2.6. Characterization methods

FTIR spectra of native and treated bacterial cellulose (BC), native and treated chitosan, and BC/chitosan membranes were recorded on a Bruker Vertex 70 FT-IR spectrophotometer with attenuated total reflectance (ATR) accessory with 32 scans and 4 cm⁻¹ resolutions in the mid-IR region. Raman spectra were recorded on a DXR Raman microscope, from Thermo Fisher Scientific. The excitation laser wavelength was 532 nm using a laser power of 14 mW. The Raman spectra were collected in the range of 100–3200 cm⁻¹ with a relevant display in the range 800–1750 cm⁻¹.

The size and morphology of the nanoparticles together with membrane morphological characteristics were investigated by Scanning Electron Microscopy (SEM) using a Quanta Inspect F50, with a field emission gun (FEG) having 1.2 nm resolution and an Energy Dispersive X-ray Spectrometer (EDXS) having 133 eV resolution at MnKα. Morphology, geometrical evaluation (size and shape) of nanostructural characteristics of the core-shell constructs were investigated by high-resolution transmission electron microscopy (HR-TEM) and selected area electron diffraction (SAED) using a TECNAI F30 G2 S-TWIN microscope operated at 300 kV with Energy Dispersive X-ray Analysis (EDAX) facility.

X-ray diffraction (XRD) spectra were registered on a Panalytical X’PERT MPD X-ray Diffractometer, in the range, 2θ = 10–80. An X-ray beam characteristic of Cu Kα radiation was used (λ = 1.5418 Å).

The swelling behavior of the biocomposites was evaluated in saline solution at 37 °C. The weight changes of the samples were recorded at regular time intervals during swelling. The swelling degree of the hydrogels was determined according to the following equation:

where W_t and W₀ denote the weight of the wet hydrogel at a predetermined time and the weight of the dry sample, respectively. The equilibrium swelling degrees (ESD) were measured until the weight of the swollen hydrogels was constant. At least three swelling measurements were performed for each sample and the mean values were reported. Furthermore, gel fraction experiments were performed by leaching tests.

The gel fraction (GF) of the samples was determined according to the following equation (Wong et al., 2015):

where W_f denotes the weight of the dried sample after water extraction.

Rheological tests have been performed with a rotational rheometer Kinexus Pro, Malvern Instruments, and a temperature control unit. In oscillating mode, a parallel plate and a geometric measuring system have been used, and the gap has been set according to the force value. The tests were performed on samples of 20 mm diameter with parallel plate geometry in a frequency range of 1–20 Hz. Shear viscosity measurements were performed at a fixed shear rate of 0.1 s⁻¹.

Mechanical tensile tests were performed on an Instron 2519-107 Universal testing machine equipped with a 5 kN load cell. The measurements were done at room temperature on dumbbell specimens with a crosshead rate of 10 mm/min. Data were collected for at least three specimens for each sample with 0.5% accuracy of force measurement and position accuracy of 0.001 mm. Specimens were prepared according to ISO 527-2012 (overall length 75 mm, gauge length 25 mm, width 5 mm, and thickness 2 mm) (Hervy et al., 2017).

The size distribution and zeta potential were investigated by Dynamic Light Scattering (DLS) using a Zetasizer Malvern DLS device. Data were collected for PNIPAM nanoparticles obtained from 0.5 and 5 wt. % polymer concentration.

The silver sulfadiazine encapsulation within both nanoparticles and BC/chitosan membranes followed two pathways: in the case of nanoparticles, the drug was dissolved into Phosphate buffer saline (potassium phosphate/sodium hydroxide) (PBS) solution (pH 7.45). The drug solution concentration was: 0.1 mg/mL for nanoparticles from 5% polymer concentration and 0.05 mg/mL for nanoparticles from 0.5% polymer concentration. The dried nanoparticles were added to the drug solution (0.1 g of nanoparticles/mL of drug solution). The resulted nanoparticle suspension was stirred for 24 h. Finally, the nanoparticles were separated by filtration. In the second case, the dried bacterial cellulose/chitosan membrane was immersed into drug solution and the encapsulation took place by swelling. For both cases, the encapsulation evaluation was done by UV–VIS analysis of the drug that remained in the solution. In vitro release behaviors of silver sulfadiazine from PNIPAM/PVA/MO nanoparticles and biocomposites were evaluated in time. Briefly, the dried loaded PNIPAM/PVA/MO nanoparticles and subsequently membrane biocomposites were entrapped in a cellulose membrane, immersed in 50 mL of PBS (0.01 M, pH 5 to reproduce the skin pH), and incubated in a precision water bath (orbital mixer Benchmark Scientific) at 400 rpm and 37 °C. Aliquots (5 mL) containing a mixture of PBS and released drug were collected at defined time points and the release medium was refreshed with the addition of an equal amount of fresh PBS after each withdrawal to maintain the total volume of the sample constant. The silver sulfadiazine release profiles from nanoparticles and biocomposites were evaluated by UV-VIS spectroscopy.