Type 1 agarose

AuNPs were synthesized using agarose (Type I Agarose, Sigma-Aldrich) and gold(iii) chloride trihydrate (Sigma-Aldrich) at various concentrations of both reagents in water. A solution of agarose (4.5 mL water, 0.025 g to 0.150 g agarose, leading to 0.5 and 3.0 m/v% concentrations calculated for the 5 mL overall solution) was heated to 90 °C and vigorously stirred in a heating magnetic stirrer at 400 rpm. When the solution of agarose became transparent (after 15 min of heating), a solution of the gold salt (0.5 mL, with concentration ranging between 0.10 and 10.0 mM, leading to final concentrations between 0.01 and 1.0 mM) was added to the stirred hot solution. After keeping the mixture at 90 °C for 30 min with a continuous stirring, it was allowed to cool down to room temperature (RT) to result in a colored hydrogel.

Quercetin (≥95 %), tannic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), (+) - 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid - Trolox (≥97 %), type I agarose and anhydrous potassium persulfate (K₂S₂O₈), dimethyl sulfoxide (DMSO) were acquired from Sigma-Aldrich (Aldrich Brasil Ltda., Brazil). l-ascorbic acid (≥99.0 %), ethanol PA (99.5 %), and anhydrous sodium carbonate Na₂CO₃ (≥99.5) were obtained from Synth (LABSYNTH, Brazil), while ultrapure monohydrate gallic acid and aluminum chloride (III) (≥99.5 %) from Vetec (VETEC-Sigma-Aldrich, Brazil). The Folin-Ciocalteu reagent and bovine serum albumin–BSA were acquired from Merck (Merck, USA). Glacial acetic acid PA and sodium hydroxide were obtained from Dinâmica (Dinâmica, Brazil. Nutrient broth, malt extract, yeast extract, potato extract, and agar were purchased from Acumedia (Acumendia, USA). DMSO‑d₆ and TMSP-d4 (2,2,3,3-d4-3- sodium trimethylsilylpropionate) were purchased from Cambridge Isotope Laboratories, Inc. (USA). The reagents used in antimicrobial susceptibility tests were ciclopirox olamine [(LOPROX®), 10 mg g⁻¹, Sanofi-Aventis, Brazil)], DMSO [(≥99.0), Sigma-Aldrich (Aldrich Brasil Ltda., Brazil], DMEM Mix F12, penicillin G, gentamicin, amphotericin B, glucose, l-glutamine, and fetal from Gibco (Thermo Fisher Scientific, Brazil).

Agarose gels were prepared by dissolving 1 g of type I agarose (Sigma-Aldrich) in 100 ml of bidistilled water. The solution was then autoclaved for 30 min. Agarose solution gelified quickly at RT; therefore, stock gels were first melted in a microwave oven at 500 W for 30 s. To produce cubic or cylindric hydrogels, hot agarose solutions were poured in polyethylene molds, which were then placed in a 4°C fridge for 2 hours. Molds containing hydrogels were rinsed in abundant water at RT, and the hydrogels were gently detached from the molds with a spatula. Hydrogel spheres were produced by dripping hot agarose solution from a 10-ml plastic syringe in an ice-cold water bath, thus obtaining 3-mm spheres.