2.2.5. Norfloxacin Release Measurements

All the release measurements were performed in sink conditions to study drug liberation from the systems independent of the concentration of the drug released during the test [20]. Two different approaches were considered: in the first one, the drug release was studied using saline solution to simulate the lesion exudates, while in the second one, the effect of lysozyme on drug release was analyzed. Each scaffold was placed in 3 mL of dissolution medium to simulate the small number of exudates generally present in the wound, and the scaffold was completely dipped in the dissolution medium to simulate the implant of the system in the lesion bed. For this purpose, two different media were considered: saline solution (NaCl 0.9% w/v) or phosphate buffer 0.05 M (pH 6.2) containing 3.3 mg/mL of lysozyme (120.530 IU/mg, Sigma-Aldrich, Milan, Italy). As for saline solution, at prefixed times, 500 μL of dissolution medium was collected and replaced with fresh medium to keep the volume constant. The samples were analyzed by means of the DAD–HPLC method (Section 2.2.5.1) [21]. When lysozyme was present, the dissolution medium was totally collected and completely substituted with fresh medium every 24 h to avoid a loss of the enzyme activity over time. Each sample was divided in two aliquots. One aliquot was assayed to quantify the norfloxacin released from each scaffold (Section 2.2.5.1) and, for this purpose, each sample was pre-processed by diluting 1:1 with 1 N perchloric acid and by centrifugation (5000 rpm for 15 min), to precipitate the lysozyme in the solution. The second aliquot was assayed to quantify the glucosamine release, as product of lysozyme activity (Section 2.2.5.2). Moreover, the morphology of scaffolds subjected to lysozyme degradation (after 10 days) was analyzed using SEM as previously described.

#### 2.2.5.1. Norfloxacin Assay

Norfloxacin released from each scaffold was determined by DAD–HPLC (Series 200 system, PerkinElmer, Milan, Italy). A Zorbax Eclipse XDB-C8 column (4.6 mm × 150 mm, silica particle size 5 μm, Agilent, Milan, Italy) was used as the stationary phase. The mobile phase was based on acetonitrile/methanol/citric acid 0.4 M, 7:15:78 (% *v*/*v*) at a flow rate of 1.0 mL/min, using 275 nm wavelength detection [21,22]. The injection volume was 10 μL. Calibration curves were obtained using norfloxacin standard solution in the mobile phase, in saline solution or processed as the samples subjected to lysozyme degradation. In every case, the method was linear from 0.08 to 200 μg/mL with an R2 value that was always higher than 0.995.

#### 2.2.5.2. Glucosamine Assay

Glucosamine released due to the lysozyme degradation of the scaffolds was quantified by means of ninhydrin assay [23].

All samples were diluted 1:1 ratio (*v*/*v*) with 400 μL of ninhydrin reagent (ninhydrin 2% *w*/*v*, hydrindantin 6.8 mg/L in 3:1 *v*/*v* dimethylsulfoxide: lithium acetate buffer 4 M, pH 5.2; Sigma-Aldrich, Milan, Italy) under a nitrogen blanket. Each sample was stirred at 100 ◦C for 8 min, and vortexed until cooling, then the samples were diluted 1:10 (*v*/*v*) with a 1:1 ethanol:water mixture and quantified by a colorimetric test at L = 570 nm using an ELISA Plate Reader (iMARK Microplate Absorbance Reader, BioRad, Milan, Italy). The calibration curve (glucosamine in phosphate buffer 0.05 M at pH 6.2) was linear in the range from 0.0125 to 0.1 μg/mL with a *R*<sup>2</sup> > 0.995.
