Extracellular vesicles (EVs) are nanometric membranous structures secreted from almost every cell and present in biofluids. Because EV composition reflects the state of its parental tissue, EVs possess an enormous diagnostic/prognostic potential to reveal pathophysiological conditions. However, a prerequisite for such usage of EVs is their detailed characterisation, including visualisation which is mainly achieved by atomic force microscopy (AFM) and electron microscopy (EM). Here we summarise the EV preparation protocols for AFM and EM bringing out the main challenges in the imaging of EVs, both in their natural environment as biofluid constituents and in a saline solution after EV isolation. In addition, we discuss approaches for EV imaging and identify the potential benefits and disadvantages when different AFM and EM methods are applied, including numerous factors that influence the morphological characterisation, standardisation, or formation of artefacts. We also demonstrate the effects of some of these factors by using cerebrospinal fluid as an example of human biofluid with a simpler composition. Here presented comparison of approaches to EV imaging should help to estimate the current state in morphology research of EVs from human biofluids and to identify the most efficient pathways towards the standardisation of sample preparation and microscopy modes.

Incorporation of Ga³⁺ions into hydroxyapatite under different conditions is studied in detail and its influence on ion-release, antibacterial and cytotoxic properties of the resulting Ga(iii)-containing hydroxyapatites is determined.

Extracellular vesicles (EVs) are membranous structures in biofluids with enormous diagnostic/prognostic potential for application in liquid biopsies. Any such downstream application requires a detailed characterization of EV concentration, size and morphology. This study aimed to observe the native morphology of EVs in human cerebrospinal fluid after traumatic brain injury. Therefore, they were separated by gravity-driven size-exclusion chromatography (SEC) and investigated by atomic force microscopy (AFM) in liquid and cryogenic transmission electron microscopy (cryo-TEM). The enrichment of EVs in early SEC fractions was confirmed by immunoblot for transmembrane proteins CD9 and CD81. These fractions were then pooled, and the concentration and particle size distribution were determined by Tunable Resistive Pulse Sensing (around 10¹⁰ particles/mL, mode 100 nm) and Nanoparticle Tracking Analysis (around 10⁹ particles/mL, mode 150 nm). Liquid AFM and cryo-TEM investigations showed mode sizes of about 60 and 90 nm, respectively, and various morphology features. AFM revealed round, concave, multilobed EV structures; and cryo-TEM identified single, double and multi-membrane EVs. By combining AFM for the surface morphology investigation and cryo-TEM for internal structure differentiation, EV morphological subpopulations in cerebrospinal fluid could be identified. These subpopulations should be further investigated because they could have different biological functions.

Extracellular vesicles (EVs) are a versatile group of cell-secreted membranous nanoparticles present in body fluids. They have an exceptional diagnostic potential due to their molecular content matching the originating cells and accessibility from body fluids. However, methods for EV isolation are still in development, with size exclusion chromatography (SEC) emerging as a preferred method. Here we compared four types of SEC to isolate EVs from the CSF of patients with severe traumatic brain injury. A pool of nine CSF samples was separated by SEC columns packed with Sepharose CL-6B, Sephacryl S-400 or Superose 6PG and a ready-to-use qEV10/70 nm column. A total of 46 fractions were collected and analysed by slot-blot followed by Ponceau staining. Immunodetection was performed for albumin, EV markers CD9, CD81, and lipoprotein markers ApoE and ApoAI. The size and concentration of nanoparticles in fractions were determined by tunable resistive pulse sensing and EVs were visualised by transmission electron microscopy. We show that all four SEC techniques enabled separation of CSF into nanoparticle- and free protein-enriched fractions. Sepharose CL-6B resulted in a significantly higher number of separated EVs while lipoproteins were eluted together with free proteins. Our data indicate that Sepharose CL-6B is suitable for isolation of EVs from CSF and their separation from lipoproteins.

Water pollution has become a serious environmental issue. The textile industries using textile dyes are considered to be one of the most polluting of all industrial sectors. The application of solar-light semiconductor catalysts in wastewater treatment, among which TiO₂ can be considered a prospective candidate, is limited by rapid recombination of photogenerated charge carriers. To address these limitations, TiO₂ was tailored with graphitic carbon nitride (g-C₃N₄) to develop a heterostructure of g-C₃N₄@TiO₂. Herein, a simple hydrothermal synthesis of TiO₂@g-C₃N₄ is presented, using titanium isopropoxide (TTIP) and urea as precursors. The morphological and optical properties and the structure of g-C₃N₄, TiO₂, and the prepared heterostructure TiO₂@g-C₃N₄ (with different wt.% up to 32%), were analyzed by various laboratory methods. The photocatalytic activity was studied through the degradation of methylene blue (MB) aqueous solution under UV-A and simulated solar irradiation. The results showed that the amount of g-C₃N₄ and the irradiation source are the most important influences on the efficiency of MB removal by g-C₃N₄@TiO₂. Photocatalytic degradation of MB was also examined in realistic conditions, such as natural sunlight and different aqueous environments. The synthesized g-C₃N₄@TiO₂ nanocomposite showed superior photocatalytic properties in comparison with pure TiO₂ and g-C₃N₄, and is thus a promising new photocatalyst for real-life implementation. The degradation mechanism was investigated using scavengers for electrons, photogenerated holes, and hydroxyl radicals to find the responsible species for MB degradation.