Ultrashort Cationic Naphthalene-Derived Self-Assembled Peptides as Antimicrobial Nanomaterials

Self-assembling dipeptides conjugated to naphthalene show considerable promise as nanomaterial structures, biomaterials, and drug delivery devices. Biomaterial infections are responsible for high rates of patient mortality and morbidity. The presence of biofilm bacteria, which thrive on implant surfaces, are a huge burden on healthcare budgets, as they are highly resistant to current therapeutic strategies. Ultrashort cationic self-assembled peptides represent a highly innovative and cost-effective strategy to form antibacterial nanomaterials. Lysine conjugated variants display the greatest potency with 2% w/v NapFFKK hydrogels significantly reducing the viable <i>Staphylococcus epidermidis</i> biofilm by 94%. Reducing the size of the R-group methylene chain on cationic moieties resulted in reduction of antibiofilm activity. The primary amine of the protruding R-group tail may not be as readily available to interact with negatively charged bacterial membranes. Cryo-SEM, FTIR, CD spectroscopy, and oscillatory rheology provided evidence of supramolecular hydrogel formation at physiological pH (pH 7.4). Cytotoxicity assays against murine fibroblast (NCTC 929) cell lines confirmed the gels possessed reduced cytotoxicity relative to bacterial cells, with limited hemolysis upon exposure to equine erythrocytes. The results presented in this paper highlight the significant potential of ultrashort cationic naphthalene peptides as future biomaterials

The plasma jet used in this study.

<p>(A) Schematic diagram of the plasma jet. (B) Photograph of the plasma jet interacting with a biofilm sample.</p

CLSM images of the plasma treated biofilms.

<p>3D rendered confocal laser scanning micrographs of 3-day <i>P. aeruginosa</i> biofilms, grown on polycarbonate coupons, exposed to the 20 kHz plasma jet for 0s (A and D), 60 s (B and E), and 240 s (C and F). Green colour indicates surviving cells whereas red colour indicates dead cells. Magnification power is 200x (a-c) and 600x (d–f).</p

D-values of 20 kHz and 40 kHz plasma jets against <i>P. aeruginosa</i> biofilm cells.

<p>D-values of 20 kHz and 40 kHz plasma jets against <i>P. aeruginosa</i> biofilm cells.</p

Percentage cell reduction curves based on colony count method vs. XTT assay.

<p>Percentage cell reduction curves of <i>P. </i>aeruginosa biofilm cells upon exposure to the 20 kHz plasma jet. The dotted line is based on the standard colony count method whereas the solid line is based on the XTT assay. (Each point represents the mean of 3 values ± SE).</p

Bacterial growth inhibition zones.

<p><i>P. aeruginosa</i> cell suspensions were spread over MHA plates (9 cm in diameter). The seeded plates were exposed to the 20 kHz plasma jet for (A) 0 s, (B) 120 s, and (C) 240 s and then incubated at 37°C for 24 hours. After incubation, photographs of agar plates, showing bacterial growth inhibition zones, were taken using a digital camera.</p

Absorbance of XTT-assay product.

<p>48-hour <i>P. aeruginosa</i> biofilms, grown on Calgary Biofilm Device, were exposed to the 20 kHz plasma jet for up to 4 minutes. After plasma exposure, bacterial cells were dislodged off the pegs into PBS buffer by sonication. 50 µl aliquots of the recovered bacterial suspensions were then mixed with 50 µl of MHB and 20 µl of XTT stock solution and incubated at 37°C for 5 hours. After incubation, the absorbance at 450 nm was measured to quantify XTT metabolic product, the intensity of which is proportional to the number of viable (respiring) cells. (Each point represents the mean of 3 values ± SE).</p

Survival curve of biofilm treated with 20 kHz plasma.

<p>48-hour <i>P. aeruginosa</i> biofilms, grown on Calgary Biofilm Device, were exposed to the 20 kHz plasma jet for up to 4 minutes. The number of biofilm surviving cells in each sample was then calculated using colony count method and used to construct the log survival curve. (Each point represents the mean of 3 values ± SE).</p