Transl Vis Sci Technol. 2025 Nov 3;14(11):16. doi: 10.1167/tvst.14.11.16.
ABSTRACT
PURPOSE: To assess the influence of varying concentrations of benzalkonium chloride (BAK), the predominant preservative utilized in ophthalmic formulations, on the barrier integrity and mitochondrial function of primary cultured human corneal epithelial cells (HCECs).
METHODS: Primary HCEC monolayers were exposed to BAK at concentrations ranging from 0.02% to 0.00002%. The barrier function was monitored using electric cell-substrate impedance sensing (ECIS), where a decrease in electrical resistance signified a loss of barrier function. Mitochondrial function was evaluated after 24 hours of BAK exposure with the Seahorse XFe96 Flux Analyzer, which measured basal respiration, adenosine triphosphate (ATP) production, and maximal respiration.
RESULTS: High BAK concentrations (≥0.02%) caused a rapid, dose-dependent decrease in resistance, exceeding 40% within 1 hour. In contrast, lower concentrations (0.00025%-0.002%) led to a delayed, gradual reduction. Specifically, 0.00025% BAK resulted in a 37% decrease in resistance by 72 hours, whereas 0.0001% caused a 26% reduction; concentrations ≤ 0.00005% had no significant effect. Increased capacitance accompanied the resistance loss, indicating membrane disturbance. Seahorse analysis revealed that BAK concentrations ≥ 0.00005% significantly reduced basal respiration and ATP production. Maximal respiration decreased at higher doses (≥0.0001%).
CONCLUSIONS: BAK induces concentration-dependent, cumulative toxicity in HCECs, causing rapid membrane disruption and irreversible barrier failure at or above its critical micelle concentration (CMC), along with ongoing sub-CMC toxicity through mitochondrial suppression at lower doses. These findings highlight the need for preservative strategies that reduce both acute and chronic epithelial damage in ophthalmic applications.
TRANSLATIONAL RELEVANCE: Real-time impedance and mitochondrial assessments determine thresholds for BAK toxicity, guiding the development of safer ophthalmic formulations to protect the ocular surface.
PMID:41235859 | DOI:10.1167/tvst.14.11.16