The efficacy of PDT is related to a number of factors which include the cellular uptake kinetics of the photosensitizer and its subcellular localization characteristics [5,7,9]. Since erythrosine is a hydrophilic dye [20], it is likely to accumulate preferentially in lysosomes and mitochondria [21]. These localization characteris- tics may help in determining the mechanism by which cell death occurs following PDT. The site of localization of a PS is usually the primary target for PDT since the ROS produced due to the PDT have a very short life-time (,0.05 ms) [5,22,23]. Upon activation using the appro- priate wavelength of light, the PS absorbs the light photon energy (h*v) and ascends from ground-state to the excited singlet state resulting in the production of ROS [7,24]. ROS production due to PDT causes a highly elevated oxidative stress in the cell, the magnitude of which depends upon the PDT dose applied and which also governs the pathway of cell-death following PDT [6,14,24,25]. The two most well-characterized cell death mech- anisms following PDT are apoptosis and (primary) necrosis [5,6,25]. Necrosis usually predominates when using high dose PDT whereas apoptosis is more usually seen with comparatively lower PDT doses. Primary necrosis is principally a passive cell death process which occurs due to extreme external physical stress or severe intracellular damage, and is irreversible [21,26]. Apoptosis (programmed cell death), on the other hand, is an active pathway of cell death which involves the activation of various cellular factors including initiator caspases (Caspase 2, 8, 9 and 10) and executioner caspases (Caspase 3 and 7) [27]. Apoptotic pathways (intrinsic and extrinsic) usually involve mitochondria, which is frequently regarded as the central processing organelle for apoptosis [25,28]. Therefore, mitochon- drial damage is often essential for the induction of apoptosis within cells following PDT [7,25]. The aim of the present investigation was to determine the efficacy of erythrosine in the photodynamic therapy of oral malignancies. To this end, we have studied the uptake kinetics of erythrosine in malignant (H357) and pre-malignant (DOK) oral epithelial cells and their susceptibility to erythrosine-based PDT. Determination of the subcellular localization of erythrosine was followed by initial investigations into the mechanism of cell killing induced at both high and low concentrations of erythrosine. Results Erythrosine Uptake Analysis At 37uC for erythrosine concentrations ranging from 71.03 mM up to 1136.5 mM, the uptake of erythrosine by DOK cells was seen to increase in an erythrosine dose-dependent manner, such that it was significantly more (*P,0.05) for all the erythrosine concen- trations than the 0 mM background (Figure 1A). A similar uptake pattern was seen for the H357 cells for erythrosine concentrations ranging from 71.03 mM to 1136.5 mM (Figure 1B). Statistical analysis showed that there was no significant difference in the uptake of erythrosine by the two cell lines at this temperature. At 4uC the erythrosine uptake was considerably slower, especially at erythrosine concentrations from 71.03 mM up to 568.27 mM (Figure 1A and 1B) for both cell lines. Moreover for both cell lines (Figure 1A and 1B) at almost all erythrosine concentrations, the erythrosine uptake at 37uC was significantly higher than at 4uC (**P,0.05). However, the overall erythrosine uptake at 4uC was higher for DOK cells (Figure 1A) when compared to H357 cells (Figure 1B). Similarly, at 1136.5 mM the erythrosine uptake was significantly higher in DOK cells than in H357 cells. Erythrosine-Based PDT of Oral Epithelial Cells PDT based Cell Killing The overall percentage (%) cell killing caused by PDT in DOK cells rose in a light/fluence and photosensitizer dose-dependent manner (Figure 2A). Low PDT doses (all irradiations for erythrosine concentrations up to 284.13 mM as well as irradiation of 40.86 J/cm2 for erythrosine concentrations of 568.27 mM and 1136.5 mM) caused less cell kill which was not significantly higher than cell killing observed at same erythrosine concentrations 2 without irradiation (i.e. fluence – 0 J/cm ). However, at higher PDT doses (irradiation fluence of 81.72 J/cm2 and/or 122.58 J/ cm2 and erythrosine concentrations of 568.27 mM and/or 1136.5 mM) the observed cell kill rose rapidly such that it was significantly more (*P,0.05) than cell killing observed at same erythrosine concentrations without irradiation (i.e. fluence – 0 J/ cm2). The maximum percentage cell kill was observed at 1136.5 mM concentration and 122.58 J/cm2 irradiation fluence (Figure 2A). The other dose showing appreciable cell killing was the 1136.5 mM concentration with 81.72 J/cm2 irradiation fluence. Overall, the LD50 for DOK cells was found to be 585.32 mM erythrosine at 122.58 J/cm2 irradiation fluence and 920.60 mM erythrosine at 81.72 J/cm2 irradiation fluence. Cell killing trends following erythrosine-PDT were similar in the H357 cells (Figure 2B) as described above for DOK cells. However, cell killing caused by PDT in H357 cells was lower than in the DOK cells (Figure 2A and 2B). Maximum cell killing was observed at 1136.5 mM concentration and 122.58 J/cm2 irradiation fluence (Figure 2B). Overall, the LD50 for PDT-treated H357 cells was found to be 818.31 mM at 122.58 J/cm2 irradiation fluence. The LD50 was not reached for the other irradiation times in H357 cells. Lastly, for erythrosine concentra- tions above 1136.5 mM (i.e. 2.27–9.09 mM), increasingly signifi- cant cytotoxicity without irradiation (dark cytotoxicity of the photosensitizer; data not shown) was observed; hence in the present study the PDT-analysis has not been performed beyond the erythrosine concentration of 1136.5 mM. Thus the therapeu- tically relevant erythrosine concentration range for PDT was less than 1136.5 mM. Sub-cellular Localization Analysis by CLSM Co-localization analysis was carried out for erythrosine with respect to dyes that stain either lysosomes (LysoTracker) or mitochondria (MitoTracker). Apart from analysis of merged channels images, co-localization was also estimated via other (statistical) parameters like co-localization frequency scatter plots (based on co-localization frequency colour ‘heat’ maps), Pearson’s coefficient and Mander’s coefficient, as described elsewhere [29]. In case of both DOK (Figure 3A) and H357 (Figure 3B) cells, erythrosine was found to co-localize more strongly with Mito- Tracker than with LysoTracker as indicated by the following four parameters – (1) higher incidence of yellow co-localization colour in the merged channels image for MitoTracker/erythrosine, (2) hotter colours in the co-localization frequency scatter plot (tending towards 100% co-localization as per the colour map in Figure 3C) for MitoTracker/erythrosine, (3) higher Pearson’s coefficient values for MitoTracker/erythrosine (r values, as indicated in the respective scatter plots) and (4) higher Mander’s coefficient for MitoTracker/erythrosine (M values, as indicated in the respective scatter plots). Based on this data, we can conclude that erythrosine localizes predominantly in the mitochondria in both the DOK as well as H357 cell lines. PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e34475