Candida albicans SC5314 was obtained from M. Ghannoum (Cleveland, OH, USA). Unless otherwise stated, the isolate was grown in yeast extract, peptone, and dextrose (YPD) broth (Difco Laboratories) containing 10 g/L yeast extract, 10 g/L peptone, and 20 g/L glucose and incubated for 24 h at 30°C in a shaker set at 150 rpm (IKA KS 4000, Cole-Parmer, Vernon Hills, IL, USA; to early stationary phase).

A hydrogel/glass composite was synthesized using a mixture of tetramethyl orthosilicate, polyethylene glycol, chitosan, glucose, and sodium nitrite in a 0.5 mm sodium phosphate buffer (pH 7). The nitrite was reduced to NO within the matrix with thermally generated electrons from glucose. After the redox reaction, the ingredients were combined and dried using a lyophilizer, and which resulted in a fine powder comprised of nanoparticles containing NO. The NO-np remains stable in this form for more than 1 year at room temperature. Once exposed to an aqueous environment, the hydrogel properties of the composite allow for an opening of the water channels inside the particles, facilitating the release of the trapped NO overextended time periods. Nanoparticles (np) were also prepared without the addition of NO, and served as a control.

To determine the impact of the NO-np on C. albicans, 1 mL of YPD broth was inoculated with a 24 h colony of the fungus grown on YPD agar. One hundred μL of C. albicans broth suspension was inoculated in each well of a 200-well plate, containing 100 μL of YPD broth with either NO-np or np (5 mg/mL; Nitric oxide kinetics release from nanoparticles: Peak state: 18.75 nM; Steady state: 12.5 nM). The inoculated plate was then incubated for 24 h at 37°C. Controls included wells containing yeast suspension alone, and yeast cells in YPD broth containing np. Growth was assessed every 30 min using a microplate reader (Bioscreen C, Growth Curves USA, Piscataway, NJ, USA) set at an OD of 600 nm.

To investigate the antifungal efficacy of NO-np in C. albicans induced burn infections, female Balb/c mice (6–8 weeks; National Cancer Institute, Frederick, MD, USA) were anesthetized (ketamine-xylazine cocktail), their back hair removed, and their exposed skin cleansed with an application of iodine. Five-mm diameter burn injuries were created applying a 60 s-heated brass bar for 45 s. Immediately after injury, C. albicans was inoculated onto the burned tissue using a 10⁷ cell suspension of the yeast prepared in PBS. Animals were individually housed to prevent crossed infection. On day 1 after infection, 5 mg of NO-np or np powder was then applied. Applications were similarly performed on days 3 and 5 after injury. Untreated, injured mice were used as additional controls. To monitor healing, photographs were taken daily. Also, a caliper was used to blindly measure the burn diameter of the injury on a daily basis, and was performed by two independent investigators. Three and 7 days after infection, mice were euthanized and tissue biopsies were taken.

At days 3 and 7 after C. albicans infection, wound tissues were excised and homogenized in sterile PBS. Samples were plated on YPD agar and fungal colony counts were determined. The results were normalized by tissue weights.

At days 3 and 7 after wounding, wound tissues were excised from euthanized mice, fixed in 10% formalin for 24 h, processed, and embedded in paraffin. Four micron vertical sections were fixed to glass slides and subjected to Hematoxylin and Eosin (H&E), Periodic acid schiff (PAS), Gomori’s trichrome, Myeloperoxidase (MPO; day 3), and Iba-1 staining to examine tissue morphology, fungal morphology, collagen deposition, neutrophil infiltration, and macrophage-like cells, respectively. Slides were examined by light microscopy with an Olympus AX70 (Melville, NY, USA) microscope, and images were obtained [QImaging Retiga 1300 digital camera (Burnaby, British Columbia, Canada)] with QCapture Suite V2.46 software (QImaging).

Candida albicans rapidly undergoes cell division by budding and forms pseudohyphae and hyphae when grown in media supplemented with fetal calf serum (FCS). Hence, the effect of NO-np on C. albicans cell division and morphological transformation was assessed using live cell imaging. Briefly, 10⁶ yeasts were plated on a polylysine coated coverslip bottom of MatTek plates. Images were collected for 2 h at 40× using a Zeiss Axiovert 200 M inverted microscope with an Axiocam MR camera controlled by the Axio Vision 4.4 software (Carl Zeiss Micro Imaging, NY). This microscope was housed in a Plexiglas box and the temperature was stabilized at 37°C, using a forced air heater system. The plate lid was kept in place to prevent evaporation, and 5% CO₂ was delivered to a chamber locally over the culture dish. Images were compiled into movies which were then used to analyze the C. albicans morphogenesis.

To assess the antifungal activity of the NO-np on invasive C. albicans filamentous form, 10⁶ yeasts were grown in 100 μL of media supplemented with FCS on a 96-well microtiter plates. Plates were incubated for 4 h at 37°C and 5% CO₂. After hyphal formation, 100 μL of media supplemented with FCS with either NO-np or np (5 mg/mL) were added to each well. Then, plates were incubated for 24 h at 37°C and 5% CO₂. Controls included wells containing untreated filamentous fungus. Metabolic activity was measured by 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay.

For filamentous C. albicans SC5314, 50 μL of XTT salt solution (1 mg/mL in PBS) and 4 μL of menadione solution (1 mM in acetone; Sigma) were added to each well. Microtiter plates were incubated at 37°C for 5 h. Mitochondrial dehydrogenases in live cells reduce XTT tetrazolium salt to XTT formazan, resulting in a colorimetric change, which was measured in a microtiter reader at 492 nm. Microtiter wells containing heat-killed fungi or only medium but no filamentous C. albicans cells were used as negative controls. The percentage of metabolic activity was determined by measuring the optical density of fungi exposed to np or NO-np relative to unexposed biofilms.

All data were subjected to statistical analysis using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA). P values were calculated by analysis of variance and were adjusted by use of the Bonferroni correction. P values of <0.05 were considered significant.

Candida albicans SC5314 growth with and without exposure to NO-np was determined in real-time for 24 h (Figure 1). NO-np significantly reduced fungal growth after 2 h co-incubation when compared with untreated controls (P < 0.001). Notably, C. albicans grown with np had significant reductions in fungal growth at 4 h, an effect that is likely due to steric interference with cell to cell contact or communication (e.g., quorum sensing). It is also possible that the chitosan incorporated into the np may offer some degree of antifungal activity; although the concentrations used to generate the np were negligible. When nano-scaled, chitosan has a higher surface to volume ratio, translating into a higher surface charge density, and increased affinity for bacteria and fungi with greater antimicrobial activity (Qi et al., 2004). Therefore at the nano-scale, the concentration used may confer some inhibitory activity. Nevertheless, NO-np significantly impeded growth relative to the np controls at >4 h (P < 0.001).

The effect of NO-np on burn healing in Balb/c mice after C. albicans infection was investigated (Figure 2). The curative process progressed most rapidly in the NO-np-treated mice (Figure 2A), with complete healing in all treated mice within 20 days following burn injury and infection (P < 0.001; Figure 2B). In contrast, the healing process of untreated or np-treated mice was significantly delayed (Figure 2A), and complete wound closure was not achieved within the 20 days following injury when mice were euthanized (Figure 2B). Notably, animals in the control groups experienced fungal transmission from the burn site to the paws, possibly by scratching of the infected injury (Figure 2C), which is consistent with higher fungal burdens and prolonged infection.

The efficacy of NO-np in killing C. albicans in the infected tissue was further explored by examining fungal burdens (Figure 3). At three and 7 days post infection, wounds were excised and plated on YPD agar. C. albicans-infected NO-np-treated burns showed significantly lower fungal burdens when compared to untreated or np-treated wounds (P < 0.01; Figure 3A).

Histological examinations (H&E staining) of day 7 untreated and np-treated C. albicans-infected burns revealed numerous pseudohyphal and hyphal forms penetrating into different tissue layers along with extensive cell necrosis (Figure 3B; insets). Tissue sections from the lesions of NO-np-treated C. albicans-infected mice revealed less suppurative inflammation along with increased fibrin deposition and no evidence of fungal hyphal structures in dermis (Figure 3B).

Time-lapse microscopy was used to examine whether NO-np interferes with C. albicans cell division and morphological transformation (Figure 4A). Untreated control yeasts displayed rapid (12 min) budding formation and morphogenesis. The control np delayed budding and transformation for up to 48 min post exposure. In contrast, NO-np arrested fungal budding and morphogenesis over the entire 60 min time course examined. At the 120 min time point, the untreated group evinced morphological changes consistent with that of candidal pseudohyphal structures while the np and NO-np-treated groups demonstrated delayed budding or no budding, respectively, without any indication of active filamentation (Figure 4B). In addition, NO-np-treated yeasts displayed a rounded shape characteristic of cellular edema which has been previously observed in studies with multi-drug S. aureus (Martinez et al., 2009). Thus, the results strongly suggest that NO-np can effectively inhibit or delay the onset of pseudohyphal formation. Together, these results suggest that the in vivo findings may in part be due to a reduction in the rate of yeast cell division and morphogenesis.

Since the filamentous stage of C. albicans invades tissue and is relevant in pathogenesis, we investigated the antifungal activity of NO-np against the mycelial form of the fungus. XTT reduction assay was utilized to quantify fungal metabolic activity. At 24 h, NO-np significantly reduced the metabolic activity of filamentous C. albicans when compared to untreated (∼65%; P < 0.001) or np (∼52%; P < 0.001; Figure 4C). Our findings suggest that NO-np are effective against the tissue invasive filamentous form of C. albicans.

The mechanisms through which the NO-np accelerates wound healing were further explored by examining the collagen content in burn wounds treated with NO-np (Figure 5). Trichrome staining dyes collagen blue. For each image, quantitative measurement of collagen intensity in 20 representative squares of the same size was performed using Image J software. Collagen content was highest in the infected wounds treated with NO-np (Figure 5A). The dispersed blue stain in these sections indicated a thicker and more mature tissue collagen formation, thus suggesting that NO-np exposure protected the dermal architecture through fungal clearance, and ultimately maintaining collagen stores while promoting formation of new collagen. Figure 5B is a morphometric analysis of the data shown in Figure 5A.

We investigated the effect of NO-np on leukocyte migration to the C. albicans-infected tissue (Figure 6). First, neutrophil infiltration was evaluated by measuring the production of MPO in the injured area. MPO is an enzyme most abundantly present in neutrophils, and the localized brown staining reaction observed indicated more neutrophil infiltration in NO-np-treated samples compared to untreated or np-treated burn tissue (Figure 6A).

Secondly, we identified macrophage-like infiltration by measuring the expression of Iba-1, which is specifically expressed and upregulated during the activation of these cells. Iba-1 is the product of the Aif1 gene (allograft inflammatory factor 1) which is located within a segment of the major histocompatibility complex class III region. Tissue sections from untreated murine burns showed only scattered macrophage-like cells whereas np and NO-np-treated mice showed massive cellular infiltrations into the injured area (Figure 6B).

Together, our data suggests NO-np can stimulate cell recruitment during burn healing and fungal cell clearance by up-regulating phagocytic cell infiltration into the injured area.