Characterization of Nanohybridosomes from Lipids and Spruce Homogenate Containing Extracellular Vesicles.

Introduction: Lipid nanovesicles associated with bioactive phytochemicals from spruce needle homogenate (here called nano-sized hybridosomes or nanohybridosomes, NSHs) were considered. Methods: We formed NSHs by mixing appropriate amounts of lecithin, glycerol and supernatant of isolation of extracellular vesicles from spruce needle homogenate. We visualized NSHs by light microscopy and cryogenic transmission electron microscopy and assessed them by flow cytometry, dynamic light scattering, ultraviolet-visual spectroscopy, interferometric light microscopy and liquid chromatography-mass spectrometry. Results: We found that the particles consisted of a bilayer membrane and a fluid-like interior. Flow cytometry and interferometric light microscopy measurements showed that the majority of the particles were nano-sized. Dynamic light scattering and interferometric light microscopy measurements agreed well on the average hydrodynamic radius of the particles Rh (between 140 and 180 nm), while the concentrations of the particles were in the range between 1013 and 1014/mL indicating that NSHs present a considerable (more than 25%) of the sample which is much more than the yield of natural extracellular vesicles (EVs) from spruce needle homogenate (estimated less than 1%). Spruce specific lipids and proteins were found in hybridosomes. Discussion: Simple and low-cost preparation method, non-demanding saving process and efficient formation procedure suggest that large-scale production of NSHs from lipids and spruce needle homogenate is feasible.

Small Cellular Particles from European Spruce Needle Homogenate.

Small cellular particles (SCPs) are being considered for their role in cell-to-cell communication. We harvested and characterized SCPs from spruce needle homogenate. SCPs were isolated by differential ultracentrifugation. They were imaged by scanning electron microscope (SEM) and cryogenic transmission electron microscope (cryo TEM), assessed for their number density and hydrodynamic diameter by interferometric light microscopy (ILM) and flow cytometry (FCM), total phenolic content (TPC) by UV-vis spectroscopy, and terpene content by gas chromatography-mass spectrometry (GC-MS). The supernatant after ultracentrifugation at 50,000× g contained bilayer-enclosed vesicles whereas in the isolate we observed small particles of other types and only a few vesicles. The number density of cell-sized particles (CSPs) (larger than 2 µm) and meso-sized particles (MSPs) (cca 400 nm-2 µm) was about four orders of magnitude lower than the number density of SCPs (sized below 500 nm). The average hydrodynamic diameter of SCPs measured in 10,029 SCPs was 161 ± 133 nm. TCP decreased considerably due to 5-day aging. Volatile terpenoid content was found in the pellet after 300× g. The above results indicate that spruce needle homogenate is a source of vesicles to be explored for potential delivery use.

Autologous Platelet and Extracellular Vesicle-Rich Plasma as Therapeutic Fluid: A Review.

The preparation of autologous platelet and extracellular vesicle-rich plasma (PVRP) has been explored in many medical fields with the aim to benefit from its healing potential. In parallel, efforts are being invested to understand the function and dynamics of PVRP that is complex in its composition and interactions. Some clinical evidence reveals beneficial effects of PVRP, while some report that there were no effects. To optimize the preparation methods, functions and mechanisms of PVRP, its constituents should be better understood. With the intention to promote further studies of autologous therapeutic PVRP, we performed a review on some topics regarding PVRP composition, harvesting, assessment and preservation, and also on clinical experience following PVRP application in humans and animals. Besides the acknowledged actions of platelets, leukocytes and different molecules, we focus on extracellular vesicles that were found abundant in PVRP.

Assessment of Small Cellular Particles from Four Different Natural Sources and Liposomes by Interferometric Light Microscopy.

Small particles in natural sources are a subject of interest for their potential role in intercellular, inter-organism, and inter-species interactions, but their harvesting and assessment present a challenge due to their small size and transient identity. We applied a recently developed interferometric light microscopy (ILM) to assess the number density and hydrodynamic radius (Rh) of isolated small cellular particles (SCPs) from blood preparations (plasma and washed erythrocytes) (B), spruce needle homogenate (S), suspension of flagellae of microalgae Tetraselmis chuii (T), conditioned culture media of microalgae Phaeodactylum tricornutum (P), and liposomes (L). The aliquots were also assessed by flow cytometry (FCM), dynamic light scattering (DLS), ultraviolet-visible spectrometry (UV-vis), and imaging by cryogenic transmission electron microscopy (cryo-TEM). In Rh, ILM showed agreement with DLS within the measurement error in 10 out of 13 samples and was the only method used here that yielded particle density. Cryo-TEM revealed that representative SCPs from Tetraselmis chuii flagella (T) did not have a globular shape, so the interpretation by Rh of the batch methods was biased. Cryo-TEM showed the presence of thin filaments in isolates from Phaeodactylum tricornutum conditioned culture media (P), which provides an explanation for the considerably larger Rh obtained by batch methods than the sizes of particles observed by cryo-TEM images. ILM proved convenient for assessment of number density and Rh of SCPs in blood preparations (e.g., plasma); therefore, its use in population and clinical studies is indicated.

Stability of Erythrocyte-Derived Nanovesicles Assessed by Light Scattering and Electron Microscopy.

Extracellular vesicles (EVs) are gaining increasing amounts of attention due to their potential use in diagnostics and therapy, but the poor reproducibility of the studies that have been conducted on these structures hinders their breakthrough into routine practice. We believe that a better understanding of EVs stability and methods to control their integrity are the key to resolving this issue. In this work, erythrocyte EVs (hbEVs) were isolated by centrifugation from suspensions of human erythrocytes that had been aged in vitro. The isolate was characterised by scanning (SEM) and cryo-transmission electron microscopy (cryo-TEM), flow cytometry (FCM), dynamic/static light scattering (LS), protein electrophoresis, and UV-V spectrometry. The hbEVs were exposed to various conditions (pH (4-10), osmolarity (50-1000 mOsm/L), temperature (15-60 °C), and surfactant Triton X-100 (10-500 µM)). Their stability was evaluated by LS by considering the hydrodynamic radius (Rh), intensity of scattered light (I), and the shape parameter (ρ). The morphology of the hbEVs that had been stored in phosphate-buffered saline with citrate (PBS-citrate) at 4 °C remained consistent for more than 6 months. A change in the media properties (50-1000 mOsm/L, pH 4-10) had no significant effect on the Rh (=100-130 nm). At pH values below 6 and above 8, at temperatures above 45 °C, and in the presence of Triton X-100, hbEVs degradation was indicated by a decrease in I of more than 20%. Due to the simple preparation, homogeneous morphology, and stability of hbEVs under a wide range of conditions, they are considered to be a suitable option for EV reference material.

Application of Dynamic and Static Light Scattering for Size and Shape Characterization of Small Extracellular Nanoparticles in Plasma and Ascites of Ovarian Cancer Patients.

In parallel to medical treatment of ovarian cancer, methods for the early detection of cancer tumors are being sought. In this contribution, the use of non-invasive static (SLS) and dynamic light scattering (DLS) for the characterization of extracellular nanoparticles (ENPs) in body fluids of advanced serous ovarian cancer (OC) and benign gynecological pathology (BP) patients is demonstrated and critically evaluated. Samples of plasma and ascites (OC patients) or plasma, peritoneal fluid, and peritoneal washing (BP patients) were analyzed. The hydrodynamic radius (Rh) and the radius of gyration (Rg) of ENPs were calculated from the angular dependency of LS intensity for two ENP subpopulations. Rh and Rg of the predominant ENP population of OC patients were in the range 20-30 nm (diameter 40-60 nm). In thawed samples, larger particles (Rh mostly above 100 nm) were detected as well. The shape parameter ρ of both particle populations was around 1, which is typical for spherical particles with mass concentrated on the rim, as in vesicles. The Rh and Rg of ENPs in BP patients were larger than in OC patients, with ρ ≈ 1.1-2, implying a more elongated/distorted shape. These results show that SLS and DLS are promising methods for the analysis of morphological features of ENPs and have the potential to discriminate between OC and BP patients. However, further development of the methodology is required.

Viscosity of Plasma as a Key Factor in Assessment of Extracellular Vesicles by Light Scattering.

Extracellular vesicles (EVs) isolated from biological samples are a promising material for use in medicine and technology. However, the assessment methods that would yield repeatable concentrations, sizes and compositions of the harvested material are missing. A plausible model for the description of EV isolates has not been developed. Furthermore, the identity and genesis of EVs are still obscure and the relevant parameters have not yet been identified. The purpose of this work is to better understand the mechanisms taking place during harvesting of EVs, in particular the role of viscosity of EV suspension. The EVs were harvested from blood plasma by repeated centrifugation and washing of samples. Their size and shape were assessed by using a combination of static and dynamic light scattering. The average shape parameter of the assessed particles was found to be ρ ~ 1 (0.94-1.1 in exosome standards and 0.7-1.2 in blood plasma and EV isolates), pertaining to spherical shells (spherical vesicles). This study has estimated the value of the viscosity coefficient of the medium in blood plasma to be 1.2 mPa/s. It can be concluded that light scattering could be a plausible method for the assessment of EVs upon considering that EVs are a dynamic material with a transient identity.

Effect of Multivalent Cations on Intermolecular Association of Isotactic and Atactic Poly(Methacrylic Acid) Chains in Aqueous Solutions.

The formation of nanoparticles of two poly(methacrylic acid) (PMA) isomers, atactic (aPMA) and isotactic (iPMA), was investigated in aqueous solutions in the presence of mono- (Na⁺) and multivalent cations (Mg2+ and La3+). Using dynamic (DLS) and static light scattering (SLS), we show that PMA nanoparticles have characteristics of microgel-like particles with a denser core and a swollen corona. iPMA aggregates are stable at a much higher degree of neutralization (αN) than the aPMA ones, indicating a much stronger association between iPMA chains. This is explained by proposing segregation of ionized and unionized carboxyl groups within the iPMA aggregates and subsequent cooperative hydrogen-bonding between COOH groups. The calculated shape parameter (ρ) suggests different behavior of both isomers in the presence of Mg2+ ions on one hand and Na⁺ and La3+ on the other. The microgel-like particles formed in the presence of Mg2+ ions have a more even mass distribution (possibly a no core-shell structure) in comparison with those in the presence of Na⁺ and La3+ ions. Differences between the aggregate structures in the presence of different ions are reflected also in calorimetric experiments and supported by pH and fluorimetric measurements. Reasons for different behavior in the presence of Mg2+ ions lie in specific properties of this cation, in particular in its strong hydration and preference towards monodentate binding to carboxylate groups.

Temperature Dependent Behavior of Isotactic and Atactic Poly(Methacrylic Acid) in the Presence of MgCl2 and CaCl2.

Temperature (T) induced nanoparticle formation of isotactic (iPMA) and atactic (aPMA) poly(methacrylic acid) chains is investigated in aqueous solutions in the presence of divalent cations (Mg2+ and Ca2+) by UV spectroscopy, visual observations and pyrene fluorimetry. In aPMA solutions, aggregation and phase separation take place with increasing T. The onset of aPMA aggregation (heating) and re-dissolution of the aggregates (cooling) is shifted to lower Ts with increasing ionic strength of the solutions. iPMA associates partly decompose upon heating, but stable nano-sized particles are present at all Ts. Structural characterization of the aggregates was carried out by dynamic and static light scattering measurements at 25 °C. Results revealed that aggregates of both PMAs with Mg2+ ions involve a lot of water and have a rather even mass distribution. This is attributed to strong hydration of Mg2+ ions and their preference towards monodentate binding to carboxylate groups. Differently, Ca2+ ions bind more strongly and in a bidentate manner, they lose the hydration water and form aggregates with several structural elements, depending on chain tacticity.

Kinetically Stable Triglyceride-Based Nanodroplets and Their Interactions with Lipid-Specific Proteins.

Understanding of the interactions between proteins and natural and artificially prepared lipid membrane surfaces and embedded nonpolar cores is important in studies of physiological processes and their pathologies and is applicable to nanotechnologies. In particular, rapidly growing interest in cellular droplets defines the need for simplified biomimetic lipid model systems to overcome in vivo complexity and variability. We present a protocol for the preparation of kinetically stable nanoemulsions with nanodroplets composed of sphingomyelin (SM) and cholesterol (Chol), as amphiphilic surfactants, and trioleoylglycerol (TOG), at various molar ratios. To prepare stable SM/Chol-coated monodisperse lipid nanodroplets, we modified a reverse phase evaporation method and combined it with ultrasonication. Lipid composition, ζ-potential, gyration and hydrodynamic radius, shape, and temporal stability of the lipid nanodroplets were characterized and compared to extruded SM/Chol large unilamellar vesicles. Lipid nanodroplets and large unilamellar vesicles with theoretical SM/Chol/TOG molar ratios of 1/1/4.7 and 4/1/11.7 were further investigated for the orientational order of their interfacial water molecules using a second harmonic scattering technique, and for interactions with the SM-binding and Chol-binding pore-forming toxins equinatoxin II and perfringolysin O, respectively. The surface characteristics (ζ-potential, orientational order of interfacial water molecules) and binding of these proteins to the nanodroplet SM/Chol monolayers were similar to those for the SM/Chol bilayers of the large unilamellar vesicles and SM/Chol Langmuir monolayers, in terms of their surface structures. We propose that such SM/Chol/TOG nanoparticles with the required lipid compositions can serve as experimental models for monolayer membrane to provide a system that imitates the natural lipid droplets.

Reappearance of slow mode in mixtures of polyethylene glycol and poly(sodium methacrylate).

Mixtures of an anionic polyelectrolyte, poly(sodium methacrylate), NaPMA, and a neutral polymer, polyethylene glycol, PEG, were investigated by dynamic and static light scattering techniques at different concentrations and chain-lengths of PEG. The NaPMA standard with a narrow molecular weight distribution was chosen for the study. The so called slow-mode behaviour, characteristic of salt-free NaPMA solutions, vanishes as the simple salt, NaCl in this case, is added in a sufficient amount. However, in NaPMA-NaCl-PEG mixtures, the slow-mode signal is observed again. We assume that the dielectric constant of the mixture with PEG, which is substantially lower than that of pure water, causes the reappearance of the slow-mode signal through reinforced electrostatic interactions between NaPMA polyions. Monte Carlo simulations of a coarse-grained model of the system confirm stronger correlations between NaPMA molecules and thus qualitatively agree with experimental results.

Pitfalls in Size Characterization of Soft Particles by Dynamic Light Scattering Online Coupled to Asymmetrical Flow Field-Flow Fractionation.

An asymmetrical flow field-flow fractionation (AF4) technique coupled to a multiangle light scattering (MALS) detector with an embedded dynamic light scattering (DLS) module was introduced to study the size characteristics and shape of soft particles of various size and type: polystyrene nanosphere size standards, lipid droplets (LDs), and large unilamellar vesicles (LUVs). A range of flow velocities through the LS detector, at which accurate hydrodynamic size can be extracted from the DLS in flow mode, was studied since the particles subjected to a longitudinal flow exhibit not only the Brownian motion due to diffusion but also the translational movement. In addition, the impact of the longitudinal flow velocity on the shape of the artificial LUV of two different sizes and two different compositions was studied by MALS. For comparison, the conventional batch DLS and static light scattering (SLS) experiments without prior sample separation by size were performed. From a combination of batch and flow light scattering results, we concluded that the passage flow velocities at the detector used in this study, 0.2, 0.5, and 1 mL/min, have no significant impact on the shape of spherical vesicles; however, the flow DLS experiments give accurate hydrodynamic radius (Rh) only at the lowest investigated passage flow rate at the detector (0.2 mL/min). With increasing rate of passage flow at the DLS detector, the error in the accuracy of the Rh determination rapidly increases. The error in Rh depends solely on the detector flow rate and particle size but not on the type of the soft particle.

Release mechanism of doxazosin from carrageenan matrix tablets: Effect of ionic strength and addition of sodium dodecyl sulphate.

The polyelectrolyte matrix tablets loaded with an oppositely charged drug exhibit complex drug-release mechanisms. In this study, the release mechanism of a cationic drug doxazosin mesylate (DM) from matrix tablets based on an anionic polyelectrolyte λ-carrageenan (λ-CARR) is investigated. The drug release rates from λ-CARR matrices are correlated with binding results based on potentiometric measurements using the DM ion-sensitive membrane electrode and with molecular characteristics of the DM-λ-CARR-complex particles through hydrodynamic size measurements. Experiments are performed in solutions with different ionic strength and with the addition of an anionic surfactant sodium dodecyl sulphate (SDS). It is demonstrated that in addition to swelling and erosion of tablets, the release rates depend strongly on cooperative interactions between DM and λ-CARR. Addition of SDS at concentrations below its critical micelle concentration (CMC) slows down the DM release through hydrophobic binding of SDS to the DM-λ-CARR complex. On the contrary, at concentrations above the CMC SDS pulls DM from the complex by forming mixed micelles with it and thus accelerates the release. Results involving SDS show that the concentration of surfactants that are naturally present in gastrointestinal environment may have a great impact on the drug release process.

Development of Cetylpyridinium-Alginate Nanoparticles: A Binding and Formulation Study.

In this study the development of stable polyelectrolyte-surfactant complex nanoparticles composed of alginate and cetylpyridinium chloride (CPC), with and without ZnCl2, for therapeutic use, is investigated. The mechanism of CPC binding by alginate was analyzed using a cetylpyridinium cation (CP(+)) selective membrane electrode. The cooperative nature of the interaction between CP(+) and alginate was underlined by the sigmoidal shape of the binding isotherms. The presence of salts was shown to weaken interactions and, moreover, ZnCl2 reduced the cooperativity of binding. The CP(+) cations in the form of micellar associates acted as multivalent crosslinkers of the alginate chains where stable dispersions of CP-alginate nanoparticles were formed in water at CP(+)/alginate charge ratios from 0.2 to 0.8. Characterization of the nanoparticles showed hydrodynamic diameters from 140 to 200nm, a polydispersity index below 0.2, a negative zeta potential and spherical morphology. The entrapment efficiency of CPC was ∼94%, the loading capacity more than 50% and prolonged release over 7days were shown. The formulations with noted charge ratios resulted in stable CP-alginate nanoparticles with a potential of treating periodontal disease.

A novel approach to the measurement of surfactant parameters in arthropod digestive juices.

In arthropods, the determination of two important parameters of digestive juices, i.e. the total surfactant concentration and the critical micelle concentration (CMC), is challenging due to small sample volumes and low surfactant concentrations. In this work, we report a successful implementation of potentiometric titrations using the surfactant ion-selective electrode (SISE) and the pyrene fluorescence method (PFM) for the determination of the total surfactant concentration and CMC in the digestive juice of terrestrial isopod crustaceans Porcellio scaber. Pooled digestive juice extracts of four (SISE) or two (PFM) animals were used per measurement run. In both cases, digestive juice extracts in 100 µL of deionized water were sufficient for one measurement run. The total surfactant concentration of P. scaber digestive juice was determined to be 9.2 ± 3.5mM and the CMC was approximately 90 µM. Our work presents an important improvement towards easy CMC determination in small volume samples in comparison with the commonly used stalagmometric technique, where much larger sample volumes are usually needed. To date, the total surfactant concentration was not measured in the digestive juices of arthropods other than Homarus vulgaris, Astacus leptodactylus and Cancer pagurus, for which complex separation and analytical techniques were required. Our results obtained by SISE and PFM therefore present the first successful quantification of surfactants and their CMC in small volumes of arthropod digestive juice without prior separation or purification techniques.

FTIR microscopy reveals distinct biomolecular profile of crustacean digestive glands upon subtoxic exposure to ZnO nanoparticles.

Biomolecular profiling with Fourier-Transform InfraRed Microscopy was performed to distinguish the Zn(2+)-mediated effects on the crustacean (Porcellio scaber) digestive glands from the ones elicited by the ZnO nanoparticles (NPs). The exposure to ZnO NPs or ZnCl2 (1500 and 4000 µg Zn/g of dry food) activated different types of metabolic pathways: some were found in the case of both substances, some only in the case of ZnCl2, and some only upon exposure to ZnO NPs. Both the ZnO NPs and the ZnCl2 increased the protein (∼1312 cm(-1); 1720-1485 cm(-1)/3000-2830 cm(-1)) and RNA concentration (∼1115 cm(-1)). At the highest exposure concentration of ZnCl2, where the effects occurred also at the organismal level, some additional changes were found that were not detected upon the ZnO NP exposure. These included changed carbohydrate (most likely glycogen) concentrations (∼1043 cm(-1)) and the desaturation of cell membrane lipids (∼3014 cm(-1)). The activation of novel metabolic pathways, as evidenced by changed proteins' structure (at 1274 cm(-1)), was found only in the case of ZnO NPs. This proves that Zn(2+) are not the only inducers of the response to ZnO NPs. Low bioavailable fraction of Zn(2+) in the digestive glands exposed to ZnO NPs further supports the role of particles in the ZnO NP-generated effects. This study provides the evidence that ZnO NPs induce their own metabolic responses in the subtoxic range.

Thermodynamic Analysis of the Conformational Transition in Aqueous Solutions of Isotactic and Atactic Poly(Methacrylic Acid) and the Hydrophobic Effect.

The affinity of amphiphilic compounds for water is important in various processes, e.g., in conformational transitions of biopolymers, protein folding/unfolding, partitioning of drugs in the living systems, and many others. Herein, we study the conformational transition of two isomer forms of poly(methacrylic acid) (PMA), isotactic (iPMA) and atactic (aPMA), in water. These isomers are chemically equivalent and differ only in the arrangement of functional groups along the chain. A complete thermodynamic analysis of the transition of the PMA chains from the compact to the extended form (comprising the conformational transition) in water in the presence of three alkali chlorides is conducted by determining the free energy, enthalpy, and entropy changes of the process as a function of temperature, and therefrom also the heat capacity change. The heat capacity change of the transition is positive (+20 J/K mol) for aPMA and negative (-50 J/K mol) for iPMA. This result suggests a different affinity of PMA isomers for water. The conformational transition of iPMA is parallel to the transfer of polar solutes into water, whereas that of aPMA agrees with the transfer of nonpolar solutes into water.

Interaction of Sodium Hyaluronate with a Biocompatible Cationic Surfactant from Lysine: A Binding Study.

Mixtures of natural and biodegradable surfactants and ionic polysaccharides have attracted considerable research interest in recent years because they prosper as antimicrobial materials for medical applications. In the present work, interactions between the lysine-derived biocompatible cationic surfactant N(ε)-myristoyl-lysine methyl ester, abbreviated as MKM, and the sodium salt of hyaluronic acid (NaHA) are investigated in aqueous media by potentiometric titrations using the surfactant-sensitive electrode and pyrene-based fluorescence spectroscopy. The critical micelle concentration in pure surfactant solutions and the critical association concentration in the presence of NaHA are determined based on their dependence on the added electrolyte (NaCl) concentration. The equilibrium between the protonated (charged) and deprotonated (neutral) forms of MKM is proposed to explain the anomalous binding isotherms observed in the presence of the polyelectrolyte. The explanation is supported by theoretical model calculations of the mixed-micelle equilibrium and the competitive binding of the two MKM forms to the surface of the electrode membrane. It is suggested that the presence of even small amounts of the deprotonated form can strongly influence the measured electrode response. Such ionic-nonionic surfactant mixtures are a special case of mixed surfactant systems where the amount of the nonionic component cannot be varied independently as was the case for some of the earlier studies.

Aggregates of Isotactic Poly(methacrylic acid) Chains in Aqueous CsCl Solutions: a Static and Dynamic Light Scattering Study.

Properties of isotactic polymethacrylic acid, iPMA, chains were studied at 25°C in aqueous solutions at various CsCl concentrations, c(s) (= 0.05-0.20 M), in dependence on degree of neutralization of the polyion's carboxyl groups, α(N), using static, SLS, and dynamic light scattering, DLS, measurements. It was demonstrated that iPMA chains with α(N) somewhat above the solubility limit of iPMA in aqueous solutions (in the present case at α(N) ≈ 0.27) are strongly aggregated. The size of the aggregates increases with increasing c(s), whereas the shape parameter, ρ, is approximately constant (ρ ≈ 0.6), irrespective of c(s). The low ρ value suggests that the aggregates have characteristics of microgel particles with a dense core surrounded by a less dense corona. The diffusion of iPMA chains was investigated also at higher α(N), up to α(N) = 1. The polyion slow mode arising from electrostatic interactions between charged chains was observed for α(N) exceeding the value 0.27 even at the highest c(s) (= 0.20 M). The diffusion coefficients for the show mode were nearly independent of α(N) and cs at the studied polymer concentration.

Size characterization and quantification of exosomes by asymmetrical-flow field-flow fractionation.

In the past few years extracellular vesicles called exosomes have gained huge interest of scientific community since they show a great potential for human diagnostic and therapeutic applications. However, an ongoing challenge is accurate size characterization and quantification of exosomes because of the lack of reliable characterization techniques. In this work, the emphasis was focused on a method development to size-separate, characterize, and quantify small amounts of exosomes by asymmetrical-flow field-flow fractionation (AF4) technique coupled to a multidetection system (UV and MALS). Batch DLS (dynamic light-scattering) and NTA (nanoparticle tracking analysis) analyses of unfractionated exosomes were also conducted to evaluate their shape and internal structure, as well as their number density. The results show significant influence of cross-flow conditions and channel thickness on fractionation quality of exosomes, whereas the focusing time has less impact. The AF4/UV-MALS and DLS results display the presence of two particles subpopulations, that is, the larger exosomes and the smaller vesicle-like particles, which coeluted in AF4 together with impurities in early eluting peak. Compared to DLS and AF4-MALS results, NTA somewhat overestimates the size and the number density for larger exosome population, but it discriminates the smaller particle population.