Vesicle Size Distribution Research Articles

A standardized method for plasma extracellular vesicle isolation and size distribution analysis.

The following protocol describes our workflow for isolation and quantification of plasma extracellular vesicles (EVs). It requires limited sample volume so that the scientific value of specimens is maximized. These steps include isolation of vesicles by automated size exclusion chromatography and quantification by tunable resistive pulse sensing. This workflow optimizes reproducibility by minimizing variations in processing, handling, and storage of EVs. EVs have significant diagnostic and therapeutic potential, but clinical application is limited by disparate methods of data collection. This standardized protocol is scalable and ensures efficient recovery of physiologically intact EVs that may be used in a variety of downstream biochemical and functional analyses. Simultaneous measurement quantifies EV concentration and size distribution absolutely. Absolute quantification corrects for variations in EV number and size, offering a novel method of standardization in downstream applications.

Improved exosome isolation methods from non-small lung cancer cells (NC1975) and their characterization using morphological and surface protein biomarker methods.

This study has demonstrated improved methods for isolating exosomes from non-small lung cancer cells, which address the problems characterized by exosome morphological and chemical methods. To improve the isolation methods, cells from the NCI 1975 cell line were used as the source for exosomes. The isolation processes were carried out using serial isolation techniques in addition to specific preservation tools. The isolated exosomes were characterized using transmission electron microscopy (TEM), and scanning electron microscopy (SEM) was added for further assurance of the investigation results. The statistical analysis results showed that the size distributions of apoptotic vesicles (APV) 450nm and necrotic bodies (NCB) 280nm (extracellular vesicles) were significantly different from exosomes (P < 0.001). In contrast, the exosome size distribution was not significantly different from the published exosome sizes, as demonstrated by statistical analysis tools. This study confirmed the improved methods for isolating exosomes that make exosomes accessible for use in the diagnosis and prognosis of non-small cell lung cancer (NSCLC).

Fluctuations of active membranes with nonlinear curvature elasticity

Biological membranes and vesicles play a vital role in critical physiological processes like endocytosis, cell division, and cell motility. Over decades, statistical and continuum mechanics studies have provided phenomenal insights into the mechanics of these membranes and their biophysical implications. However, most studies, until recently, have focused entirely on passive (or “dead”) membranes that exhibit only equilibrium thermal fluctuations. In this work, we acknowledge the growing consensus that the active nature of membranes is vital to their biophysical functioning. Active membranes contain proteins that are fueled by energy from external sources such as adinosine triphosphate hydrolysis or light. These proteins exert forces on the membrane during their activity causing the membrane to exhibit fluctuations that are non-thermal in origin, thus driving them away from equilibrium. In short, active membranes are “alive” with their own energy source capable of circumventing equilibrium considerations. In this paper, we present a theory for active membranes based on principles of continuum mechanics and a variational formulation that (we hope) provides a unifying framework to understand seemingly disparate but insightful prior works in this field. Using our developed continuum model, we study the statistical mechanics of active closed membranes or vesicles. A specific highlight of our work is that we incorporate nonlinear curvature elasticity in our continuum theory and obtain closed-form results for the fluctuation spectra for active vesicles. Our numerical results reveal a rather unanticipated interplay of active forces, surface tension as well as nonlinear elasticity for small vesicles. To gain insights into the biophysical implications of activity in vesicles, we revisit the problem of determining the vesicle size distribution by assuming that the active vesicles are close to thermal equilibrium. Our numerical calculations show that active forces may significantly impact vesicle size distribution. A tantalizing implication of this finding is that by tuning active forces, active vesicles may attain vesicle size distributions that are improbable for passive vesicles governed only by thermal fluctuations.

Longitudinal characterization of circulating extracellular vesicles and small RNA during simian immunodeficiency virus infection and antiretroviral therapy.

Latent infection by HIV hinders viral eradication despite effective antiretroviral treatment (ART). Among proposed contributors to viral latency are cellular small RNAs that have also been proposed to shuttle between cells in extracellular vesicles. Thus, we profiled extracellular vesicle small RNAs during different infection phases to understand the potential relationship between these extracellular vesicle associated small RNAs and viral infection. A well characterized simian immunodeficiency virus (SIV)/macaque model of HIV was used to profile extracellular vesicle enriched blood plasma fractions harvested during preinfection, acute infection, latent infection/ART treatment, and rebound after ART interruption. Measurement of extracellular vesicle concentration, size distribution, and morphology was complemented with qPCR array for small RNA expression, followed by individual qPCR validations. Iodixanol density gradients were used to separate extracellular vesicle subtypes and virions. Plasma extracellular vesicle particle counts correlated with viral load and peaked during acute infection. However, SIV gag RNA detection showed that virions did not fully explain this peak. Extracellular vesicle microRNAs miR-181a, miR-342-3p, and miR-29a decreased with SIV infection and remained downregulated in latency. Interestingly, small nuclear RNA U6 had a tight association with viral load peak. This study is the first to monitor how extracellular vesicle concentration and extracellular vesicle small RNA expression change dynamically in acute viral infection, latency, and rebound in a carefully controlled animal model. These changes may also reveal regulatory roles in retroviral infection and latency.

A standardized method for plasma extracellular vesicle isolation and size distribution analysis

A standardized method for plasma extracellular vesicle isolation and size distribution analysis

Complex decompression and fragmentation of mingled andesite magmas driving multi-phase Plinian eruptions at Mt. Taranaki, New Zealand

Estimating the kinetics of andesite magma vesiculation and crystallization inside volcanic plumbing systems is key for unraveling andesite Plinian eruption dynamics. The conduit kinetics provide the necessary input data for estimating the magma flow rates driving magma ascent and the fragmentation mechanisms controlling shifts in eruption explosivity and style. This information is crucial for increasing knowledge on expected hazards and for developing realistic eruption scenarios. In this work, we estimate conduit magma vesiculation and crystallization kinetics during the 3300 cal BP Upper Inglewood Plinian eruptive episode of Mount Taranaki, New Zealand. This episode comprised (i) low-intensity, conduit-opening phases of dome-collapse PDCs; (ii) pre-climactic, highly explosive phases of diverse PDCs, of up to violent 18-km-runout lateral blasts; (iii) climactic phases of steady 22-km-high Plinian eruption columns; and (iv) waning phases of column-collapse PDCs. By employing synchrotron microtomography, combined with mineral/glass chemistry and electron-microscopy, we quantified 3D vesicle and crystal size and shape distributions in juvenile pyroclasts over time, and corresponding number densities ranging from 1.1 × 105 to 2.5 × 106 mm−3 for vesicles, and from 8.0 × 104 to 5.1 × 106 mm−3 for crystals. Our results indicate that tapping of chemically alike yet rheologically contrasting magmas over a multi-phase andesite eruptive episode is linked to: (a) mafic magma recharge and differentiation in multiple storage reservoirs at distinct crustal levels, (b) stepwise to rapid magma decompression while mingling, producing variable pre- and syn-eruptive degassing and crystallization, and (c) syn-eruptive changes in melt viscosity, strain rate, localized shear deformation, and conduit geometry. The earliest and least explosive eruptive phases (≈ 2 × 106 kg s−1) were produced at the slowest rates of magma decompression (0.3–0.6 MPa s−1), ascent (0.01–0.02 m s−1) and strain (< 0.002 s−1), driven by volatile diffusion and exsolution. All subsequent pre-climactic and Plinian phases (4 × 107–1 × 108 kg s−1) were produced at either rapid or intermittent rates of magma decompression (2.0–6.0 MPa s−1), ascent (0.06–0.2 m s−1) and strain (> 0.003–0.010 s−1), powered by combined magma volatile supersaturation and delayed disequilibrium degassing, decompression-induced microlite crystallization and rapid heterogeneous vesiculation kinetics, shear deformation and magma mingling. These processes enabled complex fragmentation mechanisms of the rheologically most homogeneous magmas.

Application of Electrochemically Reduced Water for New No-Rinse Shampoo: Design and Optimization Using Response Surface Methodology

Coronavirus disease (COVID-19) cases are continuing to rise around the world, with more than 607 million confirmed cases and more than 6.51 million deaths as of September 2022. The World Health Organization (WHO) has emphasized the importance of hygiene in the ongoing COVID-19 pandemic, especially in elderly and bedridden patients. The use of no-rinse shampoo represents a simple change in hair-cleansing products. Daily hospital hair washing for the elderly, bedridden, and ICU patients would be more readily adopted. The objective of this study was to design and optimize a new no-rinse shampoo based on electrochemically reduced water (ERW) using response surface methodology. The relationship between coconut-based surfactant mixtures in a no-rinse shampoo and the resulting physicochemical properties, effectiveness (antibacterial and antifungal activity), and stability of the shampoo was investigated. The vesicle size, size distribution, zeta potential, conductivity, pH, foamability, wetting time, turbidity, and stability of the model formulation were optimized. The optimal formulation with the appropriate physicochemical properties and stability was selected. The effectiveness of the optimal formulation was compared to that of commercially available products. The dry shampoo (DS4) containing 3.5% of the mixed detergent with ERW may prevent infection by Escherichia coli, Staphylococcus epidermidis, Staphylococcus aureus, and Candida albicans. The no-rinse shampoo based on ERW successfully demonstrated good stability in addition to efficacy in terms of antibacterial and antifungal activity. The treated hair fiber was not significantly different from that of the intact hair fiber. Under the scanning electron microscopy (SEM) and the atomic force microscopy (AFM), the cuticle layer of the treated hair fiber was not damaged. Thus, no-rinse shampoos may reduce the time of hair rinsing and improve the quality of life of caregivers.

Determining small-molecule permeation through lipid membranes.

The passive permeability of cell membranes is of key importance in biology, biomedical research and biotechnology as it determines the extent to which various molecules such as drugs, products of metabolism, and toxins can enter or leave the cell unaided by dedicated transport proteins. The quantification of passive solute permeation is possible with radio-isotope distribution experiments, spectroscopic measurements and molecular dynamics simulations. This protocol describes stopped-flow fluorimetry measurements performed on lipid vesicles and living yeast cells to estimate the osmotic permeability of water and solutes across (bio)membranes. Encapsulation of the fluorescent dye calcein into lipid vesicles allows monitoring of volume changes upon osmotic shifts of the medium via (de)quenching of the fluorophore, which we interpret using a well-defined physical model that takes the dynamics of the vesicles into account to calculate the permeability coefficients of solutes. We also present analogous procedures to probe weak acid and base permeability in vesicles and cells by using the read-out of encapsulated or expressed pH-sensitive probes. We describe the preparation of synthetic vesicles of varying lipid composition and determination of vesicle size distribution by dynamic light scattering. Data on membrane permeation are obtained using either conventional or stopped-flow kinetic fluorescence measurements on instruments available in most research institutes and are analyzed with a suite of user-friendly MATLAB scripts ( https://doi.org/10.5281/zenodo.6511116 ). Collectively, these procedures provide a comprehensive toolbox for determining membrane permeability coefficients in a variety of experimental systems, and typically take 2-3 d.

The origins of transparent and non-transparent white pumice: A case study of the 52 ka Maninjau caldera-forming eruption, Indonesia

The VEI 7 (220–250 km3), a 52 ka eruption of the Maninjau caldera in Indonesia produced two distinct types of white pumices: transparent (TWP) and non-transparent (NTWP). These pumices were identified on outcrops, based on their qualitative traits, without the use of laboratory analyses (geochemical and textural). TWPs are typically fragile, irregular in shape, and have a transparent glass of vesicle walls around visible large vesicles, whereas NTWPs are relatively strong, blocky in shape, and have a non-transparent glass due to invisible small vesicles. Based on chemical and petrographical analyses, both pumice types were crystal-poor (avg. of 3.3%), with similar mineralogy, glass compositions (avg. 78.5 wt% SiO2), and plagioclase core compositions (avg. of An28). From component analysis, we found that the abundance of TWP decreased towards the upper stratigraphic ignimbrite deposits, along with an increase in NTWP, grey pumice, banded pumice, and lithic (non-juvenile) contents. We defined two vesicle populations with a threshold diameter of 0.1 mm based on bimodal vesicle size distributions by textural analysis for TWP and NTWP. Large vesicles correspond to preexisting bubbles that form in the magma chamber (pheno-vesicle, > 0.1 mm), whereas small vesicles in the groundmass (matrix-vesicle, < 0.1 mm) are attributed to second nucleation in the conduit during the eruption. We performed a comparison using vesicle data (pheno- and matrix-vesicles) for these two white pumice types. Pheno- and matrix-vesicularity showed a negative correlation. Thus, the boundary between TWP and NTWP was also quantitatively confirmed by the volume fraction ratio of the pheno- and matrix-vesicles. This evidence suggests that TWP originated from pheno-bubble-dominated magma (at least 0.1% volume fraction of pheno-bubbles in the magma chambers), whereas NTWP originated from pheno-bubble-poor magma. The variation in the abundance of TWP and NTWP in the stratigraphic positions implies the rough stratification of pre-existing bubbles (pheno-bubbles) in the pre-eruptive magma chamber, where the overpressure developed to trigger and drive the eruption. The two regimes control the formation of matrix-vesicles: (1) a pheno-bubble-controlled regime, and (2) a decompression-controlled regime. Based on a correlation between pheno-vesicle number density (PVND) and matrix-vesicle number density (MVND) together with numerical calculations based on a previous study, we suggest that the TWPs with the highest pheno-vesicularity formed under conditions corresponding to the first regimes, while the NTWPs formed in the second regime, where MVND is mostly determined by decompression rate. We also argue that NTWP is the only representative juvenile for estimating magma decompression rate, as the MVND value in TWP do not represent the actual second nucleation processes. Therefore, the occurrence of NTWP in the entire deposits with a relatively similar MVNDs value might suggest that the early and final eruption stages experienced a relatively similar magma decompression rate.

Effects of sugar concentration on the electroporation, size distribution and average size of charged giant unilamellar vesicles.

We investigated the effects of sugar concentration on the electroporation, size distribution and average size of giant unilamellar vesicles (GUVs). GUVs were prepared from 40mol% of 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) and 60mol% of 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipids. Pulsed electric field was applied to the 40%DOPG/60%DOPC-GUVs and it induced lateral electric tension (σc) in the membranes of vesicles. The σc-induced probability of rupture (Ppore) and the rate constant of rupture (kp) of GUVs under the sugar concentration, c = 40, 100 and 300mM, were determined. Both the Ppore and kp increased with the increase of σc, but higher tension was required to generate the same values of Ppore and kp with increasing c. We also investigated average sizes of GUVs from the size distribution of vesicles under various sugar concentrations. With the increase of c, the peak of the size distribution histograms shifted to the region of smaller vesicles. The average size decreased 1.6-fold when c increased from 10 to 300mM. These investigations help to understand various biomedical, biophysical, and biochemical processes in vesicles and cells. Electroporation, size distribution and average size of charged GUVs were investigated under various sugar concentrations. The sugar concentration influences the electroporation of vesicles and the average size of GUVs.

Effects of hydrocarbon chain on the vesicle size distribution, kinetics of average size, bending modulus, and elastic modulus of lipid membranes.

The effects ofthe hydrocarbon chain of lipids on the size distribution of giant unilamellar vesicles (GUVs), kinetics of average size, bending modulus, and elastic modulus of membranes have been investigated. 1,2-dioleoyl-sn-glycero-3-phosphocholine (18:1 (Δ9-Cis) PC (DOPC)), 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (16:1 (Δ9-Cis) PC), and 1,2-ditridecanoyl-sn-glycerol-3-phosphocholine (13:0 PC (DTPC)) lipids were considered. The number of hydrocarbons in a chain of the corresponding lipid was 18, 16, and 13. GUVs were prepared using the natural swelling method under incubation times of 20, 40, 60, 90, 120, and 180min. The size distribution of vesicles was fitted using the lognormal distribution. The average sizes of DOPC, 16:1 (Δ9-Cis) PC, and DTPC-GUVs increased with the incubation time until 120min, and then remained steady at 16.7 ± 0.2, 15.2 ± 0.4 and 12.0 ± 0.3µm for the corresponding lipids. The average size at equilibrium state increased with the number of hydrocarbons. The incubation time-dependent average size was fitted with an exponential growth equation, and then the kinetic constants of 0.028 ± 0.004, 0.036 ± 0.007, and 0.083 ± 0.009min-1 for DOPC, 16:1 (Δ9-Cis) PC, and DTPC-GUVs, respectively, were obtained. The equilibrium size distribution was fitted by the theoretical equation, and the bending modulus for DOPC, 16:1 (Δ9-Cis) PC, and DTPC membranes were 19.5 ± 0.2, 18.5 ± 0.1 and 14.3 ± 0.1 kBT, respectively. The bending modulus increased with the number of hydrocarbons. The elastic modulus of these membranes was 261 mN/m with a 4% fluctuation. The correlation between the average size and the square root of the bending modulus was supported by theoretical analysis.

Precision size and refractive index analysis of weakly scattering nanoparticles in polydispersions

Characterization of the size and material properties of particles in liquid suspensions is in very high demand, for example, in the analysis of colloidal samples or of bodily fluids such as urine or blood plasma. However, existing methods are limited in their ability to decipher the constituents of realistic samples. Here we introduce iNTA as a new method that combines interferometric detection of scattering with nanoparticle tracking analysis to reach unprecedented sensitivity and precision in determining the size and refractive index distributions of nanoparticles in suspensions. After benchmarking iNTA with samples of colloidal gold, we present its remarkable ability to resolve the constituents of various multicomponent and polydisperse samples of known origin. Furthermore, we showcase the method by elucidating the refractive index and size distributions of extracellular vesicles from Leishmania parasites and human urine. The current performance of iNTA already enables advances in several important applications, but we also discuss possible improvements.

Formation of Oxidation- and Acid-Sensitive Assemblies from Sterols and a Quaternary Ammonium Ferrocene Derivative: Quatsome- and Onion-like Vesicles and Extended Nanoribbons.

Quatsomes are a class of nonphospholipid vesicles in which bilayers are formed from mixtures of quaternary ammonium (QA) amphiphiles and sterols. We describe the formation of oxidation and acid-sensitive quatsome-like vesicles and other bilayer assemblies from mixtures of a ferrocenylated QA amphiphile (FTDMA) and several cholesterol derivatives. The influence of the sterol and the preparation method (extrusion or probe sonication) on the stability and morphology of the resulting vesicles is explored; a variety of structures can be obtained from small (ca. 30 nm) spherical unilamellar and oligolamellar quatsome-like vesicles to large (ca. 200 nm) multilamellar onion-like vesicles to extended nanoribbons many micrometers long. FTDMA-sterol vesicles undergo drastic shifts in vesicle and membrane structure when treated with a chemical oxidant (Frémy's salt), a feature previously observed in liposomes containing FTDMA and now confirmed in nonphospholipid vesicles. Size distributions of spherical quatsome-like vesicles obtained from cryo-TEM are examined to estimate the membrane bending rigidity, and a hypothesis is presented to explain the underlying mechanism of the profound membrane alterations observed as a consequence of ferrocene oxidation.

Transferosomes: Vesicular Carrier for both Hydrophilic and Lipophilic Drugs

The vesicular system has accessed significant importance towards a sustained drug delivery system in recent years. This article was designed to review the future of vesicular systems known as transferosomes. The vesicles are softer and highly deformable than liposomes. The word Transferosomes is derived from Latin and Greek words called “Transferre” and “soma,” respectively, where Transferre means “carry across” and Soma means “a body.” Transferosomes vesicles are carrying bodies for targeted transdermal drug delivery systems. It consists of phospholipid and an edge activator. In transferosome, phospholipids vesicles act as transdermal drug carriers. It generates an osmotic gradient at the stratum corneum and helps the transferosomes penetrate through the stratum corneum by the transcellular route. Transferosomes have a wide range of solubility, have a remarkable ability to penetrate through the skin, are compatible and biodegradable, and are highly expensive. But they are easily prone to chemical degradation and are highly expensive. The transfersomes were formulated by handshaking, vortex, and freeze-thaw techniques. Transferosomes have various Evaluation parameters such as Vesicle size distribution, the morphology of vesicle, Number of vesicles per cubic mm. Transferosomes have various applications like they can be applied in controlled release and sustained release formulation, transportation of lower and higher molecular weight drug molecules, target drug delivery system, and transdermal immunization. Transferosomes have been ultra-deformable vesicles capable of subjecting a futuristic solution to all vesicular delivery and conventional delivery complications. It can carry any drug’s molecular weight through the skin; due to its ultra-deformable characteristics feature, therefore, they are widely used to take protein and peptides. After doing all the evaluation studies, we can say that Transferosomes are the promising candidate for the vesicular system and can replace other novel vesicular systems. Thus it holds a bright future in novel drug delivery systems.

PREPARATION OF PHYTOSOME OF KERSEN LEAVES (Muntingia calabura L.) ETHANOL EXRTACT AS ANTIOXIDANT

Kersen (Muntingia calabura L.) contains flavonoids that have the potential as antioxidants. However, its hydrophilic characteristics cause poor penetration so that its bioavailability is low. Research had been carried out on the preparation of ethanol extract of cherry leaves in ethosomal vesicle carriers. This study aimed to obtain the optimal phytosome suspension formula. Methods: Sample extraction was carried out by maceration method using ethanol as a solvent and was delipidated was by liquid-liquid method with n-hexane. The characterization of the extract included organoleptic, water-soluble extract content, ethanol soluble extract content, water content, ash content, acid insoluble ash content, and residual solvent. flavonoid contents were carried out by the TLC method and antioxidant activity was determined using DPPH method. Phytosome preparation was prepared by solvent evaporation and thin film hidration with ratio of extract and phosphatidylcholine 1:1 with concentration of 0.5% (A); 1% (B); and 1.5% (C). The characterization included observing morphology of vesicles using optical microscope, determining the size distribution of vesicles using PSA, and calculating the sorption efficiency using a spectrophotometer at max 281 nm. Results: The characteristics of extract were dark green; thick; distinctive aroma; ethanol soluble content were 70.91%; water soluble content were 32.5%; water content were 1.19%; ash content were 1.25%; acid insoluble ash content were 0.49%; and the remaining solvent was 0. The identification of flavonoids showed positive results. The extract has flavonoids and strong antioxidant activity with 62.71 g/mL for IC50. The shape is single layer large vesicle (LUV), diameter was 445.7 nm (A); 420.7 nm (B); and 419.6 nm (C). Vesicle entrapment efficiency was 5.83% (A); 47.575% (B); and 68.81% (C). It can be concluded that the optimal phytosome suspension formula is C with 1.5% phosphatidylcholine and 1.5% of extract.

Differential Size Distribution and Estrogen Receptor Cargo of Oviductal Extracellular Vesicles at Various Stages of Estrous Cycle in Mice

Oviductal extracellular vesicles (OEVs) play an important role in fertilization and embryo development. However, it remains largely unknown whether the size and protein cargo of OEVs change during the estrous cycle in mice. This study analyzed the changes in the size distribution and protein cargo of OEVs at four stages of the estrous cycle in mice. The distribution widths of OEVs according to the estrous cycle stage were as follows: proestrus, 20–690 nm in diameter, with two peaks at 50 and 250 nm; estrus, 22–420 nm in diameter, with two peaks at 40 and 200 nm; metestrus, 30–70 nm diameter, with a single peak at 40 nm; and diestrus, 10–26 nm diameter, with a single peak at 20 nm. The estrogen receptor (ER) level in OEVs at the proestrus stage differed significantly from that at estrus (P = 0.013) and diestrus (P = 0.005). The levels of CD9 and Hsc70 fluctuated across the four stages, although with no significant differences. Furthermore, OEVs were observed among the cilia and microvilli of epithelial cells at the proestrus, estrus, and diestrus stages, but not at the metestrus stage. The number of observed OEVs was the highest at the proestrus stage, followed by the estrus, and the diestrus stage. Endosomes were also observed at the estrus and diestrus stages. The change of the OEV size and ER cargo is associated with the estrous cycle in mice. Our findings increase the understanding of the physiological characteristics of OEVs, which may have clinical applications.

Moxifloxacin Liposomes: Effect of Liposome Preparation Method on Physicochemical Properties and Antimicrobial Activity against Staphylococcus epidermidis.

The aim of this study was the development of optimal sustained-release moxifloxacin (MOX)-loaded liposomes as intraocular therapeutics of endophthalmitis. Two methods were compared for the preparation of MOX liposomes; the dehydration–rehydration (DRV) method and the active loading method (AL). Numerous lipid-membrane compositions were studied to determine the potential effect on MOX loading and retention in liposomes. MOX and phospholipid contents were measured by HPLC and a colorimetric assay for phospholipids, respectively. Vesicle size distribution and surface charge were measured by DLS, and morphology was evaluated by cryo-TEM. The AL method conferred liposomes with higher MOX encapsulation compared to the DRV method for all the lipid compositions used. Cryo-TEM showed that both liposome types had round vesicular structure and size around 100–150 nm, while a granular texture was evident in the entrapped aqueous compartments of most AL liposomes, but substantially less in DRV liposomes; X-ray diffraction analysis demonstrated slight crystallinity in AL liposomes, especially the ones with highest MOX encapsulation. AL liposomes retained MOX for significantly longer time periods compared to DRVs. Lipid composition did not affect MOX release from DRV liposomes but significantly altered drug loading/release in AL liposomes. Interestingly, AL liposomes demonstrated substantially higher antimicrobial potential towards S. epidermidis growth and biofilm susceptibility compared to corresponding DRV liposomes, indicating the importance of MOX retention in liposomes on their activity. In conclusion, the liposome preparation method/type determines the rate of MOX release from liposomes and modulates their antimicrobial potential, a finding that deserves further in vitro and in vivo exploitation.

Effects of cholesterol on the size distribution and bending modulus of lipid vesicles.

The influence of cholesterol fraction in the membranes of giant unilamellar vesicles (GUVs) on their size distributions and bending moduli has been investigated. The membranes of GUVs were synthesized by a mixture of two elements: electrically neutral lipid 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol and also a mixture of three elements: electrically charged lipid 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), DOPC and cholesterol. The size distributions of GUVs have been presented by a set of histograms. The classical lognormal distribution is well fitted to the histograms, from where the average size of vesicle is obtained. The increase of cholesterol content in the membranes of GUVs increases the average size of vesicles in the population. Using the framework of Helmholtz free energy of the system, the theory developed by us is extended to explain the experimental results. The theory determines the influence of cholesterol on the bending modulus of membranes from the fitting of the proper histograms. The increase of cholesterol in GUVs increases both the average size of vesicles in population and the bending modulus of membranes.

Determining the Size Distribution and Integrity of Extracellular Vesicles by Dynamic Light Scattering.

Extracellular vesicles (EVs) have emerged as significant players in intercellular communication. They carry crucial biological information, and their uptake induces changes in the biological functioning and phenotypes of the recipient cell. Thus, there has been a great deal of interest in understanding their roles in the pathobiology of benign diseases and cancer. Moreover, EVs carry the molecular signatures of the donor cells, and therefore, their utility in biomarker development is being explored. Investigations are also underway to exploit their natural property of cargo transfer from one cell to another to develop efficient, nontoxic, and nonimmunogenic drug delivery systems. EVs originate through endosomal pathways, membrane-budding, or membrane-blebbing during apoptosis. These EV subtypes are usually expected to follow a specific size and surface marker distribution reflective of their origin; however, variations are often reported, especially under pathobiological conditions. Therefore, they are categorized mainly based on their size distribution as small, medium, and large EVs. Dynamic Light Scattering (DLS) is frequently used to measure the size distribution of nanoscale particles in a solution. Moreover, it also provides data on other biophysical properties such as polydispersity, aggregation, solubility, viscosity, and stability. This chapter describes the methods for determining the size distribution and integrity of EVs using DLS along with some constraints associated with the practical use of the technology.

A bacterial membrane sculpting protein with BAR domain-like activity.

Bin/Amphiphysin/RVS (BAR) domain proteins belong to a superfamily of coiled-coil proteins influencing membrane curvature in eukaryotes and are associated with vesicle biogenesis, vesicle-mediated protein trafficking, and intracellular signaling. Here, we report a bacterial protein with BAR domain-like activity, BdpA, from Shewanella oneidensis MR-1, known to produce redox-active membrane vesicles and micrometer-scale outer membrane extensions (OMEs). BdpA is required for uniform size distribution of membrane vesicles and influences scaffolding of OMEs into a consistent diameter and curvature. Cryo-TEM reveals that a strain lacking BdpA produces lobed, disordered OMEs rather than membrane tubules or narrow chains produced by the wild-type strain. Overexpression of BdpA promotes OME formation during planktonic growth of S. oneidensis where they are not typically observed. Heterologous expression results in OME production in Marinobacter atlanticus and Escherichia coli. Based on the ability of BdpA to alter membrane architecture in vivo, we propose that BdpA and its homologs comprise a newly identified class of bacterial BAR domain-like proteins.