Sustained Nitric Oxide-Releasing Nanoparticles Induce Cell Death in Candida albicans Yeast and Hyphal Cells, Preventing Biofilm Formation In Vitro and in a Rodent Central Venous Catheter Model

Abstract

Candida albicansis a leading nosocomial pathogen. Today, candidal biofilms are a significant cause of catheter infections, and such infections are becoming increasingly responsible for the failure of medical-implanted devices.C. albicansforms biofilms in which fungal cells are encased in an autoproduced extracellular polysaccharide matrix. Consequently, the enclosed fungi are protected from antimicrobial agents and host cells, providing a unique niche conducive to robust microbial growth and a harbor for recurring infections. Here we demonstrate that a recently developed platform comprised of nanoparticles that release therapeutic levels of nitric oxide (NO-np) inhibits candidal biofilm formation, destroys the extracellular polysaccharide matrices of mature fungal biofilms, and hinders biofilm development on surface biomaterials such as the lumen of catheters. We found NO-np to decrease both the metabolic activity of biofilms and the cell viability ofC. albicansin vitroandin vivo Furthermore, flow cytometric analysis found NO-np to induce apoptosis in biofilm yeast cellsin vitro Moreover, NO-np behave synergistically when used in combination with established antifungal drug therapies. Here we propose NO-np as a novel treatment modality, especially in combination with standard antifungals, for the prevention and/or remediation of fungal biofilms on central venous catheters and other medical devices.

FIG 1

NO-np inhibit biofilm formation by Candida albicans clinical isolates. (A) Kinetics of C. albicans biofilm formation in polystyrene microtiter plates grown in the absence (untreated) and presence of 5 mg/ml of nanoparticles alone (np) or nitric oxide-releasing nanoparticles (NO-np), as determined by the XTT reduction assay. Each symbol represents the average value (n = 11 strains per time point), and error bars indicate standard deviations (SDs). P value significance (P < 0.05) was calculated by analysis of variance (ANOVA) and adjusted by use of the Bonferroni correction. * and ϕ, significantly higher optical densities (OD) than for NO-np and np groups, respectively. (B) Representative light microscopy images of untreated, np-treated, or NO-np-treated C. albicans SC5314 strain biofilms grown on microtiter plates for 2, 8, and 24 h. The pictures were taken at a magnification of ×20. Scale bar, 10 μm. All experiments were performed twice, with similar results obtained each time.

FIG 2

Mature C. albicans biofilms are susceptible to NO-np. Forty-eight-hour mature fungal biofilms were exposed to np or NO-np for 24 h, and their metabolic activities and viabilities were compared to those of untreated biofilms using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) reduction (A) and CFU (B) assays. For panels A and B, each symbol represents a C. albicans strain (three OD measurements and CFU plates per strain), bars signify the average (n = 11) for each experimental condition, and error bars indicate SDs. P value significance (*, P < 0.05; **, P < 0.001; ns, not significant) was calculated by ANOVA and adjusted by use of the Bonferroni correction. (C) Confocal microscopic examination of untreated, np-treated, or NO-np-treated C. albicans. Representative images of biofilms show metabolically active cells (FUN-1 staining, red) embedded in the polysaccharide extracellular material (ConA staining, green); the yellow-brownish areas represent metabolically inactive or nonviable cells. Scale bar, 20 μm. All experiments were performed twice, with similar results obtained each time.

FIG 3

NO-np induces apoptosis and necrosis of C. albicans cells. (A) Yeast cells were treated with np or NO-np and compared to untreated fungal cells. Apoptotic cells were analyzed by flow cytometry after being stained with annexin V-FITC together with propidium iodide (PI). The percentages of viable (live) and apoptotic or nonapoptotic dead cells are reported. (B) Fluorescence microscopy images of untreated, np-treated, and NO-np-treated filamentous C. albicans. Representative images of fungal hyphae showed viable (no fluorescence), apoptotic (annexin V; green), and dead (PI; red) cells. The pictures were taken at a magnification of ×20. Scale bar, 20 μm. All experiments were performed twice, with similar results obtained each time.

FIG 4

Reduction of C. albicans SC5314 surface-associated growth on central venous catheters (CVCs) inserted into rats' jugular veins after treatment with NO-np. Mean fungal burdens in in vitro (A) and in vivo (B) catheters infected with 10⁶ C. albicans cells are shown. The fungal burden in NO-np-treated catheters was significantly lower than that in control catheters. For panel A, each symbol represents 5-mm catheters and the bars show the averages of five catheters. In vitro experiments were performed twice, with similar results obtained each time. For panel B, in vivo experiment was performed once using five animals (average of five 5-mm pieces of catheter per rat) per group. For panels A and B, a concentration of 5 mg/ml of np or NO-np was used. In addition, statistical significance (*, P < 0.05; **, P < 0.001; ns, no significance) was calculated using ANOVA and adjusted by use of the Bonferroni correction. Error bars indicate SDs. (C to F) C. albicans SC5314 strain biofilm formation on catheters placed on the jugular vein of a Sprague-Dawley rat. (C and D) Scanning electron microscopic (SEM) examination of untreated (PBS) C. albicans biofilms; (E and F) SEM examination of C. albicans biofilms treated with 5 mg/ml of NO-np. (C) C. albicans biofilm formed on the luminal surface of the untreated catheter. (D) Higher magnification of the boxed region in panel C. Untreated biofilms showed a network comprising yeast cells (white arrowheads) and hyphae (white arrows) surrounded by large amounts of exopolymeric matrix (black arrows). (E) There was no visible candidal biofilm formation in NO-np-treated catheters. (F) Higher magnification of the boxed region in panel E, with light gray arrowheads indicating fibrous debris. Scale bar for panels C and E, 200 μm; scale bar for panels D and F, 20 μm.

FIG 5

Combination therapy of nitric oxide and antifungal drugs reduces the metabolic activity of C. albicans SC5314 biofilms. Fungal biofilms were exposed to various combinations of np with fluconazole (A) or voriconazole (C) and NO-np with fluconazole (B) or voriconazole (D). The symbol key shows the concentration of np or NO-np tested alone or in combination with the antifungal drugs. The experiment was performed twice, with similar results obtained each time.