In vitro evaluation of sodium hyaluronate protective effect against benzalkonium chloride toxicity

Abstract

Purpose: To evaluate the protective effect of different concentrations of sodium hyaluronate (SH) on the Benzalkonium chloride (BAK)‐induced toxicity using an in vitro model. Methods: The NAV14 cell line (SV40‐immortalized murine conjunctival epithelium) was used. Cell monolayers were exposed to different combinations of BAK (0.001%; 0.005%; 0.01%) and SH (0.2%; 0.3%; 0.4%) for 15 minutes; then cells were washed, and fresh culture media was added. Cell viability was evaluated after 2 h by resazurin reduction and lactate dehydrogenase (LDH) release. Also, cell migration and proliferation over 24 hours were determined by the scratch wound‐healing assay. Data were analysed by two‐way ANOVA and are shown as mean ± SD of two independent experiment with 4–6 replicates each. Results: BAK induced a concentration‐dependent decrease on cell viability (BAK 0.001%: 91 ± 14%, BAK 0.005%: 45 ± 9% and BAK 0.01%: 22 ± 10% of control cells, p < 0.001) and an increase in LDH release (no BAK:0.22 ± 0.02, BAK 0.001%: 0.31 ± 0.02, BAK 0.005%: 1.14 ± 0.05 and BAK 0.01%: 1.21 ± 0.05, p < 0.001). Conversely, SH neutralized these effects also in a concentration‐dependent manner (p < 0.001). In the presence of SH 0.4% (highest effect), cell viability was BAK 0.001%: 104 ± 22%, BAK 0.005%: 109 ± 9% and BAK 0.01%: 75 ± 13% of control cells (p < 0.001 for BAK 0.005–0.01%) while LDH release was no BAK: 0.24 ± 0.03, BAK 0.001%: 0.26 ± 0.01, BAK 0.005%: 0.37 ± 0.02 and BAK 0.01%: 0.49 ± 0.22, (vs. no SH: p < 0.001 for BAK 0.005–0.01%). BAK also reduced wound closure in vitro (after 24 h, no BAK: 76 ± 14%, BAK 0.001%: 42 ± 12%, BAK 0.005%: 17 ± 16% and BAK 0.01%: 0.0% wound closure, p < 0.001 for all BAK). Conversely, SH neutralized this effect in a concentration‐dependent fashion (p < 0.001). In the presence of SH 0.4% (highest effect), wound closure at 24 h was: no BAK: 81 ± 15%, BAK 0.001%: 58 ± 6%, BAK 0.005%: 63 ± 10%, BAK 0.01%: 60 ± 8% (vs no SH: p < 0.001 for BAK 0.005–0.01%). Conclusions: Sodium hyaluronate neutralized BAK toxicity on conjunctival epithelial cells in a concentration‐dependent manner. SH 0.4% was even protective at the highest preservative concentrations. These findings support the use of SH to mitigate BAK toxicity in long‐term antiglaucomatous treatment, although more studies are needed.