Chitosan powder

Firstly, for the preparation of the CH solution, 50 mL of 0.12 M hydrochloric acid (HCl; Fisher Scientific, Nazareth, PA, USA) was prepared in a beaker by mixing 44 mL of Milli-Q water with 6 mL of 1 M HCl [10 (link)]. The beaker was then placed under a motor stirrer set to stir slowly. Gradually, 1.665 g of chitosan powder (Sigma-Aldrich, St. Louis, MO, USA) was added into the 50 mL of 0.12 M HCl while stirring at 700–800 rpm for 2–3 h. Once the CH powder was fully dissolved, the mixture was transferred into an appropriate bottle. Finally, the solution was autoclaved at 121 °C for 20 min.
To obtain 1 mL of the CH hydrogel, 0.6 mL of the CH solution was mixed with 0.2 mL of gelling agent containing 0.5 M β-glycerophosphate disodium salt hydrate (BGP; Sigma-Aldrich, St. Louis, MO, USA) and 0.075 M sodium hydrogen-carbonate (SHC; EMSURE®, Darmstadt, Germany) as reported by Assad et al. [10 (link)]. Then, this solution was mixed with 0.2 phosphate-buffered solution (PBS; Gibco™, Thermo Fisher Scientific, Waltham, MA, USA). For the mixing procedures, 3 mL syringes containing each of the three solutions connected by a Luer-lock connector were used, as shown in Supplementary File 1. For the preparation of the CH-modified hydrogel supplemented with Azithromycin, AZI powder (TCI AMERICA, Portland, OR, USA) was weighed based on the desired concentrations (vol/wt ratio; 3 %, 5 %, 10 %, 15 %, and 25 %), added into the 0.2 mL of PBS, vortexed, and mixed with the other CH/gelling agent solution as described above (Supplementary File 2).

MNPs were synthesized using an oxidative hydrolysis approach as described previously.15 (link) In brief, 1.364 g KNO₃ (99%; Acros Organics, Belgium) and 0.486 g NaOH (≥99%; Sigma-Aldrich, St Louis, MO, USA) were mixed together, and then the mixture was dissolved in 135 mL of deionized water bubbled with N₂ flow. Then, a solution that had been previously prepared under 3 hours of N₂ flow with 15 mL of 0.01 M H₂SO₄ (96%; Panreac, Spain) solution containing 0.308 g FeSO₄·7H₂O (≥99%; Sigma-Aldrich) was added drop by drop under constant stirring. When the precipitation was finished, N₂ flow for an additional 10 minutes was allowed to pass, and the temperature was heated to 90°C for 24 hours. Finally, the product was cooled to room temperature by means of an ice bath. The synthetic black product was isolated by magnetic decantation, and then rinsed several times using deionized water to remove all residual impurities. The crystallization of MNPs was determined using X-ray diffraction (XRD) by Cu Kα X-radiation (Rigaku Miniflex, Japan), and the morphology, distribution, and average size of MNPs were examined by transmission electron microscopy (TEM; H-600; Hitachi, Japan). The chemical composition of MNPs was recorded using Fourier-transform infrared (FTIR) spectrophotometry (8400s; Shimadzu, Japan), and spectra were measured in the mid-IR range (128 scans) from 4,000 to 400 cm⁻¹ (at a resolution of 4.0 cm⁻¹). Magnetization was determined with a vibrating sample magnetometer (665; Lake Shore Cryotronics, USA).
To prepare magnetic nanocomposites (5%, 10%, 20% w/w MNPs), 5 mL of 0.1 M acetic acid aqueous solution and 100 mg chitosan powder (molecular weight >310 kD, deacetylation ≈90%, Sigma-Aldrich) were mixed well to prepare a chitosan solution. The glycerophosphate salt (Fluka, USA) solution, prepared by adding 1 mL phosphate-buffered saline (PBS) to 500 mg glycerophosphate salt, was added to the chitosan solution drop-wise with an ice bath. The final solution was homogeneous and clear, with a pH of 7.04. Then, a chitosan–glycerophosphate mixture was stored at −80°C for 24 hours and lyophilized for another 24 hours. Then, 100 mg chitosan–glycerophosphate lyophilized powder was redissolved in 5 mL of deionized water, and MNPs were added to the chitosan–glycerophosphate solution with vigorous stirring in an ice bath for 2 hours. Later, the MNP–chitosan mixture was vacuumized for 24 hours at 4°C and stored at 4°C. For different experimental purposes, the MNP–chitosan mixture was fabricated into membranes or scaffolds. For magnetic membranes, the MNP–chitosan mixture was later modeled onto a plastic dish and air-dried at room temperature for 24 hours. The dried magnetic membranes were washed using deionized water to remove spare MNPs, and air-dried again at room temperature. For magnetic scaffolds, the MNP–chitosan mixture was injected into a tailor-made mold, which was immediately stored at −80°C for 24 hours and then lyophilized for 24 hours. Then, the magnetic scaffolds were separated from the mold and cut into cylinders (2.0 cm in length, 2.0 mm in diameter). The dried magnetic scaffolds were rinsed with deionized water to remove spare MNPs, and lyophilized again for 24 hours. Ethylene dioxide gas was used to sterilize the magnetic membranes and magnetic scaffolds at 37°C according to standard industrial procedures.