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Extracellular vesicles (EVs) and, particularly, exosomes are becoming a promising material for “cell-free therapy” of many pathologic conditions. In the recent years therapeutic effects of mesenchymal stromal cells (MSCs) have been attributed to their secretory factors, including EVs. In this study we aim to investigate how EVs produced by MSCs of different tissue origin can influence myogenesis and fibrosis–the main processes that accompany skeletal muscle regeneration. Bone marrow, adipose tissue, intact muscle, and injured muscle-derived rat MSCs were obtained and cultured according to standard protocols. EVs were obtained by ultracentrifugation from the MSC-conditioned medium of cell passages 2 and 3. The effects of EVs were estimated on the in vitro models of myogenesis and fibrosis. Samples of isolated EVs contained nano-sized vesicles that carried some exosomal markers. Promyogenic microRNA were found in EVs from bone marrow and muscle MSCs. We found that MSC-derived EVs from all sources significantly increased the number of newly formed myotubes in myoblast culture in vitro and also reduced the number and size of fibrotic nodules in muscle fibroblast culture in vitro. Our results suggest that MSC-derived EVs indeed possess antifibrotic and promyogenic potentials. However, the role of microRNA in these processes is yet to be determined, and the effect of EVs on skeletal muscle regeneration is yet to be tested in vivo.

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 present work we isolated EVs from intact or SCF-overexpressing rat MSC and analyzed microarray datasets of their miRNA cargo. We uncovered a total of 95 differentially expressed miRNAs and identified 2 miRNAs significantly up-regulated in SCF-MSCs. One of them - rno-miR-344a-2, associated with aging and regulation of a-SMA was also increased in EVs from GFP-MSC that may indicate intrinsic changes in MSC after viral transduction. About 80 miRNA were downregulated in EVs from 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 consistent with our previous EVs data. Topological analyses of network also revealed among downregulated miRNAs- rno-miRNA-128-3p that regulates plenty of targets presumably associated with chemokine signaling pathways. Overall, our data suggest that genetic modification of MSC have a great impact on their miRNA composition and provide novel insights into the regulatory networks underlying EVs effects.

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.

The multi-liposomal conjugate with encapsulated enzyme laccase is described, capable of releasing the payload when changing the temperature. For this, thermosensitive cationic microgels were synthesized, differing in the molar content of cross-linker from 2 to 8 mol%. The microgels collapsed when heated beyond the volume phase transition temperature (VPTT) of 38–40 °C, while the degree of the surface area contraction (φ) decreased with increasing the cross-linker content. At lower temperature, each swollen microgel particle, ca. 320 nm in diameter, adsorbed about 200 intact 50 nm anionic liposomes. Over VPTT, the liposomes disrupted and released their payload that was visually detected via a color “development” of a loaded into liposomes laccase substrate in the surrounding solution: the higher φ, the more released enzyme and the higher colored product concentration. So, the correlation between the microgel cross-linking degree and the amount of released liposome payload was demonstrated for the first time. The results we have obtained are of interest for constructing drug carriers, catalysts and diagnostic systems.

Thermosensitive N-isopropylacrylamide based cationic microgels (μG) were synthesized and complexed with anionic liposomes containing encapsulated antitumor antibiotic Doxorubicin (Dox). Compositions of the resulting complexes, in terms of a liposome-to-μG number ratio N, varied from N = 0.5 to a saturated complex with N = 30 where the surface of μG particles is fully covered with liposomes. The microgels collapse when heated from 25 up to 50 °C and the surface area of a microgel particle decreases. The thermo-induced contraction is accompanied with release of Dox from the μG-bound liposomes while a significant fraction of DOX is released for both the unsaturated and saturated complexes. Therefore, two mechanisms are hypothesized for the thermo-induced Dox release. The first one is due to temperature-induced conformational changes of polymer chains, subsequent rearrangements at liposome-microgel interface via liposome-microgel interaction. This mechanism can work for the complexes of any composition. The second process, namely a squeezing of liposomes via liposome-liposome interaction followed by Dox release can become significant for the saturated complex. The results we have obtained are of great interest for constructing multi-liposomal drug carriers, diagnostic systems and catalysts.

In this chapter, various techniques are reviewed with focus on the identification of complex biological agents such as bacteria, viruses, proteins, and enzymes using SERS-active silver substrates. Biological targets have multiple peculiarities that add to the challenges of the SERS biosensing. In regards to the direct non-labeled sensing of bacteria, it was discovered that all bands in the registered SER spectra were generated by metabolites released from bacterial cells. It undermined the prior notion of non-labeled detection and identification of bacteria based on the presumed spectra of cellular walls. However, it also provides new opportunities for the SERS analysis of bacteria. The SERS measurements of viruses can be performed with SERS-active surfaces or colloidal solutions of silver nanoparticles. However, the use of surfaces requires extensive sample preparation and often lacks sensitivity, while colloidal SERS substrates have another problem—most types of silver nanoparticles are negatively charged and have a poor interaction with likewise predominantly negatively charged virions. Thus, a challenge is posed to develop SERS-ready positively charged silver nanoparticles or use other methods to enforce the non-specific binding of viruses to the silver surfaces. Meanwhile, SER spectra of proteins are nearly impossible to acquire at adequate sensitivity. Thus, non-direct measurements are the only way. SERS provides the most benefits when working with relatively small molecules, so small molecules serving as Raman probes can be used as an intermediary to produce SER spectra. For enzymes like butyrylcholinesterase, it means measuring SER spectra of substrates and products of the relevant reaction, while for other proteins, specialized techniques must be developed. It can be concluded that biological targets require a case-by-case approach. Prior experiences with direct SERS measurements of highly Raman-active molecules like R6G and others often used in fundamental studies might not be relevant in bioanalytics.

Novel approaches are proposed for high-throughput multiparametric characterization and quantitative detection of extracellular vesicles (EVs) based on a combination of optical and magnetic techniques. One of them, intended for EVs visualization, employs the imaging flow cytometry with submicron magnetic particles. For quantitative detection of EVs, a magnetic lateral flow assay is developed using the same submicron magnetic particles as labels.

Mesenchymal stromal cells from rat adipose tissue were transduced with adeno-associated viral (AAV) vector encoding stem cell factor SCF that stimulates proliferation of cardiac c-kit+ cells and improved cardiac function and survival of animals after myocardial infarction. Extracellular vesicles isolated from the medium conditioned by mesenchymal stromal cells by ultracentrifugation were characterized by Western blotting, transmission electron microscopy, nanoparticle tracking analysis, immunostaining, and mass spectrometry analysis. Using proteomic analysis, we identified transgenic SCF in extracellular vesicles released by AAV-modified mesenchymal stromal cells and detected some proteins specific of extracellular vesicles secreted by transduced cells. Extracellular vesicles from AAV-transduced mesenchymal stromal cells could be used for delivery of transgenic proteins as they were readily endocytosed by both cardiosphere-derived cells and cardiac-progenitor cells.

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&rsquo, 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.

Research into the molecular origin of surface-enhanced Raman spectra (SERS) of bacteria is a crucial step in assessing the future of SERS-based discrimination and identification of bacteria in clinical analysis, food quality control, etc. Previous studies have revealed that at 785 nm excitation wavelength SERS of bacterial cells placed on a solid surface functionalized with in-situ grown aggregated gold nanoparticles covered with SiO2 originate from a mixture of 6 purine derivatives (adenine, guanine, AMP, hypoxanthine, xanthine, and uric acid) that are released by the cells into the medium. The aim of the present work was to investigate whether such interpretation is possible with a different class of SERS substrates: silver nanoparticle sols at excitation wavelengths of 785 and 532 nm. The suspension of the Escherichia coli DH5α strain was used as a model bacterium. Sols of silver nanoparticles were obtained by reducing silver nitrate in the presence of alkaline hydroxylamine hydrochloride. Number-weighted mean hydrodynamic diameter of the particles was 43±2 nm. We confirm that at both excitation wavelengths the spectra can be best described as a superposition of 4 purine derivatives: adenine, guanine, hypoxanthine, and xanthine. Importantly, we have discovered that 1) the spectra of the purine mixture are characteristic of viable cells only; 2) due to the variations in the concentrations of purine metabolites released by the cells into the surrounding medium the spectra of a bacterial strain can vary significantly when a silver nanoparticle sol is used as a SERS substrate.

Using the electrostatic adsorption of anionic liposomes on the surface of cationic microparticles of ion-crosslinked chitosan, complexes in which each microparticle can bind up to 110 intact (undestroyed) liposomes are prepared. The saturated complex 350–400 nm in diameter does not dissociate to initial components in aqueous solutions with pH 7 and a NaCl concentration of 0.15 mol/L, but decomposes to 10-nm particles in the presence of proteolytic enzymes. The chitosan–liposome complex and its biodegradation products are characterized by a low cytotoxicity. The described technique may be used to obtain biodegradable multiliposomal containers for the encapsulation and delivery of drugs.

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.

Anomalous optical response for the metamaterial fabricated from silicon bar resonators is investigated. The resonators are manufactured in form of two-dimensional bars and covered by semicontinuous silver film. The calculations as well as real experiments demonstrate Wood anomalies in visible and near IR spectral ranges associated with excitation of the surface waves in metamaterial by means of diffraction of the incident light on the periodic bars. The multiple metal-dielectric resonances result in much enhanced local electromagnetic fields in-between metal particles placed on the surface of dielectric bars. The resonances can be tuned by varying angle of incidence, polarization, and geometry of the dielectric bars. It opens new venue in R&D SERS substrates including sensors detecting specific substances.

Observation of the wanted Raman signal from SERS substrates is hampered by the background luminescence. We investigate the metasurface made of periodic silicon micro bars with thin silver layer on the top. The computer simulations as well as real experiment reveal the anomalous optical response of the metasurface is due to the excitation of the metal-dielectric surface resonances. We demonstrate the enhanced Raman signal from the organic molecules of 5,5-dithio-bis[2-nitrobenzoic] acid deposited on the microbar metasurface. The microbar metasurface exhibits large Raman/luminescence (signal/noise) ratio. As result it operates as an efficient SERS substrate.

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.

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.

Microcrystals of 1,4-bis(terpyridine-4′-yl)benzene (BTB) adsorb gold nanoparticles (AuNPs) with an average size of 15 nm or 25 nm from the solution forming an organic–gold composite with ∼20 wt% of Au. The composite with 15 nm nanoparticles was characterized by thermal gravimetric analysis (TGA), transition electron microscopy (TEM), atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). Gold nanoparticles are uniformly distributed on the organic surface and retain the same size and shape distribution even after heating at 600 °C which removes the organic matrix of 1,4-bis(terpyridine-4′-yl)benzene and effectively deposits nanoparticles onto solid substrate (mica). The catalytic activity of composites was demonstrated using a model reaction of the reduction of 4-nitrophenol to 4-aminophenol. The values of the apparent rate constants at 295 K, ca. 1.9 × 10 −3 s −1 for the Au(15 nm)–BTB and 7.2 × 10 −4 s −1 for the Au(25 nm)–BTB, obtained using UV–vis spectroscopy, are comparable to those reported for other catalytic systems based on supported Au nanoparticles. The catalytic activity is found to be size-dependent; the bigger nanoparticles show lower activity and longer reaction induction period. Au–BTB catalyst can be recovered from the reaction mixture and used again.

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 Forster 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.

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.

Background: Extracellular vesicles are small membrane particles which are produced by the cells and released into the surrounding space. They can transfer biomolecules into recipient cells. The typical vesicle size is 100-1000 nm, so transmission electron microscopy (TEM) is the most suitable tool for their visualization. This is especially true for the smallest vesicles – the exosomes with the size below 200 nm. Methods: We isolated exosomes produced by MCF-7 cancer cells and the two drug-resistant MCF-7 sublines (Semina et.al., Molecules, 2018). The exosomes were isolated from the conditioned medium by ultracentrifugation, and their size was measured by nanotracking analysis (NTA) and TEM. Immunogold labelling was carried out on exosomes deposited onto carbon TEM grids. It involved the following principal steps: the vesicle deposition, sample treatment by the anti-CD9 antibodies, sample treatment by the 10 nm colloidal gold nanoparticles conjugated with protein A, staining with uranyl acetate. Washing and blocking steps were done between the principal ones. The samples were imaged using a JEM-1011 microscope at 80 kV. Results: NTA and TEM yielded similar results – the exosomes produced by the three studied cell lines had the almost equal size in the range from 50 to 200 nm. When imaged by TEM, most exosomes demonstrated the typical cup-shaped morphology. We used immunogold labelling to verify that the exosomes carried the CD9 marker on them (Fig. 1). We achieved the labelling specificity in the range of 8-10; this value was calculated as the ratio of the surface densities of the exosome-bound labels (colloidal gold nanoparticles) and the non-specific background ones. Conclusion: We hope that the sample preparation and imaging procedures that we used could be useful for the investigation of other extracellular vesicles.

Scientific Reports 5: Article number: 17319; published online: 30 November 2015; updated: 29 March 2016 The original version of this Article contained a typographical error in the spelling of the author Mikhail A. Livshits, which was incorrectly given as Mikhail A. Livshts. This has now been corrected in both the PDF and HTML versions of the Article.

Nanoparticles of various manganese dioxide crystalline modifications (amorphous, β-phase, and γ-phase) were compared with respect to their mediator properties in electrochemical detection of thiols. The highest catalytic activity towards thiocholine, cysteine, and glutathione was demonstrated by screen-printed graphite electrodes modified with γ-MnO2 at the optimized working potential of 450 mV versus the Ag/AgCl reference electrode. High sensitivity of γ-MnO2 modified electrodes towards thiocholine (345 mA cm2/M) and therefore low detection limit of butyrylcholinesterase (1 pM) enabled their use for subnanomolar detection of an organophosphate pesticide diazinon, an irreversible inhibitor of butyrylcholinesterase. The detection limit for diazinon was estimated as 0.6 nM with assay duration of less than 15 min.

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.

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.

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.

In this study we investigated the effects of monovalent anions in the series F−, Cl−, Br−, I− on the sensitivity of the biosensor assembled from polycations and ChO via theirs layer-by-layer deposition on the surface of carbon ­electrodes (CE) coated with MnO2. The efficiency of gamma-phase-MnO2 nanoparticles, deposited on screen-printed electrodes, as a new transducer element of biosensing platforms, fabricated with “layer-by-layer” technology was demonstrated. The present report describes analytical possibilities of the biosensing platform. The hydrogen peroxide registration and subsequent application for neurotoxicants assay in the environmental field. The validation of peroxide-­sensitive sensors, as a new transducer element based on MnO2 nanoparticles, for fabrication of choline oxidase biosensor has been done. The revealed analytical characteristics of the choline oxidase biosensor are perspective for highly-­sensitive monitoring of cholinesterases and their inhibitors in medical, biological and environmental samples.

This study describes the effects of F−, Cl−, Br−, and I− anions on the sensitivity of a biosensor assembled via layer-by-layer deposition of poly(diallyldimethylammonium chloride) (PDDA) and choline oxidase (ChO) on the surface of a graphite electrode coated with MnO2. A four-fold increase in sensitivity of the MnO2/PDDAKHal/ChO biosensor was observed when the adsorption of the PDDA layer was carried out from the solution containing I− in a narrow range of concentrations (20–30 mM). According to AFM and SEM data, spatial distribution of the enzyme and gold nanoparticles (AuNPs) on the surface varied with the conditions of PDDA adsorption. A fine-grain film was observed when PDDA was adsorbed from the solution containing Cl−, whereas association of ChO or AuNPs into larger domains took place in the case of I− solution. Absorption spectra of AuNPs adsorbed on a PDDAKI film evidenced aggregate formation as well. A schematic representation of the enzyme layer assembly was proposed for different ionic conditions of the polyelectrolyte pre-adsorption.