Blue Light-Based Method to Induce Oxidative Stress on Rabbit Corneal Epithelial (RCE) Cells: Development and Validation

Abstract

Daily exposure to blue light emitted by digital devices has raised concerns about oxidative stress-mediated damage to the ocular surface. Despite growing interest, validated in vitro models to study blue light-induced oxidative stress in corneal epithelial cells remain limited. A reproducible in vitro method was developed using rabbit corneal epithelial (RCE) cells exposed to blue LED light (405 nm). Irradiation parameters were optimized to induce oxidative stress without causing overt cytotoxicity. Cellular viability, intracellular ROS production, and mitochondrial oxidative stress were assessed. The model was validated using reference antioxidants (ascorbic acid and oleuropein), oleuropein formulated in a drug-in-cyclodextrin-in-liposome system (OLE-DCL), and two commercial ophthalmic formulations applied before or after irradiation. Blue light irradiation at 4.57 W/m² for 30 min significantly increased intracellular and mitochondrial ROS levels while preserving cell viability, indicating sublethal photo-oxidative stress. Ascorbic acid effectively suppressed ROS generation, whereas free oleuropein showed reduced efficacy, likely due to photosensitivity. OLE-DCL significantly enhanced antioxidant activity under irradiation. The model also discriminated between protective and restorative treatment strategies. This study establishes a validated in vitro blue light-induced oxidative stress model for corneal epithelial cells, suitable for screening antioxidant compounds, formulations, and application strategies relevant to ocular surface protection.

1. Introduction

In recent years, daily exposure to blue light from artificial sources has become unavoidable due to the widespread use of electronic devices, which has increased significantly over the past decade [1]. Patients who frequently use electronic devices often experience a range of short-term ocular and visual discomfort symptoms, including sore and dry eyes, as well as burning, itching, and stinging sensations [2].

Blue light refers to electromagnetic radiation within the visible spectrum with wavelengths ranging from 380 to 500 nm. Alongside UV-A radiation, it is classified as high-energy visible (HEV) light due to its high photon energy characteristics [3]. Because of its high energy, blue light can damage the ocular surface, penetrate the cornea and lens, reach the retina, and cause retinal cell damage. Prolonged exposure to blue light has been associated with oxidative stress, mitochondrial dysfunction, inflammation-mediated apoptosis, and DNA damage, thereby contributing to the development of ocular pathologies such as dry eye, glaucoma, and keratitis [4].

The extent of blue light-induced damage depends on several factors, including radiation intensity and wavelength, distance from the light source, and direction of exposure. Wavelengths between 400 and 455 nm are considered more harmful than those closer to 500 nm due to the higher energy of photons in the 400 nm range, which increases their ability to interact with and modify biological molecules [5,6]. The underlying mechanism primarily involves increased production of reactive oxygen species (ROS) within the cells of the ocular surface and retina. Highly reactive species such as hydrogen peroxide and superoxide anion damage macromolecules, including proteins, lipids, and DNA, ultimately leading to cellular apoptosis [7]. This cascade of events may initiate or exacerbate ocular conditions like keratitis, pterygium, cataracts, and corneal and retinal degeneration [8].

The cornea is the first structure of the eye exposed to incident light and absorbs the majority of UV light, but only about 3% of blue light [3,5]. Interaction of blue light with corneal epithelial cells triggers oxidative damage and cellular apoptosis through increased ROS production and subsequent mitochondrial dysfunction. This process is associated with elevated levels of superoxide anion and the activation of the pro-inflammatory pathway including activation of the NLRP3 inflammasome. Activation of the NLRP3 inflammasome leads to the release of active interleukin-1 and the secretion of interleukin-6, reducing mucin secretion and increasing tear film instability, thereby promoting its evaporation [6,8]. Inflammation in the corneal epithelium results in the loss of its defensive barrier functions, contributing to the pathogenesis of eye diseases such as dry eye syndrome [9]. Additionally, blue light induces an increase in the synthesis of malondialdehyde, a reactive intermediate of lipid peroxidation, causing DNA damage, and the activation of protein kinase P38, leading to apoptosis of corneal epithelial cells [8].

While the damage to the retina caused by blue light has been extensively investigated, there is still insufficient data regarding its effects on the ocular surface.

The objective of this study was to develop and validate an in vitro method to reproduce ocular damage induced by blue light irradiation using a rabbit corneal cell line as a model. The experimental protocol was optimized by determining cell viability and mitochondrial oxidative stress measurements after irradiation with blue light, modulating the irradiance through variation in the light voltage and the distance between the light source and the substrate, as well as irradiation and post-irradiation times. The effect of known antioxidants (ascorbic acid and oleuropein) and commercial products (Blu Yal A Free and Thealoz) used to mitigate or repair blue light-induced oxidative stress was evaluated under these conditions. Furthermore, this method was used to investigate the protective and repairing effects of an experimental formulation.

This study aims to fill the gap in knowledge of blue light-induced damage to the ocular surface and to provide a validated in vitro model for assessing potential protective and therapeutic interventions.

2. Materials and Methods

2.1. Materials

2.1.1. Chemicals

The following materials were used: cell proliferation reagent (WST-1; Roche Diagnostic, Monza, Italy); 2,7-dichlorofluorescein diacetate (DCF-DA), oleuropein (OLE), ascorbic acid (AA), and cholesterol, all from Sigma-Aldrich (St. Louis, MO, USA); MitoSOX™ Red assay (Life Technologies, Milan, Italy); hydroxypropyl-β-cyclodextrin parenteral grade (Kleptose^®; Roquette Freres, Lestrem, France); phosphatidylcholine (Lipoid^® S 100; Lipoid, GmbH, Ludwigshafen, Germany); Blu Yal A Free (Fidia Farmaceutici S.p.A, Abano Terme, Italy); and Thealoz (Thea Pharma S.p.A., Milan, Italy).

2.1.2. Cell Culture

For this study, a rabbit corneal epithelial cell line (RCE n. 95081046) obtained from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK) was used.

The growth medium had the following composition: Dulbecco’s modified Eagle’s medium with Ham’s nutrient mixture F12 (1:1) (DMEM/F12) with the addition of L-glutamine (1% v/v, 2 mM), penicillin (100 IU/mL), streptomycin (0.1 mg/mL), amphotericin B (0.25 µg/mL), fetal bovine serum heat-inactivated (15% v/v), insulin (5 µg/mL), and epidermal growth factor (10 ng/mL), all from Sigma-Aldrich (St. Louis, MO, USA). Cells were grown at 37 °C in a humidified atmosphere with 5% CO₂ and used at passages 13 to 17.

2.2. Methods

2.2.1. Irradiance Measurements

The experimental setup involved irradiating cells with an LED source at a wavelength of 405 nm. Although commercial digital displays typically exhibit a blue emission peak around 445–455 nm [10], a 405 nm LED was selected for this model. This wavelength lies at the boundary between violet and blue light and possesses higher photon energy compared with longer blue wavelengths, providing a conservative and effective experimental condition to induce measurable oxidative stress in vitro. This wavelength has been widely used in cellular models to generate dose-dependent reactive oxygen species (ROS) and redox-associated biochemical changes, making it a suitable and controllable tool for investigating oxidative mechanisms [11]. From a practical standpoint, narrow-band 405 nm LEDs allow precise control of irradiance and experimental stability, facilitating standardization of the irradiation protocol.

Irradiation conditions were optimized by varying parameters such as the distance between the light source and the cell surface (12.5 and 18.5 cm), the duration of irradiation (30, 60, and 180 min), and the supply voltage (8 and 16 V).

The irradiance at the surface was quantified using a photo-radiometer (HD2302.0, Delta OHM, Padua, Italy) for all combinations of lamp-to-cell distance and applied voltage.

Additionally, irradiance measurements across the entire plate helped identify a uniform and high-irradiance area.

2.2.2. Blue Light Irradiation

The irradiation protocol involved seeding RCE cells at a concentration of 3 × 10⁴ cells/well in 96-well plates followed by 24 h of incubation to achieve confluence. Subsequently, the medium was completely aspirated, substituted with fresh growth medium (L-CTR) or medium added with the product under test (PRE-treatment); then, the cells were submitted to blue light irradiation.

Throughout the experiment, the position of the plates beneath the light source was kept constant, and control columns were maintained in darkness (D-CTR) by covering them with opaque foil.

The apparatus used for blue light irradiation is schematically depicted in Figure 1. It consists of a stabilized power supply connected to an LED light source and a plate holder enclosed in an opaque wooden box. The stabilized power supply ensures controlled energy delivery to the light source, maintaining consistent irradiation conditions throughout the experiment. Blue LED exposure was performed at room temperature.

This setup provides a controlled and reproducible environment for conducting blue light irradiation experiments.

2.2.3. Cell Viability

Cell viability of RCE cells after blue light irradiation was assessed by the colorimetric method using cell proliferation reagent WST-1, a commercially available cell proliferation reagent. Being a substrate of mitochondrial dehydrogenase, WST-1 is cleaved to formazan salt, a cherry red-colored and water-soluble compound. Only viable cells possess mitochondrial dehydrogenase; therefore, the staining is proportional to the viable cell number.

The RCE cells were plated in 96-well plates and submitted to irradiation as described before. Immediately after irradiation or following a 24 h recovery period, 10 µL of WST-1 was added and the cells were incubated for 2 h at 37 °C in a humidified atmosphere with 5% CO₂. At the end of the incubation, the microplate was shaken for 1 min, and finally, the absorbance was determined at 450 nm using a microplate reader (Asys UVM 340; Biochrom, Cambridge, UK). The background absorbance was measured on wells containing only the dye solution and the culture medium, and the results were expressed as a percentage of the absorbance of irradiated versus no-irradiated (D-CTR) wells.

Cell viability was evaluated in different experimental conditions to assess the light exposure that would lead to cell alterations. For this purpose, different exposure times (30, 60 and 180 min) as well as different irradiance values (1.50, 2.61 and 4.57 W/m²) were assessed.

2.2.4. Oxidative Stress Assessment

The quantification of reactive oxygen species (ROS) generated following cell exposure to blue light was performed using DCF-DA (2,7-dichlorofluorescein diacetate) reagent. Upon cellular uptake, this reagent undergoes deacetylation by intracellular esterases, converting into a non-fluorescent compound 2,7-dichlorodihydrofluorescein. Subsequent oxidation by intracellular ROS results in the formation of the fluorescent molecule 2,7-dichlorofluorescein (DCF). Preparation of the reagent involved dissolving DCF-DA in ethanol at a concentration of 1 mM, followed by overnight stirring to ensure complete solubilization. The ethanolic solution was then diluted in isotonic phosphate buffer pH 7.4 (PBS) at a ratio of 1:100 to obtain a 10 µM dye solution.

The ROS test was performed on cells exposed to blue light at an irradiance of 4.57 W/m² for 30 min after a 24 h incubation period post irradiation. Cells were washed twice with PBS, 100 µL/well of the 10 µM DCF-DA solution was applied and the cells were incubated for 45 min. After the incubation, the fluorescence was monitored at excitation/emission wavelengths of 480/585 nm using a Varioskan multimode microplate reader (Life Technology Italia, Milan, Italy). The background absorbance was measured on wells containing only the dye solution, and the results were expressed as a percentage of the emitted fluorescence of irradiated versus no-irradiated wells.

Furthermore, mitochondrial superoxide production was measured using the MitoSOX™ Red assay (Life Technologies, Milan, Italy). After the 24 h recovery period, cells were washed twice with PBS, incubated with 1 μM MitoSOX reagent for 30 min at 37 °C in DMEM without phenol red, washed again with PBS, and supplemented with 100 μL of DMEM without phenol red. Images were acquired using an Operetta CLS high-content imaging device (PerkinElmer, Hamburg, Germany) and analyzed with Harmony 4.6 software (PerkinElmer, Hamburg, Germany). Imaging was performed at 63× magnification, capturing 48-64 fields per sample in at least three biological and technical replicates. Three channels were collected: nuclei, cytoplasmic MitoSOX fluorescence, and merged composites. Quantitative parameters extracted from Operetta analysis software included MitoSOX cytoplasmic intensity (cyt), MitoSOX spot intensity (spot) analyzed separately and expressed as Δ fluorescence relative to the respective non-irradiated control, which was set to zero.

Ratio N/C and average mitochondria per cell were analyzed as raw values to preserve their morphological significance.

The antioxidant compounds tested on RCE cells were ascorbic acid (AA, 0.1 mg/mL, reference), oleuropein (OLE, 0.2 mg/mL) and an oleuropein-in-cyclodextrin liposome complex containing 0.14, 0.20, and 0.24 mg/mL of OLE (OLE-DCL0.14, OLE-DCL0.2, and OLE-DCL0.24, respectively). AA and OLE concentrations were selected based on the previously published literature. The AA concentration was chosen in accordance with the range reported by Oh et al. [12], who demonstrated antioxidant efficacy in comparable in vitro models. The OLE concentration was selected based on our previous publication, in which 0.2 mg/mL was shown to exert significant antioxidant activity without inducing cytotoxic effects [13].

Additionally, the activity of two commercial products was evaluated, Blu Yal A Free and Thealoz. They are medical devices recommended in case of discomfort, foreign body sensation, or eye irritation due to ocular dryness. Among the causes of such symptoms listed in their leaflet, prolonged use of blue light-emitting devices, such as computers, is also referenced. In this case, the evaluation of ROS was conducted by applying commercial products after dilution in growth medium at a ratio of 1:1 either before or after blue light irradiation. This dilution ensured compatibility with cell culture conditions (nutrient availability, osmolarity, and buffering capacity) while preserving the functional characteristics of the formulations. Preliminary optimization indicated that complete replacement of culture medium with the undiluted products was not suitable for maintaining RCE viability over the experimental time frame.

2.2.5. Statistical Analysis

All experiments were performed in at least six independent biological replicates. All data are expressed as the mean ± standard error, and the analysis was performed by GraphPad Prism Software (version 8.0.2, San Diego, CA, USA).

Statistical differences were evaluated for two groups by applying Student’s two-tailed unpaired t-test. Statistical analyses for multiple groups were determined by two-way ANOVA followed by either Tukey’s or Sidak’s multiple-comparison post hoc test.

Significance thresholds were set at * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

3. Results

3.1. Irradiance Measurements

The measurement of irradiance at the plate surface enabled the determination of the values reported in

Table 1. Irradiance values (high-irradiance zone) obtained with different combinations of distance between light source and cell surface, and voltage.

Light Source–Cells Distance, cm	Voltage, V	Irradiance, W/m2 ± SE
12.5	8	2.61 ± 0.06
	16	4.57 ± 0.08
18.5	16	1.50 ± 0.09

.

Spatial mapping of irradiance across the entire plate surface enables the identification of a uniform high-irradiance area used for cell exposure, as well as a low-irradiance zone where dark control samples were positioned. The establishment of a spatially uniform irradiation zone is a crucial aspect of the experimental setup, as it ensures that all irradiated cells are exposed to comparable light intensities, thereby improving experimental reproducibility and data reliability.

Based on these measurements, the range of irradiance levels applied in this study (1.50–4.57 W/m²) was selected to generate controlled blue light exposures able to induce measurable oxidative responses in vitro, rather than to mimic typical ambient exposure conditions. Under everyday viewing scenarios, digital displays such as smartphones, tablets, and computer monitors emit substantially lower levels of blue light at the eye, typically corresponding to approximately 0.5–5 W/m² for total visible irradiance and an estimated 0.1–1 W/m² for the blue spectral component at common viewing distances (approximately 25–70 cm) [10,14].

Since in vitro cellular systems often require higher and well-defined irradiance levels to elicit detectable oxidative stress endpoints, the selected irradiance range is appropriate for investigating blue light-induced cellular responses while remaining within one order of magnitude of real-world exposures from electronic displays. This experimental approach is consistent with previous studies that employed elevated irradiance levels to investigate photochemical effects and reactive oxygen species-related mechanisms induced by blue light [15].

3.2. Cell Viability After Blue Light Treatment

The initial phase of this study aimed to assess the effects of various irradiance levels on cellular viability. Cell viability tests were performed immediately after irradiation under different conditions: 1.50, 2.61, and 4.57 W/m² extended for 30 and 60 min. The results revealed a slight decrease in cell viability following irradiation regardless of radiance and time of exposure, with a cell viability that ranged between 87.75 ± 4.02 and 92.51 ± 2.29% (Figure 2). However, these differences were not statistically significant compared with the non-irradiated control samples (two-way ANOVA followed by Tukey’s multiple-comparison post hoc test). The results suggest that these treatment conditions did not provoke any lethal damage to the cells.

To further evaluate potential delayed damage, the experiments were repeated using the same irradiation protocol followed by a 24 h recovery period after irradiation to allow the cells either to return to a physiological state or undergo apoptosis. Even under these conditions, treatment with the highest irradiance (4.57 W/m²) for 60 min did not prove cytotoxic on RCE cells, with cell viability values ranging between 86.87 ± 3.33 and 95.95 ± 3.28% without significant statistically differences (Figure 3), again without significant statistically differences compared with dark controls.

Cytotoxicity was subsequently evaluated under more extreme conditions: irradiance 4.57 W/m² for 180 min. Cell viability tended to decrease, although not significantly, reaching values of 81.61 ± 1.51%. These conditions were applied to verify whether blue light, at the highest irradiance used in this study, was harmful to cells when exposure was extremely prolonged. Nevertheless, the results obtained were not significantly different from those obtained with shorter irradiation times.

Considering that longer treatments could be extremely critical in terms of environmental conditions unsuitable for maintaining the cell line (overheating, high evaporation, non-aseptic conditions) and that no significant differences in cell viability values were found, this study continued by applying blue light at an irradiance of 4.57 W/m² for 30 min.

These results corroborate findings by Marek et al. [16] who treated human corneal epithelial (HCE) cells with blue light at 420 nm and an irradiance of 11.3 W/m² (1.13 mW/cm²) with an exposure period of 17 h. They reported a decrease in cell viability; however, after a recovery period of only four hours in the dark, the HCE showed signs of recovery from the damage. It is worth noting that any phototoxicity was detected in RCE cells in the present study, likely due to the lower irradiance and shorter exposure time compared to that of Marek and colleagues [16].

3.3. Oxidative Stress Assessment After Blue Light Treatment

In the second phase of this study, blue light-induced damage was evaluated in terms of oxidative stress. RCE cells were irradiated for 30 min at an irradiance of 4.57 W/m² to determine whether these conditions were sufficient to induce cellular stress without causing overt cell death. Under these experimental conditions, irradiated RCE cells showed a significant increase in intracellular ROS production compared to non-irradiated controls, reaching 136.47 ± 3.18% (t-test, p < 0.0001). The ROS assay was performed after a 24 h recovery period, consistently with the cell viability experiments. Since the selected irradiation conditions were demonstrated to be sublethal, the 24 h interval allowed the evaluation of sustained oxidative stress associated with cellular response and potential delayed damage, rather than the immediate transient ROS burst generated during irradiation. The persistence of significantly elevated ROS levels at this time point indicates that blue light exposure induced a stable redox imbalance rather than a short-lived photochemical event.

Mitochondria play a key role as essential organelles for cellular function and are the primary source of ROS production [17,18], so mitochondrial superoxide production was further investigated by MitoSOX-based high-content imaging. Blue light exposure resulted in a marked increase in mitochondrial oxidative stress, as evidenced by elevated cytoplasmic and mitochondrial spot fluorescence intensities (Figure 4). Notably, this increase occurred without significant alterations either in the nuclear-to-cytoplasmic (N/C) ratio or in the average number of mitochondria per cell when compared with D-CTR cells (N/C = 0.5332 ± 0.0065 and 0.5663 ± 0.0110; average mitochondria per cell = 92.01 ± 2.99 and 76.54 ± 4.94, respectively), indicating the absence of high structural or proliferative disruptions.

Morphological analysis by high-content imaging provided qualitative confirmation and mechanistic support for the quantitative data. Representative fluorescent images acquired by Operetta CLS imaging (63×) revealed evident morphological differences between dark control (D-CRT) and blue light-irradiated RCE cells (Figure 5).

In control conditions, RCE cells displayed a typical epithelial morphology characterized by high confluence, smooth and well-defined cell boundaries, and uniformly stained oval nuclei, consistent with previously reported features of healthy corneal epithelial cells in vitro [19]. Chromatin appeared homogeneously distributed, while cytoplasmic staining revealed a fine and interconnected mitochondrial reticulum, characteristic of non-stressed epithelial cells [20].

In contrast, exposure to blue light induced hallmark features of photo-oxidative damage. Nuclei appeared enlarged, irregularly shaped, and frequently lobulated, with heterogeneous chromatin distribution and bright intranuclear foci suggestive of chromatin condensation or early DNA damage, alterations widely associated with phototoxic or oxidative stress conditions [21,22,23]. The cytoplasmic fluorescence signal became more intense, granular, and heterogeneous, consistent with mitochondrial fragmentation, perinuclear clustering, and loss of reticular mitochondrial organization. These mitochondrial alterations represent a well-recognized cellular response to oxidative and phototoxic stress. In parallel, a reduction in cell density was observed, in agreement with previous reports describing light-induced cytotoxic effects in corneal epithelial cells [16,20].

Overall, these results demonstrate that blue light irradiation under the selected experimental conditions elicits a robust and reproducible oxidative stress response in RCE cells, characterized by increased intracellular and mitochondrial ROS production, mitochondrial structural remodeling [24], and clear morphological signs of photo-oxidative injury, while preserving overall cell viability. Importantly, the lack of substantial changes in the nuclear-to-cytoplasmic ratio and mitochondrial number per cell indicates that the applied irradiation protocol induces sublethal cellular damage rather than extensive cytotoxicity. This aspect is critical to set up a reliable vitro model of blue light-induced stress, as it enables the investigation of protective or modulatory interventions without confounding effects associated with massive cell loss.

On this basis, the selected irradiation conditions were considered suitable for the subsequent experimental phase aimed at validating the methodology through the evaluation of antioxidant compounds, ascorbic acid (AA) and oleuropein (OLE) [25,26], allowing an assessment of their ability to mitigate blue light-induced oxidative damage at both the cellular and mitochondrial levels.

AA and OLE are known to exert antioxidant activity through multiple and complementary mechanisms, including direct free radical scavenging, modulation of cellular redox homeostasis, and stimulation or activation of endogenous antioxidant defenses [27,28]. Ascorbic acid represents a prototypical low-molecular-weight antioxidant capable of rapidly neutralizing reactive oxygen species, thereby limiting oxidative damage at the cellular level. Oleuropein, a major phenolic compound of olive oil, also displays direct antioxidant properties and has been reported to attenuate oxidative stress by modulating intracellular redox-sensitive pathways and mitochondrial-associated ROS generation.

The combined use of AA and OLE therefore allows the validation of the proposed experimental model across distinct yet biologically relevant antioxidant mechanisms. In particular, their complementary modes of action allow for evaluating the sensitivity of the model to both immediate ROS scavenging and modulation of intracellular oxidative stress, as assessed by measuring total ROS levels and mitochondrial superoxide production.

Treatment of RCE cells with AA (0.1 mg/mL) or OLE (0.2 mg/mL) under dark conditions did not significantly modify basal ROS levels compared with untreated dark controls (Figure 6).

Importantly, the lack of significant changes in basal ROS levels upon AA or OLE treatment under dark conditions indicates that the selected concentrations do not disrupt the physiological redox state of RCE cells, supporting their use as suitable antioxidant agents to test the model under blue light-induced oxidative stress.

Blue light irradiation induced a marked increase in intracellular ROS levels in untreated RCE cells compared with the corresponding dark control (p < 0.0001, Figure 6). Pre-treatment with ascorbic acid (AA, 0.1 mg/mL) significantly attenuated blue light-induced ROS accumulation, reducing relative ROS levels to 95.86 ± 2.89% compared with irradiated control cells (p < 0.0001). Notably, ROS levels in AA-treated irradiated cells were not significantly different from those measured under dark conditions, indicating an almost complete suppression of the oxidative response induced by blue light.

Oleuropein (OLE, 0.2 mg/mL) also significantly reduced ROS levels under blue light irradiation compared with irradiated control cells, although to a lesser extent than AA. In OLE-treated irradiated cells, relative ROS levels reached 116.4 ± 2.45%, remaining significantly higher than those observed in AA-treated irradiated cells and dark control conditions (p < 0.0001). This behavior may be related to the reported photosensitivity of oleuropein, which has been shown to undergo partial photodegradation under light exposure [29,30].

Based on the limited efficacy of free oleuropein (OLE) under blue light irradiation, OLE was incorporated into a drug-in-cyclodextrin-in-liposome system (OLE-DCL), as previously reported to protect oleuropein from photodegradation [13]. Then, the antioxidant activity of OLE-DCL, at increasing OLE concentrations (0.14, 0.20 and 0.24 mg/mL), and the DCL system alone was evaluated.

Figure 7 shows that OLE incorporation into the DCL system resulted in a concentration-dependent reduction in ROS levels under irradiation. All tested treatments significantly reduced ROS levels compared to irradiated control cells (p < 0.0001). Among them, OLE-DCL0.24 produced the strongest antioxidant effect, yielding the lowest ROS levels under irradiation (78.75 ± 1.78%).

These results indicate that formulation of OLE within the DCL system preserves its antioxidant activity under photo-oxidative conditions, probably protecting the molecule from photodegradation and ensuring its availability at the cellular level.

The DCL carrier alone exerted a significant protective effect, lowering ROS to physiological levels (91.48 ± 1.83%). This behavior may be partly attributable to light attenuation mechanisms (e.g., scattering/shielding), consistent with the milky appearance of DCL dispersions, which could reduce the effective irradiance reaching the cell monolayer. Similar effects have been described for particulate and lipid-based systems and are particularly relevant in photo-oxidative experimental models [31].

Under dark conditions, ROS levels were generally comparable across treatments, confirming that neither free OLE nor OLE-DCL formulations markedly perturbed the basal redox state of RCE cells. However, the OLE-DCL0.24 group displayed lower ROS than some dark controls, suggesting a possible contribution of formulation-dependent redox modulation or assay-related effects at the highest concentration.

As a final step of this study, we evaluated whether the developed experimental protocol was able to discriminate between protective and restorative effects against blue light-induced cellular damage. To this aim, two commercially available ophthalmic products, Blu Yal A Free and Thealoz, were investigated by adding them to the culture medium either before blue light irradiation (PRE-irradiation, protective setting) or immediately after irradiation (POST-irradiation, restorative setting). Blu Yal A Free and Thealoz were selected for this study based on the indications reported in their respective product information leaflets, which describe their use in conditions associated with ocular discomfort and visual stress, including prolonged screen exposure.

The obtained results are showed in Figure 8. ROS levels observed under PRE-irradiation conditions were comparable to those of the irradiated control (L-CTR), indicating that pre-treatment appeared not to prevent blue light-induced oxidative stress. Two-way ANOVA revealed a significant main effect of the treatment timing on intracellular ROS levels, with POST-irradiation treatment resulting in lower ROS levels compared with PRE-irradiation application (p < 0.0001). A significant main effect of the product was also observed (p = 0.0395), whereas the interaction between product and treatment timing did not reach statistical significance (p = 0.0706), indicating that differences between products should be interpreted cautiously.

A post hoc Sidak’s multiple-comparison test demonstrated that POST-irradiation treatment with Thealoz resulted in a significant reduction in ROS levels compared with PRE-irradiation treatment (109.36 ± 1.77% vs. 136.64 ± 2.39%, p = 0.0001), indicating a clear restorative effect following blue light-induced oxidative stress. Blu Yal A Free also reduced ROS levels when applied after irradiation (126.81 ± 2.58% vs. 137.80 ± 7.44%), although this effect did not reach statistical significance (p = 0.1401).

The interaction plot (Figure 9) further supports these results, showing a more pronounced reduction in ROS levels from PRE- to POST-irradiation treatment for Thealoz compared with Blu Yal A Free. The results suggest a product-dependent difference in the magnitude of POST-irradiation recovery, even if the lack of a statistically significant interaction indicates that this effect should be interpreted as a tendency rather than a definitive differential response.

Overall, these results indicate that the timing of application plays a critical role in determining the efficacy of commercial formulations against blue light-induced oxidative stress. In particular, Thealoz demonstrates a significant ability to reduce intracellular ROS when applied after irradiation, consistent with a restorative mechanism that mitigates oxidative damage rather than preventing its formation. Blu Yal A Free, while showing a comparable qualitative trend, did not achieve a statistically significant effect under the same conditions.

Together, the obtained results highlight the relevance of post-irradiation treatment strategies and further support the suitability of the present in vitro model for discriminating between protective and restorative antioxidant activities.

4. Conclusions

The present study describes the development and validation of a reproducible in vitro model of blue light-induced oxidative stress in corneal epithelial cells, designed to investigate both protective and restorative strategies against photo-oxidative damage. By carefully tuning irradiance, exposure time, and recovery conditions, irradiation parameters that induce a robust oxidative response while preserving cell viability and mitochondrial integrity were established. This feature represents a critical strength of the model, as it allows the evaluation of antioxidant interventions under sublethal, biologically relevant stress conditions.

Within this experimental framework, two complementary approaches were pursued to validate the responsiveness and discriminative power of the model. The first focused on the assessment of the antioxidant activity of oleuropein, a natural polyphenol with well-documented redox-modulating properties. Oleuropein reduced blue light-induced ROS accumulation, although its efficacy was limited by its known photosensitivity [29,30]. Formulation of oleuropein into a drug-in-cyclodextrin-in-liposome (OLE-DCL) system markedly enhanced its antioxidant performance under irradiation, supporting the importance of formulation strategies in preserving the activity of photolabile antioxidants. These results highlight the suitability of the proposed model for investigating formulation-dependent modulation of antioxidant efficacy under photo-oxidative conditions.

The second approach addressed application of the model to commercially available ophthalmic medical devices. Blu Yal A Free and Thealoz were selected because their declared indications include ocular discomfort associated with visual and environmental stress, providing a rationale for testing them within a blue light irradiation context. The model successfully discriminated between protective and restorative antioxidant activities.

Overall, this study establishes a versatile in vitro platform for investigating blue light-induced oxidative stress at the ocular surface. By integrating quantitative ROS measurements, mitochondrial analysis, and controlled irradiation parameters, the proposed method provides a sensitive tool for screening antioxidant compounds, formulations, and application strategies. Beyond the specific agents tested here, this model may support future research aimed at guiding the development and optimization of therapeutic and preventive approaches against blue light-associated ocular stress.