Photoinactivation of Yeast and Biofilm Communities of Candida albicans Mediated by ZnTnHex-2-PyP4+ Porphyrin

Abstract

Candida albicans is the main cause of superficial candidiasis. While the antifungals available are defied by biofilm formation and resistance emergence, antimicrobial photodynamic inactivation (aPDI) arises as an alternative antifungal therapy. The tetracationic metalloporphyrin Zn(II) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (ZnTnHex-2-PyP⁴⁺) has high photoefficiency and improved cellular interactions. We investigated the ZnTnHex-2-PyP⁴⁺ as a photosensitizer (PS) to photoinactivate yeasts and biofilms of C. albicans strains (ATCC 10231 and ATCC 90028) using a blue light-emitting diode. The photoinactivation of yeasts was evaluated by quantifying the colony forming units. The aPDI of ATCC 90028 biofilms was assessed by the MTT assay, propidium iodide (PI) labeling, and scanning electron microscopy. Mammalian cytotoxicity was investigated in Vero cells using MTT assay. The aPDI (4.3 J/cm²) promoted eradication of yeasts at 0.8 and 1.5 µM of PS for ATCC 10231 and ATCC 90028, respectively. At 0.8 µM and same light dose, aPDI-treated biofilms showed intense PI labeling, about 89% decrease in the cell viability, and structural alterations with reduced hyphae. No considerable toxicity was observed in mammalian cells. Our results introduce the ZnTnHex-2-PyP⁴⁺ as a promising PS to photoinactivate both yeasts and biofilms of C. albicans, stimulating studies with other Candida species and resistant isolates.

Keywords: Zn(II) porphyrin; antimicrobial photodynamic inactivation; blue light; fungi; photodynamic therapy.

Figure 1

Box plots of photoinactivation of C. albicans yeasts. Two different strains were assessed, ATCC 10231 and ATCC 90028. Control: untreated group; dark: yeasts incubated with 1.5 µM of ZnP hexyl without irradiation; light: yeasts irradiated only (4.3 J/cm²). The values on the ‘x’ axis indicate the concentrations of ZnP hexyl for different aPDI groups. Differences between groups were analyzed by the Mann–Whitney test and considered significant at p < 0.05. The results are expressed as log₁₀ of the CFU/mL. At least three independent experiments were performed. *: p < 0.05 compared to control.

Figure 2

Box plot of cell viability of C. albicans ATCC 90028 biofilms assessed through MTT assay following treatment. Control: untreated biofilms; dark: biofilms incubated with 1.5 µM of ZnP hexyl without irradiation; light: biofilms irradiated only (4.3 J/cm²). aPDI was performed for biofilms incubated with 0.8 µM of ZnP hexyl for 10 min followed by irradiation (4.3 J/cm²). The results were expressed in relation to untreated biofilms. Differences between groups were analyzed by the Man–Whitney test and considered significant at p < 0.05. At least three independent experiments were performed. *: p < 0.05 compared to control.

Figure 3

Representative confocal microscopy images of C. albicans ATCC 90028 biofilms grown for 48 h and stained with PI after treatments. Control (A): biofilm without irradiation; dark (B): biofilm incubated with 1.5 µM of ZnP hexyl without irradiation; light (C): biofilm irradiated only (4.3 J/cm²); aPDI (D): biofilm incubated with 0.8 µM of ZnP hexyl for 10 min followed by irradiation (4.3 J/cm²).

Figure 4

Representative SEM images of C. albicans ATCC 90028 biofilms grown for 48 h and submitted to different treatments. Control (A): biofilm without irradiation; dark (B): biofilm incubated with 1.5 µM of ZnP hexyl without irradiation; light (C): biofilm irradiated only (4.3 J/cm²); aPDI (D): biofilm incubated with 0.8 µM of ZnP hexyl for 10 min prior to irradiation (4.3 J/cm²). Yellow arrows indicate hyphae entanglements. Yellow stars indicate the exposed substrate.

Figure 5

Box plot of cell viability of Vero cells assessed through MTT assay following treatment. Control: untreated cells; dark: cells incubated with 1.5 µM of ZnP without irradiation; light: cells irradiated in PBS only (4.3 J/cm²). Photodynamic treatment was performed for cells incubated with 0.8 or 1.5 µM of ZnP for 10 min followed by irradiation (4.3 J/cm²). The results were expressed in relation to untreated samples. Differences between groups were analyzed by Mann–Whitney test and considered significant at p < 0.05). At least three independent experiments were performed. *: p < 0.05 compared to control. ns: not significantly different, p > 0.05.