rac-POPC) and a scalemic mixture (scal-POPC) of (R)-POPC enriched with rac-POPC. Vesicle morphology and size distribution were similar among the different (R)-POPC, rac-POPC and scal-POPC, while calcein entrapments in (R)-POPC and in scal-POPC were significantly distinct by about 10%.

Preparation of Size-controlled Giant Vesicles Under Physiological Conditions.

Giant vesicles (GVs) are model cell membranes that function as tools for the study of cell membrane properties. Recently, researchers have been calling for GVs of specific sizes for use in studies with precise needs. In this paper, we report a method of forming GVs of specific sizes by using an agarose-swelling approach. The resulting GVs had a narrow size distribution and were successfully formed under physiological conditions.

Preparation and Characterization of Ascosome Vesicles Loaded with Khellin

Ascorbic acid has a unique role in the prevention and treatment of a large number of chronic diseases, including skin disorders but it can hardly penetrate the skin due to its solubility in water and its high instability. This study explored the formation of phosphatidylcholine-based vesicles upon addition of ascorbyl octanoate or decanoate, and their potential value as drug delivery systems. Khellin, a natural furanochromone with numerous applications in skin pathologies was loaded as model drug in ascosomes. Ascosomes had narrow size distribution, adequate encapsulation efficiency, long-term stability, and antioxidant properties. Increasing amounts of loaded khellin resulted in a reduction of the vesicle average size, without affecting the polydispersity, suggesting a stabilizing effect. Ascorbyl alkanoates produced remarkably different bilayer organizations and different capabilities to accommodate khellin in the hydrophobic pocket of the vesicles. The addition ascorbyl alkanoates reduced the amount of water molecules strongly bound to the polar headgroups. Moreover, the loading of khellin did not induce any significant hydration change in the unilamellar vesicular systems. The narrow size distribution, adequate encapsulation efficiency and long-term stability of ascosomes loaded with khellin, make these nanostructures suitable for dermatological use and other routes of administrations, preserving the biological properties of ascorbic acid.

Exosome-Containing Preparations From Postirradiated Mouse Melanoma Cells Delay Melanoma Growth In Vivo by a Natural Killer Cell–Dependent Mechanism

To investigate the ability of radiation to stimulate exosome release from melanoma cells and to characterize the resulting exosome-containing vesicle preparations for their ability to promote a host antitumor immune response. Cultured B16F10 murine melanoma cells or tumors were irradiated, and secreted extracellular vesicles were isolated and characterized. The exosome-containing vesicle preparations were injected into fresh tumors in syngeneic mice, and tumor growth and infiltrating T cells and natural killer (NK) cells were characterized. Irradiation stimulated exosome release from B16F10 murine melanoma cells. Exosome preparations from irradiated cell culture supernatants were biologically active, as demonstrated by uptake into recipient cells and by the ability to induce dendritic cell maturation and activation in vitro. Intratumoral injection significantly delayed tumor growth in vivo, whereas injection of similar preparations from non irradiated cells had no effect. The antitumor effect was correlated to an increase in interferon gamma-producing tumor-infiltrating NK cells. Pretreatment of the host mice with anti-NK cell antibodies abolished the effect, whereas pretreatment with anti-CD8+ T-cell antibodies did not. Exosomes from irradiated cells, or synthetic mimics, might provide an effective strategy for potentiation of NK cell-mediated host antitumor immunity.

Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment.

The neuroinflammatory state of the central nervous system (CNS) plays a key role in physiological and pathological conditions. Microglia, the resident immune cells in the brain, and sometimes the infiltrating bone marrow-derived macrophages (BMDMs), regulate the inflammatory profile of their microenvironment in the CNS. It is now accepted that the extracellular vesicle (EV) populations from immune cells act as immune mediators. Thus, their collection and isolation are important to identify their contents but also evaluate their biological effects on recipient cells. The present data highlight chronological requirements for EV isolation from microglia cells or blood macrophages including the ultracentrifugation and size-exclusion chromatography (SEC) steps. A non-targeted proteomic analysis permitted the validation of protein signatures as EV markers and characterized the biologically active EV contents. Microglia-derived EVs were also functionally used on primary culture of neurons to assess their importance as immune mediators in the neurite outgrowth. The results showed that microglia-derived EVs contribute to facilitate the neurite outgrowth in vitro. In parallel, blood macrophage-derived EVs were functionally used as immune mediators in spheroid cultures of C6 glioma cells, the results showing that these EVs control the glioma cell invasion in vitro. This report highlights the possibility to evaluate the EV-mediated immune cell functions but also understand the molecular bases of such a communication. This deciphering could promote the use of natural vesicles and/or the in vitro preparation of therapeutic vesicles in order to mimic immune properties in the microenvironment of CNS pathologies.

Coincubation of sperm with epididymal extracellular vesicle preparations from chronic intermittent ethanol-treated mice is sufficient to impart anxiety-like and ethanol-induced behaviors to adult progeny.

We previously reported that paternal preconception chronic ethanol exposure in mice imparts adult male offspring with reduced ethanol drinking preference and consumption, increased ethanol sensitivity, and attenuated stress responsivity. That same chronic ethanol exposure paradigm was later revealed to affect the sperm epigenome by altering the abundance of several small noncoding RNAs, a mechanism that mediates the intergenerational effects of numerous paternal environmental exposures. Although recent studies have revealed that the unique RNA signature of sperm is shaped during maturation in the epididymis via extracellular vesicles (EVs), formal demonstration that EVs mediate the effects of paternal preconception perturbations is lacking. Therefore, in the current study we tested the hypothesis that epididymal EV preparations are sufficient to induce intergenerational effects of paternal preconception ethanol exposure on offspring. To test this hypothesis, sperm from ethanol-naïve donors were incubated with epididymal EV preparations from chronic ethanol (Ethanol EV-donor) or control-treated (Control EV-donor) mice prior to invitro fertilization (IVF) and embryo transfer. Progeny were examined for ethanol- and stress-related behaviors in adulthood. Ethanol EV-donors imparted reduced body weight at weaning and imparted modestly increased limited access ethanol intake to male offspring. Ethanol-EV donors also imparted increased basal anxiety-like behavior and reduced sensitivity to ethanol-induced anxiolysis to female offspring. Although Ethanol EV-donor treatment did not recapitulate the ethanol- or stress-related intergenerational effects of paternal ethanol following natural mating, these results demonstrate that coincubation of sperm with epididymal EV preparations is sufficient to impart intergenerational effects of ethanol through the male germline. This mechanism may generalize to the intergenerational effects of a wide variety of paternal preconception perturbations.