Sustained release of usnic acid from graphene coatings ensures long term antibiofilm protection

Abstract

Protecting surfaces from bacterial colonization and biofilm development is an important challenge for the medical sector, particularly when it comes to biomedical devices and implants that spend longer periods in contact with the human body. A particularly difficult challenge is ensuring long-term protection, which is usually attempted by ensuring sustained release of antibacterial compounds loaded onto various coatings. Graphene have a considerable potential to reversibly interact water insoluble molecules, which makes them promising cargo systems for sustained release of such compounds. In this study, we developed graphene coatings that act as carriers capable of sustained release of usnic acid (UA), and hence enable long-term protection of surfaces against colonization by bacterial pathogens Staphylococcus aureus and Staphylococcus epidermidis. Our coatings exhibited several features that made them particularly effective for antibiofilm protection: (i) UA was successfully integrated with the graphene material, (ii) a steady release of UA was documented, (iii) steady UA release ensured strong inhibition of bacterial biofilm formation. Interestingly, even after the initial burst release of UA, the second phase of steady release was sufficient to block bacterial colonization. Based on these results, we propose that graphene coatings loaded with UA can serve as effective antibiofilm protection of biomedical surfaces.

Figure 1

Raman spectra (a) and FT-IR spectra (b) of coatings made of graphene and graphene loaded with UA, with free UA as control. (c) Morphology of surface coatings with different concentrations of graphene loaded with UA. (d) High resolution SEM images of surfaces coated with graphene and graphene loaded with UA (100 µg/mL). The red arrow is pointing to UA in crystalline form, attached to graphene flakes.

Figure 2

The release pattern of UA from the graphene coatings loaded with UA. The release pattern of UA was tested by using 200 µg/mL of UA loaded graphene coatings (15 mm of glass surface). The presented values are micrograms of UA detected in total volume of sample recovered from one coated surface. Data represent the mean ± standard deviation of two independent biological replicates.

Figure 3

CFU counts of a) S. aureus and b) S. epidermidis exposed to different concentration of graphene-UA flakes (concentrations and colour coding are indicated in the figure inset) and grown in standard cultivating conditions. Data are presented as mean ± standard deviation from three independent biological replicates.

Figure 4

Measurement of biofilm inhibitory efficiency of graphene (Gr) and UA loaded graphene coatings against (a) S. aureus and (b) S. epidermidis. Viability of bacteria, expressed as CFU counts, was determined after 24 h of growth on coated and non-coated surfaces. Data represent mean ± standard derivation from three independent biological replicates. *P < 0.05, **P < 0.0001. (c) Live/dead viability staining of biofilms of S. aureus and S. epidermidis performed on the same set of surfaces as above. Representative fluorescence microscopic images from three independent biological replicates are presented. Green color denotes live bacteria and red color denotes dead bacteria.

Figure 5

Representative scanning electron microscopic images of (a) S. aureus and (b) S. epidermidis biofilms grown on the non-coated (Control), graphene-coated (Gr) and surfaces coated with various concentrations of graphene loaded with UA. The biofilms were fixed, dehydrated and coated with gold before SEM imaging.

Figure 6

Viability of bacteria in 96 h biofilms of (a) S. aureus and (b) S. epidermidis grown on graphene-UA coatings. Data represent mean ± standard deviation from 3 independent biological replicates. **P < 0.0001.

Figure 7

Antibiofilm activity of graphene-UA coatings. Biofilms were formed on coated surfaces after the primary release of UA. After 24 h of biofilm growth, viability of bacteria, (a) S. aureus and (b) S. epidermidis, was determined by CFU counting (*P < 0.05, **P < 0.0001) and (c) live/dead viability staining. Representative fluorescence microscopic images are presented from three independent biological replicates. Green color denotes live bacteria and red color denotes dead bacteria.