Microtest

For cell viability analysis, cells were seeded in complete growth medium at a density of 9.3x10⁴ cells/cm² (Raw 264.7) or 3.1x10⁴ cells/cm² (A549) in 96-well Microtest™ plates (Falcon, Corning Inc., Corning, NY, USA). 24h after cell seeding, growth medium was replaced with fresh medium supplemented with the materials (dose range 2.5–80 μg/cm²). In all the experiments, the vehicle (0.05% BSA in PBS) was added to the control at the minimal dilution used.
Cell viability was assessed with resazurin assay. In the resazurin assay, a non-fluorescent, membrane permeant molecule is converted by intracellular enzymes in the fluorescent compound resorufin (λ_em = 572 nm) [20 (link)]. After 24, 48 and 72h of incubation with the materials, cell viability was tested replacing medium with a solution of resazurin (44 μM) in serum-free medium. After 20 min, fluorescence was measured at 572 nm with a multimode plate reader Enspire (Perkin Elmer Waltham, MA, USA). To exclude possible interference on the test by the nanomaterials, the dye was incubated with materials only (80 μg/cm²), and fluorescence measured. No fluorescence signal was detected above the background.

The human enterocyte cell line, obtained from the cell bank at the Centro de Investigaciones Biológicas Margarita Salas (Madrid, Spain), was seeded in 96-well tissue culture plates (Falcon Microtest™, USA) at a final concentration of 1.25 × 10⁵ cells mL⁻¹ and grown as monolayers of differentiated and polarized cells for 14 days as previously described (Nácher-Vázquez et al., 2017 (link)). For the adhesion assays, exponential-phase LAB cultures grown in MRS were sedimented by centrifugation (12,000 × g, 10 min, 4°C), and resuspended in the appropriate volume of Dulbecco's Modified Eagle medium (DMEM, Invitrogen) to give a final concentration of 1.25 × 10⁶ colony forming units (cfu) mL⁻¹. Each bacterial suspension (0.1 mL) was added to a well (ratio 10:1, bacteria:Caco-2 cells) and the plates were incubated for 2 h at 37°C. The non-adhered bacteria were then removed and the cell-associated bacteria processed and quantified by plate counting on MRS-agar plates, as previously described (Nácher-Vázquez et al., 2017 (link)). All adhesion assays were conducted in triplicate. The percentage of adhesion to Caco-2 cells was calculated as:
$\begin{array}{l}Adhesion(\%)=\frac{\frac{cfu}{mL}attachedtoCaco-2cells}{\frac{cfu}{mL}addedtoCaco-2cells}\times 100\end{array}$

The turbidity assay was conducted in quadruplicate in flat-bottomed 96-well assay plates (Microtest, BD Falcon). Aβ₁₋₄₂ peptide and Ru-N complexes had final concentrations of 10 μM. Ru-N complexes were dissolved in DMSO and further diluted to obtain the correct concentration. The absorbance at 500 nm was measured every 10 min for 3 h at 37°C under constant agitation using a Synergy 4 Fluorometer plate reader from BioTek. For the 20 h experiment, the samples were incubated at 37°C with constant agitation with a lid on to prevent evaporation and then the turbidity was measured.

THP-1 cells (10⁵ cells/well) in 100 μL of supplemented culture medium were added into a 96-well tissue culture plate (Falcon Microtest™, Franklin Lakes, NJ, USA) and were differentiated into macrophages as described above. Then, 10 μL of selected liposomal dispersions (L-E, L-Hx₅ or L-Ac₁₀) or 10 μL of medium (as a control) were added to the wells. Plates were incubated for 18 h, at 37 °C and 5% CO₂. The supernatant was then removed and the cells were treated with 100 μL Krebs-Henseleit buffer (Sigma-Aldrich) and 10 μL CCK-8 (Cell Counting Kit-8, Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer’s instructions. They were incubated for 2 h at 37 °C in a dark place and relative cell viability was determined spectrophotometrically at 450 nm.

The Bradford assay (Thermo Scientific) measures the absorbance at 595 nm of Coomassie brilliant blue G-250 as it binds to protein in duplicate in a flat-bottomed 96-well assay plate (Microtest, BD Falcon). Sixty microliter solutions of Aβ₁₋₄₂ in the presence of 1 eq. of Ru-N complexes were incubated at 37°C for 24 h under constant agitation. A 30 μL sample was removed at the beginning of the experiment as the 0 hour time point, and kept frozen at −80°C until time for absorbance reading. Samples were centrifuged prior to reading of the assay to remove insoluble fibrils (Mok and Howlett, 2006 (link)). Measurements of absorbance used a Synergy 4 Fluorometer plate reader from BioTek. Samples were measured in duplicate, and statistics completed using the PRYSM program and ANOVA.

After overnight incubation at 30 °C in LB medium, C. violaceum CV026 was adjusted to OD 0.5 according to McFarland scale, mixed with 3.5 nmol mL⁻¹N-hexanoyl-L-homoserinelactone, and inoculated in 96 well no tissue microtiter plates (Falcon®Micro TestTM, USA) containing LB broth with serial diluted aurantiogliocladin (180 to 3 µg mL⁻¹) dissolved in methanol. Plates were covered with a sterile adhesive porous paper (Kisker Biotech GmbH, Steinfurt, Germany) and incubated at 30 °C for 48 h. Methanol and LB broth were used as negative and tetracycline (100 µg mL⁻¹) was used as positive control. Absence of violacein indicated quorum-quenching activity.