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Human Corneal Epithelial Cells

Understanding the Effects of Benzalkonium Chloride Exposure on the Barrier Function of Human Corneal Epithelial Cells

/ by Lifeline® Cell Technology

Healthy Vision Month

It’s often said that the eyes are the window to the soul, but they are also a window to overall health. During routine eye exams, clinicians can directly observe blood vessels and nerves, offering early insight into conditions such as diabetes, cardiovascular disease, multiple sclerosis, and even certain cancers.

Our eyes are also routinely exposed to ophthalmic solutions designed to treat, clean, or lubricate, including contact lens solutions, eye drops, and ointments. Many of these products contain preservatives, such as benzalkonium chloride (BAK), to maintain sterility and extend shelf life. However, their long-term effects on delicate ocular tissues are not fully understood and may have important implications for eye health.

This has led researchers to examine how repeated, low-level exposure to these compounds impacts the integrity and function of the corneal epithelium over time. A recent study by Zheng et al. evaluated the effects of low concentrations of BAK on the health and function of human corneal epithelial cells.

Evaluating BAK Toxicity at Sub-CMC Concentrations

BAK is the most widely used preservative in ophthalmic formulations, present in over 70% of these medications at concentrations ranging from 0.005% to 0.05%. However, prolonged exposure to BAK has been associated with ocular surface damage, including dry eye symptoms, goblet cell loss, and impaired epithelial barrier function. Mechanistically, BAK toxicity arises from its interaction with negatively charged phospholipids, leading to disruption of cell membranes. Its critical micelle concentration (CMC) in aqueous media is approximately 0.01%.

While most toxicity studies have focused on concentrations that rapidly disrupt membranes, less is known about how exposure at sub-CMC levels can drive progressive toxicity. Conventional viability assays, which rely on cell death endpoints, often miss the slower, sublethal mitochondrial stress that can gradually impair epithelial function.

To address this, Zheng et al. evaluated the effects of BAK on barrier integrity and mitochondrial function using primary human corneal epithelial cells (HCECs). HCECs obtained from Lifeline Cell Technology were cultured in OcuLife® Corneal Epithelial Cell Culture Medium and exposed to BAK concentrations ranging from 0.02% to 0.00002%. Barrier function was assessed using electric cell–substrate impedance sensing (ECIS), where decreased electrical resistance indicates loss of barrier integrity. Mitochondrial function was evaluated after 24 hours using the Seahorse XFe96 Flux Analyzer, which measured basal respiration, ATP-linked respiration, and maximal respiration.

Effects of BAK Concentration on Barrier Function

High BAK concentrations (≥0.02%) caused a rapid, dose-dependent decrease in HCEC impedance, exceeding 40% within 1 hour, consistent with acute cytotoxicity. In contrast, concentrations below 0.0001% produced a gradual, delayed decline in resistance. Specifically, 0.00025% BAK resulted in a 37% decrease in resistance by 72 hours, while 0.0001% caused a 26% reduction. Concentrations ≤0.00005% had no significant effect, suggesting a threshold below which BAK fails to compromise barrier integrity under these conditions.

Phase-contrast imaging supported these findings. Cells exposed to 0.00005% to 0.0001% BAK remained largely confluent but showed subtle morphological changes, including more defined cell borders and occasional intercellular gaps, consistent with early weakening of cell–cell junctions.

Mitochondrial Bioenergetics in Response to BAK

Seahorse analysis showed that BAK affected mitochondrial function in a concentration-dependent manner after 24 hours of exposure (0.002%–0.00005%). A significant reduction in basal respiration occurred at ≥0.00005% BAK, along with consistent ATP suppression across all concentrations was observed, indicating early inhibition of oxidative phosphorylation.

Maximal respiration decreased at higher doses (≥0.0001%) whereas damage appeared more slowly at lower doses (≤0.0001%), reflecting a cumulative sub-CMC effect that did not cross a critical toxicity threshold.

Temporal analysis indicates that mitochondrial dysfunction preceded and contributed to barrier failure. Seahorse data at 24 hours revealed significant suppression of basal respiration and ATP synthesis at ≥0.00005% BAK, even at concentrations that had not yet altered ECIS impedance, suggesting that bioenergetic stress is an early driver of epithelial dysfunction.

Implications for Ophthalmic Formulations

These findings demonstrate that BAK induces concentration-dependent, cumulative toxicity in HCECs. At or above the CMC, BAK causes rapid membrane disruption and irreversible barrier failure. At sub-CMC levels, toxicity manifests more gradually through mitochondrial dysfunction, which ultimately contributes to barrier impairment.

Despite its widespread use, the dose-dependent toxicity of BAK at sub-CMC concentrations highlights the need for safer alternatives or better formulation strategies. While 0.00002% BAK falls below antimicrobial efficacy thresholds, it provides a useful reference point for understanding sublethal effects. Approaches that prioritize safe preservative levels or innovative combinations of multiple agents could help reduce cumulative damage in patients using chronic eye drops.

Importantly, this study shows the value of integrating bioenergetic profiling with impedance-based functional assays. Together, this integrated strategy provides a more sensitive and translational framework for identifying early toxicity thresholds that can inform the safety evaluation of ophthalmic formulations and preservatives and help refine concentration guidelines for clinical use.

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