Preparation Of Vesicles Research Articles

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.

Large-scale preparations of small extracellular vesicles from conditioned media of mesenchymal stromal cells modulate therapeutic impacts on a newly established Graft-versus-Host-Disease model in batch dependent manners

Background & Aim Extracellular vesicles (EVs) harvested from supernatants of humane adult bone marrow-derived mesenchymal stem/stromal cells (MSCs) can suppress acute inflammatory cues in a variety of different diseases, including Graft-versus-Host Disease (GvHD) and ischemic stroke. Furthermore, they can promote regeneration of affected tissues. Following a successful clinical treatment attempt of a steroid refractory GvHD patient, we intend to optimize MSC-EV production strategies for further clinical applications. As we observed functional differences of independent MSC-EV preparations in vitro, we aimed to adopt an in vivo GvHD model for the more advanced functional testing of different MSC-EV preparations. Methods, Results & Conclusion To this end we set up a bone marrow transplantation mouse model in which endogenous bone marrow was myeloablated by ionizing irradiation (IIR). GvHD was induced by the transplantation of major histocompatibility mismatched allogeneic spleen-derived murine T cells. If not treated otherwise, myeloablated mice developed severe GvHD symptoms. The GvHD symptoms were effectively suppressed, when MSC-EV preparations were applied at 3 consecutive days, which exerted immune modulatory effects in a mixed-lymphocyte reaction assay. MSC-EV preparations lacking in vitro immune modulating activities, however, hardly improved the symptoms of the GvHD mice. Thus, our results demonstrate that not all MSC-EV preparations harvested from adult bone marrow-derived MSCs contain the same therapeutic potential. Thus, successful transplantation of MSC-EVs into the clinics requires a platform allowing identification of MSC-EV preparations with sufficient therapeutic, most probably immune modulating activities.

Thermoresponsive Block Copolymer Vesicles by Visible Light-Initiated Seeded Polymerization-Induced Self-Assembly for Temperature-Regulated Enzymatic Nanoreactors.

Block copolymer vesicles loaded with active compounds have been employed as decent candidates to mimic complex biological systems that attract considerable interest in different research communities. We herein report a visible light-initiated seeded reversible addition-fragmentation chain transfer (RAFT)-mediated polymerization-induced self-assembly (PISA) for in situ preparation of enzyme-loaded cross-linked block copolymer vesicles without compromising the bioactivity. Permeability of the vesicular membrane can be regulated through changing the solution temperature, allowing further control over the enzymatic reaction rate of enzyme-loaded vesicles. Finally, non-cross-linked thermoresponsive block copolymer vesicles that can transform into worm-like micelles at low temperature are also prepared by this method, allowing the release of bimacromolecules from the vesicles under mild conditions.

Nanodomains Persist to much Higher Temperatures than Large Scale Phase Separation in Giant Plasma Membrane Vesicles and Can Respond Differently to Alterations of Plasma Membrane Lipid Composition

The formation and properties of liquid ordered (Lo) lipid domains (rafts) in the plasma membrane is still poorly understood. Giant plasma membrane vesicles (GPMV) derived from mammalian cells can undergo large scale phase separations into co-existing Lo and disordered lipid (Ld) domains with properties similar to domains in artificial lipid vesicles containing simple lipid mixtures. However, large scale phase separation in GPMV detected by light microscopy only occurs at low temperature. We compared large domain formation detected by light microscopy to nanodomain formation in GPMV using a FRET assay. We found that nanodomains can persist to physiological temperatures, much higher temperatures than large scale phase separation. Altering sterol or phospholipid structure in the plasma membrane outer leaflet of intact mammalian cells by cyclodextrin-catalyzed lipid exchange altered both GPMV lipid composition and the ability of GPMV to form ordered domains. Phospholipids and sterols that either stabilize or destabilize ordered domain formation in artificial lipid vesicles had a similar effect on ordered domain stability in GPMV. Nanodomain and large scale domain formation were generally stabilized or destabilized by lipid substitutions to a similar (but not identical) extent. These results show it is possible to alter the lipid composition and properties of membrane vesicles derived from plasma membrane. Other experiments found that after transfection and expression of soluble GFP in mammalian cells, GPMVs contained GFP in their lumen. Because membrane vesicles can be used to deliver biomolecules to cells, combining an analogous expression step with lipid exchange might allow preparation of plasma membrane vesicles with improved abilities to deliver cytosolic molecules to other cells.

Charge Detection Mass Spectrometry Measurements of Exosomes and other Extracellular Particles Enriched from Bovine Milk.

The masses of particles in a bovine milk extracellular vesicle (EV) preparation enriched for exosomes were directly determined for the first time by charge detection mass spectrometry (CDMS). In CDMS, both the mass-to-charge ratio (m/z) and z are determined simultaneously for individual particles, enabling mass determinations for particles that are far beyond the mass limit (∼1.0 MDa) of conventional mass spectrometry (MS). Particle masses and charges span a wide range from m ∼ 2 to ∼90 MDa and z ∼ 50 to ∼1300 e (elementary charges) and are highly dependent upon the conditions used to extract and isolate the EVs. EV particles span a continuum of masses, reflecting the highly heterogeneous nature of these samples. However, evidence for unique populations of particles is obtained from correlation of the charges and masses. An analysis that uses a two-dimensional Gaussian model, provides evidence for six families of particles, four of which having masses in the range expected for exosomes. Complementary proteomics measurements and electron microscopy (EM) imaging are used to further characterize the EVs and confirm that these samples have been enriched in exosomes. The ability to characterize such extremely heterogeneous mixtures of large particles with rapid, sensitive, and high-resolution MS techniques is critical to ongoing analytical efforts to separate and purify exosomes and exosome subpopulations. Direct measurement of each particle's mass and charge is a new means of characterizing the physical and chemical properties of exosomes and other EVs.

Supported Lipid Bilayer Formation: Beyond Vesicle Fusion.

Supported lipid bilayers (SLBs) are cell-membrane-mimicking platforms that can be formed on solid surfaces and integrated with a wide range of surface-sensitive measurement techniques. SLBs are useful for unravelling details of fundamental membrane biology and biophysics as well as for various medical, biotechnology, and environmental science applications. Thus, there is high interest in developing simple and robust methods to fabricate SLBs. Currently, vesicle fusion is a popular method to form SLBs and involves the adsorption and spontaneous rupture of lipid vesicles on a solid surface. However, successful vesicle fusion depends on high-quality vesicle preparation, and it typically works with a narrow range of material supports and lipid compositions. In this Feature Article, we summarize current progress in developing two new SLB fabrication techniques termed the solvent-assisted lipid bilayer (SALB) and bicelle methods, which have compelling advantages such as simple sample preparation and compatibility with a wide range of material supports and lipid compositions. The molecular self-assembly principles underpinning the two strategies and important experimental parameters are critically discussed, and recent application examples are presented. Looking forward, we envision that these emerging SLB fabrication strategies can be widely adopted by specialists and nonspecialists alike, paving the way to enriching our understanding of lipid membrane properties and realizing new application possibilities.

Measuring Lipid Transfer Protein Activity Using Bicelle-Dilution Model Membranes.

In vitro assessment of lipid intermembrane transfer activity by cellular proteins typically involves measurement of either radiolabeled or fluorescently labeled lipid trafficking between vesicle model membranes. Use of bilayer vesicles in lipid transfer assays usually comes with inherent challenges because of complexities associated with the preparation of vesicles and their rather short "shelf life". Such issues necessitate the laborious task of fresh vesicle preparation to achieve lipid transfer assays of high quality, precision, and reproducibility. To overcome these limitations, we have assessed model membrane generation by bicelle dilution for monitoring the transfer rates and specificity of various BODIPY-labeled sphingolipids by different glycolipid transfer protein (GLTP) superfamily members using a sensitive fluorescence resonance energy transfer approach. Robust, protein-selective sphingolipid transfer is observed using donor and acceptor model membranes generated by dilution of 0.5 q-value mixtures. The sphingolipid transfer rates are comparable to those observed between small bilayer vesicles produced by sonication or ethanol injection. Among the notable advantages of using bicelle-generated model membranes are (i) easy and straightforward preparation by means that avoid lipid fluorophore degradation and (ii) long "shelf life" after production (≥6 days) and resilience to freeze-thaw storage. The bicelle-dilution-based assay is sufficiently robust, sensitive, and stable for application, not only to purified LTPs but also for LTP activity detection in crude cytosolic fractions of cell homogenates.

Preparation of Bacterial Outer Membrane Vesicles for Characterisation of Periplasmic Proteins in Their Native Environment.

Bacterial outer membrane vesicles (OMVs) are naturally formed by budding from the outer membrane of Gram-negative bacteria. OMVs consist of a lipid bilayer identical in composition to the original outer membrane and contain periplasmic content within their lumen. Enriched with specific envelope proteins, OMVs make for an excellent native-like platform to study these proteins in-situ using biophysical methods. Here, we describe in detail the preparation of OMVs from Escherichia coli, which are luminally enriched with periplasmic proteins and uniformly labeled with stable isotopes (2H and 15N), suitable for the subsequent characterisation of proteins at atomic resolution in their native environment by solution-state NMR spectroscopy. The ability to perform structural studies of periplasmic components in-situ clears the way to reaching an in-depth understanding of the functional and mechanistic details of this unique cellular compartment.

Differential or Curvature Stress? Modus Vivendi

Differential or Curvature Stress? Modus Vivendi

A novel method to prepare lipid vesicles as carrier of hydrophilic bioactive substances

A novel method was presented to synthesize self-assembly lipid vesicles as carrier of hydrophilic bioactive substances. Based on the interaction of nonpolar groups of surfactants in the transmembrane process, the surface of particles was reassembled in the particles transfer process. In this method, water droplets loaded with hydrophilic bioactive substances in inverted microemulsion were transformed into surfactant solution through an oil/water interface contained with rich surfactants by using centrifugal casting process, and then unilamellar nanoscale lipid vesicles loaded with hydrophilic bioactive substances were formed. The water droplets in the inverted microemulsion were used as soft template to load hydrophilic substances. The dimensions of the lipid vesicles can be controlled by adjusting size of droplets in the microemulsion. Some important parameters for the preparation of lipid vesicles were optimized. The encapsulation efficiency of hydrophilic bioactive substances in the lipid vesicles could be as high as 50%, and it is much higher than that prepared by other method. This method could be used to synthesize size-control lipid vesicles, and the lipid vesicles possess high encapsulation efficiency of bioactive substances and high stability.

Lipid Vesicle Preparation Using W/O/W Multiple Emulsions Via Solvent Evaporation: The Effect of Emulsifiers on the Entrapment Yield of Hydrophilic Materials

AbstractWe studied the effects of emulsifiers on the entrapment yields of a hydrophilic material, calcein, in lipid vesicles formed using the multiple emulsion method. The primary emulsion (W1/O) was prepared via sonication while the secondary emulsification that produced the W1/O/W2 multiple emulsions was achieved using the microchannel (MC) emulsification technique. The emulsifiers used in the continuous (W2) phase were Tween® 80, Pluronic® F68, and bovine serum albumin (BSA). Lipid vesicles formed via solvent evaporation of the multiple emulsion droplets had an average diameter of about 180 nm, similar to the size of the water droplets in the primary W1/O emulsions. The entrapment yields of calcein increased with decreasing concentrations of Tween® 80 but decreased with decreasing concentrations of Pluronic® F68 and BSA. The effects of type and concentration of emulsifier were considered in relation to three possible mechanisms: (i) destabilization/solubilization of lipid bilayers by emulsifiers, (ii) reversed‐micellar transport of W1 contents from internal to external water phases through the O phase, and (iii) release of inner water (W1) contents into the continuous (W2) phase via the instability of W1/O/W2 and leakage of W1 contents. Using the food grade emulsifier Tween® 80 at a low concentration of about 0.1 or 0.05 wt%, calcein as a model hydrophilic material could be efficiently entrapped (ca. 80%) in homogenously dispersed lipid vesicles.

Characterization of phospholipid vesicles containing lauric acid: physicochemical basis for process and product development

Lauric acid (LAH) strongly inhibits the growth of acne-causing bacteria. LAH is essentially water-insoluble and the solubility of laurate (LA) salts are medium and temperature dependent. Hence, LAH/LA preparations are difficult to formulate. Here we fully characterized phospholipid vesicles containing up to 50 mol% LAH. Vesicles of dipalmitoylphosphatidylcholine (DPPC) containing LAH, at pHs 7.4 and 5.0, were characterized measuring size, charge, bilayer phase transition temperature (Tm) and permeability of water-soluble probes. Small angle X-ray scattering and cryotransmission electron microscopy showed multilamellar vesicles at low LAH %. Increasing LAH % had a negligible effect on particle size. An internal aqueous compartment in all vesicle's preparations, even at equimolar DPPC: LAH fractions, was demonstrated using water-soluble probes. At pH 5.0, the interaction between DPPC and LAH increased the Tm and phase transition cooperativity showing a single lipid phase formed by hydrogen-bonded DPPC: LAH complexes. At pH 7.4, vesicles containing 50 mol% LAH exhibited distinct phases, ascribed to complex formation between LAH and LA or LAH and DPPC. LAH incorporated in the vesicles minimally permeated a skin preparation at both pHs, indicating that the primary sites of LAH solubilization were the skin layers. These results provide the foundations for developing processes and products containing DPPC: LAH.

Preparation and Properties of Semi-Self-Assembled Lipopeptide Vesicles.

Novel lipopeptide vesicles are prepared from self-assembled nanomembranes through an extrusion method. The size of vesicles can be controlled by the pore diameter of the extrusion filter. The vesicles are rather stable because hydrogen bonds exist among the peptide headgroups. When doxorubicin hydrochloride (DOX·HCl) is encapsulated in the vesicles, it could be released sustainably, and its side effect would also be reduced due to encapsulation. The leakage rate of DOX·HCl depends on the pH via charge regulation. As drug carriers, lipopeptide vesicles have been proved to have nontoxicity to normal cells. A magnetic surfactant CH3(CH2)14CH2N(CH3)3+ [FeCl3Br]- (CTAFe) was mixed with lipopeptide to modify the vesicles. Also, the results demonstrated that the vesicles is endowed with magnetic property after the addition of CTAFe. We believe that the strategy of lipopeptide vesicle preparation would enrich the drug carrier family and expand the application of lipopeptide materials.

Purification of High Yield Extracellular Vesicle Preparations Away from Virus.

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

Direct Measurement of Kinetic Parameters of ABCG2-Dependent Transport of Natural Flavonoids Using a Fluorogenic Substrate.

Flavonoids are an important part of the human diet since plant-derived polyphenols and the mechanisms governing their pharmacokinetics are important both due to their own nutriceutical activity and the potential for food-drug interactions. A central determinant of absorption and distribution of flavonoids in the human body is the ATP-binding cassette transporter ABCG2, expressed in gut epithelium and other barrier tissues. While flavonoids were previously identified as substrates and/or inhibitors of this protein, precise enzyme kinetic calculations of affinity and activity parameters are rare due to the lack of suitable experimental models. We present a novel method that allows the direct measurement of kinetic constants for ABCG2-mediated cellular efflux of natural flavonoids thanks to the application of fluorogenic 2-aminoethyl diphenylborinate, which reacts with intracellular flavonoids forming a fluorescent, nonmembrane-permeable conjugate, thus making it possible to measure the intracellular substrate concentration throughout the experiment. Our studies were performed in Madin-Darby canine kidney II-derived cell lines expressing human ABCG2 and involve substrate efflux from whole, unmodified cells, precluding the need for plasma membrane vesicle preparation. We present methods for calculation of enzyme kinetic constants by measuring substrate concentration at efflux-influx equilibrium or during efflux from preloaded cells, and we obtained K m values of 137 µM for quercetin, 36 µM for kaempferol, and 348 µM for luteolin. Our method also allows direct verification of the transport inhibition mechanism and potentially the structure-activity relationship in substrates. SIGNIFICANCE STATEMENT: The study presents the first direct calculation of kinetic constants for enzyme-mediated active transport of natural flavonoids in a whole-cell assay, using a fluorogenic compound to measure intracellular substrate concentrations at specific time points. It has implications for nutriceutical use of polyphenols, mechanisms of food-drug interactions, and studies on absorption/distribution-determining membrane transporters, allowing a quantitative approach to pharmacokinetics of flavonoid transport across barrier tissues.

Effect of Core Cross-linking on the Physical Properties of Poly(dimethylsiloxane)-Based Diblock Copolymer Worms Prepared in Silicone Oil

A trithiocarbonate-capped poly(dimethylsiloxane) (PDMS) precursor is chain-extended via reversible addition–fragmentation chain transfer dispersion polymerization of 2-(dimethylamino)ethyl methacrylate (DMA) in decamethylcyclopentasiloxane (D5) silicone oil at 90 °C. For a fixed mean degree of polymerization (DP) of 66 for the PDMS steric stabilizer block, targeting core-forming PDMA block DPs of between 105 and 190 enables the preparation of either well-defined worms or vesicles at a copolymer concentration of 25% w/w. The as-synthesized linear PDMS66–PDMA100 worms exhibit thermoresponsive behavior in D5, undergoing a worm-to-sphere transition on heating to 100 °C. Variable temperature 1H NMR spectroscopy indicates that this thermal transition is driven by reversible solvent plasticization of the PDMA cores. This change in copolymer morphology is characterized by transmission electron microscopy (TEM) studies, variable temperature dynamic light scattering and small-angle X-ray scattering experiments. Oscillatory rheology studies indicate that degelation occurs at 32 °C, but shear-induced polarized light imaging measurements suggest that full conversion of worms into spheres requires significantly higher temperatures (∼110 °C). 1,2-Bis(2-iodoethoxy)ethane (BIEE) is evaluated as a cross-linker for PDMS66–PDMAx diblock copolymer nano-objects in D5. This bifunctional reagent quaternizes the tertiary amine groups on the DMA residues within the worm cores, introducing cross-links via the Menshutkin reaction. TEM studies confirm that such covalently-stabilized worms no longer undergo a worm-to-sphere transition when heated to 100 °C. Kinetic studies performed on PDMS66–PDMA176 vesicles suggest that cross-linking requires approximately 13 h at 20 °C to ensure that these nano-objects remain intact when dispersed in chloroform, which is a good solvent for both blocks. Oscillatory rheology studies of a PDMS66–PDMA100 worm gel indicated that covalent stabilization using a BIEE/DMA molar ratio of 0.15 increased its dynamic elastic modulus (G′) by almost two orders of magnitude. Furthermore, such cross-linked worms exhibit a much lower critical gelation concentration (∼2% w/w) compared to that of the linear precursor worms (∼12% w/w).

Multi-drug hybrid delivery systems with distinct release profiles based on gelatin/collagen containing vesicles derived from block copolymers

Hybrid delivery systems can release multiple drugs with different profiles and have several applications, including skin dressing. In this work, the co-solvent technique was used for the preparation of nanometric vesicles based on poly(styrene-b-ethylene oxide) block copolymer (BCPVs) containing adapalene (AD). The BCPVs were incorporated into collagen and gelatin matrices together with free AD and silver sulfadiazine (SSD). The AD content of BCPVs and their release capacity were analyzed by using ultraviolet-visible spectroscopy (UV–Vis). The gelatin and collagen matrices were evaluated for their ability to release AD and SSD through an in vitro release study. The obtained results confirmed that the production of empty and AD-loaded BCPVs was viable. The degree of AD encapsulation in BCPVs was 9.0% and the in vitro test revealed a constant, slow, and prolonged release of AD content from AD-loaded BCPVs. The combination of free and encapsulated multiple drugs in hybrid delivery systems based on gelatin and collagen matrices was shown to act as a skin dressing that combined the progressive release of large amounts of drugs within the first hours of use (to restrict infection) with a more prolonged and slow release of AD to enhance skin healing.

The Application of Microfluidics in Preparing Nano Drug Delivery Systems

The Application of Microfluidics in Preparing Nano Drug Delivery Systems

Circulating microRNAs in adrenal tumors.

Circulating microRNAs represent promising minimally invasive markers of several diseases including tumors. As the preoperative diagnosis of different adrenal tumors is difficult, for example, diagnosis of adrenocortical or adrenomedullary malignancy, circulating microRNAs might be helpful in their clinical management. Observations regarding the applicability of circulating microRNAs isolated both from unfractionated plasma or serum and from extracellular vesicle preparations for the diagnosis of adrenocortical malignancy have been published. Data show that circulating microRNA might be exploited for monitoring adrenocortical cancer progression. Circulating microRNA profiles of adrenal myelolipoma have also been published that might be useful for differentiating adrenocortical cancer and adrenal myelolipoma in dubious cases. In this review, recent advances in the field of circulating microRNAs in adrenal tumors are discussed.