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Nanomaterials 2020, 10, 390 3 of 16 2. Materials and Methods 2.1. Materials Sodium alginate (Protonal®RF6650, FMC Corporation, Philadelphia, Pennsylvania, USA) was donated by FMC (Campinas, São Paulo, Brazil). Silver nitrate was purchased from Química Contemporânea (Indaiatuba, São Paulo, Brazil). Bovine gelatin was obtained from Gelnex (Itá, Santa Catarina, Brazil). Other chemicals of analytical grade were obtained from Sigma-Aldrich (Darmstadt, Germany). Water from Millipore Milli®-Q system (Merck KGaA, Darmstadt, Germany), home supplied, was used for the preparation of the aqueous solutions. 2.2. Production of AgNPs-Composing Alginate/Gelatin Hydrogel The production of AgNPs-composing alginate/gelatin hydrogel was adapted from Rescignano et al. [25]. Briefly, sodium alginate and gelatin were firstly solubilized, separately, in water at the concentration of 2% (m/v) each, followed by the production of a solution of sodium alginate and gelatin in different proportions (i.e., 80:20, 50:50 or 20:80) by homogenization for 2 h at 500 rpm with a mechanical stirrer (Biomixer 78HW-1, Biomex Biotecnologia, Ribeirão Preto, São Paulo, Brazil), to obtain a hydrogel. AgNO3 was simultaneously dissolved in water in different concentrations (1.0, 2.0, and 4.0 mM) and slowly added to the hydrogel, under mechanical stirring for 2 h at 500 rpm to produce AgNPs. 2.3. UV–VIS Spectrophotometer Analysis UV–VIS absorption spectra were recorded on a UV–Vis spectrophotometer (FEMTO 800Xi, Femto Indústria e Comércio de Instrumentos Ltda., São Paulo, Brazil) in a scan range of 300–700 nm. All samples were previously dispersed in water, and analysis was run at room temperature. 2.4. Fourier-Transform Infra-Red Analysis Fourier Transform Infra-red (FT–IR) analysis was used to investigate the chemical interaction between components of the formulations (Agilent, Cary 630, Santa Clara, CA, USA) with 32 accumulations of 4000–500 cm−1, with resolution 2 cm−1 in potassium bromide (KBr) tablets. 2.5. Thermal Analysis The thermal behavior was assessed by DSC (DSC Q20 TA Instruments, New Castle, DE, USA). The samples were previously weighted (3–5 mg) in an aluminum pan and sealed hermetically. Analyses were carried out under an inert atmosphere (45 mL/min of N2). DSC thermograms were scanned in the first heating run at a constant rate of 10 ◦C/min and a temperature range of 25–500 ◦C. TGA was conducted in a dynamic nitrogen atmosphere (50 mL/min) at a heating rate of 10 ◦C/min (Shimadzu DTG 60, Tokyo, Japan). The percentage of mass loss was recorded from the ambient temperature up to 800 ◦C. 2.6. Transmission Electron Microscopy (TEM) Micrographs were obtained by TEM-MSC JEOL 2100 (JEOL Ltd., Tokyo, Japan) operating with 200 kV acceleration. Aqueous dispersions were dripped directly onto the copper grid, while the semi-solid hydrogels were previously dispersed in water, applying ultrasounds for 60 min and then dripped onto carbon-coated (carbon electrolyte 400 mesh Copper grids) (~4 nm). 2.7. Biological Assays 2.7.1. Viability Assays The test was performed based on ISO 10993-5 [26]. The cytotoxicity assay was carried out against human fibroblast L929 cell lines [27,28]. Samples of AgNO3, pure gelatin, pure alginate,PDF Image | Wound Healing Silver Nanoparticles-Composing Hydrogel
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