Em 900

EVs were prepared for electron microscopy as described previously [36 (link)] with few modifications: Purified EVs were fixed with an equal amount of 4% PFA. A small volume (5–7 µL) of this suspension was transferred to a formvar/carbon-coated meshed copper grid (Ted Pella Inc., Redding, CA, USA) and air-dried for 20 min. The grids were washed with sterile-filtered PBS and fixed for 5 min with 1% glutaraldehyde. After 8 washing steps (2 min each) with distilled water, the sample was incubated with 1% uranyl acetate for 5 min and then transferred to 2% methylcellulose supplemented with 4% uranyl acetate (ratio 9:1) and incubated for 10 min on ice. Excess fluid was removed, and the grid was air-dried for up to 10 min. Images were taken with a Zeiss EM 900 (Zeiss, Jena, Germany) at 80 kV equipped with a 2k slow-scan CCD camera (TRS-STAR GmbH, Stutensee, Germany).

The size and morphological features of the obtained FA-DEX-VBL-SPION nanocomposite were estimated using TEM (Zeiss EM900, Carl Zeiss AG, Jena, Germany) and SEM (Hitachi S-3000 SEM, Tokyo, Japan) at a voltage of 30.0 kV and 30 mA. Zeta potential and dynamic light scattering (DLS) (Nano S, Malvern, UK) were performed after resuspension in ultrapure water and dilution to an appropriate concentration. FTIR spectra were recorded on a Thermo Nicolet 6700 instrument (AEM, Madison WI, USA) within the range of 400–4000 cm⁻¹. Moreover, VSM (Lakeshore 7404, LakeShore, MI, USA) was performed to evaluate the magnetic properties of the particles.

A nano zinc oxide dispersion (20 wt.% in H₂O) was purchased from Sigma (Sigma‐Aldrich, Louis, MO, USA). The nanoparticles were mostly spherical with an average size less than 50 nm (characterized by a transmission electron microscope, Zeiss EM900, Carl Zeiss, Oberkochen, Germany). A stock solution of 5 × 10³ mg/L Zn was prepared by sonication in an ultrasonic bath (Elmasonic S40, Elma^®, Germany) for 30 min and stored at 4°C under darkness. Each time, a test solution was made, and the stock solution was ultrasonicated for 30 min at 20 kHz with a maximum power output of 400 W to eliminate aggregates. A sublethal exposure concentration of nZnO (86 µg/L Zn) was used. This concentration corresponds to 10% of the EC_{50, 48 hr} immobilization for D. magna neonates based on a pilot range‐finding experiment. The EC_{50, 48 hr} immobilization is defined as the concentration at which half of the Daphnia individuals were not moving anymore after 48 hr of exposure. This concentration is similar to chronic EC₅₀ values of nZnO for D. magna reproduction (Adam et al., 2014) and is environmentally relevant as the estimated nZnO concentrations in UK waterbodies go up to 100 μg/L (Boxall, Tiede, & Chaudhry, 2007).

TEM was conducted to investigate AuNP internalization in BeWo cells. Therefore, cells were cultivated on inserts for 3 days as described above and treated with 50 µg/mL Au-4-COONa or 25 µg/mL Au-3-PEG for 24 h at 37 °C and 5% CO₂. Cells were detached from 11 inserts per condition (without NPs, Au-3-PEG, Au-4-COONa) using 0.5% Trypsin–EDTA, pelleted by centrifugation (200 g, 5 min) and sucked up into a capillary tube (Leica-Microsystems). Cells enriched in the capillaries were immediately fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer and washed in 0.2 M sodium cacodylate buffer. After a post-fixation step in 2% osmium tetroxide in 0.1 M sodium cacodylate buffer, samples were dehydrated through a graded ethanol series followed by acetone and finally embedded in Epon resin (Sigma-Aldrich, Buchs, Switzerland). Ultrathin sections were contrasted with 2% uranyl acetate and lead citrate (Reynolds 1963) before imaged in a Zeiss EM 900 (Carl Zeiss Microscopy GmbH, Germany) at 80 kV.

For light and electron microscopic examination, the embryonic body wall with limb buds (including differentiated muscle tissue) was fixed in modified Karnovsky fixative (1% paraformaldehyde [PFA] and 1% glutaraldehyde, in a 0.1 M phosphate buffer pH 7.2) for 24 h at 4 °C. The material was repeatedly rinsed with the same buffer and was postfixed for 2 h in a 1:1 mixture of osmium tetroxide-potassium ferricyanide (OsO₄-K₃Fe(CN)₆). Following rinsing in the phosphate buffer, the material was dehydrated, first in a graded alcohol series and then in acetone, and was then embedded in epoxy resin Epon 812 (Sigma-Aldrich) (Luft 1961 (link)). The Epon blocks were cut on Leica Ultracut UCT (Leica, Wetzlar, Germany). Semithin sections (0.6 μm) were collected on glass slides and were stained with methylene blue in a 1% borax solution. The stained material was examined under an Olympus BX60 light microscope (Olympus). The ultrathin sections were collected on 200-mesh copper grids and were stained with uranyl acetate and lead citrate according to the standard protocol (Reynolds 1963 (link)), before being examined under the transmission electron microscope (TEM), Zeiss EM 900 (Carl Zeiss AG, Oberkochen, Germany; 80 kV).