Ns300 particle analyzer

Complete medium was replaced by basic medium without FBS when cells were cultured about 80–90% confluence. Cell supernatants containing sEVs were collected after incubation for 48 h. The sEV pellets from cell supernatants were purified by a series of centrifugation. Briefly, cell supernatants were centrifuged at 300 g for 10 min to remove cells and then at 16,000 g for 30 min to remove cell debris. After filtration with 0.22-μm filter membrane (Millipore, Cork, Ireland), cleared supernatants were centrifuged at 120,000 g for 70 min using a type 70Ti rotor (Beckman Coulter, Brea, CA, United States). All centrifuge steps were kept at 4°C. The sEV pellets were resuspended in filtered phosphate-buffered saline (PBS; pH 7.4).
The particle size distribution of sEVs was measured by nanoparticle tracking analysis (NTA) using a NanoSight NS300 particle analyzer (NanoSight NTA version 2.3) following the manufacturer’s instruction (Malvern Instruments Ltd., Worcestershire, United Kingdom). Pellets of sEVs were fixed and examined using a JEM-1400 (JEOL, Tokyo, Japan) transmission electron microscopy (TEM) at an acceleration voltage of 80 kV as described (Zhang et al., 2021 (link)).

The introduction of the organic phase into the aqueous phase was carried out using an Instilar 1438 Dixion infusion pump (Moscow, Russia). Magnetic stirrers and a Unimax 1010 thermoshaker from Heidolph (Schwabach, Germany) were used for mixing. A VaCo 5–II lyophilizer Zirbus (Bad Grund, Germany) was used to dry the obtained samples. The hydrodynamic radius and ζ-potential of the particles were analyzed (at a scattering angle of 173 °C) using a Zetasizer Nano ZS Malvern Panalytical particle analyzer (Malvern, UK) and a NanoSight NS300 particle analyzer (Malvern, UK). UV absorption measurements were carried out on a UV–1800 Shimadzu spectrophotometer (Kyoto, Japan). SEC analyses were performed on a Shimadzu LC-20 Prominence chromatograph with an RID 10-A refractometric detector (Kyoto, Japan) equipped with a tandem of two Agilent PLgel MIXED-D columns (5 µm, 7.5 mm × 300 mm, Agilent Technologies, Santa-Clara, CA, USA). NMR spectroscopy was carried out using a Bruker Avance III WB at 400 MHz (Karlsruhe, Germany).