Millex gp

Stock solutions of 0.1 M and 0.01 M NiCl₂·6H₂O (n = 237.7 g/mol) were prepared with deionized water and filter-sterilized prior to use (0.22 µm, diam. 33 mm, Millex-GP, Sigma-Aldrich).

Polyethyleneimine (PEI) was obtained from Sigma-Aldrich (St. Louis, MO, USA) and diluted to 5% (v/v) with MilliQ water. Poly-L-lysine hydrochloride (PLL, molecular weight 1.6 kDa, equals 12–13 lysine units) was bought from Alamanda Polymers (Huntsville, AL, USA). The aggrecanase-labile peptide sequence “KKKK-NITEGE↓ARGSV-KKKK-carboxyl” (NITEGE) was prepared at the Helmholtz Centre for Infection Research (Braunschweig, Germany), according to standard solid-phase peptide synthesis protocols. All solutions were filtered through 0.22 µm Millex–GP (polyethersulfon; Sigma-Aldrich) filters before use. Glycosylated IFN-β was purchased from PBL Interferon Source (Piscataway, NJ, USA) and was treated according to the manufacturer’s instructions. Trypsin, thermolysin, and alginate-lyase were all obtained from Sigma-Aldrich and used as received.
Titanium alloy substrates (1 mm thick, Goodfellow, Hamburg, Germany) were cut into 1 × 1 cm² squares and treated as described below. Acetone, methanol, ethanol, and dichloromethane were distilled before use.

(1R,8S,9S)-Bicyclo-[6.1.0]-non-4-yn-9-ol, polyethylenimine (PEI), pyridine, alginate, ammonium chloride, trypsin, N-methylmorpholine (NMM), sodium sulfate (Na₂SO₄), ciprofloxacin, N,N-dimethylformamide (DMF), 1-hydroxy-1H-benzotriazol (HOBt), trifluoroacetic acid (TFA), N,N-diisopropylethylamine (DiPEA) and diethyl ether were purchased from Sigma-Aldrich (Steinheim, Germany) and used as received. Amino acids, 2-Chlorotrityl chloride resin and N,N′-diisopropylcarbodiimide were purchased from Carbolution Chemicals (St. Ingbert, Germany). Sodium chloride (NaCl), ethyl acetate (EtOAc), and petroleum ether (PE) were purchased from Fisher scientific (Schwerte, Germany). Dichloromethane (CH₂Cl₂) and methanol were purchased from VWR (Darmstadt, Germany). All solutions were filtered through 0.22-µm Millex-GP (polyethersulfon; Sigma-Aldrich) filters before use. Titanium alloy substrates were cut into 1 × 1 cm² squares. Dichloromethane, PE, DMF, diethyl ether were purified with a Solvent Purification System SPS-800 from MBraun (München, Germany) before use.

For preparing methanolic extracts, 4 g samples of lyophilized (Labconco, Kansas City, MO, USA) and pulverized material (biomass of each species) were weighed. The material was then extracted with 50 mL methanol (STANLAB, Lublin, Poland) by sonication in an ultrasonic bath (POLSONIC 2, Warsaw, Poland) for 30 min. Next, the extracts were centrifuged (MPW-342, Med. Instruments, Warsaw, Poland) for 10 min at 4300 rpm. methanol in the supernatant was allowed to evaporate at 22 ± 2 °C. After evaporation, the dry residue was dissolved quantitatively in HPLC-grade methanol. Before the HPLC analysis, the obtained extracts were filtered through sterilized syringe filters (0.22 μm, Millex^®GP, Millipore).