Effects Of 5-Aminolevulinic Photodynamic Therapy (ALA-PDT) On Candida Albicans Biofilms

Objectives:To evaluate the antifungal effects of 5-aminolevulinic photodynamic therapy (ALA-PDT) on the cell activity and structure of Candida albicans biofilms based on an in vitro model, and to determine the optimal parameters including ALA incubation time, ALA concentration and light dose.Methods:1. Formation of Candida albicans biofilms model in vitroThe formation of Candida albicans ATCC 90028 biofilms were obtained in 96-well microtiter plates and glass bottom cell culture discs. Crystal violet staining, fluorescence dual staining and XTT reduction assay were utilized to investigate the kinetics and ultrastructures of formation under the observation of light microscopy and confocal laser scanning microscopy (CLSM).2. Study of optimal parameters of ALA on Candida albicans biofilmsFluorescence intensity of Protopophrin IX (PpIX) in ALA-treated Candida albicans biofilms were examined and measured by fluorescence spectrophotometry and the optimal ALA incubation time and ALA concentration were settled.3. Evaluation of antifungal effects of ALA-PDT on Candida albicans biofilmsThe antifungal effects of ALA-PDT on the cell activity and structure of Candida albicans biofilms were evaluated by calculating the changes of cell inhibition rates and the average biofilm thickness under the observation of CLSM and the optimal irradiation dose was then settled.Results:1. Formation of Candida albicans biofilms model in vitroMature biofilms of Candida albicans ATCC 90028 biofilms were formed after 48 hours of inoculation. Under the observation of crystal violet staining and light microscopy, the biofilms were attached to the bottom of the carriers and remained intact under blowing and PBS flushing. Typical biofilm characteristics were seen under fluorescence dual staining and CLSM observation that the biofilm was an intense membrane-like structure with a certain thickness and mainly consisted of extracelluar matrix. XTT reduction assay revealed that the growth of biofilms was saturated after 48 hours of incubation, indicating that the biofilms were in a mature and stable state2. Optimal parameters of ALA on Candida albicans biofilmsUnder CLSM observation of ALA-treated Candida albicans biofilms, brick-red fluorescence was seen, indicating that Candida alicansbiofilms absorbed ALA and converted into PpIX. Fluorescence intensity of PpIX was in an incubation time-dependent manner but was not in a concentration-dependent manner. The optimal ALA incubation time was 5h, and the optimal ALA concentration was 15mM.3. Antifungal effects of ALA-PDT on Candida albicans biofilmsUnder fluorescence dual staining and CLSM observation, when the light dose increased, dead cells in the biofilms increased. The biofilms structure became looser and the average biofilm thickness became smaller when the light dose increased. Cell inhibition rate was nearly 80%and the average biofilm thickness decreased more than half when the light dose was 300 J/cm2.Conclusions:1. Candida albicans ATCC 90028 can form stable in vitro biofilm model after 48 hours of inoculation.2. Candida alicans biofilms can absorb ALA and convert into photosensitizer PpIX.3. ALA-PDT can inhibit cell activity in Candida albicans biofilms and can be destructive to the biofilms themselves.4. The optimal parameters of ALA-PDT on Candida alicans biofilms are as follows:ALA incubation time is 5h, ALA concentration is 15mM, and light dose of ALA-PDT is 300J/cm2.