Composites Based on Gellan Gum, Alginate and Nisin-Enriched Lipid Nanoparticles for the Treatment of Infected Wounds

Abstract

Due to growing antimicrobial resistance to antibiotics, novel methods of treatment of infected wounds are being searched for. The aim of this research was to develop a composite wound dressing based on natural polysaccharides, i.e., gellan gum (GG) and a mixture of GG and alginate (GG/Alg), containing lipid nanoparticles loaded with antibacterial peptide-nisin (NSN). NSN-loaded stearic acid-based nanoparticles (NP_NSN) were spherical with an average particle size of around 300 nm and were cytocompatible with L929 fibroblasts for up to 500 µg/mL. GG and GG/Alg sponges containing either free NSN (GG + NSN and GG/Alg + NSN) or NP_NSN (GG + NP_NSN and GG/Alg + NP_NSN) were highly porous with a high swelling capacity (swelling ratio above 2000%). Encapsulation of NSN within lipid nanoparticles significantly slowed down NSN release from GG-based samples for up to 24 h (as compared to GG + NSN). The most effective antimicrobial activity against Gram-positive Streptococcus pyogenes was observed for GG + NP_NSN, while in GG/Alg it was decreased by interactions between NSN and Alg, leading to NSN retention within the hydrogel matrix. All materials, except GG/Alg + NP_NSN, were cytocompatible with L929 fibroblasts and did not cause an observable delay in wound healing. We believe that the developed materials are promising for wound healing application and the treatment of bacterial infections in wounds.

Keywords: antibacterial wound dressings; nisin; solid lipid nanoparticles.

Figure 1

Nisin purification: HPLC chromatogram of NSN after reverse-phase purification (a), the mass spectrum of NSN (b), pictures of S. pyogenes growth inhibition zones (c) and diameters of S. pyogenes growth inhibition zones (d).

Figure 2

Characterization of unloaded NP and NSN-loaded NP: AFM images (a), particle size distribution (b), surface zeta potential (c), and metabolic activity of L929 fibroblasts incubated for 24 h in presence of unloaded and NSN-loaded NP (d). Statistically significant differences at * p < 0.05 and ** p < 0.01.

Figure 3

Physico-chemical characterization of GG and GG/Alg hydrogels containing NSN or NP_NSN: rheological characteristics (a), dry mass percentage (b), gross morphology in dry and hydrated (wet) state (c) and SEM images at magnification of 200× and 6000× (d).

Figure 4

Swelling ratio (a) and pH change (b) during the incubation in PBS buffer, mass loss after 48 h of incubation (c) and NSN release profile (d) of GG and GG/Alg hydrogels containing NSN or NP_NSN. Statistically significant differences at * p < 0.05.

Figure 5

Antibacterial efficacy of GG and GG/Alg hydrogels containing NSN or NP + NSN: pictures of S. pyogenes growth inhibition zones (a) and diameters of S. pyogenes growth inhibition zones (b). Statistically significant differences at * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 6

Cytocompatibility of GG and GG/Alg hydrogels containing NSN or NP + NSN: metabolic activity (a) and live/dead staining (b) of L929 cells incubated for 24 h in presence of sample extracts, wound healing assay performed for up to 3 days in presence of sample extracts (c) and evaluation of cell adhesion to the samples 24 h after cell seeding (d). Statistically significant differences at * p < 0.05.