Safire2

The possible cytotoxic effects of compound 17 and nitroxoline in the cell lines used were determined by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymetoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] colorimetric assay. U-87 MG (3 × 10⁴ and 5 × 10³ for 24 and 72 hours respectively), LPB-1 (1 × 10⁵ and 2.5 × 10³ for 24 and 72 h, respectively), U373 (3 × 10⁴ cells for 72 h) cells and MSCs (3 × 10⁴ for 72 h) were seeded into wells of a 96-well microplate and allowed to attach overnight. They were then treated with 100 μl of medium containing 1.25, 2.5 or 5 μM of inhibitor or DMSO (0.05%) and incubated for 24 or 72 h. 10 μl of MTS (Promega, Madison, WI, USA) was then added to the walls of a 96-well microplate and, after incubation, absorbance of formazan was measured at 490 nm on a Tecan Safire²™ (Tecan, Mannedorf, Switzerland). Cell viability (%) was expressed as the ratio of absorbance obtained in the presence of compounds to that in DMSO alone. All assays were performed in quadruplicate and repeated twice.

Culture samples were collected from bioreactors after 32 h of cultivation; a rotating disk shear device [52 (link)] was used to determine the relative robustness of E. coli cells [53 (link)]. 20 mL of cell broth were exposed for 20 s to a rotation speed of 233 revolutions per second in the device, which corresponds to an energy dissipation rate of 0.75 × 10⁶ W/kg [52 (link)]. This represents the energy normally generated in high shear producing devices such as pumps and process scale centrifuges. Pre and post-shearing samples were centrifuged at 17,000×g for 10 min, the supernatants collected and frozen at −20 °C. The supernatants were thawed and the total protein content in sheared and non-sheared samples was measured via Bradford assay (Thermo Scientific, IL, USA) in 96 well plate at A 595 nm (Tecan Safire2, Tecan, Reading, UK).

TEM images (HT7700, Hitachi, Tokyo, Japan) was used to observe the morphology and mesoporous structure of the nanoparticles. The nitrogen adsorption analysis analyzer BSD-1 (Beishide Instrument Technology Co., Ltd., Beijing, China) was used to measure the surface area and pore size distribution of the samples. The particle sizes and Zeta potentials were measured in pH 7.4 PBS with a Zetasizer Nano-ZS90 Nanosizer (Malvern Ltd., Leamington Spa, UK). The fluorescence spectrum of the GQDs was detected using a microplate reader (Tecan Safire 2, Tecan Ltd., Männedorf, Switzerland). The infrared spectrogram of the sample was measured using an Equinox 55 Fourier Transform Infrared Spectrometer (Bruker, Germany).