3.3.2. Features of NPs Interaction with Spheroids Cells

Examination of ultrathin sections of HepG2 spheroids incubated with AuNPs showed inability of these cells to internalize these NPs, which were localized only on spheroid surface during 4 h (Figure 8A–C). At the same time, ultrathin sections of HEK293 spheroids showed signs of both clathrin-mediated endocytosis and macropinocytosis of AuNPs which were found mostly in late endosomes starting from 1 h of incubation (Figure 8D–H). HEK293 cells containing AuNPs were located in spheroid external zone (about 35–40 μm from the surface).

Thus, both HepG2 and HEK293 cultures in form of spheroid (3D-culture) kept the character of interaction with AuNPs observed in their monolayers. The same phenomenon occurred during incubation with AuBSA-NPs: ultrathin sections of both HepG2 and HEK293 spheroids showed adsorption of NPs on plasmalemma and presence of individual particles in late endosomes (Figure 9). The cells containing AuBSA-NPs were located in external zone (about 35–40 μm from the surface) of HepG2 and HEK293 spheroids.

AuPEI-NPs were observed on basal plasmalemma of cells forming outer surface of HepG2 and HEK293 spheroids, and in the spaces between lateral cell surfaces, and in pseudosinusoids of HepG2 spheroids (Figure 10A–D). As in the case of a monolayer, AuPEI-NPs more actively penetrated the cells than other studied NPs and accumulated in late endosomes of both HepG2 and HEK293 cells (Figure 10D,E). AuPEI-NPs visually were more abundant in a tissue of HepG2 and HEK293 spheroids than AuBSA-NPs, however, their penetration depth was similar: 35–40 μm. Thus, positive net charge of AuPEI-NPs did not influence the depth of NPs penetration into spheroids, although it increased their accumulation inside spheroids, as well as in monolayer HepG2 and HEK293 cells.

**Figure 7.** Representative images of NPs penetration into monolayer cells. Adsorption of single AuBSA-NP (**A**) and small cluster (**B**) of AuNPs on plasmalemma (HepG2); (**C**) coated pit containing AuNP and (**D**) coated vesicle containing AuPEI-NP (HEK293); (**E**) endocytotic vesicle containing AuBSA-NPs (HepG2). (**F**) Early endosome receives AuNP in vesicle; arrows show vesicles fusing with endosome body (HEK293). (**G**) AuNPs in endosome cavity, arrow shows NP in tubule (HEK293). (**H**) AuPEI-NPs in late endosome and a vesicle (HepG2). (**I**) AuNPs in late endosome (HEK293). (**J**,**K**) Macropinocytosis of AuPEI-NPs (HEK293). TEM, ultrathin sections. Bars correspond to 100 nm.

**Figure 8.** Interaction of AuNPs with cells of different spheroids. (**A**–**C**) adsorption of AuNPs on plasmalemma (HepG2 cells). (**D**–**H**) Penetration of AuNPs into HEK293 cells. (**E**) Adsorption of AuNPs on plasmalemma; (**F**) Macropinocytosis of AuNPs; (**G**,**H**) AuNPs in late endosomes. 1—cytoplasm; 2—late endosome; asterisks show external space and arrows show plasmalemma. Bars correspond to 100 nm.

**Figure 9.** Interaction of AuBSA-NPs with cells of different spheroids. (**A**–**D**) Penetration of AuBSA-NPs into HepG2 cells. (**A**,**B**) adsorption of NPs on plasmalemma, insert shows NP adsorption at high magnification. (**C**,**D**) AuBSA-NPs in late endosomes. (**E**,**F**) Penetration of AuBSA-NPs into HEK293 cells. (**E**) NPs on plasmalemma and in late endosome. (**F**) AuBSA-NPs in late endosomes and in a vesicle. 1—cytoplasm; 2—late endosomes; 3—cell outgrowths; asterisks show external space and arrows show plasmalemma. Bars correspond to 100 nm.

**Figure 10.** Interaction of AuPEI-NPs with cells of different spheroids. (**A**) Adsorption of AuPEI-NPs on HepG2 cell plasmalemma. Images in insert, (**B**,**C**) show NP adsorption at high magnification, HepG2 cells. (**D**) AuPEI-NPs in late endosomes of HepG2 cells. (**E**) Penetration of AuPEI-NPs into HEK293 cell, insert shows NPs in late endosome at high magnification, yellow arrows show NPs between spheroid cells. 1—cytoplasm; 2—late endosomes; asterisks show external space and arrows show plasmalemma. Bars correspond to 100 nm.
