β casein from

Citric acid (251,275), urea (U5128), PBS Tablet (524,650), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (03450), N-Hydroxysuccinimide (130,672), lactoferrin from bovine colostrum (L4765), glucose oxidase from aspergillus niger (G7141), concanavalin A-peroxidase from Canavalia ensiformis (Jack bean) (L6397), β-casein from bovine milk (C6905), lysozyme human (L1667), bovine serum albumin (B4287), potassium permanganate (223,468) and hydrochloric acid (320,331) were purchased from Sigma-Aldrich. Graphite flake (43,209) used for fabricating GONSs was purchased from Alfa Aesar. Sulphuric acid and magnesium chloride (105,672) were purchased from Caledon Laboratory Chemicals. The lactoferrin aptamer was purchased from Integrated DNA Technologies^{31 (link),36 (link)}.

The conditioned medium collected from the several cell cultures, which were grown 6-well plates coated with collagen type I, was analysed for proteinases activity using β-casein zymography. Samples were mixed with sample buffer [0.03% bromophenol blue, 0.25 M Tris-HCl pH 6.8, 10% SDS (w/v) and 4% sucrose (w/v)] and electrophoresed, under non-reducing conditions, on 10% polyacrilamide gels containing 0.1% (w/v) β-casein from bovine milk (Sigma). After electrophoresis, gels were washed twice, for 30 min, in 2% (v/v) Triton X-100 (Sigma) at room temperature, in order to remove SDS. β-casein gels were then incubated in a substrate reaction buffer [0.2 M NaCl, 5 mM CaCl₂, 1% (v/v) Triton X-100 in 50 mM Tris-HCl, pH 7.4], during 72 h, and finally stained with Coomassie Blue Staining Solution [0.1% (w/v) Coomassie Blue R250 in 10% (v/v) acetic acid and 40% (v/v) methanol], for 25 min. The gels destaining was performed in a solution with 20% methanol and 10% acetic acid, until bands start to become visible. Enzymatic activity was visualized as a clear band against the blue background of stained casein gels, and MMPs were identified by their molecular weight. Quantification of band density was carried out using the Quantity One software (version 4.0, BioRad, Hercules, CA, USA).