Wga 647

An evaluation of the free zinc cations,
due to intracellularly dissolved NPs after their internalization and
in the absence of any mechanical treatment, was initially performed.
The cells were plated in chamber slides, treated with Fe:ZnO NPs,
and fixed (see the details above). The intracellular free zinc cations
were labeled through the FluoZin-3 AM (Thermo Fisher) probe: the cells
were washed twice with PBS and a 1 μM solution of FluoZin-3
AM fluorescent dye in PBS was administered. After 30 min of incubation
at 37 °C, the excess of dye was removed by two washing steps
with PBS. Then, cell lysosomes were labeled by substituting the PBS
with a 1 μM dye solution (LysoTracker Red DND-99, Thermo Fisher)
in PBS and incubating the cells for 30 min at room temperature. After
two further washing steps, the cell membranes and cell nuclei were
stained with WGA-647 (Thermo Fisher) and Hoechst, respectively, following
the same procedure reported above. The images were collected with
a spinning disk confocal microscope, keeping the exposure times and
laser powers constant among the different samples. They were then
post-processed in the same way to allow a direct comparison between
the samples in terms of free zinc intensity fluorescence too.

The samples were stained for 10 minutes with 10 μg/ml wheat germ agglutinin conjugated with Alexa Fluor 647 (WGA-647) (ThermoFisher, Waltham, MA). Samples were then washed with PBS and imaged. Images were acquired at 0.39 μm z-steps on a Zeiss LSM 710 scanning head confocal microscope equipped with an Airyscan detector (Carl Zeiss MicroImaging GmbH, Jena, Germany) with a Zeiss a Plan-Apochromat 100x/1.46 DIC M27 75 mm objective. Excitation lasers were 405 and 633 nm for the green and red emission channels, respectively. GFP was detected with a 495–550 nm filter and WGA-647 was detected with a 645 nm LP filter. Laser dwell times were 1.65 μs for both channels. Image analysis (2D and 3D) was conducted using Volocity (PerkinElmer, Waltham, MA).

For the fluorescence microscopy analysis, EVs were labelled with Wheat Germ Agglutinin (WGA) conjugated with Alexa Fluor 647 (WGA647, λ_Ex = 650 nm, Thermo Fisher), ZnO NCs with Atto 550-NHS ester (λ_Ex = 554 nm, ATTO-Tech), and the TNH^CD20 nanoconstruct was assembled using the AMCA AffiniPure F(ab′)₂ Fragment Goat Anti-Human IgG, Fcγ fragment specific as secondary antibody.
Samples were treated with the same protocol used for the cytofluorimetric analysis and plated in a volume of 100 μL. After 24 and 48 h of culturing at 37 °C, 5% CO₂ in 96 well plates, the content of each well was collected, centrifuged, resuspended in 40 μL of the correspondent medium. The 40 μL drop was spotted in a 8-well chamber slide (Thermo Scientific™ Nunc™ Lab-Tek™ II CC2™ Chamber Slide System) and placed at 37 °C, 5% CO₂ for 30 min to allow the attachment of the cells. After that, cells were fixed using 250 μL of Image-iT™ Fixative Solution (4% formaldehyde, methanol-free, Thermo Scientific) for 10 min, washed in PBS and cells’ membranes were labelled by incubating cells with 1.25 μL of WGA conjugated with Alexa Fluor 488 (WGA488, λ_ex = 495 nm, Thermo Fisher) for 10 min and washed two other times in PBS. Images were acquired using a wide-field fluorescence-inverted microscope using an immersion oil 100× objective.