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Nanomaterials (Basel)
2020 Feb 23;102:. doi: 10.3390/nano10020390.
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Silver Nanoparticles-Composing Alginate/Gelatine Hydrogel Improves Wound Healing In Vivo.
Diniz FR
,
Maia RCAP
,
Rannier L
,
Andrade LN
,
V Chaud M
,
da Silva CF
,
Corrêa CB
,
de Albuquerque Junior RLC
,
P da Costa L
,
Shin SR
,
Hassan S
,
Sanchez-Lopez E
,
Souto EB
,
Severino P
.
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Polymer hydrogels have been suggested as dressing materials for the treatment of cutaneous wounds and tissue revitalization. In this work, we report the development of a hydrogel composed of natural polymers (sodium alginate and gelatin) and silver nanoparticles (AgNPs) with recognized antimicrobial activity for healing cutaneous lesions. For the development of the hydrogel, different ratios of sodium alginate and gelatin have been tested, while different concentrations of AgNO3 precursor (1.0, 2.0, and 4.0 mM) were assayed for the production of AgNPs. The obtained AgNPs exhibited a characteristic peak between 430-450 nm in the ultraviolet-visible (UV-Vis) spectrum suggesting a spheroidal form, which was confirmed by Transmission Electron Microscopy (TEM). Fourier Transform Infra-red (FT-IR) analysis suggested the formation of strong intermolecular interactions as hydrogen bonds and electrostatic attractions between polymers, showing bands at 2920, 2852, 1500, and 1640 cm-1. Significant bactericidal activity was observed for the hydrogel, with a Minimum Inhibitory Concentration (MIC) of 0.50 µg/mL against Pseudomonas aeruginosa and 53.0 µg/mL against Staphylococcus aureus. AgNPs were shown to be non-cytotoxic against fibroblast cells. The in vivo studies in female Wister rats confirmed the capacity of the AgNP-loaded hydrogels to reduce the wound size compared to uncoated injuries promoting histological changes in the healing tissue over the time course of wound healing, as in earlier development and maturation of granulation tissue. The developed hydrogel with AgNPs has healing potential for clinical applications.
M-ERA-NET/0004/2015 (PAIRED) and UIDB/04469/2020 Portuguese Science and Technology Foundation (FCT), CNPq, #443238/2014-6, #470388/2014-5 Coordenação Aperfeiçoamento de Pessoal de Nivel Superior (CAPES), Fundação de Amparo à Pesquisa do Estado de Sergipe (FAPITEC), Conselho Nacional de Desenvolvimento Científico e Tecnológico
Figure 1. Ultraviolet-visible (UV–Vis) absorption spectra of sodium alginate, gelatin, and hydrogel with silver nanoparticles at different concentrations (1 mM, 2 mM, and 4 mM).
Figure 2. Fourier Transform Infra-red (FT–IR) spectra of (A) sodium alginate, (B) gelatin, and of hydrogels with silver nanoparticles at different concentrations (C) 1 mM, (D) 2 mM, and (E) 4 mM. Arrows of spectrum A correspond to the bands around 1650 cm−1 (C=C stretching), 1250 cm−1 (C–O–C stretching); arrows of spectrum B correspond to 1640 cm−1 and 1510 cm−1 of amide carbonyl (C=O and C=N stretching vibration); spectra of the hydrogel (square of C, D, and E) are the bands around 1600–1500 cm−1.
Figure 3. Differential Scanning Calorimetry (DSC) analysis (left-hand panel) of the (A) hydrogel alginate/gelatin (80:20) and (B) hydrogel with silver nanoparticles (AgNPs) (4 mM); Thermogravimetric (TGA) analysis (right-hand panel) of (A) hydrogel alginate/gelatin (80:20), and (B) hydrogel with AgNPs (4 mM).
Figure 4. Electron micrographs of the hydrogels with silver nanoparticles at different concentrations: 1 mM on the 200 nm scale (a) and 50 nm scale (b); 4 mM on the 200 nm (c) and 100 nm scale (d).
Figure 5. Cell viability assay of gelatin, hydrogel containing 4 mM of AgNPs, sodium alginate, and silver nitrate of human L929 fibroblasts, determined by the methyl-thiazolyl-tetrazolium (MTT) assay after 24 h of incubation. The vehicle used to dilute the drug (dimethyl sulfoxide, DMSO 5%) was used as the negative control (100% viability). The data correspond to the mean ± SEM of four independent experiments. * p < 0.05 compared to the control group using one-way analysis of variance followed by Tukey’s test.
Figure 6. Non-splinted model showing the percentage of the non-epithelialized surface of the wound of the groups: GCTR (Control Group), GH (Group with hydrogel sodium alginate/gelatin (80:20), and GHP (Group hydrogel with AgNP 4 mM AgNO3). All values are mean ± S.E. Statistical analysis comprised ANOVA followed by Tukey’s test. * P < 0.05 in relation to GCTR, GH, and GHP groups, respectively (n = 21/group).
Figure 7. Photomicrographs of hematoxylin/eosin-stained histological sections representative of histological wound healing versus the time course of the experiment. Day 3: Wounds present intense edema (ed) and infiltration of polymorphonuclear neutrophils; note the lymphocyte-rich infiltrate (lym) and immature granulation tissue (igt) in the bottom of GH and GHP, respectively (100×). Polymorphonuclear neutrophil (small lobular nuclei) and lymphocytes (dark round nuclei) are highlighted in higher magnification (800×). Day 7: Thick strips of granulation tissue are observed in all groups (dashed arrows); irregular and slit-shaped capillary blood vessels concentrated in the edge are seen in GCTR (thin arrows), whereas dilated hyperemic vessels (thick arrows) are observed throughout the wound areas in GH and GHP (100×). Note the lower content of inflammatory cells in GHP. Stromal spindle cells (fibroblast and endothelial-like cells) are highlighted at higher magnification (800×). Day 14: Residual vascular granulation tissue (right) is observed in GCTR, but a cellular primary fibrous scar (cfb) is seen in GH and GHP. Epithelial buddings (compatible with rudimentary cutaneous appendages) (white arrows) are found in the edges of the wound area in GH but over the full epithelial surface in GHP (100×). Stromal spindle cells (fibroblast-like cells) are highlighted in higher magnification (800×). GCTR (Control Group), GH (Group with hydrogel sodium alginate/gelatin (80:20), and GHP (Group hydrogel with AgNP 4 mM AgNO3).
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