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Extracellular nanoparticles (EPs) are a subject of increasing interest for their biological role as mediators in cell–cell communication; however, their harvesting and assessment from bodily fluids are challenging, as processing can significantly affect samples. With the aim of minimizing processing artifacts, we assessed the number density (n) and hydrodynamic diameter (Dh) of EPs directly in diluted plasma and blood using the following recently developed technique: interferometric light microscopy (ILM). We analyzed 613 blood and plasma samples from human patients with inflammatory bowel disease (IBD), collected in trisodium citrate and ethylenediaminetetraacetic acid (EDTA) anticoagulants, and 163 blood and plasma samples from canine patients with brachycephalic obstructive airway syndrome (BOAS). We found a highly statistically significant correlation between n in the plasma and n in the blood only in the human (i.e., but not canine) blood samples, between the samples with trisodium citrate and EDTA, and between the respective Dh for both species (all p < 10−3). In the human plasma, the average was 139 ± 31 nm; in the human blood, was 158 ± 11 nm; in the canine plasma, was 155 ± 32 nm; and in the canine blood, was 171 ± 33 nm. The differences within species were statistically significant (p < 10−2), with sufficient statistical power (P > 0.8). For , we found no statistically significant differences between the human plasma and blood samples or between the samples with trisodium citrate and EDTA. Our results prove that measuring n and Dh of EPs in minimally processed fresh blood and in diluted fresh plasma by means of ILM is feasible for large populations of samples.

Nano- and micro-sized vesicular and colloidal structures mediate cell–cell communication. They are important players in the physiology of plants, animals, and humans, and are a subject of increasing interest. We investigated the effect of three surfactants, N-cetylpyridinium chloride (CPC), sodium dodecyl sulfate (SDS), and Triton X-100 (TX100), and two anionic polyelectrolytes, sodium polystyrene sulfonate (NaPSS) and sodium polymethacrylate (NaPMA), on nanoliposomes. In addition, the effect of SDS and TX100 on selected biological membranes (erythrocytes and microalgae) was investigated. The liposomes were produced by extrusion and evaluated by microcalorimetry and light scattering, based on the total intensity of the scattered light (Itot), hydrodynamic radius (Rh), radius of gyration (Rg), shape parameter p (=Rh/Rg,0), and polydispersity index. The EPs shed from erythrocytes and microalgae Dunaliella tertiolecta and Phaeodactylum tricornutum were visualized by scanning electron microscopy (SEM) and analyzed by flow cytometry (FCM). The Rh and Itot values in POPC liposome suspensions with added CPC, SDS, and TX100 were roughly constant up to the respective critical micelle concentrations (CMCs) of the surfactants. At higher compound concentrations, Itot dropped towards zero, whereas Rh increased to values higher than in pure POPC suspensions (Rh ≈ 60–70 nm), indicating the disintegration of liposomes and formation of larger particles, i.e., various POPC–S aggregates. Nanoliposomes were stable upon the addition of NaPSS and NaPMA, as indicated by the constant Rh and Itot values. The interaction of CPC, SDS, or TX100 with liposomes was exothermic, while there were no measurable heat effects with NaPSS or NaPMA. The SDS and TX100 increased the number density of EPs several-fold in erythrocyte suspensions and up to 30-fold in the conditioned media of Dunaliella tertiolecta at the expense of the number density of cells, which decreased to less than 5% in erythrocytes and several-fold in Dunaliella tertiolecta. The SDS and TX100 did not affect the number density of the microalgae Phaeodactylum tricornutum, while the number density of EPs was lower in the conditioned media than in the control, but increased several-fold in a concentration-dependent manner. Our results indicate that amphiphilic molecules need to be organized in nanosized particles to match the local curvature of the membrane for facilitated uptake. To pursue this hypothesis, other surfactants and biological membranes should be studied in the future for more general conclusions.

Nicotine activates nicotinic acetylcholine receptors (nAChRs), which are overexpressed in numerous cancer types, leading to signaling pathways that increase lung cancer invasiveness and resistance to chemotherapeutic agents. In this study, the effects of APS12-2, a synthetic analog of marine sponge toxin that acts as an antagonist of nAChRs, was investigated in vitro on A549 human lung adenocarcinoma cells and non-tumorigenic human lung epithelial BEAS-2B cells. In addition, gelatin nanoparticles (GNPs) loaded with APS12-2 (APS12-2-GNPs) were prepared and their effects were compared with those of free APS12-2. Nicotine reduced cytotoxicity, the formation of reactive oxygen species, and the formation of lipid droplets caused by cisplatin on A549 cells. The effects of nicotine on the decreased efficacy of cisplatin were reduced by APS12-2 and APS12-2-GNPs. APS12-2-GNPs showed a substantial advantage compared with free APS12-2; the cytotoxicity of APS12-2 on BEAS-2B cells was greatly reduced when APS12-2 was loaded in GNPs, whereas the cytotoxicity on A549 cells was only slightly reduced. Our results suggest that both APS12-2 and APS12-2-GNPs hold promise as supportive agents in the cisplatin-based chemotherapy of lung cancer.

In order to prepare optimal platelet and extracellular vesicle (EV)-rich plasma for the treatment of chronic temporal bone inflammation, we studied effects of centrifugation parameters on redistribution of blood constituents in blood samples of 23 patients and 20 volunteers with no record of disease. Concentrations of blood cells and EVs were measured by flow cytometry. Sample content was inspected by scanning electron microscopy. A mathematical model was constructed to interpret the experimental results. The observed enrichment of plasma in platelets and EVs after a single spin of blood depended on the erythrocyte sedimentation rate, thereby indicating the presence of a flow of plasma that carried platelets and EVs in the direction opposite to settling of erythrocytes. Prolonged handling time correlated with the decrease of concentration of platelets and larger EVs in platelet and EV-rich plasma (PVRP), R = −0.538, p = 0.003, indicating cell fragmentation during the processing of samples. In further centrifugation of the obtained plasma, platelet and EV enrichment depended on the average distance of the sample from the centrifuge rotor axis. Based on the agreement of the model predictions with observations, we propose the centrifugation protocol optimal for platelet and EV enrichment and recovery in an individual sample, adjusted to the dimensions of the centrifuge rotor, volume of blood and erythrocyte sedimentation rate.

Electrochemical-based biosensors have the potential to be a fast, label-free, simple approach to detecting the effects of cytotoxic substances in liquid media. In the work presented here, a cell-based electrochemical biosensor was developed and evaluated to detect the cytotoxic effects of Zn2+ ions in a solution as a reference test chemical. A549 cells were attached to the surface of stainless-steel electrodes. After treatment with ZnCl2, the morphological changes of the cells and, ultimately, their death and detachment from the electrode surface as cytotoxic effects were detected through changes in the electrical signal. Electrochemical cell-based impedance spectroscopy (ECIS) measurements were conducted with cytotoxicity tests and microscopic observation to investigate the behavior of the A549 cells. As expected, the Zn2+ ions caused changes in cell confluency and spreading, which were checked by light microscopy, while the cell morphology and attachment pattern were explored by scanning electron microscopy (SEM). The ECIS measurements confirmed the ability of the biosensor to detect the effects of Zn2+ ions on A549 cells attached to the low-cost stainless-steel surfaces and its potential for use as an inexpensive detector for a broad range of chemicals and nanomaterials in their cytotoxic concentrations.

Humans are frequently exposed to environmental hepatotoxins, which can lead to liver failure. Biosensors may be the best candidate for the detection of hepatotoxins because of their high sensitivity and specificity, convenience, time-saving, low cost, and extremely low detection limit. To investigate suitability of HepG2 cells for biosensor use, different methods of adhesion on stainless steel surfaces were investigated, with three groups of experiments performed in vitro. Cytotoxicity assays, which include the resazurin assay, the neutral red assay (NR), and the Coomassie Brilliant Blue (CBB) assay, were used to determine the viability of HepG2 cells exposed to various concentrations of aflatoxin B1 (AFB1) and isoniazid (INH) in parallel. The viability of the HepG2 cells on the stainless steel surface was quantitatively and qualitatively examined with different microscopy techniques. A simple cell-based electrochemical biosensor was developed by evaluating the viability of the HepG2 cells on the stainless steel surface when exposed to various concentrations of AFB1 and INH by using electrochemical impedance spectroscopy (EIS). The results showed that HepG2 cells can adhere to the metal surface and could be used as part of the biosensor to determine simple hepatotoxic samples.

Plant-derived nanovesicles (NVs) have attracted interest due to their anti-inflammatory, anticancer and antioxidative properties and their efficient uptake by human intestinal epithelial cells. Previously we showed that tomato (Solanum lycopersicum L.) fruit is one of the interesting plant resources from which NVs can be obtained at a high yield. In the course of the isolation of NVs from different batches of tomatoes, using the established differential ultracentrifugation or size-exclusion chromatography methods, we occasionally observed the co-isolation of viral particles. Density gradient ultracentrifugation (gUC), using sucrose or iodixanol gradient materials, turned out to be efficient in the separation of NVs from the viral particles. We applied cryogenic transmission electron microscopy (cryo-TEM), scanning electron microscopy (SEM) for the morphological assessment and LC–MS/MS-based proteomics for the protein identification of the gradient fractions. Cryo-TEM showed that a low-density gUC fraction was enriched in membrane-enclosed NVs, while the high-density fractions were rich in rod-shaped objects. Mass spectrometry–based proteomic analysis identified capsid proteins of tomato brown rugose fruit virus, tomato mosaic virus and tomato mottle mosaic virus. In another batch of tomatoes, we isolated tomato spotted wilt virus, potato virus Y and southern tomato virus in the vesicle sample. Our results show the frequent co-isolation of plant viruses with NVs and the utility of the combination of cryo-TEM, SEM and proteomics in the detection of possible viral contamination.

Nanoparticles (NPs) show various properties depending on their composition, size, and surface coating, which shape their interactions with biological systems. In particular, NPs have been shown to interact with immune cells, that represent a sensitive surveillance system of external and internal stimuli. In this light, in vitro models represent useful tools for investigating nano-bio-interactions in immune cells of different organisms, including invertebrates. In this work, the effects of selected types of NPs with different core composition, size and functionalization (custom-made PVP-AuNP and commercial nanopolystyrenes PS-NH2 and PS-COOH) were investigated in the hemocytes of the marine bivalve Mytilus galloprovincialis. The role of exposure medium was evaluated using either artificial seawater (ASW) or hemolymph serum (HS). Hemocyte morphology was investigated by scanning electron microscopy (SEM) and different functional parameters (lysosomal membrane stability, phagocytosis, and lysozyme release) were evaluated. The results show distinct morphological and functional changes induced in mussel hemocytes depending on the NP type and exposure medium. Mussel hemocytes may represent a powerful alternative in vitro model for a rapid pre-screening strategy for NPs, whose utilization will contribute to the understanding of the possible impact of environmental exposure to NPs in marine invertebrates.