Disodium hydrogen phosphate

Glu (98%, lot no. 140690-200401) was purchased from National Standard Material Centre (Beijing, China); Homoserine (IS) (99%, lot no. 5005F23A) was purchased from Alfa Aesar Chemical Co. Ltd (Shanghai, China); Gln (99%, lot no. BCBH4247V), GABA (97%, lot no. BCBN4574V), o-phthalaldehyde (OPA), β-mercaptoethanol, disodium hydrogen phosphate, sodium borate and perchloric acid were purchased from Sigma Aldrich Chemie (Steinheim, Germany). HPLC-grade acetonitrile and methanol were purchased from Merck (Darmstadt, Germany). Water was purified by means of a Milli-Q plus system from Millipore (Bedford, MA).

The lyophilized powder of DNA oligonucleotide (5'- GTC GTA CTG ATA CAT GAG CC –'3; 6117 Da) was the product of Genomed (Warsaw, Poland) or Syntol (Moscow, Russia). It was used as an aqueous solution prepared in accordance with the producer recommendations and stored at –20 °C no longer than 1 month. Indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] was synthesized at the University of Vienna (see the Electronic Supplementary Material (ESM) for chemical formula). The stock solution of the Ru drug (1 mM) was prepared daily in 100 mM NaCl. Human transferrin (>98%), glutathione (>98%), and L-ascorbic acid (>99.5%) were purchased from Sigma-Aldrich (St. Louis, USA). Sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, also from Sigma-Aldrich (St. Louis, USA), were of analytical reagent grade. Sodium hydroxide and citric acid (>99.5%) were obtained from Fluka (Buchs, Switzerland). High-purity water (18 MΩ cm^–1) used throughout this work was obtained from a Milli-Q water purification system (Millipore Elix 3; Saint-Quentin, France).

Haloperidol, carvacrol, phytohaemagglutinin (PHA), cytochalasin B, thiobarbituric acid, Tris-HCl, Tris ammonium, MgCl_2, Disodium hydrogen phosphate, TCA, sucrose, EDTA, sodium acetate, Triton X-100, and Giemsa stain were from Sigma; DMEM cell culture and phosphate buffered saline (PBS) were from Gibco. Phosphoric acid, methanol, acetic acid glacial, potassium chloride, n-Butanol, sodium chloride, DTNB, Na₂EDTA, sodium hydroxide, sodium lauroyl sarcosinate, DMSO, and normal melting point agarose were from Merck. The low melting point (LMP) agarose was from Cleaver Scientific.

The following materials were used: sucrose (Sigma-Aldrich), polysorbate 80 (Tween 80, Sigma-Aldrich), inulin (4kD; Sensus, Roosendaal), dextran (5kD; Pharmacosmos Denmark), sodium dihydrogen phosphate (Sigma-Aldrich), disodium hydrogen phosphate (Sigma-Aldrich) and 5 mg/mL of Infliximab antibody bulk (5 mg/ml in 5% sucrose and 0.005% (polysorbate 80, pH 7.2) (obtained from AIMM therapeutics).

Three types of extracellular matrix models were crafted for this study. Collagens solely comprised of rat tail monomers (4 g/l rat collagen type I, SERVA, Heidelberg, Germany), collagens containing only bovine skin monomers (4 g/l bovine collagen type I, Biochrom, Berlin, Germany) and collagens composed of a mixture of both collagen monomer types (rat tail and bovine skin) in a mass fraction of 1:2 were used for all collagen related experiments (Fischer et al., 2017 (link), 2019 (link), 2020 (link); Kunschmann et al., 2019 (link)). For the polymerization of the monomer solution, a 1 M phosphate buffered solution containing disodium hydrogen phosphate (Sigma Aldrich, Cat. No. 71636), sodium dihydrogen phosphate (Sigma Aldrich, Cat. No. 71507) and ultrapure water were mixed keeping stable conditions (pH value 7.4, ionic strength 0.7, final phosphate concentration 200 mM). The components were kept at 0°C for mixing and finally added to 6-well plates for invasion assays, Petri dishes for elasticity measurements, 96-well plates for investigating the polymerization dynamics, or ibidi 24-well μ-plates for studying the pore size characteristics. Due to differences in the polymerization times (Supplementary Figure S5) R and RB collagen matrices were incubated for 2 h whereas B collagens were incubated for 5 h under normal conditions.

All the reagents and chemicals used in this study were of analytical grade. Standard reference amoxicillin (99% purity) Sigma-Aldrich Chemical Company (St. Louis, USA) was kindly provided by the Ethiopian Food and Drug Administration (EFDA). The chemical and structural formula of amoxicillin is illustrated in Figure 1. Both methanol and acetonitrile (>99%) were HPLC grade solvents, obtained from Merck (Germany). Disodium hydrogen phosphate (>99%), orthophosphoric acid (>85%), and sodium hydroxide (>85%) were purchased from Sigma-Aldrich (USA). The blank chicken sample, without any antibiotics including amoxicillin, is referred to as tissue performance negative concentration CHARM II kit, lot TPNC-053, was a kind donation of the Ethiopian Public Health Institute (EPHI) and Water Still, 4 LPH, double distilled, 240 VAC, 50/60 Hz from Stuart Aquatron (USA) used to purify the water throughout the experiment.