Incorporation of antimicrobial peptides on electrospun nanofibres for biomedical applications

Abstract

The aim of this work was to immobilize antimicrobial peptides onto a fibrous scaffold to create functional wound dressings. The scaffold was produced by electrospinning from a mixture of the water soluble polymers poly(acrylic acid) and poly(vinyl alcohol) and subsequently heat cured at 140 °C to produce a stable material with fibre diameter below micron size. The peptides were incorporated into the negatively charged scaffold by electrostatic interaction. The best results were obtained for lysozyme impregnated at pH 7, which rendered a loading of up to 3.0 × 10^-4 mmol mg^-1. The dressings were characterized using SEM, ATR-FTIR, elemental analysis, ζ-potential and confocal microscopy using fluorescamine as an amine-reactive probe. The dressings preserved their fibrous structure after impregnation and peptides were distributed homogeneously throughout the fibrous network. The antibacterial activity was assessed by solid agar diffusion tests and growth inhibition in liquid cultures using Staphylococcus aureus, a pathogenic strain generally found in infected wounds. The antibacterial activity caused clear halo inhibition zones for lysozyme-loaded dressings and a 4-fold decrease in S. aureus viable colonies after two weeks of contact of dressings with bacterial liquid cultures. The release profile in different media showed sustained release in acidic environments, and a rapid discharge at high pH values. The incorporation of lysozyme resulted in dressing surfaces essentially free of microbial growth after 14 days of contact with bacteria at pH 7.4 attributed to the peptide that remained attached to the dressing surface.

Fig. 1. FTIR-ATR spectra of PAA–PVA functionalized dressings. Nisin (A) and lysozyme (B) functionalized at pH 7 (the spectra for dressings functionalized at pH 10, are shown in Fig. S1†).

Fig. 2. SEM images of PAA–PVA fibres before impregnation (A), and dressings after impregnation (pH 7) nisin (N_2@PAA–PVA, (B)) and lysozyme (L_2@PAA–PVA, (C)).

Fig. 3. Confocal laser scanning microscopy images of PAA–PVA fibres (A), nisin-loaded PAA–PVA (N_3@PAA–PVA, pH 7, (B)) and lysozyme-loaded PAA–PVA (L_3@PAA–PVA, pH 7, (C)) after conjugation with fluorescamine.

Fig. 4. Halo inhibition zone expressed in mm² for L_1@PAA–PVA, L_2@PAA–PVA and L_3@PAA–PVA (pH 7) along 14 day experiments with cultures of S. aureus in agar plates at 37 °C (A). Representative images of inhibition experiments corresponding to neat PAA–PAA, N_2@PAA–PVA at pH 7 and L_2@PAA–PVA at pH 7 (B).

Fig. 5. Colony-forming units (CFU) in liquid media in contact with PAA–PVA and L_1@PAA–PVA dressings (A) and for microorganisms detached from dressings (same dressings, after 24 h and 14 days, (B)).

Fig. 6. SEM micrographs of PAA–PVA (A) and lysozyme loaded L_1@PAA–PVA (B) dressings after 24 h and 14 days in contact with S. aureus cultures at 37 °C.

Fig. 7. Release of lysozyme in different media: phosphate buffer saline pH 7.4 (●), carbonate buffer pH 10.0 (□) and acetate buffer pH 3.5 (○). The dashed line represents the lysozyme loading of specimens L_1@PAA–PVA.