Rapid and accurate analysis of stem cell-derived extracellular vesicles with super resolution microscopy and live imaging

Extracellular vesicles (EVs) have prevalent roles in cancer biology and regenerative medicine. Conventional techniques for characterising EVs including electron microscopy (EM), nanoparticle tracking analysis (NTA) and tuneable resistive pulse sensing (TRPS), have been reported to produce high variability in particle count (EM) and poor sensitivity in detecting EVs below 50?nm in size (NTA and TRPS), making accurate and unbiased EV analysis technically challenging. This study introduces direct stochastic optical reconstruction microscopy (d-STORM) as an efficient and reliable characterisation approach for stem cell-derived EVs. Using a photo-switchable lipid dye, d-STORM imaging enabled rapid detection of EVs down to 20–30?nm in size with higher sensitivity and lower variability compared to EM, NTA and TRPS techniques. Imaging of EV uptake by live stem cells in culture further confirmed the potential of this approach for downstream cell biology applications and for the analysis of vesicle-based cell-cell communication.     

Vesicle sizing: Number distributions by dynamic light scattering

A procedure is described which optimizes nonnegative least squares and exponential sampling fitting methods for analysis of dynamic light scattering (DLS) data from aqueous suspensions of vesicle/liposome systems. This approach utilizes a Rayleigh-Gans-Debye form factor for a coated sphere and yields number distributions which can be compared directly to distributions obtained by freeze-fracture electron microscopy (EM). Excellent agreement between the DLS and EM results are obtained for vesicle size distributions in the 100-200-nm range.     

Mechanical properties of vesicles. I. Coordinated analysis of osmotic swelling and lysis.

To determine how transmembrane osmotic gradients perturb the structure and dynamics of biological membranes, we examined the effects of medium dilution on the structures of osmolyte-loaded lipid vesicles. Our preparations were characterized by dynamic light scattering (DLS) and nuclear magnetic resonance (NMR) spectroscopies. Populations of Escherichia coli phosphatidylethanolamine (PE) or dioleoylphosphatidylglycerol (DOPG) vesicles prepared by the pH jump technique were variable and polymodal in size distribution. Complex and variable structural changes occurred when PE vesicles were diluted with hypotonic buffer. Such vesicles could not be used as model systems for the analysis of membrane mechanical properties. NaCl-loaded, DOPG vesicles prepared by extrusion through 100 nm (diameter) pores were reproducible and monomodal in size distribution and unilamellar, whereas those prepared by extrusion through 200-, 400-, or 600-nm pores were variable and polymodal in size distribution and/or multilamellar. Time and pressure regimes associated with osmotic lysis of extruded vesicles were defined by monitoring release of carboxyfluorescein, a self-quenching fluorescent dye. Corresponding effects of medium dilution on vesicle structure were assessed by DLS spectroscopy. These experiments and the accompanying analysis (Hallett, F.R., J. Marsh, B.G. Nickel, and J.M. Wood. 1993. Biophys. J. 64:000-000) revealed conditions under which vesicles are expected to reside in a consistently strained state.     

Isolation of Extracellular Microvesicles from Cell Culture Medium: Comparative Evaluation of Methods

Extracellular vesicles (EV) are secreted by cells of multicellular organisms. EV mediate specific mode of intercellular communication by “horizontal” exchange of substances and information. This phenomenon seems to have an essential biological significance and became a subject of intensive research. Biogenesis, structural and functional EV features are usually studied in vitro. Several methods of EV isolation from cell culture medium are currently used; however, selection of a particular method may have a significant impact on obtained results. The choice of the optimal method is usually determined by the amount of starting biomaterial and the aims of the research. We have performed a comparative analysis of four different methods of EV isolation from cell culture medium: differential ultracentrifugation, ultracentrifugation with 30% sucrose/D₂O “cushion,” precipitation with plant proteins and latex-based immunoaffinity capturing. EV isolated from several human glial cell lines by different approaches were compared in terms of the following parameters: size, concentration, EV morphology, contamination by non-vesicular particles, content of exosomal tetraspanins on the EV surface, content of total proteins, total RNA, and several glioma-associated miRNAs. The applied methods included nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), cryo-electron microscopy, flow cytometry and RT-qPCR. Based on the obtained results, we have developed practical recommendations that may help researchers to make the best choice of the EV isolation method.     

Ultrasonic storage modulus as a novel parameter for analyzing protein-protein interactions in high protein concentration solutions: correlation with static and dynamic light scattering measurements

The purpose of this work was to establish ultrasonic storage modulus (G') as a novel parameter for characterizing protein-protein interactions (PPI) in high concentration protein solutions. Using an indigenously developed ultrasonic shear rheometer, G' for 20-120 mg/ml solutions of a monoclonal antibody (IgG(2)), between pH 3.0 and 9.0 at 4 mM ionic strength, was measured at frequency of 10 MHz. Our understanding of ultrasonic rheology indicated decrease in repulsive and increase in attractive PPI with increasing solution pH. To confirm this behavior, dynamic (DLS) and static (SLS) light scattering measurements were conducted in dilute solutions. Due to technical limitations, light scattering measurements could not be conducted in concentrated solutions. Mutual-diffusion coefficient, measured by DLS, increased with IgG(2) concentration at pH 4.0 and this trend reversed as pH was increased to 9.0. Second virial coefficient, measured by SLS, decreased with increasing pH. These observations were consistent with the nature of PPI understood from G' measurements. Ultrasonic rheology, DLS, and SLS measurements were also conducted under conditions of increased ionic strength. The consistency between rheology and light scattering analysis under various solution conditions established the utility of ultrasonic G' measurements as a novel tool for analyzing PPI in high protein concentration systems.     

Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry

We provide a protocol for a high-resolution flow cytometry-based method for quantitative and qualitative analysis of individual nano-sized vesicles released by cells, as developed and previously described by our group. The method involves (i) bright fluorescent labeling of cell-derived vesicles and (ii) flow cytometric analysis of these vesicles using an optimized configuration of the commercially available BD Influx flow cytometer. The method allows the detection and analysis of fluorescent cell-derived vesicles of ～100 nm. Integrated information can be obtained regarding the light scattering, quantity, buoyant density and surface proteins of these nano-sized vesicles. This method can be applied in nanobiology to study basic aspects of cell-derived vesicles. Potential clinical applications include the detailed analysis of vesicle-based biomarkers in body fluids and quality control analysis of (biological) vesicles used as therapeutic agents. Isolation, fluorescent labeling and purification of vesicles can be done within 24 h. Flow cytometer setup, calibration and subsequent data acquisition can be done within 2-4 h by an experienced flow cytometer operator.     

Study and formation of vesicle systems with low polydispersity index by ultrasound method

The formation of liposomes with low polydispersity index by application of ultrasounds was investigated considering methodology specifications such as sonication time and sonication power. Phosphatidylcholine (PC) liposomes were formed by the evaporation–hydration method. The vesicles were sonicated using several sonication conditions. The liposomes were then characterized by dynamic light scattering (DLS) and freeze-fracture electron microscopy (FFEM). Correlation functions from DLS were treated by cumulants method and GENDIST to obtain the mean radius and polydispersity index. These calculations allowed to fix an optimal sonication time (3000 s) and a useful interval of ultrasound power between 39 and 91 W. DLS and FFEM results confirmed that vesicle size, lamellarity and the polydispersity index decreased with the increase of sonication power. Thus, we propose a systematic method to form liposomes in which the physical characteristics of the vesicles may be controlled as a function of sonication time and power.     

What can light scattering spectroscopy do for membrane-active peptide studies?

Highly charged peptides are important components of the immune system and belong to an important family of antibiotics. Although their therapeutic activity is known, most of the molecular level mechanisms are controversial. A wide variety of different approaches are usually applied to understand their mechanisms, but light scattering techniques are frequently overlooked. Yet, light scattering is a noninvasive technique that allows insights both on the peptide mechanism of action as well as on the development of new antibiotics. Dynamic light scattering (DLS) and static light scattering (SLS) are used to measure the aggregation process of lipid vesicles upon addition of peptides and molecular properties (shape, molecular weight). The high charge of these peptides allows electrostatic attraction toward charged lipid vesicles, which is studied by zeta potential (zeta-potential) measurements.     

Light scattering from suspensions of membrane fragments derived from sonication of beef heart mitochondria.

The intensity of light scattering from suspensions of membrane fragments prepared by sonication of beef heart mitochondria in the presence of EDTA at alkaline pH (ESMP) was determined at 45, 90, and 135 degrees with light of wavelength 546 nm. The dissymmetry ratio Z = I45 degrees c/I135 degrees c, where I45 degrees c and I135 degrees c are the scattering intensities at 45 and 135 degrees extrapolated to zero particle concentration and corrected for reflectance effects, was used to calculate particle size from the Rayleigh-Gans-Debye theory. An average particle diameter D of 184-190 nm was obtained, within the range of particle diameter 50-300 nm determined previously by electron microscopy. This average diameter determined by light scattering is a useful parameter for characterization of ESMP particle size. We propose the term: light scattering average particle diameter, DLS, for this parameter. The refractive index of ESMP was determined to be 1.443 by measurement of scattering intensity in buffer solutions of varying sucrose concentration. The value of Z was independent of sucrose concentration in this determination, showing that the particles are osmotically inactive toward sucrose. The values of average particle diameter DLS and of refractive index fall within the range of validity of the Rayleigh-Gans-Debye theory, for which light scattering changes are attributable solely to dimension change, rather than to change in particle refractive index. Uptake of water accompanying energy-linked salt uptake in ESMP was calculated from light scattering changes to be 0.18 mul of H2O/mg of protein, compared with 0.49 mul of H2O/mg of protein measured by dextran inaccessibility. Measurement of light scattering changes provides a rapid and sensitive method for determining volume changes of ESMP. The magnitude of the volume change observed during energy-linked water and salt uptake and the initial degree of hydration suggests that ESMP are analogous to polyelectrolyte gels with regard to sorption of strong electrolytes and that the Donnan formulation for ion exchange equilibria may be usefully applied to these processes in ESMP.     

Lysozyme effect on oleic acid/oleate vesicles

We have analysed by means of turbidimetric, dynamic light scattering (DLS), and fluorimetric techniques the effect of lysozyme on negatively charged oleic acid/oleate vesicles. The addition of lysozyme brings about a decrease in optical density of the vesicle population, which finally results in a size distribution of oleate vesicles shifted toward smaller mean diameters. On the contrary, (a) when phosphatidylserine vesicles were used, lysozyme induces an increase of turbidity and a shift toward larger vesicle sizes; and (b) the addition of histone H1 or poly-L-lysine produces an aggregative behavior both in oleate and in phosphatidylserine vesicles. Experiments carried out with calcein-containing vesicles indicate that the observed changes in the lysozyme/oleate system occur with partial leakage of the vesicle content. All this is taken to suggest that the interaction between lysozyme and oleate vesicles is of quite specific nature, and certainly not just due to electrostatic interactions.     

Lysozyme Effect on Oleic Acid/Oleate Vesicles

We have analysed by means of turbidimetric, dynamic light scattering (DLS), and fluorimetric techniques the effect of lysozyme on negatively charged oleic acid/oleate vesicles. The addition of lysozyme brings about a decrease in optical density of the vesicle population, which finally results in a size distribution of oleate vesicles shifted toward smaller mean diameters. On the contrary, (a) when phosphatidylserine vesicles were used, lysozyme induces an increase of turbidity and a shift toward larger vesicle sizes; and (b) the addition of histone H1 or poly-L-lysine produces an aggregative behavior both in oleate and in phosphatidylserine vesicles. Experiments carried out with calcein-containing vesicles indicate that the observed changes in the lysozyme/oleate system occur with partial leakage of the vesicle content. All this is taken to suggest that the interaction between lysozyme and oleate vesicles is of quite specific nature, and certainly not just due to electrostatic interactions.     

The SPA1-like proteins SPA3 and SPA4 repress photomorphogenesis in the light

Suppressor of phyA-105 (SPA1) is a phytochrome A-specific signaling intermediate that acts as a light-dependent repressor of photomorphogenesis in Arabidopsis seedlings. SPA1 is part of a small gene family comprising three genes: SPA1-related 2 (SPA2), SPA1-related 3 (SPA3), and SPA1-related 4 (SPA4). Here, we investigate the functions of SPA3 and SPA4, two very closely related genes coding for proteins with 74% identical amino acids. Seedlings with mutations in SPA3 or SPA4 exhibit enhanced photomorphogenesis in the light, but show no phenotype in darkness. While there are small differences between the effects of spa3 and spa4 mutations, it is apparent that SPA3 and SPA4 function to inhibit light responses in continuous far-red, red, and blue light. Phytochrome A is necessary for all aspects of the spa4 mutant phenotype, suggesting that SPA4, like SPA1, acts specifically in phytochrome A signaling. Enhanced photoresponsiveness of spa3 mutants is also fully dependent on phytochrome A in far-red and blue light, but not in red light. Hence, SPA3 function in red light may be dependent on other phytochromes in addition to phytochrome A. Using yeast two-hybrid and in vitro interaction assays, we further show that SPA3 as well as SPA4 can physically interact with the constitutive repressor of light signaling COP1. Deletion analyses suggest that SPA3 and SPA4, like SPA1, bind to the coiled-coil domain of COP1. Taken together, our results have identified two new loci coding for negative regulators that may be involved in fine tuning of light responses by interacting with COP1.     

Interaction of apolipoprotein A-I with lecithin-cholesterol vesicles in the presence of phospholipase C

Here we study the anti-nucleating mechanism of apolipoprotein A-I (apo A-I) on model biliary vesicles in the presence of phospholipase C (PLC) utilizing dynamic light scattering (DLS), steady-state fluorescence spectroscopy, cryogenic transmission electron microscopy (cryo-TEM), and UV/Vis spectroscopy. PLC induces aggregation of cholesterol-free lecithin vesicles from an initial, average size of 100 nm to a maximal size of 600 nm. The presence of apo A-I likely inhibits vesicle aggregation by shielding the PLC-generated hydrophobic moieties, which results in vesicles of an average size of 200 nm. A similar phenomenon is observed in cholesterol-enriched lecithin vesicles. Whereas PLC alone produces aggregates of 300 nm, no aggregation is observed when apo A-I is present along with PLC. However, the ability of apo A-I to inhibit aggregation is temporary, and after 8 h, a broad particle size distribution with sizes as high as 800 nm is observed. Apo A-I possibly induces the formation of small apo A-I/lecithin/cholesterol complexes of about 5-20 nm similar to the discoidal pre-HDL complexes found in blood when it can no longer effectively shield all the DAG molecules. Concomitant with formation of complexes, DAG molecules coalesce into large oil droplets, which account for the large particles observed by light scattering. Thus, apo A-I acts as an anti-nucleating agent by two mechanisms, anti-aggregation and microstructural transition. The mode of protection is dependent on the cholesterol content and the relative amounts of DAG and apo A-I present. This study supports the possibility of apo A-I solubilizing lipids in bile in a similar fashion as it does in blood and also delineates the mechanism of formation of the complexes.     

Silica nanoparticles for the layer-by-layer assembly of fully electro-active cytochrome c multilayers

Background

Gold nanoparticles (AuNPs) scatter light intensely at or near their surface plasmon wavelength region. Using AuNPs coupled with dynamic light scattering (DLS) detection, we developed a facile nanoparticle immunoassay for serum protein biomarker detection and analysis. A serum sample was first mixed with a citrate-protected AuNP solution. Proteins from the serum were adsorbed to the AuNPs to form a protein corona on the nanoparticle surface. An antibody solution was then added to the assay solution to analyze the target proteins of interest that are present in the protein corona. The protein corona formation and the subsequent binding of antibody to the target proteins in the protein corona were detected by DLS.

Results

Using this simple assay, we discovered multiple molecular aberrations associated with prostate cancer from both mice and human blood serum samples. From the mice serum study, we observed difference in the size of the protein corona and mouse IgG level between different mice groups (i.e., mice with aggressive or less aggressive prostate cancer, and normal healthy controls). Furthermore, it was found from both the mice model and the human serum sample study that the level of vascular endothelial growth factor (VEGF, a protein that is associated with tumor angiogenesis) adsorbed to the AuNPs is decreased in cancer samples compared to non-cancerous or less malignant cancer samples.

Conclusion

The molecular aberrations observed from this study may become new biomarkers for prostate cancer detection. The nanoparticle immunoassay reported here can be used as a convenient and general tool to screen and analyze serum proteins and to discover new biomarkers associated with cancer and other human diseases.     

Size analysis and stability study of lipid vesicles by high-performance gel exclusion chromatography, turbidity, and dynamic light scattering

Vesicles of egg phosphatidylcholine (EPC) and phosphatidic acid (EPA) were prepared by reverse-phase evaporation (REV) followed either by sequential extrusion through polycarbonate membranes with pore diameters of 0.8, 0.4, 0.2, 0.1, and 0.05 micron or by filtration through 0.8-micron cellulosic or 0.22-micron polyvinylidene fluoride (PVF) membranes. The resulting vesicles ranging from 130 to 640 nm in mean diameter (REVs) were characterized by high-performance liquid chromatography (HPLC) using a TSK G6000 PW gel exclusion column. The efficiency of this technique to determine vesicle size parameters was studied by the analysis of the chromatograms in combination with dynamic light scattering (DLS) determination of the mean diameters (MD) of the fractionated vesicles in the region of the elution profile maxima. The HPLC TSK G6000 PW gel exclusion provides a reproducible and fast method of size characterization for lipid vesicles having MD up to 1 micron, the best selectivity being obtained in the 20- to 500-nm MD range. HPLC analysis of REV's demonstrates that: (i) both the average size and polydispersity of the vesicles decrease with decreasing pore size of the membranes, cellulosic or PVF "tortuous" ones being less efficient than "straight bores" polycarbonate ones; (ii) mixed EPC/EPA REVs sequentially extruded down through 0.2-micron polycarbonate membranes are highly deformable without rupture of the bilayer; and (iii) the mean size of extruded REV's is stable for at least 1 week. The role of EPA on the size stability of mixed EPC/EPA vesicles was studied by coupling HPLC gel exclusion and turbidity analysis of pure EPC and EPC/EPA (mole ratio: 91/9) sonicated small unilamellar vesicles as a function of time. The apparent size variation of EPC vesicles observed over a week, is mainly due to their aggregation which is significantly reduced by the introduction of a small amount of EPA in the vesicle membrane.     

Mechanism of thermal aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase

Thermal denaturation and aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been studied using differential scanning calorimetry (DSC), dynamic light scattering (DLS), and analytical ultracentrifugation. The maximum of the protein thermal transition (T(m)) increased with increasing the protein concentration, suggesting that the denaturation process involves the stage of reversible dissociation of the enzyme tetramer into the oligomeric forms of lesser size. The dissociation of the enzyme tetramer was shown by sedimentation velocity at 45 degrees C. The DLS data support the mechanism of protein aggregation that involves a stage of the formation of the start aggregates followed by their sticking together. The hydrodynamic radius of the start aggregates remained constant in the temperature interval from 37 to 55 degrees C and was independent of the protein concentration (R(h,0) approximately 21 nm; 10 mM sodium phosphate, pH 7.5). A strict correlation between thermal aggregation of GAPDH registered by the increase in the light scattering intensity and protein denaturation characterized by DSC has been proved.     

Size and structure of antigen-antibody complexes. Electron microscopy and light scattering studies

Size parameters of model antigen-antibody (Ag-Ab) complexes formed by the interaction of bovine serum albumin (BSA) and pairs of monoclonal anti-BSA antibodies (mAb) were evaluated by quasielastic light scattering, classical light scattering, and electron microscopy (EM). Mean values for the hydrodynamic radius, radius of gyration, and molecular weight were determined by light scattering. Detailed information regarding the molecular weight distribution and the presence of cycles or open chains was obtained with EM. Average molecular weights were calculated from the EM data, and the Porod-Kratky wormlike chain theory was used to model the conformational behavior of the Ag-mAb complexes. Ag-mAb complexes prepared from three different mAb pairs displayed significantly different properties as assessed by each of the techniques employed. Observations and size parameter calculations from EM photomicrographs were consistent with the results from light scattering. The differences observed between the mab pairs would not have been predicted by idealized thermodynamic models. These results suggest that the geometric constraints imposed by the individual epitope environment and/or the relative epitope location are important in determining the average size of complexes and the ratio of linear to cyclic complexes.     

pH-sensitive niosomes: Effects on cytotoxicity and on inflammation and pain in murine models

pH-sensitive nonionic surfactant vesicles (niosomes) by polysorbate-20 (Tween-20) or polysorbate-20 derivatized by glycine (added as pH sensitive agent), were developed to deliver Ibuprofen (IBU) and Lidocaine (LID). For the physical-chemical characterization of vesicles (mean size, size distribution, zeta potential, vesicle morphology, bilayer properties and stability) dynamic light scattering (DLS), small angle X-ray scattering and fluorescence studies were performed. Potential cytotoxicity was evaluated on immortalized human keratinocyte cells (HaCaT) and on immortalized mouse fibroblasts Balb/3T3. In vivo antinociceptive activity (formalin test) and anti-inflammatory activity tests (paw edema induced by zymosan) in murine models were performed on drug-loaded niosomes. pH-sensitive niosomes were stable in the presence of 0 and 10% fetal bovine serum, non-cytotoxic and able to modify IBU or LID pharmacological activity in vivo. The synthesis of stimuli responsive surfactant, as an alternative to add pH-sensitive molecules to niosomes, could represent a promising delivery strategy for anesthetic and anti-inflammatory drugs.     

Bis(monoacylglycero)phosphate and ganglioside GM1 spontaneously form small homogeneous vesicles at specific concentrations

The morphology and size of hydrated lipid dispersions of bis(monoacylglycero)phosphate (BMP) mixed with varying mole percentages of the ganglioside GM1 were investigated by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Electron paramagnetic resonance (EPR) spectroscopy of these same mixtures, doped at 0.5 mol% with doxyl labeled lipids, was used to investigate acyl-chain packing. Results show that for 20-30% GM1, hydrated BMP:GM1 mixtures spontaneously form small spherical vesicles with diameters ∼100 nm and a narrow size distribution profile. For other concentrations of GM1, hydrated dispersions with BMP have non-spherical shapes and heterogeneous size profiles, with average vesicle diameters >400 nm. All samples were prepared at pH 5.5 to mimic the lumen acidity of the late endosome where BMP is an essential component of intraendosomal vesicle budding, lipid sorting and trafficking. These findings indicate that GM1 and BMP under a limited concentration range spontaneously form small vesicles of homogeneous size in an energy independent manner without the need of protein templating. Because BMP is essential for intraendosomal vesicle formation, these results imply that lipid-lipid interactions may play a critical role in the endosomal process of lipid sorting and trafficking.