Increased anti-biofilm efficacy of toluidine blue on Staphylococcus species after nano-encapsulation

Abstract

Background: Photodynamic therapy has been studied as a method for inactivating bacterial growth. Workers have used planktonic bacterial as well as biofilm bacterial cultures to evaluate the potential of photodynamic therapy in inactivating bacteria. However, almost all the studies use a photosensitiser in aqueous solution, which could be detrimental to the efficiency of photodynamic therapy.

Methods: In this study, the photodynamic killing effect of toluidine blue O (TBO) has been investigated on Staphylococcal biofilms in-vitro. The sensitivity of the in-vitro biofilms to photodynamic killing action was compared using different formulations of TBO, different dosages of photosensitiser and different light irradiation strengths. Effect of TBO formulations on bacterial quorum sensing system was evaluated using a colorimetric assay. Finally, dual staining using hoechst and propidium iodide stains was carried out on the photodynamically treated biofilms to visualise and compare the effects of photodynamic therapy. Scanning electron microscope imagery was also carried out to evaluate the photodynamic killing effect on the in-vitro biofilms.

Results: The sensitivity of biofilms to the photodynamic killing effect increased proportionally with the photosensitiser dosage and the light irradiation duration. TBO encapsulated in microemulsion was more effective in killing the biofilm bacteria than only TBO in water. The combination of TBO in microemulsion with EDTA was another effective way of increasing the photodynamic killing effect on the bacterial biofilms. Effect of encapsulated TBO on the quorum sensing system of bacteria was greater than the effect of aqueous solution of TBO. The in-vitro Staphylococcal biofilms could thus be inhibited by the photodynamic effect, and TBO encapsulated in microemulsion was much more effective than only TBO in water.

Conclusions: The encapsulation of a photosensitiser is an effective way of increasing the likelihood of the complete and successful inactivation of the biofilm growth. The encapsulated photosensitiser achieves higher inactivation of the bacterial biofilm than that of the aqueous solution of a photosensitiser.

Keywords: Antimicrobial photodynamic therapy; Biofilm; Microemulsion; Photodynamic therapy; Photosensitiser; Toluidine blue O.