Formvar carbon grid

Nanoparticles were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential measurements. Both zeta potential and hydrodynamic diameter of nanoparticles were measured by a Malvern Zetasizer Nano (Malvern Instrument Ltd., UK) with a laser wavelength of 633 nm in 10 mM sodium phosphate buffer at pH 7.4. For the calculation of zeta potential, data processing was done by using Smoluchowski model⁴⁸ . For TEM analysis, nanoparticles were loaded onto glow‐discharged 200-mesh copper grids (Formvar/carbon grids, Ted Pella) for 20 s, blot-dried, and then stained three times with uranyl formate and dried. TEM imaging was performed using a Tecnai G2 Spirit BioTWIN (FEI) operating at 120-kV acceleration. Images were obtained on a TemCam‐F416(R) (TVIPS) CMOS camera. To estimate the size distribution of nanoparticle–corona complexes, the size of at least 150 particles was measured by Fiji/ImageJ. Nanoparticle sizes were determined in aqueous conditions by dynamic light scattering (DLS).

4T1 tumor cell and 4T1-2H11 tumor-endothelial cell spheroids grown in hanging drops for 10 days were transferred to Formvar/Carbon grids (Ted Pella Inc, Redding, CA). The spheroid containing grids were imaged with an S-3200-N Hitachi scanning electron microscope at an accelerating voltage of 16 kV to observe the spheroid compactness or integrity and extracellular matrix formation.

The purified bEV samples were diluted 50 times with PBS and applied to 200-mesh Formvar/Carbon grids (Ted Pella, Inc., Redding, CA, USA). Samples were dried on the grids and viewed with a transmission electron microscope (HT7700, Hitachi, Tokyo, Japan) operated at 100 kV.