*2.5. Incubation of Cells and Spheroids with the NPs*

Cells of the HepG2 and HEK293 lines were sown in Petri dishes (40 mm diameter, TPP Techno Plastic Products AG, Trasadingen, Switzerland), 105 cells per dish. After reaching 70% coverage, the monolayers were washed with a culture medium, and AuNPs or AuBSA-NPs, or AuPEI-NPs suspended in an IMDM (for HepG2) or DMEM (for HEK293) were added to cells. Final concentration of AuNPs in the medium was 1 nM.

The cells were incubated for 15 min, 30 min, 1, 2 and 4 h in a medium without serum. Then the cells were rinsed three times with PBS, removed with trypsin, sedimented by centrifugation (5 min at 3000 rpm), and fixed with 4% paraformaldehyde for TEM studies.

Seven-day spheroids of HepG2 and HEK 293 cells cultured in 96-well plates were washed with a culture medium. The AuNPs or AuBSA-NPs or AuPEI-NPs were added to spheroids in an IMDM (HepG2-spheroids) or DMEM (HEK293 spheroids); final concentration of AuNPs was 1 nM. The spheroids were incubated with NPs for 1, 2 and 4 h without serum, and then fixed with 4% paraformaldehyde.
