Ethanol

The Ti_0.8Sn_0.2O₂-C composite supports were prepared using Black Pearls 2000 active carbon (Cabot Corporation, Boston, MA, USA). For the composite synthesis, titanium isopropoxide (Sigma-Aldrich, St. Louis, MO, USA, 97%), tin (IV) chloride-5-hydrate (Honeywell Riedel-de Haën GmbH, Seelze, Niedersachsen Germany, 98%), nitric acid (65%, a.r., Molar Chemicals, Halásztelek, Hungary) and ultrapure water (18 MΩ cm, produced by Millipore equipment, (Burlington, MA, USA)) were used. The Pt precursor for electrocatalyst synthesis was H₂PtCl₆·6H₂O (Sigma-Aldrich, St. Louis, MO, USA, 37.5% Pt). Further chemicals used for the Pt loading were ethanol (99.55%), ethylene glycol (99.8%), HCl (37%) and NaBH₄ (99.95%) (all obtained from Molar Chemicals). The catalyst ink for the electrochemical studies was prepared using a Nafion solution (DuPont™ Nafion^® PFSA Polymer Dispersions DE 520, The Chemours Company, Wilmington, DE, USA), isopropanol (Molar Chemicals) and ultrapure water (see above).

To determine the antimicrobial activity of the mouthwashes, first we performed a disc diffusion assay. Adequate agar medium, such as Mueller–Hinton (Sigma-Aldrich, Munich, Germany), blood agar (Sigma-Aldrich, Munich, Germany), Sabouraud agar (Sigma-Aldrich, Munich, Germany), Rogosa agar (Sigma-Aldrich, Munich, Germany) was used with 1.8% agar concentration.
Our controls were 40 mg/mL Gentamicin (Sandoz, Basel, Switzerland), 2% chlorhexidine-digluconate (CHX, Molar Chemicals, Halásztelek, Hungary), and 10% ethanol (Molar Chemicals, Halásztelek, Hungary). Experiments were performed in five technical replicates.
10 µL of the microbial suspensions with 0.4 optical density (measured at 590 nm) were dispensed respectively on the agar surface on a 92 × 16 mm dish. Following 30 min incubation, a single sterile paper disc was placed on the agars loaded with 10 µL of the test samples. After 72 h incubation at 37 °C, the plates were scanned and digitally evaluated with a densitometry software using a Kodak Image Station 2000R (Kodak, Rochester, NY, USA). The largest diameter of inhibition zone was determined [72 (link)].

For protein stability assay, transfection medium was replaced after overnight incubation at 37 °C with 1 mL DMEM containing the translational inhibitor cycloheximide (50 μg/mL) and the cells were incubated for 1, 2, 4, or 6 h in 12-well plates. Non-transfected samples as well as cells transfected with pcDNA3.1(–) “empty” vector were used as control in all experiments.
For mRNA stability assay, transfection medium was replaced after overnight incubation at 37 °C with 1 mL DMEM containing the mRNA synthesis inhibitor actinomycin D (5 μg/mL) and the cells were incubated for 0, 1, 2, 4, 8, or 12 h in 12-well plates.
Oleate, palmitate, palmitOleate, linOleate, and stearate (Sigma-Aldrich, St. Louis, MO, USA) were diluted in ethanol (Molar Chemicals, Halásztelek, Hungary) to a final concentration of 50 mM, conjugated with 4.16 mM FA free BSA (Sigma-Aldrich, St. Louis, MO, USA) in 1:4 ratio, at 50 °C for 1 h. The working solution for FA treatments was prepared freshly in FBS-free and antibiotic-free medium at 100 µM final concentration. The culture medium had been replaced by FBS-free and antibiotic-free medium 1 h before the cells were treated with FA. The FA-treatment was carried out for 6 h in 12-well plates.

The active pharmaceutical, aceclofenac (Figure 1a) was provided by Richter Gedeon Plc. (Budapest, Hungary). For the preparation of the viscous polymeric solutions, poly(vinyl-pyrrolidone) (PVP K90, Kollidon K90, Figure 1b) was used, which was obtained from Sigma Aldrich (Merck, Darmstadt, Germany). Triethanolamine (trolamine) and ethanol were from Molar Chemicals (Budapest, Hungary). Ultrapure, distilled water was prepared in-house by a MilliQ water purification system. Potassium dihydrogen phosphate and disodium hydrogen phosphate (Merck KGaA, Darmstadt, Germany) were employed for the preparation of the phosphate buffer solution (1 M, pH 6.8) used for the in vitro dissolution study.

LOM (≥98%), PG (≥98%), and phosphatidylcholine (PCL, ≥99%) from soybean were purchased from Sigma-Aldrich Co. Ltd. (Budapest, Hungary). Cholesterol (CHL, ≥99%), ethanol (96% v/v), and sodium chloride physiological solution (0.9% w/w) were obtained from Molar Chemicals Ltd., (Budapest, Hungary). All cell lines were purchased from the European Collection of Cell Cultures (Salisbury, UK). All reagents for cell line studies were purchased from Sigma–Aldrich Co. Ltd. (Budapest, Hungary) if not indicated otherwise.

Oleate, palmitate, stearate, linoleate, elaidate, and vaccenate were diluted in ethanol (Molar Chemicals, Halásztelek, Hungary) to a final concentration of 50 mM and conjugated with 20% FA-free BSA in 1:4 ratio at 50 °C for 1 h. The working solution for FA treatments was prepared freshly in FBS-free and antibiotic-free medium at 100 µM final concentration. The FA treatment was carried out for 24 h in 12-well plates. For luciferase assay, the culture medium was replaced 5 h after transfection and the cells were incubated for a further 24 h.