*3.1. Physicochemical Characteristics of the NPs*

We prepared AuNPs and covered them with PEI or BSA for cell studies. Values of polydispersity indexes evidenced that NP preparations represent well dispersed aqueous suspensions (Table 1). Examination in TEM revealed spherical naked particles of high electron density (d = 12.0 ± 0.1 nm) in sample of AuNPs, and the same particles surrounded with "corona" of PEI or BSA having middle electron density, in the samples of AuPEI-NPs and AuBSA-NPs (Figure 1A).


**Table 1.** Physicochemical characteristics of AuNPs, AuPEI-NPs and AuBSA-NPs.

**Figure 1.** Physicochemical characterization of the NPs. (**A**) TEM images of AuNP (top), AuPEI-NP (middle) and AuBSA-NP (bottom). Negative staining with phosphotungstic acid. Bars correspond to 10 nm. (**B**) Image of the gel after electrophoretic analysis of AuNPs, AuMUA-NPs, AuPEI-NPs and AuBSA-NPs. (**C**) Optical extinction spectra of AuNPs, AuMUA-NPs, AuPEI-NPs and AuBSA-NPs in water. We used samples with different concentrations to separate the curve maximums for clear visualization.

To prepare AuPEI-NPs we modified previously described layer-by-layer method, which uses MUA for stabilization of AuNPs [31]. We introduced a step of AuNPs preliminary incubation with ON (anyone with length 20–30 n.) [29]. This ON layer allowed to increase colloid stability of the NPs at subsequent stages of interaction with MUA and PEI. The resulting AuPEI-NPs had a positive net charge, unlike AuNPs and AuBSA-NPs, which have a negative net charge (Zeta potential) (Table 1). Optical extinction spectra did not critically change after covering of AuNPs with PEI or BSA (Figure 1C).

Thus, we obtained three types of well dispersed NPs to study their interaction with cells: AuNPs, AuPEI-NPs and AuBSA-NPs, similar by size, and differing in the magnitude of net charge (Table 1, Figure 1B). Detailed presentation of physico-chemical properties of NPs, similar to studied in this work could be found in our previous studies [32,33]. Absence of noticeable cytotoxicity of all studied NPs was shown in our previous work using two different cell cultures [32].
