Fabrication and characterization of an antibacterial chitosan/silk fibroin electrospun nanofiber loaded with a cationic peptide for wound-dressing application

Abstract

Wound infections are still problematic in many cases and demand new alternatives for current treatment strategies. In recent years, biomaterials-based wound dressings have received much attention due to their potentials and many studies have been performed based on them. Accordingly, in this study, we fabricated and optimized an antibacterial chitosan/silk fibroin (CS/SF) electrospun nanofiber bilayer containing different concentrations of a cationic antimicrobial peptide (AMP) for wound dressing applications. The fabricated CS/SF nanofiber was fully characterized and compared to the electrospun silk fibroin and electrospun chitosan alone in vitro. Then, the release rate of different concentrations of peptide (16, 32, and 64 µg/ml) from peptide-loaded CS/SF nanofiber was investigated. Finally, based on cytotoxic activity, the antibacterial activity of scaffolds containing 16 and 32 µg/ml of the peptide was evaluated against standard and multi-drug resistant strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa isolated from burn patients. The peptide-loaded CS/SF nanofiber displayed appropriate mechanical properties, high water uptake, suitable biodegradation rate, a controlled release without cytotoxicity on Hu02 human foreskin fibroblast cells at the 16 and 32 µg/ml concentrations of peptide. The optimized CS/SF containing 32 μg/ml peptide showed strong antibacterial activity against all experimental strains from standard to resistance. The results showed that the fabricated antimicrobial nanofiber has the potential to be applied as a wound dressing for infected wound healing, although further studies are needed in vivo.

Fig. 1

FTIR spectra of silk fibroin. FTIR spectra of silk fibroin.The bands at 1648 cm⁻¹, 1513 cm⁻¹, and 1241 cm⁻¹ represent amide I (C=O stretching), amide II (secondary N-H bending) and amid III (C–N and N–H functionalities) groups, respectively

Fig. 2

The ultrastructure of the electrospun CS and electrospun SF after treating with ethanol observed under SEM

Fig. 3

The biodegradation rate of the scaffolds after 35 days. CS significantly degrades faster from day 1 to 35 compared to other samples in all time intervals (P < 0.05). There are no significant differences between SF and CS/SF scaffolds during 21 days (P > 0.05). CS/SF sample degrades significantly faster than SF alone from day 21 to 35 in all-time intervals (P < 0.05)

Fig. 4

The swelling rate of scaffolds. The figure shows a similar swelling pattern among all samples within 24 h. However, CS shows a significant water uptake in all time intervals compared with SF and CS/SF samples (P < 0.0001). There are no considerable differences between SF and CS/SF samples in all predetermined time intervals. * represents a significant difference with SF and CS/SF (P < 0.0001)

Fig. 5

The release pattern of CM11 peptide from the scaffolds. A burst release of the peptide at day 1, and an increase in a linear manner until day 8 is evident in the figure in all samples. Approximately 90% of the peptide is released through 8 consecutive days from all three experimental samples

Fig. 6

A Cell viability results of the 4 investigated bilayer scaffolds with different concentrations of the peptide, show a significant difference in fibroblasts’ growth of peptide-loaded CS/SF (64 µg/ml peptide) scaffold and the control in days 3 and 7 post-seeding. B adhesion of a fibroblast cell to the surface of the bilayer fibers. White and red arrows represent the fibers and cell filopodia, respectively

Fig. 7

Assessment of antibacterial activity peptide-loaded CS/FS by disk diffusion assay. A The antibacterial activity peptide-loaded CS/SF (16 & 32 µg/ml peptide, respectively) bilayers are compared with control (Cnt). A halo around peptide-loaded bilayers is visible after 24 h in standard strains of S. aureus, E. coli, and P. aeruginosa MHA plates compared to control disks. In resistant strains, an inhibition zone is observable only around CS/SF containing 32 µg/ml CM11 peptide. B The diameter of the growth inhibition zone recorded by a ruler. C Diameters around peptide-loaded CS/SF (16 µg/ml) disks are compared to peptide-loaded CS/SF (32 µg/ml). There are significant differences in the scaffolds containing higher concentrations of CM11 peptide (32 µg/ml) with peptide-loaded CS/SF containing 16 µg/ml peptide in both standard strains (P < 0.05). Results from resistant strains and all controls are excluded as no diameters have formed and there is nothing to compare