Erythrosine b

Cells were cultured until 70–80% confluence, then washed with phosphate-buffered saline (PBS) and incubated for two consecutive periods (18 h and additional 18 h) with IMDM supplemented with 10% FBS depleted of extracellular nanovesicles obtained via ultracentrifugation [18 (link)]. Cell density and viability were assessed by the erythrosine B (Sigma-Aldrich) dye exclusion method [19 (link)]. EVs pellet was obtained from the supernatant collected from MG-63, MG-63DXR30, 143B, and ADMSC cells grown on 15 Petri dishes (diameter 150 mm, 18 mL/Petri). The EVs were concentrated by a series of differential centrifugations: 500× g for 10 min (twice), 2000× g for 15 min (twice), and 10,000× g for 30 min (twice) at 4 °C to remove floating cells and cellular debris. The supernatant was then ultracentrifuged at 110,000× g for 1 h at 4 °C (Beckman Coulter, Milan, Italy). The EVs pellet was washed (110,000× g for 1 h at 4 °C), resuspended in PBS, and stored at −80 °C until use. EVs quantity was determined by the Bradford method (Bio-Rad, Milan, Italy). The EVs derived from the supernatant of MG-63 and from the medium of MG-63DXR30 were named EVs/s and EVs/DXR, respectively.

The photosensitiser, erythrosine-B (Sigma-Aldrich, Germany), was prepared as a filter-sterilised 1mg/ml stock solution in Reduced Transport Fluid (RTF) and stored in the dark at 4 °C. It was then diluted to the required 220 μM concentration, which was found by Tahmassebi et al. (2015 (link)) to be the most effective bactericidal concentration for PDT on in vivo-formed oral biofilms. This was used for all PDT treated groups in this study.
A 400 W-Tungsten filament lamp (Aurora, USA), white light source, was used to activate erythrosine. It emitted 22.7 mW/cm² of light intensity in the wavelength range 500–550 nm (the region of maximal absorption by erythrosine). Bacterial samples were placed at 30 cm from the lamp and the heat-dissipating water bath (Stuart, SBS40, UK) was positioned between the samples and the lamp to prevent heating.

hASCs, seeded in a 48-well plate (Corning Incorporated, Corning, NY, USA), were exposed for 24, 72 h (3 days), 6 or 14 days to CB or DMSO. Moreover, hASC recovery was assessed after 24 or 48 h from the end of 24 h CB treatment. At each time point, cells were detached (2 wells/condition for each experimental point) by trypsin–EDTA and counted, as previously described [45 (link)]. Briefly, cells were resuspended in a medium with 50% erythrosine B (Sigma-Aldrich Co., St. Louis, MO, USA) and 0.2% red dye in PBS. Not stained viable cells and red stained dead cells were manually counted, at least twice for each condition, using the Neubauer hemocytometer (BRAND GmbH, Wertheim, Germany) and a light microscope (Nikon Eclipse TS100, Nikon Instruments, Melville, NY, USA). The total numbers of viable and dead cells were calculated according to the manufacturer’s instructions; for each sample, cell viability was obtained calculating the percentage of living cells compared to the total number of cells. Moreover, cell growth rate (gr) was calculated using the following formula: gr = (ln (N(t)/N(0))/t, where N(t) = the number of cells counted after 24 h or 48 h recovery upon CB removal; N(0) = the number of cells counted after 24 h of CB treatment; t = time passed (24 or 48 h).

To evaluate the possible cytotoxic effect of Fragaria-derived EPDENs, ADMSCs were treated with nanovesicles at different concentrations (0–2–4–9 µg/1 × 10⁴ cells) for 48 h and 120 h. ADMSCs were harvested, and the number of viable cells was evaluated by the erythrosine B (Sigma-Aldrich) dye vital staining. All experiments were performed two times in triplicate.

Polyvinyl alcohol (PVA), N-isopropyl acrylamide (NIPA), N,N-Methylene bisacrylamide (BA), N-Phenylglycine (NPG), Erythrosine B (Er B) and citric acid (CA) were purchased from Sigma-Aldrich, St. Louis, MO, USA. Glycerol was purchased from Fisher Scientific, Hampton, NH, USA, while Fe₃O₄ 20 nm and Fe₂O₃ 20–30 nm MNPs were purchased from GetNanoMaterials, Saint-Cannat, France. All chemicals were used without further modification.
The stock solution of Polyvinyl alcohol (PVA) and Erythrosine B (Er B) sensitising dye was prepared as previously reported [71 (link)]. For PVA solution (10% wt./vol), 10 g of PVA powder was dissolved in 100 mL of deionised (DI) water through magnetic stirring and heating at 75 °C. A dye solution of concentration (0.11% wt./vol) was obtained by dissolving 0.11 g of Er B dye in 100 mL of DI water through magnetic stirring at room temperature.