(J-390) Methylene blue-loaded liposomes photodynamic therapy effect against Candida albicans biofilms

Introduction:: Candida albicans is an opportunistic fungal pathogen that can cause invasive infections especially in immunocompromised patients and is often associated with high mortality rates. One of the main challenges in treating candidiasis is that C. albicans has the ability to form biofilms which are complex structures formed by yeasts, hyphae, and pseudohyphae embedded in extracellular polymeric substances (EPS). This structure makes it difficult for antifungal drugs to penetrate the biofilm. Due to the increased resistance of Candida biofilms against antifungal agents and the host immune system, new therapeutic approaches are needed. Photodynamic therapy (PDT) is a promising alternative antimicrobial treatment in which a photosensitizer is activated by a light source at a specific wavelength and interacts with oxygen to yield reactive oxygen species (ROS), such as superoxide and singlet oxygen, which can be cytotoxic. PDT has been shown to be effective against planktonic C. albicans, but must be improved for use against biofilms. One approach to enhance the efficacy of PDT against biofilms is to use drug delivery systems to improve the penetration of the photosensitizer into the biofilm matrix. Liposomes in particular, which are lipid-based nanoparticles, have exhibited potential to increase antimicrobial agent activity against biofilms by enhancing biofilm penetration. Here we aimed to develop liposomes loaded with methylene blue (MB), a widely used PDT photosensitizer, to increase the penetration of MB inside the C. albicans biofilm and improve antifungal activity.

Materials and Methods:: MB at a concentration of 1 mg/mL was used for encapsulation into liposomes by the thin-film hydration method and extrusion using 100 nm pore size polycarbonate membranes. Liposomes were formed with 4:1 (w/w) 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol. Dynamic light scattering (DLS) measurements were performed on a Zetasizer Nano ZS90 to obtain Z-average size, polydispersity index (PDI), and zeta-potential. After removing the free MB via centrifugation, the encapsulation efficiency (EE) was determined by lysing MB-loaded liposomes with 70% (v/v) methanol and obtaining MB absorbance intensity at 660 nm (Cytation 3, BioTek, Winooski, Vermont). C. albicans SC5314 was used as a reference strain to evaluate the antifungal activity of MB-loaded liposomes. Antifungal efficacy of MB and MB-loaded liposomes against planktonic and 24 h aged C. albicans biofilms was assessed by colony forming unit (CFU) enumeration. To evaluate the PDT efficacy, a light-emitting diode with a wavelength at 660 nm (LED, BlackBox smart®, Biolambda, Brazil) was used as the light source with a power density of 28.56 J/cm². For this, the 96-well plates containing the test samples were irradiated for 714 seconds. For each experimental group, non-irradiated groups were included as controls (data not shown). Statistical significance was analyzed using GraphPad Prism 5 with one-way analysis of variance (ANOVA) with α =0.05.

Results, Conclusions, and Discussions:: In this work, we developed MB-loaded liposomes (Figure 1a) for treatment of C. albicans biofilms. These liposomes had an average hydrodynamic diameter of 123.3 ± 1.5 nm with PDI of 0.54 ± 0.08 and zeta-potential of -1.42 ± 0.09 mV. The EE was found to be ~11% (Figure 1b). MB-loaded liposomes efficacy was evaluated against planktonic and biofilm C. albicans. MB and MB-loaded liposomes were both highly effective against planktonic C. albicans at a MB concentration of 0.5 µg/mL and were able to completely eradicate growth (Figure 1c). However, when we tested MB antifungal efficacy against C. albicans biofilms at different concentrations from 16 to 64 µg/mL, PDT had no effect on fungal growth, suggesting that MB was unable to penetrate the biofilm, which is known to challenge antifungal treatment (Figure 1d).^1,2 However, at a MB concentration of 32 µg/mL, MB loaded liposome treatment resulted in a ~3 log reduction in CFU compared with untreated C. albicans biofilms (Figure 1d). This result suggests that MB-loaded liposomes were more effective in penetrating C. albicans biofilms than free MB, resulting in greater infection eradication. These MB-loaded liposomes have the potential to significantly improve candidiasis treatment.


Acknowledgements (Optional): : We gratefully acknowledge the financial support provided by Coordination for the Improvement of Higher Education Personnel (CAPES) during the course of this research.

References (Optional): : 1. Yang, Y. T. et al. Photodynamic inactivation of chlorin e6-loaded CTAB-liposomes against Candida albicans. Lasers Surg Med 45, 175–185 (2013).

2. Vera-González, N. et al. Anidulafungin liposomes nanoparticles exhibit antifungal activity against planktonic and biofilm Candida albicans. J Biomed Mater Res A 108, 2263–2276 (2020).