Peg 6000

Edible sunflower oil (Zvijezda plus d.o.o., Zagreb, Croatia) was purchased from a local supermarket. Polyethylene glycols with average molecular weights of 1500 g/mol (PEG 1500) and 6000 g/mol (PEG 6000) were purchased from Acros Organics (Geel, Belgium), while the 20,000 g/mol polyethylene glycol (PEG 20,000) was obtained from Sigma-Aldrich (Taufkirchen, Germany). Dried mint leaves (Mentha piperita L.) were purchased from Suban, Croatia. Plant materials were collected during the flowering season of 2019 in the north-western part of Croatia, dried naturally, and stored in ambient conditions before use.

CPC scaffolds with interconnected pores (diameter 5mm, thickness 2 mm) were printed by a semi-solid extrusion 3D printer (Innotere, Radebeul, Germany) using calcium phosphate semi-solid paste (α-tricalcium phosphate and calcium-deficient hydroxyapatite). All 3D printed scaffolds were subjected to coating using a polymeric solution of a model anti-cancer drug 5-Fluorouracil (5-FU, Acros Organics™, 99%, Fair Lawn, NJ, USA). Soluplus^® (BASF Ltd., London, UK) and polyethylene glycol (PEG 6000, Acros Organics™, Ludwigshafen, Germany) were used. We used 0.9% NaCl solution as the dissolution medium in the dissolution test. Cancer and transformed cell lines, HeLa and HEK293T, stably expressing GFP constructs were generated at the GDSC, University of Sussex, Brighton, UK. Cell lines were maintained in DMEM supplemented with 10% FCS, penicillin/streptomycin and L-Glutamine at 37 °C and 5% CO₂. PBS (pH = 7.4, Fisher Scientific, Loughborough, UK) was used for washing.

L-Proline (>99%), PEG 6000, ninhydrin, 2,2′-azobis(2 methylpropionamidine)dihydrochloride (AAPH), (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) and potassium ferricyanide (K₃[Fe(CN)₆]) were acquired from Acros Organics (Geel, Germany). Lactic acid, and fluorescein were purchased from Panreac (Barcelona, Spain). Sodium carbonate anhydrous (Na₂CO₃), ferric chloride (FeCl₃), Folin-Ciocalteu’s reagent, and gallic acid were acquired from VWR (Leuven, Belgium). Ascorbic acid, acetic acid, and potassium iodide were acquired from Merck (Darmstadt, Germany). Salicylic acid (SA), methyl jasmonate (MeJA), Fe₃O₄ nanoparticles (50–100 nm), hydrogen peroxide (H₂O₂), trichloroacetic acid (TCA), L- proline (≥99%), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 3,4-dihydroxy-L-phenylalanine (L-DOPA), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt tablets (ABTS), mushroom tyrosinase (EC 1.14.18.1), kojic acid, potassium persulfate (K₂S₂O₈), potassium bromide, HPLC-MS-grade water, HPLC-MS-grade acetonitrile, luteolin, epigallocatechin gallate, protocatechuic acid, and formic acid were obtained from Sigma–Aldrich (Steinheim, Germany). Rosmarinic acid and quercetin were supplied by Extrasynthese (Genay, France), and ρ-coumaric, caffeic acid, and catechin were provided by AASC Ltd. (Southhampton, UK).

Unless specified, all chemicals were obtained from Sigma-Aldrich. PEG-6000 (molecular weight: 6 kDa) was purchased from Fisher (Pittsburg, PA). A 50% (w/v) stock solution of PEG-6000 was prepared in nuclease-free water before being used in experiments. BSA was purchased from New England Biolabs. GroEL/ES were obtained from Takara; DnaJ was obtained from Accurate Chemical & Scientific Corporation, DnaK and GrpE were obtained from Novus Biologicals. Tryptone and yeast extract were obtained from BD Difco. For protein purification, liquid cultures of all strains were grown in SB media (24 g/L tryptone, 12 g/L yeast extract, 5 g/L glucose, 2 g/L NaH₂PO₄, 16.4 g/L K₂HPO₄-3H₂O, 4 mL/L glycerol). Protein purifications were carried out with ÄKTAprime (GE Healthcare) equipped with 5 mL HisTrap HP column (GE Healthcare). Purified protein concentrations were determined by standard Bradford assay (Bio-Rad).

The hyperimmune egg yolk generated in our recent study (9 (link)) was used for IgY purification following a published protocol (10 (link)) with slight modifications. Briefly, one volume of egg yolk was diluted with four volumes of PBS and then mixed with 3.5% (w/v) of polyethylene glycol 6000 (PEG 6000, Fisher Scientific, MA, USA) for lipid removal. After 20 min of incubation at room temperature, the mixture was centrifuged at 5,000g for 20 min. Subsequently, the supernatant was added with 12% (w/v) of PEG 6000 for IgY precipitation and the mixture was incubated at room temperature for 10 min. Following centrifugation at 5,000g for 20 min, the precipitate was dissolved in PBS and added with 12% (w/v) of PEG 6000 again for another round of IgY precipitation. After centrifugation, the precipitate was dissolved in a small volume of PBS, added with equal volume of precooled (−20°C) ethanol, and centrifuged at 5,000g and 4°C for 20 min to remove PEG 6000. Lastly, the precipitate containing a high proportion of IgY was dissolved in a small volume of PBS and the solution was dialyzed against PBS. Initial egg yolk, the supernatant and precipitate fractions obtained from the centrifugation of 12% PEG 6000 mixture, and the final dialyzed IgY extract were subjected to SDS-PAGE analysis.