Photodynamic inactivation of Candida albicans with imidazoacridinones : influence of irradiance, photosensitizer uptake and reactive oxygen species generation

The increasing applicability of antifungal treatments, the limited range of available drug classes and the emergence of drug resistance in Candida spp. suggest the need for new treatment options. To explore the applicability of C. albicans photoinactivation, we examined nine structurally different imidazoacridinone derivatives as photosensitizing agents. The most effective derivatives showed a >10(4)-fold reduction of viable cell numbers. The fungicidal action of the three most active compounds was compared at different radiant powers (3.5 to 63 mW/cm2), and this analysis indicated that 7 mW/cm2 was the most efficient. The intracellular accumulation of these compounds in fungal cells correlated with the fungicidal activity of all 9 derivatives. The lack of effect of verapamil, an inhibitor targeting Candida ABC efflux pumps, suggests that these imidazoacridinones are not substrates for ABC transporters. Thus, unlike azoles, a major class of antifungals used against Candida, ABC transporter-mediated resistance is unlikely. Electron paramagnetic resonance (EPR)-spin trapping data suggested that the fungicidal light-induced action of these derivatives might depend on the production of superoxide anion. The highest generation rate of superoxide anion was observed for 1330H, 1610H, and 1611. Singlet oxygen production was also detected upon the irradiation of imidazoacridinone derivatives with UV laser light, with a low to moderate yield, depending on the type of compound. Thus, imidazoacridinone derivatives examined in the present study might act via mixed type I/type II photodynamic mechanism. The presented data indicate lack of direct correlation between the structures of studied imidazoacridinones, cell killing ability, and ROS production. However, we showed for the first time that for imidazoacridinones not only intracellular accumulation is necessary prerequisite of lethal photosensitization of C. albicans, but also localization within particular cellular structures. Our findings present IA derivatives as efficient antifungal photosensitizers with a potential to be used in local treatment of Candida infection

Antimicrobial photodynamic therapy with fulleropyrrolidine : photoinactivation mechanism of Staphylococcus aureus, in vitro and in vivo studies

A family of N-methylpyrrolidinium fullerene iodide salts has been intensively studied to determine their applicability in antimicrobial photodynamic therapy (APDT). This study examined in vitro the efficacy of a C60 fullerene functionalized with one methylpyrrolidinium group to kill upon irradiation with white light gram-negative and gram-positive bacteria, as well as fungal cells, and the corresponding mechanism of the fullerene bactericidal action. The in vitro studies revealed that the high antistaphylococcal efficacy of functionalized fullerene could be linked to their ability to photogenerate singlet oxygen and superoxide anion. Following Staphylococcus aureus photoinactivation, no modifications of its genomic DNA were detected. In contrast, photodamage of the cell envelope seemed to be a dominant mechanism of bactericidal action. In in vivo studies, a 2 log10 reduction in the average bioluminescent radiance between treated and non-treated mice was reached. One day post APDT treatment, moist and abundant growth of bacteria could be observed on wounds of non-fulleropyrrolidine and dark control mice. APDT-treated wounds stayed visibly clear up to the third day. Moreover, cytotoxicity test on human dermal keratinocytes revealed great safety of using the sensitizer toward eukaryotic cells. These data indicate potential application of functionalized fullerene as antistaphylococcal sensitizer for superficial infections

Accumulation of imidazoacridinone derivatives in <i>Candida albicans</i> ATCC 10231 cells in the presence of verapamil.

<p>The cells (10⁶ CFU/ml) were incubated with 50 μM of a particular imidazoacridinone derivative (indicated on the X-axis) in the dark, and the accumulation was measured through spectrophotometry as described in Materials and Methods (<b>black bars</b>). The cells treated as described, but pretreated with 200 μM of verapamil before incubation with the imidazoacridinone derivative, are represented with <b>grey bars</b>. The mean values from three independent biological experiments are shown and the error bars represent the standard deviation.*, significant at the level of P value of < 0.05.</p

Survival of <i>Candida albicans</i> ATCC 10231 cells after photodynamic treatment with various irradiance power and imidazoacridinone derivatives.

<p>Three representative imidazoacridinone derivatives are shown here: <b>a.</b> 1330, <b>b.</b> 1558, and <b>c.</b> 1610H. The cells were incubated with the following concentrations of each imidazoacridinone derivative: 0, 50, and 100 μM. Following incubation, a total light dose (LED light 405 nm) of 20 J/cm² (<b>left panel</b>) or 30 J/cm² (<b>right panel</b>) was applied, at various irradiance power as indicated on the X-axis. The value of ‘0 mW’ represents control experiments, i.e., cells incubated in the dark in the absence (0 μM) or presence (50 μM or 100 μM) of IA derivative, and not subjected to illumination. The remaining values of ‘3.5 mW’ up to ‘63 mW’ represent the light-treated samples at a given irradiance. The statistical significance of the reduction in survival was calculated for each IA-treated sample with respect to the light only treated samples (black bars). The number of surviving cells presented on the Y-axis represents the mean value of three independent biological replicates, and the error bars represent the standard deviation. *, significant at the level of P value of < 0.05.</p

Electron Paramagnetic Resonance (EPR)-spin trapping experiments.

<p><b>a.</b> An example of an EPR spectrum of superoxide anion created upon photoexcitation of imidazoacridinone 1330 alone and in the presence of NADH. <b>b.</b> Comparison of the <i>coefficient a</i> indicating the efficiency in superoxide anion creation derived from the EPR spectra of different irradiated imidazoacridinone derivatives (indicated in X-axis) in the presence and absence of NADH. Riboflavin was used as a positive control photosensitizer that efficiently generates superoxide anion in the absence of NADH.</p

A representative single Electron Paramagnetic Resonance (EPR) spectrum obtained upon photoexcitation of imidazoacridione derivative 1330.

<p>The predicted (inlet) and experimental spectra of photoexcited imidazoacridinone 1330 in a DMSO/DMPO mixture, upon exposure to light at a wavelength range of 540–740 nm are shown.</p