Lilium candidum Extract Loaded in Alginate Hydrogel Beads for Chronic Wound Healing

Abstract

Chronic wounds are a major health problem, affecting millions of people worldwide. Resistance to treatment is frequently observed, requiring an extension of the wound healing time, and improper care can lead to more problems in patients. Smart wound dressings that provide a controlled drug release can significantly improve the healing process. In this paper, alginate beads with white lily leaf extract were prepared and tested for chronic wound healing. The obtained beads were characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Also, the efficiency of extract encapsulation in alginate was determined as being of. The obtained hydrogel was tested on two normal human cell lines, respectively, dermal fibroblasts (BJ-CRL-2522-ATCC) and endothelial cells (human umbilical vein endothelial cells-HUVEC 2). The longer release of bioactive compounds from plant extract loaded in the alginate hydrogel resulted in more effective wound closure, compared to the extract alone, and scar formation, compared to the alginate hydrogel. Therefore, the effect of the white lily extract in combination with that of sodium alginate hydrogel improves the biological activity of the alginate hydrogel and increases the wound healing properties of the alginate.

Keywords: alginate beads; antioxidant capacity; chronic wound; polyphenols; white lily.

Figure 1

SEM image of the (a) microencapsulated Ext sample and (b) alginate beads.

Figure 2

The FTIR spectra of sodium alginate (Alg), white lily extract (Ext), and microencapsulated extract (Alg-Ext).

Figure 3

Viability assay. Dermal fibroblasts (BJ-upper panels) and endothelial cells (HUVECs-lower panels) were treated for 24 h with medium extract of alginate hydrogel formulations w/o the plant extract in different dilutions (left panels) and, respectively, different polyphenol concentrations of the plant extract (right panels). The resulting data is presented as a percentage of untreated control, average (n = 3) ± SD (standard deviation).

Figure 4

Comparative microscopy aspect of the wounds at different time points (initial, at 8, 24, 48, 72 h) in experimental groups: 1. Control, 2. LPS (bacterial lipopolysaccharide), 3. LPS + A (lipopolysaccharide + alginate hydrogel), 4. LPS + AE (lipopolysaccharide + alginate hydrogel with plant extract), 5. LPS + E (lipopolysaccharide + plant extract), original magnification, objective 4×, bar = 10 µm.

Figure 5

Wound area was measured at different time points (8, 24, 48, and 72 h) for each experimental group: 1. Control, 2. LPS (bacterial lipopolysaccharide), 3. LPS + A (lipopolysaccharide + alginate hydrogel), 4. LPS + AE (lipopolysaccharide + alginate hydrogel with plant extract), 5. LPS + E (lipopolysaccharide + plant extract), using the Image J software 1.8.0 and MiToBo plugging, data are presented as % of remaining wound area from the initial wound area, mean (n = 5) ± SD. * p < 0.05, ** p < 0.01.

Figure 6

Viable cell count was estimated from the level of ATP measured in the cell cultures at 72 h in the wound scratch assay wells for each experimental group: 1. Control, 2. LPS (bacterial lipopolysaccharide), 3. LPS + A (lipopolysaccharide + alginate hydrogel), 4. LPS + AE (lipopolysaccharide + alginate hydrogel with plant extract), 5. LPS + E (lipopolysaccharide + plant extract), results are presented as % of controls, mean (n = 3) ± SD. * p < 0.05.

Figure 7

Western blot analysis of the protein levels of MMP9, MMP2, TIMP1, collagen 1, and caspase 3 for each experimental group: 1. Control, 2. LPS (bacterial lipopolysaccharide), 3. LPS + A (lipopolysaccharide + alginate hydrogel), 4. LPS + AE (lipopolysaccharide + alginate hydrogel with plant extract), 5. LPS + E (lipopolysaccharide + plant extract); WB bands quantification was conducted by densitometry, and for normalization, β actin was used. Data are presented as mean (n = 3) ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA.

Figure 8

Oxidative stress parameters measurement. Malondialdehyde (MDA). Superoxide dismutase (SOD). Catalase (CAT). Experimental groups: 1. Control, 2. LPS (bacterial lipopolysaccharide), 3. LPS + A (lipopolysaccharide + alginate hydrogel), 4. LPS + AE (lipopolysaccharide + alginate hydrogel with plant extract), 5. LPS + E (lipopolysaccharide + plant extract). Data are presented as mean (n = 3 ± SD), * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA.

Figure 9

Pro-inflammatory cytokine IL1 β and IL6 levels. Experimental groups: 1. Control, 2. LPS (bacterial lipopolysaccharide), 3. LPS + A (lipopolysaccharide + alginate hydrogel), 4. LPS + AE (lipopolysaccharide + alginate hydrogel with plant extract), 5. LPS + E (lipopolysaccharide + plant extract). Data are presented as mean (n = 3 ± SD), ** p < 0.01, *** p < 0.001, one-way ANOVA.