Ciprofloxacin hydrochloride

In total, 5 mg/mL ciprofloxacin (CP) stock was prepared by dissolving ciprofloxacin hydrochloride (MP Biomedicals, Santa Ana, CA, USA) in de-ionized water. The stock was filtered with a 0.22 μm PES membrane sterile filter (Millex^®GP filter unit) and stored at 2 °C.

Polymyxin B sulfate salt (Sigma Aldrich, Singapore) and Ciprofloxacin Hydrochloride (MP Biomedicals, LLC, France) were prepared in sterile deionized water in the range of a final concentration of 4–1024 µg/mL. Tannic acid powder and tannic-acid-stabilized AgNPs were provided by Prime Nanotechnology Co., Ltd. (Bangkok, Thailand), our collaborator, with a stock concentration of 10,000 mg/L. AgNPs solution for use was the same preparation as the method above.

Collagen-based sponges were prepared starting from a collagen gel of 1.91% and acidic pH. This gel was obtained from bovine derma using a current technology at INCDTP—Division Leather and Footwear Research Institute, Collagen Department (Bucharest, Romania) [40 (link),41 ]. Ciprofloxacin Hydrochloride (MP Biomedicals, LCC, Solon, OH, USA) was added in concentration of 0.5; 0.75 and 1 g/g to a collagen solution (1%, pH 7.4) prepared from the initial gel with 1 M sodium hydroxide. The obtained solutions were further cross-linked with 0.5% glutaraldehyde reported to dry collagen. The sponge materials were obtained by lyophilization process consisting of three steps: (i) freezing at −55 °C for 1 min; (ii) drying at −55 °C for 15 h—time in which approximately 90% of water is sublimated; and (iii) final-drying at −40 °C for 10 min. The sublimation process took place under vacuum and was performed using Delta 2-24 LSC (Martin Christ, Osterode am Harz, Germany) equipment. All obtained collagen sponges of 2 cm diameter and 0.5 cm thickness were packed in polyethylene bags and sterilized at 254 nm using Vilber–Lourmat equipment (Suebia, Germany).

A total of 5 mg mL⁻¹ ciprofloxacin stock was prepared by dissolving ciprofloxacin hydrochloride (MP Biomedicals, Santa Ana, CA, USA) in deionized water. Tobramycin stock was prepared following the same procedure by dissolving Tobramycin sulfate salt (Sigma Aldrich, St. Louis, MO, USA). Erythromycin was obtained from MP Biomedicals, and the stock was prepared by mixing 1.5 mg mL⁻¹ Erythromycin into deionized water. Stocks were filtrated using a 0.22 μm pore PES membrane sterile filter (Millex^®GP filter unit) and stored at 2 °C.