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The Journal of Veterinary Medical Science logoLink to The Journal of Veterinary Medical Science
. 2026 Aug 4;88(10):1537–1545. doi: 10.1292/jvms.26-0107

In vitro evaluation of cytotoxicity of TONS504 and blue light irradiation on porcine corneal epithelial cells

Tomoya FURUJO 1, Norihiko ITO 1, Isao SAKATA 2, Tomohiro OSAKI 1,*
PMCID: PMC13680716  PMID: 42557041

Abstract

Photodynamic antimicrobial chemotherapy (PACT) for infectious keratitis has attracted increasing attention, and the clinical application of PACT with TONS504, a newly developed chlorin derivative, is anticipated. However, studies evaluating the cytotoxicity of TONS504 combined with light exposure on corneal cells are lacking. We aimed to investigate cytotoxic effects on the corneal epithelium and examined safe irradiation conditions by evaluating the cytotoxicity of TONS504, blue light (405 nm), and their combination using porcine corneal epithelium (PCEP). Additionally, the synergistic cytotoxicity of TONS504 with ethylenediaminetetraacetic acid (EDTA) under blue light—previously shown to have a synergistic antimicrobial effect with PACT—was assessed. Viability of PCEP cells exposed to various TONS504 concentrations (1–100 mg/L), irradiation intensities (5–30 J/cm2, 20 mW/cm2, 405 nm LED), and their combinations were evaluated using the Cell Counting Kit-8 assay. Inhibition of PCEP cell migration under these conditions was assessed using a primary porcine corneal explant model. The synergistic cytotoxicity of TONS504 with EDTA (0.015 M) under blue light irradiation at 5 J/cm2 was also evaluated similarly. TONS504 alone demonstrated low cytotoxicity (up to 10 mg/L), whereas the combination of TONS504 with blue light showed concentration-dependent cytotoxicity or cytotoxicity at high light irradiation intensity against PCEP cells. No clear synergistic cytotoxicity was observed between TONS504 and EDTA under blue light. These findings provide fundamental data for establishing a blue light-based TONS504-PACT protocol.

Keywords: blue light, corneal epithelium, ethylenediaminetetraacetic acid, phototoxicity, porcine

INTRODUCTION

The corneal epithelium is the outermost mechanical barrier of the eye and serves as a defensive shield against microbial infection. Various chemical, physical, and pathological stimuli can damage the corneal epithelium, resulting in a compromised barrier and epithelial wounding [15, 39]. As a self-renewing tissue, the corneal epithelium contains stem cell reservoir at the corneoscleral junction, which supplies proliferating cells for continuous epithelial regeneration throughout life [15, 39]. The corneal epithelial wound healing process involves several coordinated events, including cell migration, proliferation, adhesion, and differentiation accompanied by stratification of the epithelial layers [15, 39]. Proper corneal epithelial wound healing is crucial for maintaining the integrity and health of the corneal surface, preserving corneal transparency and vision [15, 39]. Therefore, eye drops and other treatments used in ophthalmology are evaluated for cytotoxicity to corneal epithelial cells.

The standard treatments for bacterial keratitis, a common eye disease in humans and dogs that can cause blindness [10, 28], are antibiotic and anti-collagenase agents. However, multidrug resistance remains a major treatment hindrance, complicating the treatment of bacterial keratitis in both humans and dogs [10, 28].

Photodynamic antimicrobial chemotherapy (PACT), also referred to as antimicrobial photodynamic therapy or antibacterial photodynamic inactivation, represents a promising alternative to antibiotic therapy for infectious diseases [9, 23, 38]. The antimicrobial effect of PACT relies on reactive oxygen species (ROS) generated through the synergistic interaction of photosensitizers, light of a specific wavelength that activates the photosensitizer, and molecular oxygen [9, 23, 38]. PACT offers a broad antimicrobial spectrum and functions through a mechanism distinct from that of conventional antibiotics, making this treatment promising for infections caused by multidrug-resistant bacteria [9, 23, 38]. Previous studies have explored the application of PACT in ophthalmology [17, 27, 35, 44].

The initial use of PACT for managing infectious keratitis involved riboflavin and ultraviolet A (UVA) light, originally developed for treating keratoconus by inducing collagen cross-linking in the cornea [17, 27, 35]. This treatment protocol includes soaking the de-epithelialized cornea in riboflavin, followed by UVA exposure [17, 27, 35]. Many studies have assessed the efficacy and safety of this treatment to establish appropriate clinical protocols [17, 37, 40,41,42]. Although this method has been applied as an adjunctive therapy for infectious keratitis, its antimicrobial effectiveness remains limited [35]. The development of new PACT approaches with stronger antimicrobial activity remains a critical need.

Several studies have reported the effectiveness of PACT using a light-emitting diode (LED) and TONS504—13,17-bis (1-carboxyethyl)-carbamoyl-(3-methylpyridine)-3-(1,3-dioxane-2-yl) methylidene-8-ethenyl-2-hydroxy-2,7,12,18-tetramethyl chlorin, di-N-methyl iodide (C51H58N8O5I2)—against various microorganisms that cause infectious keratitis [7, 8, 13, 21, 29, 30, 33, 34]. TONS504 is a hydrophilic and cationic chlorin derivative, which has two visible light absorption peaks at approximately 420 and 660 nm [30]. A previous in vitro study demonstrated that PACT with TONS504 (TONS504-PACT) using blue light achieved a sufficient antimicrobial effect [7, 8]. For instance, TONS504-PACT (10 mg/L) combined with blue light (30 J/cm2) significantly reduced the survival fraction of Pseudomonas aeruginosa (ATCC27853) to 3.5 × 10−5, and the addition of 0.015 M ethylenediaminetetraacetic acid (EDTA) markedly enhanced efficacy even at a light intensity of 5 J/cm2 [8]. Furthermore, against multidrug-resistant canine keratitis isolates, TONS504-PACT using blue light at 30 J/cm2 completely eradicated methicillin-resistant Staphylococcus pseudintermedius at concentrations as low as 0.1 mg/L, achieving an approximately 108 CFU/mL reduction [7]. Under the same irradiation conditions, maximal efficacy against extended-spectrum β-lactamase-producing Escherichia coli and P. aeruginosa was observed at 10 mg/L, reducing survival fractions to 3.2 × 10−5 and 2.6 × 10−4, respectively [7]. Owing to its high antimicrobial effects in vitro, clinical application of TONS504-PACT is expected. To establish a treatment protocol for TONS504-PACT, assessing both antimicrobial efficacy and cytotoxicity to normal cells is essential. However, no published studies have evaluated the cytotoxicity of TONS504, light irradiation, or their combination on normal cells.

We aimed to investigate the cytotoxicity of TONS504, blue light irradiation, and their combination on normal corneal cells. Evaluating the impact of PACT on corneal epithelial wound healing is important, as removal of the corneal epithelium during PACT facilitates deeper penetration of the photosensitizer and may contribute to postoperative pain. Therefore, in this study, the cytotoxicity of the condition of TONS504-PACT using blue light, which has been previously reported [7, 8, 29, 30], was assessed on primary porcine corneal epithelium (PCEP) by measuring cell viability using the Cell Counting Kit-8 (CCK-8) assay. Moreover, we evaluated the inhibition of cell migration and proliferation in PCEP cells under various conditions—TONS504 alone, blue light alone, and their combination—using a primary porcine corneal explant assay [36]. The synergistic cytotoxic effect of TONS504 with EDTA under blue light irradiation was also examined using both the CCK-8 assay and primary porcine corneal explant model.

MATERIALS AND METHODS

Photosensitizer

Stock solutions (1,000 mg/L) of TONS504 (JS-Stage Inc., Okayama, Japan) were prepared by dissolving it in phosphate-buffered saline (PBS) (Gibco, Grand Island, NY, USA). Each solution was wrapped in aluminum foil to prevent ambient light exposure and stored at 4°C. Before each experiment, the stock solution was diluted in PBS to final concentrations of 1, 10, and 100 mg/L.

LED system

The light source was obtained from Pleiades Technology, LLC (Fukuoka, Japan). The light source comprised an LED array with a wavelength of 405 nm. The LED device was designed for direct attachment to a cell culture plate, and a ventilator was installed to prevent temperature elevation during use. An optical power meter (Optical power meter 3664. HIOKI, Nagano, Japan) and sensor (Optical sensor 9742, HIOKI) were used to confirm that a power density of 20 mW/cm2 was achieved by irradiating the entire bottom surface of the plate within the light irradiation range. Light irradiation from the bottom of the plate achieved a power density of 20 mW/cm2. When irradiated from the top of the plate for 250, 500, and 1,500 sec, the total light energies delivered were 5, 10, and 30 J/cm2, respectively. The plates were placed on a stand during irradiation to prevent medium heating. Temperature measurements (Center521, Sato Shouji Inc., Tokyo, Japan) confirmed that the TONS504 solution temperature without cells did not increase following light exposure.

Culture medium

All primary cultures and serial passaging were maintained in a growth medium comprising defined keratinocyte serum-free medium (Gibco) and Dulbecco’s modified Eagles medium Ham’s F-12 (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), supplemented with 3% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), epidermal growth factor (Gibco), and antibiotics (gatifloxacin and amphotericin B), which was developed by incorporating improvements as previously reported [36].

Porcine corneal epithelial cell culture

A modified version of the expanded primary corneal limbal explant culture model was developed using a previously described method [12]. Porcine eyes were obtained from a local slaughterhouse and transported under cooled conditions. Upon arrival in the laboratory, the eyes were rinsed with saline and iodine. Corneal limbal tissues measuring 3 × 3 mm were excised using an ophthalmic knife and microscissors and transferred into 35-mm cell dishes coated with type I collagen gel (0.3% Cellmatrix I-P; Nitta Gelatin, Tokyo, Japan). The dishes were filled with growth medium and incubated at 37°C in a humidified atmosphere with 5% carbon dioxide (CO2). The growth medium was replaced every 24 hr. Corneal epithelial cells proliferated from the margins of the explants. After 7–10 days, the cells reached confluence and were used for subsequent experiments.

Intracellular uptake of TONS504

The cell discs were punched out by φ2 mm biopsy trephine from confluent cells, followed by transferring into a new cell dish. After overnight incubation, the discs were exposed to 10 mg/L of TONS504 for 5 min. Following PBS rinsing, intracellular uptake was visualized using an all-in-one microscope (BZ-X810; Keyence, Osaka, Japan). Images were captured using 405/20 and 630/30 filters for excitation and emission, respectively.

Cell viability assay

Corneal limbus pieces were removed, and the PCEP cells were dispersed with 0.25% trypsin and 1 mmol/L EDTA tetrasodium salt solution with phenol red (FUJIFILM Wako Pure Chemical Corp.) at 37°C for 10 min, followed by collagenase treatment at 37°C for 20 min. The resulting cell suspension was seeded into 96-well microplates for subsequent viability assays. Cell viability was assessed using the CCK-8 assay (Dojindo Molecular Technologies, Inc., Tokyo, Japan). In total, 2.0 × 104 cells/well were seeded into 96-well microplates and allowed to adhere overnight. After removing the culture medium, cells were incubated with various concentrations of TONS504 (0, 1, 10, or 100 mg/L) or benzalkonium chloride (BAC) (FUJIFILM Wako Pure Chemical Corp.) (0.01, 0.001, or 0.0001%) at room temperature (26–28°C) for 5 min. BAC is commonly used in eye drops; however, owing to its cytotoxic effects on corneal epithelial cells, it was used as a positive control for cytotoxicity [5, 36]. Following PBS rinsing and replacement with new PBS (100 μL), the cells were exposed to 405 nm LED at 0, 5, 10, or 30 J/cm2. After irradiation, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for another 4 hr at 37°C with 5% CO2 in the dark. Absorbance was measured at 450 nm using ChroMate 4300 (Awareness Technology, Palm City, FL, USA). The relative cell viability of each group was expressed as a percentage of the control (TONS504 0 mg/L under blue light irradiation intensity of 0 J/cm2). These experiments were repeated three times.

Cell viability at various TONS504 concentrations (0, 1, 10, or 100 mg/L) with or without EDTA (0.015 M) under a blue light irradiation intensity of 5 J/cm2 was evaluated. EDTA was obtained from FUJIFILM Wako Pure Chemical Corp. The solution was diluted in PBS to a final concentration of 0.015 M before use, as previously reported [8]. The cell viability assay was performed as described previously. Cells were exposed to TONS504 (0, 1, 10, or 100 mg/L) with or without EDTA (0.015 M) at room temperature for 5 min. Following PBS rinsing and replacement with fresh PBS, the cells were irradiated with a 405-nm LED at 5 J/cm2. Simultaneously, another plate containing cells exposed to 0 mg/L TONS504 without EDTA and without light irradiation served as the control. Cell viability was expressed as a percentage relative to this control. These experiments were repeated three times.

Primary porcine corneal explant model

A modified version of the primary porcine corneal explant model was developed based on a previously reported method [36]. Briefly, porcine eyes were prepared as described above. Corneal epithelial and stromal sheets (160 μm thickness, φ9.5 mm) were excised from the ocular surface using a Nidek MK-2000 microkeratome (NIDEK Co., Ltd, Kyoto, Japan) and microscissors. The discs were punched out with a φ2 mm biopsy trepan from the corneal sheets, and then four discs were placed on 6-well plates coated with type I collagen gel. The discs were observed and photographed (−18 hr) and incubated at 37°C with 5% CO2 for 18 hr. Once epithelial cells extended from the disc edges (defined as 0 hr), a cell extension assay was performed. The discs were exposed to various concentrations of TONS504 (0, 1, 10, or 100 mg/L) or BAC (0.001 or 0.0001%) as a positive control for 5 min in the dark. Following PBS rinsing and replacement with fresh PBS, the discs were irradiated with a 405-nm LED at 0, 5, 10, or 30 J/cm2. After irradiation, PBS was removed, and the plate was filled with the growth medium and incubated for 24 hr at 37°C with 5% CO2. The discs were imaged again at 24 hr. Cell extension was quantified using ImageJ software (National Institutes of Health; http://rsb.info.nih.gov/ij/). The cell extension rate was calculated by dividing the number of pixels at 24 hr by the number at 0 hr. These experiments were repeated three times.

The cell extension rate of porcine corneal discs exposed to various TONS504 concentrations (0, 1, 10, or 100 mg/L), with or without EDTA (0.015 M), under a blue light irradiation intensity of 5 J/cm2 was evaluated. The primary corneal explant assay was performed as described previously. The discs were exposed to various concentrations of TONS504 (0, 1, 10, or 100 mg/L) with or without EDTA (0.015 M) at room temperature for 5 min. Following PBS rinsing and replacement with fresh PBS, the discs were irradiated with a 405-nm LED at 5 J/cm2. Simultaneously, the discs in another plate were exposed to 0 mg/L TONS504 without EDTA under no blue light irradiation as a control. The cell extension rate was calculated as described previously. These experiments were repeated three times.

Statistical analyses

All statistical analyses were performed using GraphPad Prism software version 10.0 (GraphPad Software Inc., La Jolla, CA, USA). Data were shown as the mean ± standard deviation (sample size: n=3, cell viability assay; n=4, cell extension assay). Multiple group comparisons were performed using one-way analysis of variance with a post hoc Tukey’s test. To compare TONS504 cytotoxicity with or without EDTA at each concentration, Student’s t-test was applied. Statistical significance was set at P<0.05.

RESULTS

Intracellular uptake of TONS504

Figure 1 shows the fluorescence microscopy images of PCEP cells exposed to 10 mg/L TONS504 for 5 min. Although some uneven fluorescence was observed at low magnification, most cells were fluorescein negative, but TONS504 was taken up by some cells and localized predominantly in the cytoplasm.

Fig. 1.

Fig. 1.

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Intracellular uptake of TONS504. Intracellular uptake of TONS504 was observed using a fluorescence microscope. Cultured porcine corneal epithelial cells were exposed to 10 mg/L of TONS504 for 5 min. Although some fluorescence unevenness was noted at low magnification, TONS504 was taken up by the cells and localized in the cytoplasm. (a): phase-contrast image, (b): fluorescence image, and (c): merge image.

Cell viability assay

The viability of PCEP cells exposed to various TONS504 or BAC concentrations under different light irradiation intensities is shown in Fig. 2. Cell viability was dependent on TONS504 concentration and light irradiation intensity. At 0 J/cm2, the cell viability at 100 mg/L TONS504 and BAC 0.01% was significantly lower than that at control conditions (0 mg/L TONS504 at 0 J/cm2) (P=0.0075 and <0.0001, respectively). At 5 J/cm2, the cell viability at 10 and 100 mg/L TONS504 and BAC 0.001 and 0.01% was significantly lower than that at control conditions (P<0.0001, <0.0001, =0.043, and <0.0001, respectively). At 10 J/cm2, the cell viability at all TONS504 and BAC concentrations was significantly lower than that at control conditions (P<0.0001 for all except at BAC 0.0001%, P=0.0001). At 30 J/cm2, the cell viability at all TONS504 and BAC concentrations was significantly lower than that at control conditions (P<0.0001 for all except at BAC 0.0001%, P=0.0035). Compared to 0 mg/L at the same light irradiation intensity, at 5 J/cm2, the cell viability at 10 and 100 mg/L TONS504 and BAC 0.01% was significantly lower (P<0.0001 for all). At 10 J/cm2, the cell viability at 10 and 100 mg/L TONS504 and BAC 0.01% was significantly lower than that in PBS (P=0.0003, 0.0012, and 0.005, respectively). At 30 J/cm2, the cell viability at 10 and 100 mg/L TONS504 and BAC 0.01% was significantly lower than that in PBS (P<0.0001, <0.0001, and=0.0003, respectively).

Fig. 2.

Fig. 2.

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Cell viability after exposure to TONS504, blue light irradiation, or their combination. Cell viability of cultured porcine corneal epithelial cells exposed to various concentrations of benzalkonium chloride (BAC) or TONS504 under different light irradiation intensities is shown. Cell viability was dependent on TONS504 concentration and light irradiation intensity. Data are shown as mean ± standard deviation (n=3). Multiple comparisons were performed using a one-way analysis of variance with a post hoc Tukey’s test. Statistical significance was set at P<0.05. *P<0.05 compared to the control (0 mg/L at 0 J/cm2). #P<0.05 compared to 0 mg/L at the same light irradiation intensity.

The viability of PCEP cells exposed to various concentrations of TONS504, with or without EDTA, under blue light irradiation at 5 J/cm2 is shown in Fig. 3. Cell viability was dependent on TONS504 concentration. However, no significant differences were observed between the EDTA-treated and non-EDTA-treated groups at any TONS504 concentration (0 mg/L: P=0.058, 1 mg/L: P>0.99, 10 mg/L: P=0.57, and 100 mg/L: P=0.50).

Fig. 3.

Fig. 3.

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Cell viability after exposure to TONS504 with or without EDTA under blue light irradiation intensity of 5 J/cm2. Cell viability of cultured porcine corneal epithelial cells exposed to TONS504 (1, 10, or 100 mg/L) with or without ethylenediaminetetraacetic acid (EDTA, 0.015 M) under blue light irradiation at 5 J/cm2. Data are shown as mean ± standard deviation (n=3) and expressed as a percentage relative to the control (0 mg/L TONS504, 0 M EDTA, 0 J/cm2 light). Comparisons of the same TONS504 concentration with and without EDTA were performed using Student’s t-test. P<0.05 was considered statistically significant. No significant differences were observed between EDTA-treated and untreated groups at the same TONS504 concentrations.

Primary porcine corneal epithelial cell assay

Representative images of PCEP cell discs cultured for 24 hr after exposure to various TONS504 concentrations under different light irradiation intensities, along with BAC as a positive control, are shown in Fig. 4A. In the discs exposed to BAC, a concentration-dependent inhibition of cell extension was observed. At 0 J/cm2, TONS504 did not inhibit cell extension at any concentration. However, under blue light irradiation, TONS504 caused a concentration-dependent inhibition of cell extension.

Fig. 4.

Fig. 4.

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Primary porcine corneal explant model after exposure to various TONS504 concentrations under varying light irradiation intensities. A. Representative images of corneal epithelial cell discs cultured for 24 hr after exposure to various concentrations of benzalkonium chloride (BAC) (0.01 or 0.001%) or TONS504 (0, 1, 10, or 100 mg/L) under varying light irradiation intensities (0, 5, 10, or 30 J/cm2) are shown. Under blue light irradiation, TONS504 exhibited a concentration-dependent inhibitory effect on cell extension. B. Cell extension rate of porcine corneal epithelial cell discs exposed to various BAC (0.01 or 0.001%) or TONS504 (0, 1, 10, or 100 mg/L) concentrations under various blue light irradiation intensities (0, 5, 10, or 30 J/cm2) is shown. Cell extension rate was dependent on both TONS504 concentration and blue light irradiation intensity. Decreased cell extension rate was observed at ≥10 mg/L combined with ≥10 J/cm2. Data are shown as mean ± standard deviation (n=4). Multiple comparisons were performed using a one-way analysis of variance with a post hoc Tukey’s test. Statistical significance was set at P<0.05. *P<0.05 compared to the control (0 mg/L at 0 J/cm2), #P<0.05 compared to 0 mg/L at the same light irradiation intensity.

The cell extension rates of PCEP cell discs exposed to various TONS504 and BAC concentrations under different light irradiation intensities are illustrated in Fig. 4B. The cell extension rate was dependent on TONS504 concentration and light irradiation intensity. Compared to the control (0 mg/L TONS504 at 0 J/cm2), the cell extension rates at BAC 0.001 and 0.0001% were significantly lower (P=0.0046 and 0.013, respectively). At 5 J/cm2, the cell extension rates at 10 and 100 mg/L TONS504 were significantly lower than those in the control (P=0.032 and <0.0001, respectively). At 10 J/cm2, the cell extension rates at 10 and 100 mg/L TONS504 were significantly lower than those of the control (P=0.0002 and <0.0001, respectively). At 30 J/cm2, the cell extension rates at all TONS504 concentrations were significantly lower than that of the control (P=0.027, <0.0001, and <0.0001, respectively). Compared to 0 mg/L at the same light irradiation intensity of 5 J/cm2, the cell extension rate at 100 mg/L TONS504 was significantly lower (P=0.0005). At 10 J/cm2, the cell extension rates at 10 and 100 mg/L TONS504 were significantly lower than those at 0 mg/L (P=0.0012 and <0.0001, respectively). At 30 J/cm2, the cell extension rates at 10 and 100 mg/L of TONS504 were significantly lower than those at 0 mg/L (P=0.047 and 0.023, respectively).

The cell extension rate of PCEP cell discs exposed to various TONS504 concentrations with or without EDTA under blue light irradiation intensity of 5 J/cm2 is illustrated in Fig. 5. The cell extension rate was dependent on TONS504 concentration. At 1 mg/L TONS504, EDTA co-treatment significantly reduced the extension rate compared to treatment without EDTA (P=0.026). At other TONS504 concentrations (0, 10, and 100 mg/L), no significant differences were observed between EDTA-treated and untreated groups (P=0.70, 0.092, and 0.064, respectively).

Fig. 5.

Fig. 5.

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Cell extension rate of porcine epithelial cell discs exposed to various TONS504 concentrations with or without EDTA under blue light irradiation intensity of 5 J/cm2. Cell extension rate of porcine corneal epithelial cell discs exposed to TONS504 (1, 10, or 100 mg/L) with or without ethylenediaminetetraacetic acid (EDTA) (0.015 M) under blue light irradiation at 5 J/cm2 is shown. Data are presented as mean ± standard deviation (n=4). The control was defined as 0 mg/L TONS504 and 0 M EDTA at 0 J/cm2. Comparisons at the same TONS504 concentrations with and without EDTA were analyzed using Student’s t-test. P<0.05 was considered statistically significant. The cell extension rate at 1 mg/L TONS504 with EDTA was significantly lower than that without EDTA (P=0.026). *P<0.05.

DISCUSSION

To date, many studies have investigated the antimicrobial effects of PACT using various photosensitizers, light wavelengths, and light sources [3, 4, 23, 38, 44, 45]. TONS504-PACT has shown promising antimicrobial activity against various microorganisms in vitro [7, 8, 13, 21, 29, 30, 33, 34], suggesting its potential as a novel treatment for infectious keratitis. Although the cytotoxicity of TONS504-based photodynamic therapy has been evaluated in mouse mammary tumor cells [20], no prior studies have assessed its cytotoxic effects on normal cells such as corneal epithelial cells, keratocytes, and endothelial cells. To establish safe treatment protocols for TONS504-PACT in clinical practice, its cytotoxicity on corneal cells must be evaluated. Previous studies have examined the PACT cytotoxicity using riboflavin or rose Bengal on corneal cells, and their findings have contributed to the development of safe therapeutic conditions [19, 37, 40, 41]. The present study is the first to evaluate the cytotoxic effects of the combination of TONS504 and blue light irradiation on corneal epithelial cells.

In this study, we used a primary porcine corneal explant model to assess cytotoxicity. This method reflects the effects of a single exposure to TONS504 and blue light irradiation. It reportedly offers advantages over conventional methods, including higher sensitivity, the ability to simultaneously evaluate multiple test drugs, and procedural simplicity [36]. Furthermore, the migration and proliferation ability of corneal epithelial cells exposed to test drugs can be evaluated in terms of cytotoxicity [36]. Similar to this model, the usefulness of ocular toxicity assessment using primary corneal epithelial explant models, such as rabbit and bovine explants, has been reported [22, 26, 31]. Establishing a treatment protocol that preserves wound healing in corneal epithelial cells after PACT remains essential. The porcine explant model serves as an appropriate method for evaluating the cytotoxicity of PACT on corneal epithelial cells.

TONS504 was accumulated in a subset of PCEP cells with only 5 min of exposure (Fig. 1). In mouse mammary tumor cells, TONS504 was accumulated in the mitochondria and lysosomes after 4 hr of exposure [20]. The mechanism and rate of TONS504 cellular uptake remain unclear. Additionally, intracellular metabolism and excretion of TONS504 have not been clarified. Corneal epithelial cells that take up TONS504 are thought to sustain damage when exposed to specific wavelengths of light. However, in the treatment of infectious keratitis using PACT, the removal of central corneal epithelial cells allows photosensitizers to penetrate the cornea [43, 44]. Cellular damage can be mitigated by preventing TONS504 from contacting limbal and peripheral corneal epithelial cells, which remain intact, or by avoiding limbal irradiation at specific wavelengths.

Photosensitizers must not generate excessive ROS and should exhibit low cytotoxicity to host cells in the absence of light irradiation at specific wavelengths [3, 38]. The present study suggested that TONS504 alone (up to 10 mg/L) exhibits low cytotoxicity against corneal epithelial cells and minimally inhibits corneal epithelial wound healing (Figs. 2 and 4). Eye drops are diluted by tears; therefore, further in vivo studies remain warranted to determine dosages that account for cytotoxicity and tear dilution.

Blue light exerts greater cytotoxic effects on corneal and conjunctival epithelial cells than do other wavelengths of visible light [14, 16]. Additionally, blue light has a stronger antimicrobial effect than that of other visible light wavelengths [1, 11, 14]. ROS production has been reported as one of the causes of cytotoxicity [14, 16]. Although the degree of cytotoxicity is influenced by various factors, including wavelength, type of light source, or exposure time, even at the same irradiation intensity, a decrease in human corneal epithelial cell viability at doses ≥10 J/cm2 using blue LED light (410 nm, 10.75 mW/cm2) has also been reported [14, 16]. In the present study, high-intensity blue light alone showed a significant decrease in cell viability compared to controls (Fig. 2). Although blue light alone showed no significant reduction in the corneal epithelial extension rate compared to controls (P=0.99 for 5 J/cm2, P>0.99 for 10 J/cm2, and P=0.068 for 30 J/cm2), a downward trend was observed in cell extension under high-intensity blue light irradiation, consistent with previous reports (Fig. 4) [14, 16]. During PACT application, the corneal epithelial cells are removed. Therefore, the inhibitory effect on wound healing, which causes postoperative pain, was considered more important than the viability of corneal epithelial cells. Light irradiation intensity should be kept as low as possible while still ensuring sufficient antimicrobial efficacy. This study suggested that blue light irradiation at ≤5 J/cm2 is advisable.

This study evaluated cytotoxicity against PCEP cells at various TONS504 concentrations and blue light irradiation intensities. The results provided foundational data for establishing a safe protocol for TONS504-PACT using blue light. Cytotoxicity of TONS504-PACT using blue light was observed in a concentration-dependent manner or at high light irradiation intensity (Figs. 2 and 4). Furthermore, inhibition of cell migration and proliferation was observed, suggesting the possibility that wound healing may be inhibited after TONS504-PACT using blue light. The light irradiation intensity and TONS504 concentration must be optimized to minimize inhibition of corneal epithelial wound healing. This study showed that conditions set at 1 mg/L and 5 J/cm2 provided better outcomes for preserving corneal epithelial cell migration and proliferation (Fig. 4). However, under these conditions, the antimicrobial effect against P. aeruginosa resulted in only a 101 reduction, which may be insufficient [8]. Further investigation is needed to enhance the antimicrobial efficacy of TONS504-PACT, such as combining with additional preservatives or antibiotics, as reported for other photosensitizers [6, 43].

Studies have described combining TONS504 with EDTA or BAC to enhance the antimicrobial effect of PACT [8, 29]. EDTA reportedly has a stronger synergistic effect than that of BAC against P. aeruginosa in TONS504-PACT using red light [29]. Furthermore, TONS504-PACT containing 0.015 M EDTA produced antimicrobial effects at a blue light intensity of 5 J/cm2 that were equivalent to or greater than those achieved at 30 J/cm2 [8]. EDTA destabilizes the bacterial wall and releases lipopolysaccharides, increasing the permeability of photosensitizers and enhancing the antimicrobial efficacy of PACT [8, 29]. Using EDTA in combination with TONS504 is expected to achieve sufficient antimicrobial effects even under lower light irradiation intensity. Additionally, EDTA has lower cytotoxicity than that of BAC in corneal epithelial cells, although EDTA affects cell membranes at high concentrations [5]. Under blue light irradiation at 5 J/cm2, no significant difference in cell viability was observed between combinations with or without 0.015 M EDTA across TONS504 concentrations of 0, 1, 10, or 100 mg/L (Fig. 3). However, the cell extension rate of TONS504 at 1 mg/L with EDTA (0.015 M) significantly decreased compared with that at the same concentration of TONS504 without EDTA under blue light at 5 J/cm2 (Fig. 5). At the same concentration of TONS504, TONS504-PACT with EDTA (0.015 M) may have more cytotoxicity against corneal epithelial cells than treatment without EDTA. However, no clear synergistic cytotoxicity of TONS504 and EDTA under 5 J/cm2 blue light irradiation was observed. Although EDTA reportedly affects intercellular adhesion molecules and temporarily increases paracellular permeability [5], our results indicated that 0.015 M EDTA did not promote the transcellular internalization or intercellular accumulation of TONS504 under the conditions tested. It is presumed that higher EDTA concentrations may enhance the intracellular uptake of TONS504, potentially exacerbating cytotoxicity. Therefore, using concentrations >0.015 M, as in this study, is not recommended. Since the results in this study were verified only under limited conditions, future evaluations should assess cytotoxicity at various EDTA concentrations and irradiation intensities. BAC was used only as a positive control for cytotoxicity in this study. Evaluation of the cytotoxic effects of TONS504 combined with BAC under blue light irradiation on corneal epithelial cells remains necessary in future studies.

This study had some limitations. First, the results were based on a small sample size, necessitating additional studies with larger sample sizes. Investigating more stable cell culture methods is necessary. Second, specific factors responsible for the cytotoxicity of TONS504-PACT could not be identified. A more detailed evaluation of cytotoxicity and wound healing inhibition would have been possible by measuring the amount of ROS generated and levels of cytokines secreted by corneal epithelial cells [19, 25, 32, 37]. Third, the cytotoxicity of TONS504-PACT was evaluated using PCEP cells. Previous studies have reported that sensitivity to drug-induced cytotoxicity varies depending on the animal species or cell type [2, 24]. Future studies should evaluate the feasibility of extrapolating these results to human and canine corneal epithelial cells. Fourth, evaluation of whether the PCEP cells used maintain the properties of corneal epithelial cells is lacking. Notably, the properties of corneal epithelial cells change with repeated subculture [2, 18]. Furthermore, the explant model may contain both epithelial and stromal cells. Although this assay may more closely reflect in vivo conditions, these results should be interpreted with caution. In both experimental systems, it was necessary to confirm that the cells were corneal epithelial cells via immunostaining. Fifth, this study was performed in presumably healthy corneal cells. In diseased cornea, there would likely be additional factors, including inflammation, corneal opacity, and vascularization. Future studies should evaluate the cytotoxicity of TONS504 and blue light against corneal epithelial cells in bacterial keratitis corneas.

In conclusion, the cytotoxicity of TONS504 combined with blue light irradiation was evaluated using PCEP cells. The results demonstrated concentration-dependent cytotoxicity or cytotoxicity at high light intensities, providing fundamental data for establishing a protocol for TONS504-PACT using blue light. Based on the findings, treatment with 1 mg/L TONS504 under 5 J/cm2 blue light is recommended. Additionally, the synergistic cytotoxicity of TONS504 and EDTA under blue light irradiation was assessed. Although greater cytotoxicity against corneal epithelial cells was observed with the combination of TONS504 and EDTA, no clear synergistic cytotoxicity was found. Further studies should evaluate the feasibility of extrapolating these results to human and canine corneal epithelial cells for establishing a protocol for TONS504-PACT using blue light.

Conflicts of Interest

The authors declare no conflict of interest.

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