2.4. Characterization of Nanostructures and Electrospun Fibers

The pure CTS and CTS-Ag NPs samples were optically and structurally characterized by Fourier Transform Infrared (FTIR) spectroscopy, X-Ray diffraction (XRD), ultraviolet-visible (UV-Vis) spectroscopy and dynamic light scattering (DLS), respectively; while the electrospun PVA-CTS and PVA-CTS-Ag NPs fibers were characterized by FTIR, morphologically by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and their mechanical properties were analyzed by nanoindentation. Additionally, the antibacterial activity of the electrospun fibers was assessed by the Kirby Bauer method (disc diffusion method).

FTIR measurements were performed to determine the interaction between the polymeric matrix and NPs. The pure CTS (solid) analysis was carried out through pellet method with potassium bromide (KBr) using a 1:100 ratio. On the other side, for analysis of the CTS-Ag NPs compound, films were obtained from colloidal solutions and analyzed without any additional preparation using the ATR mode. The electrospun fibers were analyzed directly, without previous preparation, using an air blank. For analysis, a Perkin Elmer spectrophotometer, model Spectrum One, was used (Waltham, MA, USA). Measurements were carried out in a frequency range of 4000–400 cm⁻¹ with a resolution of 2 cm⁻¹. The structural analysis of the samples was carried out using a diffractometer Bruker D8 Advance (Billerica, MA, USA), which operates with Bragg-Brentano geometry (θ–2θ), voltage 40 kV, current 40 μA and a Kα radiation of Cu (λ = 1.5406 Å) in a 2θ range from 10° to 90°. UV-Vis analysis was performed to confirm the formation of the nanoparticles into the CTS matrix. For this purpose, an Agilent Technologies spectrophotometer was used, series Cary 5000 (Santa Clara, CA, USA), in absorbance mode for CTS-Ag NPs compound (keeping constant volume). The analysis was carried out in a wavelength range of 200–800 nm.

The mean particle size (hydrodynamic radius) and polydispersity index (PDI) of CTS-Ag NPs solution colloids were measured using a Delsa Nano system (Beckman Coulter, UK). Measurement parameters were as follows: a laser wavelength of 658 nm and a medium refractive index of 1.3328. Before DLS measurement, the colloid was dissolved in 3 mL of deionized water and passed through a 0.22 μm polyethersulfone (PES) membrane. The sample was loaded into disposable microcuvette, and five measurements were performed, for which the mean result was recorded.

The morphology of the nanocomposite fibers was observed using a Carl Zeiss Field Emission Gun Scanning Electron Microscopy (FEG-SEM), model (LEO) 1530 VP (Jena, Germany). The samples were fitted on a conductive carbon ribbon and coated with Au/Pd using an Emitech SC7640 cathodic coating device, for 90 s. The microstructure and the dispersion of the inorganic NPs in the synthetized samples were evaluated through TEM. The samples were collected during the electrospinning process by placing a grid close to a rotating collector. The samples were analyzed using a JEOL-2000 FX-II microscope (Tokyo, Japan), operating at an accelerating voltage of 200 kV. The hardness and elastic modulus of electrospun fibers were evaluated through nanoindentation. NHT3 nanoindentation equipment, brand Anton Paar (Graz, Austria), Berkovich-type pyramidal geometry diamond indenter was used, operating at a load of 5 mN. The sample mats were discs of 3 cm of diameter.

The antibacterial assessment was carried out through the disc diffusion technique (Kirby Bauer). The bacterial strains E coli ATCC (American Type Culture Collection) 25922 and S. aureus ATCC 25923, common bacteria in wounds, were used. Separately, colonies of E. coli (Gram negative) and S. aureus (Gram positive) were cultivated on a Mueller-Hinton agar medium. Bacteria were swabbed uniformly onto the petri dish using sterile cotton swabs. Subsequently, the 2 cm diameter nanofiber mat discs were placed on a Mueller Hinton agar medium containing bacterial culture. Finally, the plates were incubated at 37 °C for 24 h. For each sample, the inhibition halo was measured in millimeters (mm) using a ruler and compared with the control (a culture not exposed to the fibers) for each strain. Three replicates were carried out under the same conditions for each sample.