*3.9. Cytotoxic Activity*

The cytotoxic activity of PldA-GFP IBs was determined by MTT method. After incubation of mouse neuroblastoma Neuro-2a cells with IBs in 96-well microplate for 24 h at 37 ◦C and 5% CO2, the supernatant was replaced with pure medium. Then, 5 mg/mL MTT reagent solution (Sigma-Aldrich, St. Louis, MO, USA) was added to each well and microplate was incubated for additional 4 h, after which SDS-HCl solution (1 g SDS/10 mL dH2O/17 μL 6 N HCl) was added and incubated at 37 ◦C for 4–18 h. Absorption was measured at a wavelength of 570 nm using a Multiskan FC spectrophotometer (Thermo Scientific, Canada). The cytotoxic activity of IBs was expressed as the concentration of EC50 at which the metabolic activity of cells is inhibited by 50%.

#### *3.10. Dynamic Light Scattering (DLS)*

The size of PldA-GFP IBs was determined using the DLS technique with a ZetaSizer Nano ZS instrument (Malvern, UK) equipped with a He-Ne-laser (λ 633 nm, 4 mW) at 173◦ angle. The hydrodynamic radius (RH) of the protein particles was calculated using the software supplied with the instrument. IBs samples (0.1–0.6 mg/mL) were suspended in PBS, pH 7.5, by passing 10 times through a syringe microneedle and incubation at room temperature for a specified time with mixing. Data was accumulated for 5–60 min. Measurements were conducted in a 10 mm × 10 mm cuvette. Time of data accumulation for the correlation function was selected automatically via the instrument software, and it was 5–30 min. All measurements were replicated 2–3 times.
