3.3.1. Ultrastructural Features of NPs Interaction with the Cells in Monolayer

Study of NPs uptake by HepG2 and HEK293 cells were performed in culture medium without serum during 4 h to prevent "corona" formation by serum proteins, because "corona" forms differently on differently charged NPs, and it can alter their behavior by unknown way [54,55].

First, we examined interaction of AuNPs with HepG2 cells and unexpectedly found that all AuNPs remained on cell surface, no signs of the NPs penetration into cells were observed during 4 h of incubation (Figure 6A). In contrast, HEK293 cells readily internalized the same AuNPs, which were observed in structures associated with endocytosis after 15 min of incubation, and accumulated in cells up to 4 h (Figure 6B). At the same time, HepG2 cells actively engulfed AuNPs covered with PEI or BSA, as HEK293 cells did (Figure 6C–F). The accumulation of AuBSA-NPs was detected later than AuPEI-NPs (first AuBSA-NPs were found in both cell cultures after 2 h of incubation, the particles were dispersed in endosomes, and this reflects endocytosis of single particles (Figure 6C,D). The cells are able to accumulate many AuBSA-NPs in late endosomes (Figure S4), and it is easy to imagine the huge amount of NPs accumulated inside the cell, remembering that one ultra-thin section is about 70 nm thick, and the cell diameter is more than 10 microns. Previously long-term flotation of AuBSA-NPs was observed on HeLa cells [32], what is similar to current observation and obviously associated with particle negative net charge (Table 1). Penetration of AuPEI-NPs into both cell lines visually was highest, and numerous particles were found in endosomes after 15 minutes of incubation (Figure 6 E,F), obviously due to their positive net charge (Table 1). It is interesting, that all kinds of the NPs had

different behavior inside endosomes of HepG2 and HEK293 cells: AuNPs formed loose aggregations of various shape, AuBSA-NPs localized individually; and AuNP-PEI formed compact aggregations or were located separately (Figure 6).

**Figure 6.** Representative TEM-images of NPs uptake by cells. (**A**) AuNPs on HepG2 cell surface are shown with arrows; inserts show AuNPs aggregations on plasmalemma at high magnification. 4 h incubation. (**B**) Penetration of AuNPs into HEK293 cells. Arrows show endocytosis-associated structures containing AuNPs; left insert shows two enlarged endosomes; right insert shows early endosome containing AuNPs. 30 min incubation. (**C**) AuBSA-NPs inside late endosomes of HepG2 cells. 4 h incubation. (**D**) AuBSA-NPs inside HEK293 cells. 4 h incubation. (**E**) AuPEI-NPs in endosomes of HepG2 cells; insert shows enlarged particles inside endosome, PEI looks as grey material around gold core. 1 h incubation. (**F**) AuPEI-NPs in endosomes of HEK293 cells; insert shows aggregates of AuPEI-NPs inside endosome. 1 h incubation. Bars in inserts correspond to 100 nm.

Undoubtedly, most interesting of our findings is absence of AuNPs uptake by HepG2 cells in contrast with active internalization of the same AuNPs covered with BSA or PEI. We did not find any study confirming or neglecting our results. Uptake of AuNPs (20 nm) by HepG2 cells was detected by inductively coupled plasma mass spectrometry [26], however, this method did not provide reliable results because it operates with a whole mass of the cells dissolved in 3% HNO3, so it is impossible say were AuNPs inside the cells or were they adsorbed on cell surface [25]. The TEM of ultrathin sections is the only method that unambiguously demonstrates the penetration of metal NPs into cells and allows identification of cell structures without a special labeling. Most published studies skip TEM examination of cell-NPs interaction. A number of studies provide images of the final stages of NPs accumulation in cells after several days of incubation; such data only confirm the presence of NPs in cells without bringing details of their penetration and interaction with cellular structures [22,23]. Meanwhile, understanding of the pathways of NPs internalization is necessary for biomedicine and nanotoxicology, current knowledge in the field is comprehensively analyzed in recent reviews, which noted advantages of TEM: possibility of direct and simultaneous visualization of NPs and cell structures on ultrathin sections [4,55,56].

Our examination of ultrathin sections in TEM revealed that clathrin-mediated endocytosis is main way to enter both HepG2 and HEK293 cells for all studied types of NPs. Figure 7 demonstrates sequential steps of clathrin-mediated endocytosis: adsorption, transfer by vesicle to early endosome and accumulation in late endosomes. All types of NPs were observed only in membrane-bound structures (endosomes and lysosomes); no signs of NPs cytoplasmic localization were detected during 4 h of incubation. Our previous studies showed that AuNPs stay inside late endosomes and lysosomes of HeLa cells at least for 72 h [32].

HEK293 cells also showed the signs of macrpinocytosis of all studied NPs, cells developed long outgrowths and macropinocytic cups (Figure 7J,K). Morphological signs of macropinocytosis in HEK293 cells were identical to earlier published TEM data on this type of endocytosis, the authors noted independence of macropinocytosis and clathrin-mediated endocytosis [57–59].

Clathrin-mediated endocytosis was examined in HepG2 cells incubated with AuNPs coated with human ferrotransferrin (8 nm) or asialoorosomucoid (20 nm) in TEM and the data showed all subsequent steps of the endocytosis [60], identical to our images, however, author did not study interaction of HepG2 cells with AuNPs.
