*3.1. Chemico-Physical Characterization*

Preliminarily, to scaffold preparation and cross-linking by heating, the drug stability in the heating process was assessed. For this purpose, the drug was subjected to a heating treatment in the same conditions used for the scaffold cross-linking (1 h at 150 ◦C). The heating treatment did not cause the drug degradation; in fact, after the process, the N content was 99.21% *w*/*w* (SD = 2.04) compared to the active ingredient in standard storage conditions. Figure 1 reports the UV spectra of N, and N subjected to the heating treatment at the maximum of the chromatographic peak (N retention time).

**Figure 1.** UV spectra of N (red line) and N subjected to heat treatment (1 h at 150 ◦C) (black line) obtained from the HPLC analysis at N peak maximum.

The complete overlapping of the spectra supported the stability of the drug in the heating treatment. In a previous work [8], norfloxacin was loaded in montmorillonite, a phyllosilicate widely used in pharmaceutical field, to obtain a nanocomposite. This was prepared by means of the adsorption mechanism, as one single process, and the clay–drug adsorption isotherm was calculated. The solid-state analysis (XRPD, FTIR, thermal analysis—differential scanning calorimetry/ thermogravimetric analysis DSC/TGA, HRTEM) evidenced that protonated norfloxacin molecules interact with the active sites of montmorillonite located at its edges and within its interlayer space, thus forming a drug monolayer onto the clay mineral interlayer surface. Norfloxacin in the nanocomposite was proved in an amorphous state, and its loading (16% *w*/*w* of total nanocomposite weight) is homogeneous and causes an expansion of montmorillonite interlayer spaces. Moreover, the nanocomposite causes a prolonged norfloxacin release over time. Moreover, the nanocomposite was characterized by good biocompatibility in vitro toward fibroblasts, and it was able to increase the antimicrobial potency of the free drug against *P. aeruginosa* and *S. aureus*, Gram-negative and Gram-positive bacteria, respectively, both of which are often concurrent causes of wound chronicization, leading to the possible impairment of the healing

path and, finally, to nonhealing wounds. Montmorillonite norfloxacin nanocomposite was loaded into scaffolds and their performance was compared to those loaded with the free drug scaffolds.

Figure 2 reports SEM microphotographs of CH, CH/CS or CH/HA scaffolds loaded with 1% or 2% norfloxacin, as a free drug (1% N or 2% N), or loaded with VHS-N nanocomposite (1% H or 2% H).

**Figure 2.** SEM microphotographs of chitosan-based (CH), chitosan/chondroitin sulfate-based (CH/CS) and chitosan/hyaluronic acid-based (CH/HA) scaffolds loaded with 1% or 2% norfloxacin, as a free drug (1% N or 2% N), or in VHS-N nanocomposite. In each image, the nanofiber diameters (nm, mean values ± SD; n = 30) and Si content for hybrid scaffolds are reported. Statistics: Mann–Whitney (Wilcoxon) W test *p* < 0.05: CH2H vs. CH/CS2H; CH/CS2H vs. CH/HA2H; CH/HA1H vs. CH/HA2H; CH/HA2H vs. CH/HA2N (scale bar 5 μm).

The N scaffolds, loaded with N as a free drug, were characterized by a regular structure with a smooth surface where no ribbon could be detected, independent of drug concentration. The H scaffolds, loaded with N in nanocomposite, presented nanofiber portions with a regular, smooth surface spaced out in broadened parts, with knots and a scattered structure. These conceivably could be related to the montmorillonite–norfloxacin (VHS-N) nanocomposite. Moreover, the presence of glycosaminoglycans (CS or HA) in the scaffolds caused a certain degree of surface roughness (probably due to chitosan and

glycosaminoglycan interaction [9]) and this was more evident due to the increasing drug concentration in the fibers.

Nanofiber diameters were generally smaller when norfloxacin was loaded as a free drug (around 500 nm), independent of the drug concentration, although the differences were not statistically significant. On the contrary, H scaffolds were characterized by nanofibers with higher diameters (around 500 nm for 1% scaffold and around 1000 nm for 2% scaffolds) compared to those containing 1% of the drug, although this was significant only for the CH/HA scaffold; in this case, HA's high molecular weight was ten folds greater than that of CS and could cause the formation of fibers with greater diameters. On the contrary, H scaffold containing chondroitin sulfate and loaded with 2% of the drug showed similar nanofiber diameters to those loaded with the free drug. The content of Si, an element characteristic of montmorillonite, was consistent with the nanocomposite concentration in each scaffold [23].

The analysis of system viscosity previously performed on the blank systems [9], stated that chondroitin sulfate (negatively charged) conceivably interacted with chitosan (positively charged) and this could be due to the high charge density of sulfate groups greater than those of the carboxylic moieties of hyaluronic acid. However, the presence of particles in suspension, as was the case in nanocomposite, could cause unbalanced particle charge density that generally increases the conductivity, influencing fiber diameter during electrospinning [24]. Moreover, the acid environment of the polymer blends, due to the 45% *v*/*v* acetic acid in the medium, conceivably prevented the interactions between the various moieties and drug precipitation [25,26].

Figure 3 reports the HR-TEM microphotographs and EDX spectra obtained for CH (A–C), CH/CS (G–I) and CH/HA (D–F) H scaffolds, loaded with N in the nanocomposite at 2%.

**Figure 3.** Transmission electron microscope (TEM) microphotographs and EDX spectra for CH, CH/CS and CH/HA scaffolds loaded with 2% norfloxacin in nanocomposite CH (**A**–**C**), CH/CS (**G**–**I**) and CH/HA (**D**–**F**).

The EDX analysis performed in the marked zone (red square) confirms that there was the presence of C, O and N (typical of organic elements) and characteristic elements of montmorillonite (Si, Al, Mg) in the broad, interwoven knots. This was observed for all the scaffolds, independent of their polymeric composition. At a higher magnification (Figure 3C,F,I), it was possible to identify the typical lamellar structure of montmorillonite (red arrows).

Figure 4 reports FTIR spectra evaluated for norfloxacin-loaded scaffolds (CH-N2, CH/CS-N2, CH/HA-N2) and VHS-N loaded scaffolds (CH-H2, CH/CS-H2, CH/HA-H2), both types containing 2% *w*/*w* norfloxacin.

**Figure 4.** Fourier transform infrared spectroscopy (FTIR) spectra evaluated for norfloxacin-loaded scaffolds (CH-N2, CH/CS-N2, CH/HA-N2) and VHS-N loaded scaffolds (CH-H2, CH/CS-H2, CH/HA-H2), both types containing 2% *w*/*w* norfloxacin in norfloxacin–montmorillonite nanocomposite (VHS-N).

Independent of the polysaccharide composition and loading type, either with N (free drug, norfloxacin) or H (VHS-N nanocomposite), the typical polysaccharide signals (hydrogen bonds of –OH and –NH2 groups) (pullulan: 3331 cm−<sup>1</sup> and chitosan: 3355 cm−1) hid the drug and nanocomposite-related peaks [27]. In fact, the VHS spectrum should present a band around 1017 cm−<sup>1</sup> due to the vibrational band of the silicates.

Figure 5 reports the XRPD patters of the scaffolds loaded with VHS-N nanocomposite containing norfloxacin at 2% compared to VHS-N, the nanocomposite and the unloaded CH scaffold.

**Figure 5.** X-ray powder diffraction (XRPD) diffractograms of the scaffolds loaded with VHS-N at 2% norfloxacin (CH-H2, CH/CS-H2, CH/HA-H2) compared to unloaded CH scaffolds (CH) and VHS-N.

All the unloaded scaffolds were characterized by amorphous behavior (CH pattern is reported in Figure 5 as example) and no paracrystallinity could be detected. This was probably related to the electrospinning process. For all the scaffolds, the diffractograms were characterized by a hump between 20◦ and 29◦ 2θ, which was probably due to the presence of the polysaccharides. The reflection peak centered at 6◦ 2θ, which was probably due to the nanocomposite (VHS-NF) since it coincided with the d001 of montmorillonite once NF located in the interlayer space (5.94◦ 2θ). Similar XRD results were obtained by Rabbani et al. (2016) [28]. Moreover, there was an absence of other intense peaks that could be attributable to the nanocomposite (VHS-N), probably due to the nanocomposite concentration in the scaffolds, which was too low.

## *3.2. Mechanical Properties*

Figure 6 reports the mechanical properties (force at break mN, a–b; elongation %, c–d; Young's modulus mN·cm2, e–f) of scaffolds loaded with 1% or 2% of norfloxacin as a free drug (N) or as a nanocomposite (H), in dry (a, c, e) or wet (b, d, f) conditions.

In a dry state, the increase in N concentration in the scaffolds caused an increase in the force at break, except for the scaffold containing hyaluronic acid (Figure 3a). This was less evident when norfloxacin was loaded as a nanocomposite: it is conceivable that the effect of montmorillonite, which altered the entanglement of polymer chains in the scaffolds, causing lower resistance to break, prevailed over the effect attributable to the free drug, which seems to reinforce the structure. In this condition, the N scaffolds were less deformable than H scaffolds and the N concentration at 1% in H scaffolds was responsible for a higher deformability (Figure 3c). Moreover, the free drug seems to increase

scaffold elasticity, especially for scaffolds containing chondroitin sulfate (Figure 3e). The hydration of the scaffolds, which simulates the application/implant in the lesion, dramatically changed the scaffold mechanical properties. N scaffolds, loaded with N as a free drug, were characterized by slightly higher resistance to break with respect to H scaffolds, confirming the behavior of the dry state (Figure 3b), while the scaffolds were simultaneously characterized by a higher degree of deformability (Figure 3d), which could be advantageous for wound bed application, and low elasticity (Figure 3f). The hydration caused a remarkable decrease in resistance to break, an increase in deformability and a loss of elasticity.

**Figure 6.** Mechanical properties (force at break mN, a-b; elongation %, c-d; Young's modulus mN.cm2, e-f) for dry (**a**,**c**,**e**) and wet (**b**,**d**,**f**) scaffolds loaded with 1% (**a**) or 2% (**b**) of norfloxacin as a free drug (N) or as nanocomposite (H) (mean values ± SD; *n* = 3). Statistics: \* = Mann–Whitney (Wilcoxon) W test *p* < 0.05.

The presence of montmorillonite in the hybrid scaffolds seems to weaken the scaffold structure, and this was probably due to the presence of particles embedded into the polymeric matrix that could disrupt the polymer chain entanglements, causing a significant decrease in the scaffold elasticity, and mechanical resistance, and a directly related increase in the deformability: this was more evident when 2% of drug in the nanocomposite was loaded in the scaffolds compared to scaffolds loaded with the free drug.

The mechanical properties are key features for the success of scaffold implants and their integration with the surrounding tissue. In fact, the native skin is characterized by tensile strength values approximately between 5.0 and 30.0 MPa (5000–30,000 mN/mm2), the Young's modulus in the range of 4.6–20.0 MPa (46–200 mN/cm2) and the elongation at break of about 35.0–115.0% [29]. Clearly, the ranges of the reference values are wide since the mechanical properties of the skin are strictly related to age and body lines (static lines, as described by Langer, Kraissl's lines or Borge's lines) [30]. In particular, force at break (mechanical strength) is related to the scaffold's capability to maintain its integrity during implantation, which should occur in the dry state, while the elongation and the Young's modulus are mainly related to the scaffold performance upon implantation. The scaffolds developed in the present work were characterized by force at break in the dry state close to the skin,

especially for CH and CH/CS scaffolds, when loaded with norfloxacin at 2% as a free drug. Moreover, upon hydration, all the scaffolds were characterized by elongation superimposable to that of native skin. Furthermore, as for the Young's modulus, the scaffolds were characterized by the stiffness/elasticity closest to that of the skin, both in dry and hydrated states. Moreover, there is evidence in the literature that correlates the fibroblast adhesion and proliferation to substrate stiffness [31]; stiff matrices with a 2 MPa Young's modulus enhanced fibroblast proliferation much more than an elastic substrate (0,042 MPa). In fact, in the literature, there is evidence that the fibroblasts of granulation tissue are proliferative and motile, while those of the dermis are in a quiescent and stationary state [32]. Moreover, stiff substrates were demonstrated to sustain cell spreading and to facilitate guiding the pro-angiogenic signaling of fibroblasts [33].
