Highly Sensitive Measurement of Horseradish Peroxidase Using Surface-Enhanced Raman Scattering of 2,3-Diaminophenazine.

The development of various enzyme-linked immunosorbent assays (ELISAs) coupled with surface-enhanced Raman scattering (SERS) detection is a growing area in analytical chemistry due to their potentially high sensitivity. A SERS-based ELISA with horseradish peroxidase (HRP) as an enzymatic label, an o-phenylenediamine (oPD) substrate, and a 2,3-diaminophenazine (DAP) enzymatic product was one of the first examples of such a system. However, the full capabilities of this long-known approach have yet to be revealed. The current study addresses a previously unrecognized problem of SERS detection stage performance. Using silver nanoparticles and model mixtures of oPD and DAP, the effects of the pH, the concentration of the aggregating agent, and the particle surface chloride stabilizer were extensively evaluated. At the optimal mildly acidic pH of 3, a 0.93 to 1 M citrate buffer, and AgNPs stabilized with 20 mM chloride, a two orders of magnitude advantage in the limits of detection (LODs) for SERS compared to colorimetry was demonstrated for both DAP and HRP. The resulting LOD for HRP of 0.067 pmol/L (1.3 amol per assay) underscores that the developed approach is a highly sensitive technique. We suppose that this improved detection system could become a useful tool for the development of SERS-based ELISA protocols.

Stomatin is highly expressed in exosomes of different origin and is a promising candidate as an exosomal marker.

Proteins involved in the organizing of lipid rafts can be found in exosomes, as shown for caveolin-1, and they could contribute to exosomal cargo sorting, as shown for flotillins. Stomatin belongs to the same stomatin/prohibitin/flotillin/HflK/C family of lipid rafts proteins, but it has never been studied in exosomes except for extracellular vesicles (EVs) originating from blood cells. Here we first show the presence of stomatin in exosomes produced by epithelial cancer cells (non-small cell lung cancer, breast, and ovarian cancer cells) as well as in EVs from biological fluids, including blood plasma, ascitic fluids, and uterine flushings. A high abundance of stomatin in EVs of various origins and its enrichment in exosomes make stomatin a promising exosomal marker. Comparison with other lipid raft proteins and exosomal markers showed that the level of stomatin protein in exosomes from different sources corresponds well to that of CD9, while it differs essentially from flotillin-1 and flotillin-2 homologs, which in turn are present in exosomes in nearly equal proportions. In contrast, the level of vesicular caveolin-1 as well as its EV-to-cellular ratio vary drastically depending on cell type.

Exosome-Mediated Transfer of Cancer Cell Resistance to Antiestrogen Drugs.

Exosomes are small vesicles which are produced by the cells and released into the surrounding space. They can transfer biomolecules into recipient cells. The main goal of the work was to study the exosome involvement in the cell transfer of hormonal resistance. The experiments were performed on in vitro cultured estrogen-dependent MCF-7 breast cancer cells and MCF-7 sublines resistant to SERM tamoxifen and/or biguanide metformin, which exerts its anti-proliferative effect, at least in a part, via the suppression of estrogen machinery. The exosomes were purified by differential ultracentrifugation, cell response to tamoxifen was determined by MTT test, and the level and activity of signaling proteins were determined by Western blot and reporter analysis. We found that the treatment of the parent MCF-7 cells with exosomes from the resistant cells within 14 days lead to the partial resistance of the MCF-7 cells to antiestrogen drugs. The primary resistant cells and the cells with the exosome-induced resistance were characterized with these common features: decrease in ERα activity and parallel activation of Akt and AP-1, NF-κB, and SNAIL1 transcriptional factors. In general, we evaluate the established results as the evidence of the possible exosome involvement in the transferring of the hormone/metformin resistance in breast cancer cells.

Proteome-metabolome profiling of ovarian cancer ascites reveals novel components involved in intercellular communication.

Ovarian cancer ascites is a native medium for cancer cells that allows investigation of their secretome in a natural environment. This medium is of interest as a promising source of potential biomarkers, and also as a medium for cell-cell communication. The aim of this study was to elucidate specific features of the malignant ascites metabolome and proteome. In order to omit components of the systemic response to ascites formation, we compared malignant ascites with cirrhosis ascites. Metabolome analysis revealed 41 components that differed significantly between malignant and cirrhosis ascites. Most of the identified cancer-specific metabolites are known to be important signaling molecules. Proteomic analysis identified 2096 and 1855 proteins in the ovarian cancer and cirrhosis ascites, respectively; 424 proteins were specific for the malignant ascites. Functional analysis of the proteome demonstrated that the major differences between cirrhosis and malignant ascites were observed for the cluster of spliceosomal proteins. Additionally, we demonstrate that several splicing RNAs were exclusively detected in malignant ascites, where they probably existed within protein complexes. This result was confirmed in vitro using an ovarian cancer cell line. Identification of spliceosomal proteins and RNAs in an extracellular medium is of particular interest; the finding suggests that they might play a role in the communication between cancer cells. In addition, malignant ascites contains a high number of exosomes that are known to play an important role in signal transduction. Thus our study reveals the specific features of malignant ascites that are associated with its function as a medium of intercellular communication.

Engineering Systems with Spatially Separated Enzymes via Dual-Stimuli-Sensitive Properties of Microgels.

This work examines the adsorption regime and the properties of microgel/enzyme thin films deposited onto conductive graphite-based substrates. The films were formed via two-step sequential adsorption. A temperature- and pH-sensitive poly(N-isopropylacrylamide)-co-(3-(N,N-dimethylamino)propylmethacrylamide) microgel (poly(NIPAM-co-DMAPMA microgel) was adsorbed first, followed by its interaction with the enzymes, choline oxidase (ChO), butyrylcholinesterase (BChE), or mixtures thereof. By temperature-induced stimulating both (i) poly(NIPAM-co-DMAPMA) microgel adsorption at T > VPTT followed by short washing and drying and then (ii) enzyme loading at T < VPTT, we can effectively control the amount of the microgel adsorbed on a hydrophobic interface as well as the amount and the spatial localization of the enzyme interacted with the microgel film. Depending on the biomolecule size, enzyme molecules can (in the case for ChO) or cannot (in the case for BChE) penetrate into the microgel interior and be localized inside/outside the microgel particles. Different spatial localization, however, does not affect the specific enzymatic responses of ChO or BChE and does not prevent cascade enzymatic reaction involving both BChE and ChO as well. This was shown by the methods of electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM), and amperometric analysis of enzymatic responses of immobilized enzymes. Thus, a novel simple and fast strategy for physical entrapment of biomolecules by the polymeric matrix was proposed, which can be used for engineering systems with spatially separated enzymes of different types.

Therapy-induced secretion of spliceosomal components mediates pro-survival crosstalk between ovarian cancer cells.

Ovarian cancer often develops resistance to conventional therapies, hampering their effectiveness. Here, using ex vivo paired ovarian cancer ascites obtained before and after chemotherapy and in vitro therapy-induced secretomes, we show that molecules secreted by ovarian cancer cells upon therapy promote cisplatin resistance and enhance DNA damage repair in recipient cancer cells. Even a short-term incubation of chemonaive ovarian cancer cells with therapy-induced secretomes induces changes resembling those that are observed in chemoresistant patient-derived tumor cells after long-term therapy. Using integrative omics techniques, we find that both ex vivo and in vitro therapy-induced secretomes are enriched with spliceosomal components, which relocalize from the nucleus to the cytoplasm and subsequently into the extracellular vesicles upon treatment. We demonstrate that these molecules substantially contribute to the phenotypic effects of therapy-induced secretomes. Thus, SNU13 and SYNCRIP spliceosomal proteins promote therapy resistance, while the exogenous U12 and U6atac snRNAs stimulate tumor growth. These findings demonstrate the significance of spliceosomal network perturbation during therapy and further highlight that extracellular signaling might be a key factor contributing to the emergence of ovarian cancer therapy resistance.

Investigating volatile compounds in the Bacteroides secretome.

Microorganisms and their hosts communicate with each other by secreting numerous components. This cross-kingdom cell-to-cell signaling involves proteins and small molecules, such as metabolites. These compounds can be secreted across the membrane via numerous transporters and may also be packaged in outer membrane vesicles (OMVs). Among the secreted components, volatile compounds (VOCs) are of particular interest, including butyrate and propionate, which have proven effects on intestinal, immune, and stem cells. Besides short fatty acids, other groups of volatile compounds can be either freely secreted or contained in OMVs. As vesicles might extend their activity far beyond the gastrointestinal tract, study of their cargo, including VOCs, is even more pertinent. This paper is devoted to the VOCs secretome of the Bacteroides genus. Although these bacteria are highly presented in the intestinal microbiota and are known to influence human physiology, their volatile secretome has been studied relatively poorly. The 16 most well-represented Bacteroides species were cultivated; their OMVs were isolated and characterized by NTA and TEM to determine particle morphology and their concentration. In order to analyze the VOCs secretome, we propose a headspace extraction with GC-MS analysis as a new tool for sample preparation and analysis of volatile compounds in culture media and isolated bacterial OMVs. A wide range of released VOCs, both previously characterized and newly described, have been revealed in media after cultivation. We identified more than 60 components of the volatile metabolome in bacterial media, including fatty acids, amino acids, and phenol derivatives, aldehydes and other components. We found active butyrate and indol producers among the analyzed Bacteroides species. For a number of Bacteroides species, OMVs have been isolated and characterized here for the first time as well as volatile compounds analysis in OMVs. We observed a completely different distribution of VOC in vesicles compared to the bacterial media for all analyzed Bacteroides species, including almost complete absence of fatty acids in vesicles. This article provides a comprehensive analysis of the VOCs secreted by Bacteroides species and explores new perspectives in the study of bacterial secretomes in relation the intercellular communication.

Highly Sensitive Nanomagnetic Quantification of Extracellular Vesicles by Immunochromatographic Strips: A Tool for Liquid Biopsy.

Extracellular vesicles (EVs) are promising agents for liquid biopsy-a non-invasive approach for the diagnosis of cancer and evaluation of therapy response. However, EV potential is limited by the lack of sufficiently sensitive, time-, and cost-efficient methods for their registration. This research aimed at developing a highly sensitive and easy-to-use immunochromatographic tool based on magnetic nanoparticles for EV quantification. The tool is demonstrated by detection of EVs isolated from cell culture supernatants and various body fluids using characteristic biomarkers, CD9 and CD81, and a tumor-associated marker-epithelial cell adhesion molecules. The detection limit of 3.7 × 105 EV/µL is one to two orders better than the most sensitive traditional lateral flow system and commercial ELISA kits. The detection specificity is ensured by an isotype control line on the test strip. The tool's advantages are due to the spatial quantification of EV-bound magnetic nanolabels within the strip volume by an original electronic technique. The inexpensive tool, promising for liquid biopsy in daily clinical routines, can be extended to other relevant biomarkers.

Analysis of MicroRNA Profile Alterations in Extracellular Vesicles From Mesenchymal Stromal Cells Overexpressing Stem Cell Factor.

Mesenchymal stem/stromal cells (MSCs) represent a promising tool to treat cardiovascular diseases. One mode of action through which MSCs exert their protective effects is secretion of extracellular vesicles (EVs). Recently, we demonstrated that rat adipose-derived MSC-overexpressing stem cell factor (SCF) can induce endogenous regenerative processes and improve cardiac function. In the present work, we isolated EVs from intact, GFP- or SCF-overexpressing rat MSC and analyzed microarray datasets of their miRNA cargo. We uncovered a total of 95 differentially expressed miRNAs. We did not observe significant differences between EVs from GFP-MSC and SCF-MSC that may indicate intrinsic changes in MSC after viral transduction. About 80 miRNAs were downregulated in EVs from both SCF- or GFP-MSC. We assembled the miRNA-based network and found several nodes of target genes among which Vim Sept3 and Vsnl1 are involved in regulation of cellular migration that is consistent with our previous EVs data. Topological analyses of the network also revealed that among the downregulated miRNA-rno-miRNA-128-3p that regulates plenty of targets is presumably associated with chemokine signaling pathways. Overall, our data suggest that genetic modification of MSC has a great impact on their miRNA composition and provide novel insights into the regulatory networks underlying EV effects.

Tropism of Extracellular Vesicles and Cell-Derived Nanovesicles to Normal and Cancer Cells: New Perspectives in Tumor-Targeted Nucleic Acid Delivery.

The main advantage of extracellular vesicles (EVs) as a drug carrier system is their low immunogenicity and internalization by mammalian cells. EVs are often considered a cell-specific delivery system, but the production of preparative amounts of EVs for therapeutic applications is challenging due to their laborious isolation and purification procedures. Alternatively, mimetic vesicles prepared from the cellular plasma membrane can be used in the same way as natural EVs. For example, a cytoskeleton-destabilizing agent, such as cytochalasin B, allows the preparation of membrane vesicles by a series of centrifugations. Here, we prepared cytochalasin-B-inducible nanovesicles (CINVs) of various cellular origins and studied their tropism in different mammalian cells. We observed that CINVs derived from human endometrial mesenchymal stem cells exhibited an enhanced affinity to epithelial cancer cells compared to myeloid, lymphoid or neuroblastoma cancer cells. The dendritic cell-derived CINVs were taken up by all studied cell lines with a similar efficiency that differed from the behavior of DC-derived EVs. The ability of cancer cells to internalize CINVs was mainly determined by the properties of recipient cells, and the cellular origin of CINVs was less important. In addition, receptor-mediated interactions were shown to be necessary for the efficient uptake of CINVs. We found that CINVs, derived from late apoptotic/necrotic cells (aCINVs) are internalized by in myelogenous (K562) 10-fold more efficiently than CINVs, and interact much less efficiently with melanocytic (B16) or epithelial (KB-3-1) cancer cells. Finally, we found that CINVs caused a temporal and reversible drop of the rate of cell division, which restored to the level of control cells with a 24 h delay.

Adsorption of extracellular vesicles onto the tube walls during storage in solution.

Short term storage of extracellular vesicle (EV) solutions at +4°C is a common practice, but the stability of EVs during this procedure has not been fully understood yet. Using nanoparticle tracking analysis, we have shown that EVs isolated from the conditioned medium of HT-29 cells exhibit a pronounced concentration decrease when stored in PBS in ordinary polypropylene tubes within the range of (0.5-2.1) × 1010 particles/ml. EV losses reach 51±3% for 0.5 ml of EVs in Eppendorf 2 ml tube at 48 hours of storage at +4°C. Around 2/3 of the observed losses have been attributed to the adsorption of vesicles onto tube walls. This result shows that the lower part (up to at least 2 × 1010 particles/ml) of the practically relevant concentration range for purified EVs is prone to adsorption losses at +4°C. Total particle losses could be reduced to 18-21% at 48 hours by using either Eppendorf Protein LoBind tubes or ordinary tubes with the surface blocked with bovine serum albumin or EVs. Reduction of losses to 15% has been shown for isolated EVs dissolved in the supernatant after 100 000 g centrifugation as a model of conditioned medium. Also, a previously unknown feature of diffusion-controlled adsorption was revealed for EVs. In addition to the decrease in particle count, this process causes the predominant losses of smaller particles.

Secretome of Mesenchymal Stromal Cells Prevents Myofibroblasts Differentiation by Transferring Fibrosis-Associated microRNAs within Extracellular Vesicles.

Fibroblasts differentiation into myofibroblasts is a central event of tissue fibrosis. Multipotent mesenchymal stromal cells (MSCs) secretome can interfere with fibrosis development; despite precise underlying mechanisms remain unclear. In this study, we tested the hypothesis that MSC secretome can affect fibroblast' differentiation into myofibroblasts by delivering regulatory RNAs, including microRNAs to these cells. Using the model of transforming growth factor-beta (TGFbeta)-induced fibroblast differentiation into myofibroblasts, we tested the activity of human MSC secretome components, specifically extracellular vesicles (MSC-EV). We showed that MSC-EV down-regulated secretion of extracellular matrix proteins by fibroblasts as well as suppressed their contractility resulting in prevention as well as reversion of fibroblasts differentiation to myofibroblasts. High-throughput sequencing of RNAs extracted from MSC-EV has revealed many fibrosis-associated microRNAs. Fibroblast treatment with MSC-EV led to direct transfer of microRNAs, which resulted in the elevation of most prominent fibrosis-associated microRNAs, including microRNA-21 and microRNA-29c. Using MSC-EV transfection by antagomirs to these microRNAs we demonstrated their involvement in the suppression of fibroblast differentiation in our model. Taken together, MSC secretome can suppress fibrosis by prevention of fibroblast differentiation into myofibroblasts as well as induce de-differentiation of the latter by direct transfer of specific microRNAs.

Effect of MSCs and MSC-Derived Extracellular Vesicles on Human Blood Coagulation.

Mesenchymal stem cells (MSCs) have emerged as a potent therapeutic tool for the treatment of a number of pathologies, including immune pathologies. However, unwelcome effects of MSCs on blood coagulation have been reported, motivating us to explore the thrombotic properties of human MSCs from the umbilical cord. We revealed strong procoagulant effects of MSCs on human blood and platelet-free plasma using rotational thromboelastometry and thrombodynamic tests. A similar potentiation of clotting was demonstrated for MSC-derived extracellular vesicles (EVs). To offer approaches to avoid unwanted effects, we studied the impact of a heparin supplement on MSC procoagulative properties. However, MSCs still retained procoagulant activity toward blood from children receiving a therapeutic dose of unfractionated heparin. An analysis of the mechanisms responsible for the procoagulant effect of MSCs/EVs revealed the presence of tissue factor and other proteins involved in coagulation-associated pathways. Also, we found that some MSCs and EVs were positive for annexin V, which implies the presence of phosphatidylserine on their surfaces, which can potentiate clot formation. Thus, we revealed procoagulant activity of MSCs/EVs associated with the presence of phosphatidylserine and tissue factor, which requires further analysis to avoid adverse effects of MSC therapy in patients with a risk of thrombosis.

Influence of stabilizing components on the integrity of antitumor liposomes loaded with lipophilic prodrug in the bilayer.

Previously, we proposed a liposomal formulation of melphalan (Mlph)-a chemotherapeutic alkylating agent-incorporated in a fluid lipid bilayer in the form of dioleoylglyceride ester. In this work, we compared the stabilizing effect of different amphiphiles included in the Mlph-liposomes, such as phosphatidylinositol (PI), ganglioside GM1, a conjugate of N-carboxymethyl-modified oligoglycine with dioleoylphosphatidylethanolamine (acidic lipopeptide), and polyethylene glycol (2000 Da) conjugated with dipalmitoylphosphatidylethanolamine (PEG-lipid), upon incubation in human serum. Mean hydrodynamic diameter values (86-90 nm) were similar among different liposome samples, while zeta potential values considerably varied. The formulations were incubated in human serum at 37 °C for different time intervals up to 24 h. Liposome integrity was evaluated by changes in fluorescence upon leakage of calcein or disruption of Förster resonance energy transfer between donor and acceptor fluorescent lipid probes in the bilayer. The best stabilization of liposomes was achieved upon the addition of ganglioside GM1 or the acidic lipopeptide. Inclusion of 10 mol% PI improved liposome stability only for the first 4 h of incubation. Pegylated liposomal formulations of melphalan lipophilic prodrug with fluid phase bilayer were the least stable, which is probably due to the propensity of the PEG-lipid to exit liposome membranes. Cholesterol-containing bilayers of liquid ordered phase, supplemented with sufficient amounts of the PEG-lipid, showed good stability in serum.

Control of optical transparency and infrared laser heating of costal cartilage via injection of iohexol.

Infrared (IR) laser impact has no analogues for rapid and safe cartilage reshaping. For better penetration of radiation optical clearing agents (OCAs) can be applied. In present work, the effect of low-osmolality agent iohexol on costal cartilage is studied. Specifically, it is shown that ½ of total increase of optical transparency occurs in 20 minutes of immersion. Maximally, cartilage transparency on 1560 nm can be increased in 1.5 times. Injection of iohexol results in increased tissue hygroscopicity, lower drying rate and higher percentage of bound water. Effective diffusion coefficients of water liberation at 21°C are (5.3 ± 0.4) × 10-7 and (3.3 ± 0.1) × 10-7 cm2 /s for untreated and iohexol-modified tissue, respectively. Raman spectroscopy of irradiated iohexol solution reveals its photo and thermo-stability under clinically used IR laser energies up to 350 W/cm2 for exposure times of several seconds. At energies higher than 500 W/cm2 [Correction added on 5 September 2018, after first online publication: This unit has been changed] decomposition of iohexol occurs rapidly through formation of molecular iodine and fluorescent residue.

Controlling the near-infrared transparency of costal cartilage by impregnation with clearing agents and magnetite nanoparticles.

Penetration depth of near-infrared laser radiation to costal cartilage is controlled by the tissue absorption and scattering, and it is the critical parameter to provide the relaxation of mechanical stress throughout the whole thickness of cartilage implant. To enhance the penetration for the laser radiation on 1.56 µm, the optical clearing solutions of glycerol and fructose of various concentrations are tested. The effective and reversible tissue clearance was achieved. However, the increasing absorption of radiation should be concerned: 5°C-8°C increase of tissue temperature was detected. Laser parameters used for stress relaxation in cartilage should be optimized when applying optical clearing agents. To concentrate the absorption in the superficial tissue layers, magnetite nanoparticle (NP) dispersions with the mean size 95 ± 5 nm and concentration 3.9 ± 1.1 × 1011 particles/mL are applied. The significant increase in the tissue heating rate was observed along with the decrease in its transparency. Using NPs the respective laser power can be decreased, allowing us to obtain the working temperature locally with reduced thermal effect on the surrounding tissue.

Apoptotic Cell-Derived Extracellular Vesicles Promote Malignancy of Glioblastoma Via Intercellular Transfer of Splicing Factors.

Aggressive cancers such as glioblastoma (GBM) contain intermingled apoptotic cells adjacent to proliferating tumor cells. Nonetheless, intercellular signaling between apoptotic and surviving cancer cells remain elusive. In this study, we demonstrate that apoptotic GBM cells paradoxically promote proliferation and therapy resistance of surviving tumor cells by secreting apoptotic extracellular vesicles (apoEVs) enriched with various components of spliceosomes. apoEVs alter RNA splicing in recipient cells, thereby promoting their therapy resistance and aggressive migratory phenotype. Mechanistically, we identified RBM11 as a representative splicing factor that is upregulated in tumors after therapy and shed in extracellular vesicles upon induction of apoptosis. Once internalized in recipient cells, exogenous RBM11 switches splicing of MDM4 and Cyclin D1 toward the expression of more oncogenic isoforms.

Comparative Study of Non-Enveloped Icosahedral Viruses Size.

Now, as before, transmission electron microscopy (TEM) is a widely used technique for the determination of virions size. In some studies, dynamic light scattering (DLS) has also been applied for this purpose. Data obtained by different authors and using different methods could vary significantly. The process of TEM sample preparation involves drying on the substrate, which can cause virions to undergo morphology changes. Therefore, other techniques should be used for measurements of virions size in liquid, (i.e. under conditions closer to native). DLS and nanoparticle tracking analysis (NTA) provide supplementary data about the virions hydrodynamic diameter and aggregation state in liquid. In contrast to DLS, NTA data have a higher resolution and also are less sensitive to minor admixtures. In the present work, the size of non-enveloped icosahedral viruses of different nature was analyzed by TEM, DLS and NTA: the viruses used were the encephalomyocarditis virus (animal virus), and cauliflower mosaic virus, brome mosaic virus and bean mild mosaic virus (plant viruses). The same, freshly purified, samples of each virus were used for analysis using the different techniques. The results were compared with earlier published data and description databases. DLS data about the hydrodynamic diameter of bean mild mosaic virus, and NTA data for all examined viruses, were obtained for the first time. For all virus samples, the values of size obtained by TEM were less than virions sizes determined by DLS and NTA. The contribution of the electrical double layer (EDL) in virions hydrodynamic diameter was evaluated. DLS and NTA data adjusted for EDL thickness were in better agreement with TEM results.

Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol.

Exosomes, small (40-100 nm) extracellular membranous vesicles, attract enormous research interest because they are carriers of disease markers and a prospective delivery system for therapeutic agents. Differential centrifugation, the prevalent method of exosome isolation, frequently produces dissimilar and improper results because of the faulty practice of using a common centrifugation protocol with different rotors. Moreover, as recommended by suppliers, adjusting the centrifugation duration according to rotor K-factors does not work for "fixed-angle" rotors. For both types of rotors--"swinging bucket" and "fixed-angle"--we express the theoretically expected proportion of pelleted vesicles of a given size and the "cut-off" size of completely sedimented vesicles as dependent on the centrifugation force and duration and the sedimentation path-lengths. The proper centrifugation conditions can be selected using relatively simple theoretical estimates of the "cut-off" sizes of vesicles. Experimental verification on exosomes isolated from HT29 cell culture supernatant confirmed the main theoretical statements. Measured by the nanoparticle tracking analysis (NTA) technique, the concentration and size distribution of the vesicles after centrifugation agree with those theoretically expected. To simplify this "cut-off"-size-based adjustment of centrifugation protocol for any rotor, we developed a web-calculator.