Potassium chloride (kcl)

The carrier for the AF4 was prepared from analytical grade sodium chloride (prod. no. 27788.297, VWR), di-sodium phosphate dihydrate (prod. no. 28029.292, VWR), potassium phosphate (prod. no. 26936.293, VWR), potassium chloride (prod. no. 26764.298, VWR) and sodium azide (prod. no. 103692K, VWR). For the aggregation buffer, sodium acetate (prod. no. A13184, Alfa Aesar) was used. Water used for the preparation was purified on an ion-exchange unit (Purelab Ultra, ELGA). Standard proteins myoglobin (prod. no. M1882), ovalbumin (prod. no. A5503), bovine serum albumin (prod. no. A3059), immunoglobulin G (prod. no. 56834) and thyroglobulin (prod. no. T1001) as well as the human serum (prod. no. H4522) were obtained from Sigma-Aldrich.
The trastuzumab was obtained from a Swedish pharmacy (Apoteket AB). Purified HER-2 was acquired in two modified forms from Sino Biological Inc.: His-tagged (prod. no. 10004-H08H) and biotinylated and His-tagged (prod. no. 10004-H08H-B). The molecular weight of the HER-2 protein was 71 kDa, and after reconstitution, the concentration of both stock solutions was 250 μg/mL.
Biotinylated Au sensors, NTA-modified carboxymethyldextran sensors (CMD-NTA) and unmodified carboxymethyldextran sensors (CMD) were provided by BioNavis Ltd.

The chemicals used during the experiments were of analytical grade and were not further purified before usage. Graphite rods (6 mm diameter, 99.995% purity); potassium chloride (KCl, 99.98%); sodium dihydrogen phosphate (NaH₂PO₄, 100%) and di-sodium hydrogen phosphate anhydrous (Na₂HPO₄, 99.7%) were supplied by VWR Chemicals (Leuven, Belgium). Sodium acetate anhydrous (CH₃COONa, ≥99.0%); ammonium sulphate ((NH₄)₂SO₄, ≥99.0%), sodium chloride (NaCl, 99.5%); sodium carbonate anhydrous (Na₂CO₃, 99.3%), magnesium chloride hexahydrate (MgCl₂∙6H₂O, 99–102%); magnesium sulphate (MgSO₄, 99%); magnesium nitrate hexahydrate (Mg(NO₃)₂∙6H₂O, 99%) were provided by REACTIVUL Bucuresti (Bucharest, Romania). Potassium ferrocyanide K₄[Fe(CN)₆] and L(+)-Ascorbic acid (C₆H₈O₆, AA, ≥99%) were acquired from Merck (Darmstadt, Germany). Sodium acetate anhydrous (CH₃COONa, ≥99.0%) was purchased from ChimReactiv SRL (Bucharest, Romania). N,N-dimethylformamide (DMF) was acquired from Fluka Chemie GmbH (Buchs, Switzerland), while boric acid (H₃BO₃, 99%) was provided by Andra Chim SRL (Bucharest, Romania). The MQuant^® Nitrite Test (Merck KGaA, Darmstadt, Germany) was employed for UV-Vis investigation of nitrite solutions. Deionized water with a resistivity of at least 18.2 MΩ × cm was used to prepare all solutions.

High molecular weight (HMW) sodium hyaluronate (1.8–2.2 MDa), was a kind gift from Inquiaroma and N-hydroxymethylglycinate 50% (Suttocide) from Ashland (Covington, Kentucky, EUA, Brussels, Belgium). Potassium chloride, sodium chloride, sodium tetraborate and Ethylenediamine Tetraacetic Acid (EDTA) were purchased form VWR International (Carnaxide, Portugal), calcium chloride.6H₂O was purchased from José Manuel Gomes Santos (Odivelas, Portugal), magnesium chloride.6H₂O was purchased from Sigma-Aldrich Quimica (Sintra, Portugal) and boric acid was purchased from LaborSpirit (Loures, Portugal). In addition, for the mucoadhesive studies, it was used dried mucin from porcine stomach type II (Sigma-Aldrich, Saint Louis, MI, USA). Human retinal pigment epithelial cell lines ARPE-19 (ATCC^® CRL-2302™) were obtained from American Type Cell Culture collection (Manassas, VA, USA), and they were used for cell viability and dry eye assays. Cell culture medium and supplements were from Gibco (Thermo Fisher Scientific, Rochford, UK).

Unless otherwise specified, all reagents were purchased from Millipore-Sigma (Oakville, ON, Canada). The BCA protein assay reagent (bicinchoninic acid), NuPAGE® 4–12% Bis-Tris gels, NuPAGE® MES SDS running buffer, NuPAGE® sample reducing agent, NuPAGE® antioxidant, NuPAGE® LDS sample buffer, prestained protein standard, methanol, sodium chloride, and Corning clear polystyrene 96-well microplates were purchased from ThermoFisher Scientific (Nepean, ON). Glacial acetic acid, glycerol, sodium phosphate dibasic, and trizma base were from Bioshop Canada (Burlington, ON, Canada). Bromophenol blue, Coomassie blue R250, potassium chloride, potassium phosphate monobasic, and sodium dodecyl sulfate were from VWR (Mississauga, ON, Canada). Petri dishes (150 mm) were purchased from Ultident Scientific (St. Laurent, QC, Canada).

Magnolol and honokiol (98% purity) were acquired from New Natural Biotechnology (Shanghai, China). Phosphotungstic acid solution, sodium phosphate monobasic and potassium phosphate monobasic were purchased from Sigma-Aldrich (Madrid, Spain). N-octanol, acetonitrile, ethyl alcohol, and potassium chloride were obtained from VWR chemicals (Barcelona, Spain). Hydrochloric acid 37% and sodium hydroxide were purchased from Scharlab (Barcelona, Spain). Hydrogen peroxide 30% and boric acid were obtained from Merck (Barcelona, Spain). Phospholipon 90 G was provided by Lipoid GmbH (Ludwigshafen, Germany).

All reagents and solvents were either HPLC or analytical grade. Moreover, 2,2-diphenyl-1-picrylhydrazyl (DPPH), gallic acid (98%), quercetin (98%), artemisinin (98%), Folin–Ciocalteu reagent, ascorbic acid, β-carotene, and linoleic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anhydrous sodium sulfate, potassium ferricyanide, trichloroacetic acid (TCA), ferric chloride, sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, and hydrochloric acid were bought from VWR Chemicals (Radnor, PA, USA). Hexane, dichloromethane (CHCl₃), ethyl acetate (EtOAc) and methanol (technical and HPLC) were purchased from VWR International (Fontenay-sous-Bois, Val-de-Marne, France).