**1. Introduction**

Gold nanoparticles have a number of unique physical and chemical properties that, together with a good biocompatibility, makes them a promising tool for nanomedicine. Advantages of using gold nanoparticles (AuNPs) and their various modifications in the treatment and diagnosis of diseases are being actively studied; a number of comprehensive detailed reviews is devoted to this issue [1–6]. Similar to other NPs, AuNPs are studied in cell cultures and in laboratory animals; and in last decade a new experimental model has been developed: multicellular spheroids or micro-tissues (cell cultures in 3D-form); the advantages of spheroids are described in details [7–10]. Spheroids that mimic the structure and functions of various tissues have shown their suitability for studies of different problems in modern biomedicine, including the effects of drugs, drug damage to the liver, toxicity of chemical compounds, and human hepatocarcinoma (HepG2) spheroids are considered in such studies as a practically adequate replacement of primary hepatocytes [11–15]. The advent of commercially available

devices for cultivation of spheroids has transformed their obtaining from "high art" into affordable technology, which expanded the scope of their application. Various approaches for obtaining spheroids are reported, which roughly can be divided to scaffold-based and scaffold-free; see reviews [8,12,16–18].

The number of published works on cellular spheroids is already in the thousands, but many details of their structure remain unknown, including the structure of their external surface and the morphological substrate of contact with the environment. Meanwhile, structure of the region adjacent to spheroids surface determine the nature of interaction not only with the culture medium, but also with soluble preparations and NPs containing in that medium. Morphological changes in spheroids treated with NPs or chemical compounds are studied mainly in transmitted light and various fluorescence methods [9,19–21]. The use of electron microscopy is rare and mostly is limited to registration of NPs presence in a cell [22–24] or TEM-illustration of NPs used in a study [9,15,21,25,26]. However, the size of NPs requires studying their interaction with cells at subcellular level, which is realized in a transmission electron microscopy (TEM) of ultrathin sections.

In this work, we examined and compared the morphology of HepG2 and human embryo kidney (HEK293) cell monolayers and spheroids with TEM and scanning electron microscopy (SEM), because we found out an insufficiency of published data. Both cell lines are epithelial in nature; however, HepG2 is well-differentiated line, which possesses structural and morphologic characteristics of hepatocytes, while morphology of HEK 293 cell line does not show tissue-specific features. In this work, we describe structural organization of the spheroids and point out the features specific for each cell type.

It was interesting to find out how HepG2 and HEK293 epithelial cell lines interact with the same NPs in monolayer and spheroids. We incubated the cells in monolayer and spheroids with synthesized AuNPs and their modified variants coated with protein (bovine serum albumin, BSA) or polymer (polyethylenimine, PEI). Here, we present data on features of the penetration of these NPs into HepG2 and HEK293 cells and compare those in spheroids and monolayer.

In common, in this work we present new comparative data on morphology of HepG2 and HEK293 cells in monolayer and spheroids, and their interaction with AuNPs, AuPEI and AuBSA-NPs.
