Copper-Containing Anti-Biofilm Nanofiber Scaffolds as a Wound Dressing Material

Abstract

Copper particles were incorporated into nanofibers during the electrospinning of poly-D,L-lactide (PDLLA) and poly(ethylene oxide) (PEO). The ability of the nanofibers to prevent Pseudomonas aeruginosa PA01 and Staphylococcus aureus (strain Xen 30) to form biofilms was tested. Nanofibers containing copper particles (Cu-F) were thinner (326 ± 149 nm in diameter), compared to nanofibers without copper (CF; 445 ± 93 nm in diameter). The crystalline structure of the copper particles in Cu-F was confirmed by X-ray diffraction (XRD). Copper crystals were encapsulated, but also attached to the surface of Cu-F, as shown scanning transmission electron microscopy (STEM) and transmission electron microscopy (TEM), respectively. The copper particles had no effect on the thermal degradation and thermal behaviour of Cu-F, as shown by thermogravimetric analysis (TGA) and differential scanning calorimeter (DSC). After 48 h in the presence of Cu-F, biofilm formation by P. aeruginosa PA01 and S. aureus Xen 30 was reduced by 41% and 50%, respectively. Reduction in biofilm formation was ascribed to copper released from the nanofibers. Copper-containing nanofibers may be incorporated into wound dressings.

Fig 1

Scanning electron microscopy (SEM) images of (a) control nanofibers (CF), (b) copper-containing nanofibers (Cu-F) and (c) copper particles before electrospinning. Digital images of the electrospun nanofiber mats and copper powder are shown as inserts. The TEM image of Cu-F (d) shows the encapsulation of copper crystals (right arrow) in nanofibers and the attachment of crystals on the surface (left arrow) of the fibers. Image (e) is the same as (c), but visualised with TEM. Image (f) is the same as (d), but recorded with STEM and shows the inclusion of copper crystals of different sizes in Cu-F (two arrows).

Fig 2

SEM-energy-dispersive X-ray (EDX) analysis of (a) nanofibers without copper (CF), (b) copper-containing nanofibers (Cu-F) and (c) copper particles. The EDX scan spectra of selected areas pointed out in images (a), (b) and (c) are shown in (d), (e) and (f), respectively. The percentages shown for each of the elements are the mean of three selected areas from SEM images.

Fig 3. X-ray diffraction (XRD) patterns of nanofibers with copper particles (Cu-F), nanofibers without copper particles (CF) and copper particles (Cu).

Fig 4

(a) Thermal behaviour of control nanofibers (CF), copper-containing nanofibers (Cu-F) and copper particles (Cu). (b) Differential scanning calorimetric (DSC) thermograms of CF, Cu-F and Cu.

Fig 5. Changes in viable cell numbers and biofilm formation by P. aeruginosa PA01 and S. aureus Xen 30 in the presence of copper particles electrospun into nanofibers (Cu-F) and nanofibers without copper particles (CF).

Cells not treated with copper and not cultured in the presence of nanofibers serves as control (labelled c). Incubation was at 37°C. Readings were taken at 3, 6, 24 and 48h. (a) Number of viable cells of P. aeruginosa PA01, (b) total biofilm formation by cells of P. aeruginosa PA01, (c) number of viable cells of S. aureus Xen 30 and (d) total biofilm formation by cells of S. aureus Xen 30. Biofilm formation was determined by staining with crystal violet and recording OD readings at 595 nm. Data points presented are the average of three independent experiments (mean ± standard deviation). * p< 0.05.

Fig 6. Scanning electron microscopy (SEM) images of nanofibers collected from 48-h-old biofilms.

Images (a) and (c) show cell growth of P. aeruginosa PA01 and S. aureus Xen 30, respectively, on the surface of CF. Images (b) and (d) show cell growth of P. aeruginosa PA01 and S. aureus Xen 30, respectively, on the surface of Cu-F.

Fig 7

LIVE/DEAD® Baclight^TM and FilmTracer^TM SYPRO® Ruby Biofilm Matrix Stain of 48-h-old biofilms of P. aeruginosa PA01 (A) and S. aureus Xen 30 (B). For each strain, images were taken of biofilms that formed in (a) the absence of nanofibers and copper, (b) nanofibers without copper particles (CF) and (c) copper-containing nanofibers (Cu-F). The images in column 1 is after propidium iodide staining, column 2 after ruby staining and column 3 after SYTO® 9 staining. The image in column 4 is an overlap of all stains.

Fig 8

A: Scanning electron microscopy (SEM) images of P. aeruginosa PA01 biofilms (a, b and c) and S. aureus Xen 30 biofilms (d, e and f) that formed after 48 h on the polymer surfaces of the wells. B: Atomic force microscopy (AFM) images of P. aeruginosa PA01 biofilms (a, b and c) and S. aureus Xen 30 biofilms (d, e and f) that formed after 48 h on the polymer surfaces of the wells. Images a and d were taken from biofilms that formed in the absence of nanofibers and copper particles, b and e from biofilms that formed in the presence of CF, and c and f from biofilms that formed in the presence of Cu-F.

Fig 9

A: Release of copper from PDLLA/PEO nanofibers. A SEM image of the electrospun nanofibers after the release of copper particles is shown in the insert. B: Percentage of MCF-12A breast epithelial cells that survived exposure to CF and Cu-F. Sterile culture medium served as negative control (NC). Data points presented are the average of three independent experiments (mean ± standard deviation). * p < 0.05.