Fibroblast growth medium

Reagents: poly(ethyleneimine), branched, Mn ~60,000, Mw 750,000, analytical standard, 50% (w/v) in H₂O (Sigma-Aldrich, München, Germany); silver nanoparticles, 100 ppm, of 10 nm size, stabilized with sodium citrate, in 0.01% Tween 20 (University Technology Transfer Centre of the University of Warsaw (UTTC UW): Bell Synthesis, Poland, EU); golden nanoparticles, 100 ppm, of 10 nm size, stabilized with sodium citrate, in 0.01% Tween 20 (UTTC UW: Bell Synthesis, Warsaw, Poland); hydroxyapatite, aqueous paste, particle size < 50 nm, 30 wt.%, spec. surface area ≥ 80 m²/g (Sigma-Aldrich, München, Germany); propidium iodide, ≥94.0% (HPLC) (Sigma-Aldrich, München, Germany); trypsin EDTA Solution C (0.5%), EDTA 0.2% (10X) (Biological Industries, Israel); phosphate-buffered saline (PBS) (Biomed Lublin, Poland, UE); water MilliQ.
Media: Fibroblast Growth Medium (FGM) (Sigma-Aldrich, München, Germany).
Cells: Cell line of Human Dermal Fibroblasts (HDF) (Sigma-Aldrich, München, Germany).

L-lysine, propylene carbonate, NaOH, chitosan, HCl, DPPH (2,2-diphenyl-1-picrylhydrazyl) were all purchased from Sigma-Aldrich, Poznań, Poland. XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) assay, human dermal fibroblasts (HDF), and fibroblast growth medium (FGM) were also bought from Sigma-Aldrich, Poland. Multi-hole plates (96 holes) were baught from Nest, GenoPlast, Rokocin, Poland.

Adult Human Primary Normal Gingival Fibroblast cells (HGF, ATCC® PCS-201-018™) obtained from the American Type Culture Collection (Manassas, VA, USA), was cultured in Fibroblast growth medium (#116-500, Sigma-Aldrich Inc.). Human OSCC cell line with high metastatic potential HSC-3 (#JCRB0623) and SAS (#JCRB0260) were purchased from the National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN)-Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan), and cultured in Gibco™ Dulbecco’s Modified Eagle Medium (DMEM, #11995065, Thermo Fisher Scientific Inc., Bartlesville, OK, USA), supplemented with 10% fetal bovine serum (FBS, #26140079) and 1% penicillin-streptomycin at 37 °C, in 5% humidified CO₂ incubator. Cells used in the present study were all ≤ passage number 3 (P.3) and were sub-cultured when 90% confluent and media changed every 48 h.

Human breast cancer MC7-7 and MDA-MB-231 cells, as well as human melanoma C32 cells and COLO 829 cells, were obtained from the American Type Culture Collection (ATCC). The base medium for MCF-7, MDA-MB-231, C32 and A375 was Dulbecco’s modified Eagle’s medium (DMEM), from Cell Applications, San Diego, CA, USA. The base medium for COLO 829 was Roswell Park Memorial Institute (RPMI) 1640 (Cell Applications). To make the complete growth media, the following components were added: fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) to a final concentration of 10%, penicillin G (final concentration: 100 U/mL), neomycin (final concentration: 10 µg/mL) and amphotericin B (final concentration: 0.25 µg/mL). Normal dermal human fibroblasts were obtained from Sigma-Aldrich and cultured in Fibroblast Growth Medium (Sigma Aldrich, Saint Louis, MO, USA). All cells were maintained at 37 °C in humidified incubators with 5% carbon dioxide. The cells were passaged when there was ca. 80% confluence.

Lomefloxacin hydrochloride, penicillin G, amphotericin B, H2DCFDA (2′,7′-dichlorofluorescein diacetate), SIGMAFAST™ Protease Inhibitor Cocktail Tablet, and Phosphatase Inhibitor Cocktail 3, Dulbecco’s phosphate-buffered saline (DPBS) with MgCl₂ and CaCl₂, phosphate buffered saline (PBS),and Fibroblast Growth Medium were obtained from Sigma Aldrich Inc. (St. Louis, MO, USA). A Pierce BCA Protein Assay Kit, ECL Western Blotting Substrate, and Hoechst 33342, CellROX™ Green Reagent were obtained from Thermo Fisher Scientific (Waltham, MA, USA). GAPDH (14C10) Rabbit mAb, SOD1 (71G8) Mouse mAb, SOD2 (D9V9C) Rabbit mAb, Catalase (D4P7B) Rabbit mAb, and GPx1 (C8C4) Rabbit mAb were obtained from Cell Signaling (Danvers, MA, USA), and Anti-Rabbit IgG (A154), Anti-Mouse IgG, Tween-20, RIPABuffer and PVDF membranes were obtained from Sigma-Aldrich Inc. (St. Louis, MO, USA). Neomycin sulfate was obtained from Amara (Kraków, Poland). Trypsin/EDTA solution was purchased from Cascade Biologics/Gibco (Carlsbad, CA, USA). Solution 3 (1 μg/ mL DAPI, 0.1% triton X-100 in PBS), Solution 5 (VB-48TM, propidium iodide-PI, acridine orange—AO), NC-Slide A8 and Via-1-Cassette (AO and DAPI fluorophores) were obtained from ChemoMetec (Lillerød, Denmark). Cell Proliferation Reagent WST-1 was produced by Roche GmbH (Mannheim, Germany). Other chemicals were from POCH S.A. (Gliwice, Poland).

The in vitro cytotoxicity of the gold complexes was evaluated in adult human dermal fibroblasts (HDF) (Sigma-Aldrich) using the MTT assay as previously described [29 ]. Cells were cultured in Fibroblast Growth Medium (Sigma-Aldrich) following the instructions of the supplier for these cells. Cells laid in 96 well plates were incubated with serial dilutions of the compounds for 24 h. Each compound’s dilution was performed in four wells. For each assay controls (cells without test compound) were done. At least two independent experiments were performed for each cytotoxicity analysis. The cytotoxicity of each compound was expressed by the IC₅₀, the concentration of compound causing 50% decrease of cellular viability.