Sinergy ht

Cell proliferation was spectrophotometrically assessed as described previously [59 (link)]. Briefly, Caki-1 cells (500 cells/well) were seeded in 96-well plates and cultured with McCoy’s 5A with L-glutamine supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, in the presence of vehicle (DMSO, 0.1%), TRAM-34 (1 μM), Paxilline (10 μM), or a combination. Final DMSO concentrations were the same for all conditions. Non-heat inactivated calf serum served as standard mitogenic stimulus. Cells were fixed with formalin (10% in phosphate-buffered saline) at days 0,1,2,3, and 4, and stained for 5 min with 0.3% Janus B Green dye at room temperature with constant stirring. Cells were then de-stained with water. Dye was eluted with 200 μl/well of 0.5 M HCl for 15 min and absorbance at 595 nm was determined using a microplate reader (Sinergy HT, Biotek, USA). Absorbance values lower than control indicated less cell proliferation.

Production of colonic IL-1β was determined using commercial enzyme-linked immunosorbent assay (ELISA) kits (Murine IL-1β, PeproTech, Cranbury, USA) according to the manufacturer’s protocol. Absorbance determined at 415 nm with a microplate reader (Sinergy HT, Biotek®, Bad Friedrichshall, Germany). Levels of this cytokine were determined in duplicate and expressed as picograms per milligram of wet weight tissue (pg/mg tissue).

HaCaT cells were seeded at 5 × 10⁵ cells/well in 6-well plates, incubated for 24 h and treated with either RA (5 µM), FX (5 µM) or M2 (5 µM RA plus 5 μM FX), for 1 h. Then, the cells were irradiated at 100 mJ/cm², and incubated with fresh medium for 24 h. Supernatant fluids were collected and stored at −80 °C until use. Commercial enzyme-linked immunosorbent assay (ELISA) kits (Diaclone GEN-PROBE, Besançon cedex, France) was used to quantify IL-1β production according to the manufacturer’s protocol. The absorbance at 450 nm was read by a microplate reader (Sinergy HT, Biotek^®, Bad Friedrichshall, Germany). To calculate the concentration of IL-1β (pg/mL), a standard curve was constructed using serial dilutions of cytokine standards provided with the kit.

P*(0.5)AA50-AEDP-DOX cytotoxic effect was evaluated on MC3T3-E1 and Hela cells by Alamar Blue assay (Sigma Aldrich). Both cell lines were seeded into a 96-multiwell at 6 × 10³ cells per well, in complete DMEM for 24 h and then treated with 10 mM GSH-OET for 2 h at 37 °C. Therefore, Hela cells were incubated with P*(0.5)AA50-AEDP-DOX nanogels or free DOX as control at the final DOX concentration of 1 μg/mL for 24 and 48 h; while MC3T3-E1 cells were treated with drug alone (as control) or conjugated with NGs at the final DOX concentration of 0.3 μg/mL for 48 and 72 h. Cells GSH-OET untreated and incubated with NGs were used as another control. After treatment, cells were incubated with Alamar Blue solution (10% in culture medium), a reagent for evaluating cellular health, for 4 h at 37 °C. Fluorescence intensity (λexc 530/25 nm and λemm 590/35 nm) was read through spectrofluorometer (SinergyHT, BioTek, Winooski, VT, USA), which changes according to the degree of cell viability. The results were expressed as the percentage ratio of the fluorescence between the treated sample with respect to untreated cells, used as negative control.

SRB assay was used for determining the viability of THP-1 macrophages and HaCaT cells upon exposure to FX (Sigma-Aldrich, St. Louis, MO, USA) [51 (link)]. Firstly, for differentiation into macrophages, THP-1 cells were seeded into 96-well plates at 10⁴ cells/well in presence of phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, St. Louis, MO, USA) for a final concentration of 0.2 µM for 72 h in 96-well plates and HaCaT cells were seeded into 96-well plates in the growth medium at 10⁴ cells/well for 24 h to ensure the adherence. Both cellular types were incubated in a humidified atmosphere of 5% CO₂ at 37 °C. After that, cells were treated with FX at final concentrations range of 10–100 µM in DMSO 0.1% (v/v) and the cytotoxicity was measured after 24, 48 and 72 h of incubation. The absorbance was determined at 492 nm in a microplate spectrophotometer (Sinergy HT, Biotek^®, Bad Friedrichshall, Germany).

The cell growth inhibitory activity of gemcitabine in Caco-2 cells was assessed with the MTT assay. Briefly, a growth curve was traced to determine the best cell density for the assay. Cells were seeded in 96-well plates (growth surface 0.322 cm², from TPP®, Product No. 92696) with an initial cell density of 9.3 x 10³ cells/mL (200 μL per well). Cells were allowed to attach for 24 h and were then either left untreated (culture medium was replaced for fresh medium) or treated with gemcitabine (0.1, 1, 5, 10, 20, 50, 100, 1000, 10000 and 100000 μM). Following 72 h incubation, cell medium was removed and 100 μL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution were added per well. Cells were then incubated for another 4 h protected from light. Finally, MTT solution was removed, DMSO (100 μL per well) was added to solubilize the formazan crystals formed by viable cells and absorbance was measured at 570 nm in an automated microplate reader (Sinergy HT, Biotek Instruments Inc, Vermont, USA). All conditions were performed in triplicate.
Gemcitabine cytotoxicity results were compared with the untreated control (mean of values was set to 100 %) and expressed as mean ± SEM. The statistical significance between different gemcitabine concentrations was analyzed in GraphPad Prism 7 (San Diego, EUA) using one-way ANOVA (p<0.05).

To verify the orientation of the carbohydrate moieties associated with the surface of the F/C nanoparticles, an agglutination assay was performed. For this effect, 500 µL of nanoparticles were diluted in 500 µL of double-deionized water and centrifuged at 10,000× g for about 20 min. The supernatant was removed, and the pellet was re-suspended in 1 mL of PBS, 10 mM at pH 7. A volume of 200 µL of each type of nanoparticles under study were incubated with 50 µL of concanavalin A solution (1 mg mL⁻¹). The time-dependent increase in the turbidity at 405 and 550 nm was monitored spectrophotometrically (Sinergy HT, BioTek^®; Bedfordshire, UK) for 90 min at 25 °C.