*3.3. Norfloxacin Release Properties*

Figure 7 reports the release profiles of norfloxacin in saline solution. As for H scaffolds (N loaded as nanocomposite), independent of the drug loading, the profiles reached plateau values at 20% of the drug released after 3 h.

**Figure 7.** Release profiles (%) of norfloxacin from the N or H scaffolds loaded with 1% (**a**) or 2% (**b**) as a free drug (N) or as nanocomposite (H), in saline solution (mean values ± SD; *n* = 3).

As for N scaffolds (N loaded as a free drug), independent of the drug loading, the release profiles reached plateau values after 5 h; CH/CS scaffolds were characterized by their higher profile (50% and 57% for 1% and 2% N loading, respectively) followed by CH/HA scaffolds (about 40% and 50% for 1% and 2% N loading, respectively) and finally by CH scaffolds (33% and 50% for 1% and 2% N loading, respectively). When norfloxacin was loaded in the scaffolds as a nanocomposite (H scaffolds), the release was lower than when the scaffolds contained the free drug, and this seems to be independent of scaffold polymer composition. On the contrary, when norfloxacin was loaded as a free drug (N scaffolds), the presence of glycosaminoglycans markedly influenced norfloxacin release. This could be due to an interaction between anionic glycosaminoglycans and cationic chitosan forming a polyelectrolyte complex, which could make the fibrous structure less entangled and, therefore, more available to interact with the dissolution medium and to allow drug diffusion through the polymer matrix and, consequently, its release. In fact, scaffolds containing chondroitin sulfate, characterized by a charge density greater than hyaluronic acid, were characterized by a higher release profile. Chondroitin sulfate is characterized by the sulfate groups having an acid behavior greater than the carboxylic groups of hyaluronic acid. Consequently, the interaction between chondroitin sulfate and chitosan could cause a coiled structure less prone to polymer chain entanglements [34].

In any case, in the scaffolds loaded with higher concentrations of the drug, this difference was less evident with respect to those with lower drug loadings.

Figure 8 reports the norfloxacin release profiles (a and b) and glucosamine release profiles (c and d) of scaffolds subjected to lysozyme degradation.

**Figure 8.** Norfloxacin released (%) in lysozyme from the scaffolds loaded with 1% (**a**) or 2% (**b**) of norfloxacin as a free drug (N) or as nanocomposite (H) and glucosamine released (%) from the scaffolds loaded with 1% (**c**) or 2% (**d**) of norfloxacin as a free drug (N) or as nanocomposite (H) subjected to lysozyme activity (mean values ± SD; n = 3)

Independent of norfloxacin concentration in the N scaffolds (free drug loading), the activity of lysozyme markedly increased the drug release: CH scaffolds containing chitosan, without glycosaminoglycans, showed higher release profiles, reaching, in almost 24 h, 100% of the drug being released; scaffolds based on CH/HA showed 80% of the drug being released in 48 h, while CH/CS scaffolds were characterized by a lower release close to 40–50% of the drug being released in 72 h, for 1% or 2% norfloxacin loading, and these profiles were similar to those obtained in saline solution. These behaviors could be explained considering that the activity of lysozyme on the scaffold matrices:

CH scaffolds completely lost their nanofibrous structure in contact with lysozyme (Figure 9). On the contrary, after 10 days of lysozyme activity, the CH/CS scaffold and, mainly, the CH/HA scaffold showed a residual of nanofibrous structure, submerged in a non-structured material. It is conceivable that the interaction of chitosan amino groups (positively charged) with either sulfate groups of chondroitin sulfate or the carboxylic ones of hyaluronic acid (both negatively charged) conferred a higher resistance against enzyme degradation, probably hindering interaction with the substrate. Moreover, chitosan/glycosaminoglycan interactions could partially prevent the loss of the system morphology, decreasing drug release.

**Figure 9.** SEM microphotographs of the scaffolds loaded with 1% or 2% of norfloxacin as a free drug (N) or as nanocomposite (H) subjected to lysozyme activity for 10 days (scale bar: 5 μm).

In hybrid H scaffolds, loaded with norfloxacin in the montmorillonite nanocomposite, the profiles of norfloxacin released in the presence of the lysozyme were higher than those obtained in saline solution, although no difference could be evidenced, considering both the scaffold composition and the percentage of drug loaded, and all the scaffolds were characterized by release profiles reaching drug loading of 50% in 72 h.

However, in all cases, the glucosamine release profiles suggest that the enzymatic degradation of chitosan occurred, independent of system composition and percentage of drug loaded. CH scaffolds were characterized by their higher profiles, followed by H/HA scaffolds and CH/CS ones. Generally, the presence of norfloxacin–montmorillonite nanocomposite seems to decrease the lysozyme activity and the profiles of glucosamine (degradation product) were consistent with the norfloxacin release ones. Furthermore, the drug loading seems to have a negative impact on enzymatic activity and the glucosamine release profiles were higher in 1% loaded systems than in 2% ones. It is reported in the literature that lysozyme interacts with quinolones and this supports that there is a competition between norfloxacin and chitosan, as enzyme substrates, decreasing the enzymatic activity towards chitosan degradation when norfloxacin is at higher concentrations [35]. Moreover, the presence of montmorillonite in the scaffolds could impair lysozyme activity, probably due to a certain degree of interaction between montmorillonite and chitosan, which could prevent chitosan interaction with the enzyme. Furthermore, the interaction between chitosan and either chondroitin sulfate or hyaluronic acid could render chitosan, as lysozyme substrate, less prone to interaction with lysozyme, resulting in less efficient degradation activity towards chitosan [36].

Similar norfloxacin release profiles were observed by Dua et al. [37] for semisolid systems loaded with 1% norfloxacin. Dependent of the type of system, drug release ranged from 70% to 41% in 7 h. The highest drug release was observed for Carbopol-based gel (about 70%) followed by polyethylene glycol-based formulation (66%), HPMC-based gel (45%) and, finally, the slowest release was evidenced in the case of an ointment. Analogous behavior was observed by Denkba¸s et al. [38] and Mahmoud and Salama [21] for chitosan and chitosan collagen sponge-like dressings loaded with norfloxacin. In those cases, the norfloxacin release was mainly related to system swelling that controlled the drug diffusion for an extended time of up to 4 days.

However comparing the features of the nanofibrous scaffolds presented in this work with those of the systems in the literature, the capability of the scaffolds based on chitosan or glycosaminoglycan (either chondroitin sulfate or hyaluronic acid) associated with chitosan (CH, CH/CS and CH/HA) to possess minimal swelling (as shown by SEM images after 6 days of hydration in aqueous environment) and controlled norfloxacin release, tuned up by both the hydration and the activity of lysozyme (secreted during the inflammatory phase of wound healing), confer the ideal properties of these systems. Indeed, as soon as the systems can be implanted, norfloxacin release should occur due to the hydration of exudate from the lesions; subsequently, the inflammatory phase, preceding the proliferative one, should lead to a further release of the drug to support the whole healing process.

Figure 9 reports SEM microphotographs of all the scaffolds subjected to 10 days of enzymatic degradation by lysozyme. These images are in agreement with the glucosamine release profiles (Figure 8c,d). In fact, the higher degree of scaffold degradation (loss of nanofibrous structure) was associated with a higher glucosamine release profile. Independent of drug concentrations in CH scaffolds containing chitosan, without glycosaminoglycans, and loaded with norfloxacin as a free drug, the nanofibrous structure was no longer visible, while CH scaffolds loaded with norfloxacin in nanocomposite were characterized by a nanofibrous structure, partially covered by spherical particles, reported in the literature as lysozyme molecules attached to the biopolymer matrix [36]. The presence of glycosaminoglycans in the scaffolds determined a higher resistance against enzymatic activity. In some cases, as for the CH/CS-N2 scaffold, nanofibers were partially broken, swollen, and partially fused. Long-lasting scaffold degradation could be advantageous, especially in deep/cavity wounds, since this should allow the gradual replacement of the scaffold matrix with native tissue, due to the production of the extracellular matrix by fibroblasts.
