Sodium hydroxide (naoh)

The following chemicals were used in this research: aceton (Lach-Ner, Neratovice, Czech Republic); acetonitrile (Applichem, Darmstadt, Germany); ammonium formate (GramMol, Zagreb, Croatia); argon, 99.9995% (Messer, Bad Soden am Taunus, Germany), certified element reference material (CPAChem, Bogomilovo, Bulgaria); distilled water and nitric acid (Gram-Mol, Zagreb, Croatia); helium, 6.0 (Messer, Bad Soden am Taunus, Germany); methanol (GramMol, Zagreb, Croatia); mixture of magnesium sulphate, sodium chloride, and citrate salts (Citrate-Kit-01, BEKOlut, Bruchmühlbach-Miesau, Germany); mixture of magnesium sulphate salt, primary secondary amine, and GCB (PSA-Kit-06, BEKOlut, Bruchmühlbach-Miesau, Germany); sodium hydroxide (T.T.T., Sveta Nedjelja, Croatia); petrol ether (Applichem, Chicago, IL, USA); sulfuric acid (Chemistry, Zagreb, Croatia); and hydrogen peroxide (Gram-Mol, Zagreb, Croatia).

The bare iron oxide nanoparticles employed for this study were synthesised by co-precipitation of Fe²⁺ and Fe³⁺ in alkaline aqueous solutions according to our previously optimised procedure^{66 (link)}. 21.2 g of FeCl₃ × 6 H₂O and 8.3 g of FeCl₂ × 4 H₂O were dissolved in 200 mL of deionised, degassed water resulting in a Fe(III): Fe(II) ratio of 1.9: 1. This iron chloride solution was added to 1 L of 1 M NaOH prepared with deionised, degassed water stirring at 250 rpm in a reaction vessel. The reaction mixture was kept under a nitrogen atmosphere at 25 °C and stirred for an additional 30 minutes before the resulting nanoparticles were washed with deionised water until the conductivity of the MNP solution was below 200 µS cm⁻¹. In order to separate the particles, the mixture was placed on a NdFeB permanent magnet. Suspensions were lyophilised with an ALPHA 1-2LD plus from Martin Christ Gefriertrocknungsanlagen GmbH, Germany in order to obtain solid particles. FeCl₃ × 6 H₂O and sodium hydroxide were purchased from AppliChem GmbH, Germany in the highest purity available. FeCl₂ × 4 H₂O extra pure was obtained from Merck KGaA, Germany.

The polymer polyvinyl alcohol EMPROVE^® ESSENTIAL PVA 18-88 (Merck KGaA, Darmstadt, Germany) was used to prepare the films. Anhydrous glycerol (AppliChem GmbH, Darmstadt, Germany) served as plasticizer. Demineralized water was used as solvent.
Potassium dihydrogen phosphate (neoFroxx GmbH, Einhausen, Germany), sodium hydroxide (AppliChem GmbH, Darmstadt, Germany) and deionized water were used to prepare the phosphate buffer pH 7.4 USP. Both chemicals were used in analytical grade.
Agar for microbiology (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and mucin 75–95% for biochemistry (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) as well as demineralized water were used to prepare biomimetic gels.
For testing mucoadhesion on porcine intestine, porcine small intestine was obtained from a local slaughterhouse (female, 12–15 weeks of age, 35–50 kg, n = 3). The small intestine was examined after collection in the unprocessed and processed state. The preparation of the tissue is explained in more detail in Section 2.2.3 Preparation of animal tissue. Furthermore, prepared tissue sections were additionally deep-frozen at −20 °C, thawed for the measurements and subsequently examined.

High purity (>99%) FLU, OA, OFL, ENR, and CIP were purchased from Sigma-Aldrich. NaOH, H₂SO₄ (96%), glacial acetic acid, and UHPLC-grade methanol and acetonitrile were provided by AppliChem-Panreac. Ammonium acetate was provided by Scharlau.
Respective (F)Q 250 µM stock solutions were prepared in basic media; all solutions were stable at dark conditions (no hydrolysis was observed).

By inoculation of the preculture in 1500 mL of the same medium in a bioreactor (Biostat A plus, Sartorius AG, Göttingen, DE) cultivation was started at 37 °C and constant oxygen supply of 2 L/min filtered air. The pH was kept constant by automatic addition of 0.5 M NaOH (AppliChem, Darmstadt, DE) or 0.5 M HCl (Sigma Aldrich, Darmstadt, DE) with a control unit (Biostat A plus DCU, Sartorius AG, Göttingen, DE). The pH levels were varied between 7.00 and 9.00 in steps of pH 0.50. After 48 h the fermentation was stopped, microorganisms harvested and washed as described above. All spores were subsequently heat activated at 80 °C for 20 min. The viable amount of spores was examined by serial decimal dilutions in Ringer’s solution (Merck, Darmstadt, DE). 100 µL of the appropriate dilutions were plated on plate count agar (for 1000 mL:5.0 g peptone from caseine (Gerbu, Heidelberg, DE), 2.5 g yeast extract (Sigma Aldrich, Darmstadt, DE), 1.0 g glucose (Merck, Darmstadt, DE), 15 g agar–agar (Fisher Scientific, Schwerte, DE)) and incubated at 30 ° for 24 h.