Combating Concomitant Bacterial and Fungal Infections via Codelivery of Nitric Oxide and Fluconazole

Abstract

Device-associated infections are a major challenge for healthcare and cause patient morbidity and mortality as well as pose a significant economic burden. Infection-causing bacteria and fungi are equally notorious and responsible for biofilm formation and the development of antibiotic and antifungal-resistant strains. Biomaterials resisting bacterial and fungal adhesion can address device-associated infections more safely and efficiently than conventional systemic antibiotic therapies. Herein, we present a combination of potent antibacterial nitric oxide (NO) with antifungal fluconazole codelivery system from a polymeric matrix to combat bacterial and fungal infections simultaneously. The NO donor S-nitroso-N-acetyl-penicillamine (SNAP)-blended low-water-uptake polycarbonate urethane (TSPCU) was dip-coated with high-water-uptake polyether urethane (TPU) containing fluconazole to have an antibacterial and antifungal surface. The composites were characterized for surface wettability and coating stability using water contact angle (WCA) analysis. The real-time NO release (72 h) was evaluated using a chemiluminescence-based nitric oxide analyzer which showed physiologically relevant levels of NO released. The composites released fluconazole for 72 h under physiological conditions. Antibacterial analysis demonstrated a > 3-log reduction of viable Staphylococcus aureus and >2-log reduction of viable Escherichia coli compared to controls. The antifungal evaluation resulted in ∼98% reduction in adhered and ∼92% reduction in planktonic Candida albicans. The SNAP-fluconazole composites also showed biocompatibility against mouse fibroblast cells. This novel preventative strategy to combat bacterial and fungal infections may offer a promising tool for further translational research.

Keywords: antibacterial; antifungal; drug delivery; fluconazole; hospital-acquired infections; nitric oxide.

Figure 1

Characterization of the polymeric composites. (A) Surface wettability of the fabricated composites. WCA was measured using 10 μL of water. (B) Percent water uptake of TSPCU composites. (C) X-ray diffraction spectra of fluconazole incorporated TPU. (D) Coating stability studies for TSPCU + TPU composites over 24 h. Data represent mean ± standard deviation, n > 3. Statistical analysis: One-way ANOVA coupled with Tukey’s test; * indicates p < 0.05, ** indicates p < 0.001, *** indicates p < 0.0005, **** indicates p < 0.0001.

Figure 2

Representative scanning electron microscopy of the polymeric substrates showing surface morphology. Scale bar: 100 μm, inset images 10 μm.

Figure 3

Fluconazole and SNAP release from SNAP-Fluconazole composites. Cumulative release of (A) fluconazole, (B) SNAP, and SNAP-fluconazole composites over 3 days under physiological conditions (37 °C, pH 7.4, submerged in PBS-EDTA). Data represent mean ± standard deviation, n > 3.

Figure 4

Real-time NO release kinetics from SNAP + fluconazole composites. (A) Structure of NO donor S-nitroso-N-acetylpenicillamine (SNAP). (B) Schematic of NO release from SNAP catalyzed by light, heat, enzymes, and metal ions. (C) Prolonged NO release from SNAP + fluconazole composites under physiological conditions (37 °C, pH 7.4, submerged in PBS). Data represent mean ± standard deviation, n > 3.

Figure 5

Antifungal evaluation of SNAP-fluconazole composites. (A) Mechanism of fluconazole antifungal action. Fluconazole inactivates cytochrome P₄₅₀ enzyme 14 α-demethylase which inhibits synthesis of cell membrane component ergosterol from lanosterol, causing membrane degradation and cell death. (B) Structure of the drug fluconazole. (C) Reduction in adhered C. albicans after 24 h under physiological conditions. (D) Reduction in planktonic C. albicans after 24 h under physiological conditions (37 °C, pH 7.4, PBS). Data represent mean ± standard deviation, n = 3. Statistical analysis: One-way ANOVA coupled with Tukey’s test; * indicates statistical significance of p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

Figure 6

Antibacterial evaluation of SNAP-fluconazole composites. Reduction in viable bacteria after 24 h under physiological conditions (37 °C, pH 7.4, PBS) (A) adhered E. coli, (B) planktonic E. coli, (C) adhered S. aureus, and (D) planktonic S. aureus. Data presents mean ± standard deviation, n = 3. Statistical analysis: One-way ANOVA coupled with Tukey’s test; * indicates statistical significance of p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

Figure 7

Cytocompatibility evaluation of the SNAP-fluconazole composites. 3T3 cells exposed to leachates for 24 h under physiological conditions followed by viability estimation using CCK-8 kit. Data represent mean ± standard deviation, n = 5.