Lactoferrin-loaded contact lenses counteract cytotoxicity caused in vitro by keratoconic tears

Lactoferrin (LF), an iron-binding protein with antioxidant activity, is significantly reduced in the lacrimal film of patients affected by keratoconus (KC) compared to healthy subjects, and this is supposed to be the cause for tear iron increase and consequent iron deposition and oxygen free radical accumulation in the cornea. We decided to study if LF-loaded contact lenses (LF-CLs) could exert antioxidant activity on epithelial cells incubated in tears collected from two patients affected by keratoconus (KC1 and KC2). Moreover through this model we indirectly estimated iron concentration in the tears of healthy and KC subjects. Reflex tears were collected during the first 2 min of light or onion-induced lacrimation and stored at -20 °C. After incubation in tears for 18 h, mortality of epithelial cells was investigated by trypan blue exclusion test. Successively, LF-CLs were deposed on cells incubated in KC1 tears. For the indirect determination of iron content, cells were incubated with LF 1.2 mg/mL and different FeSO₄ concentrations, and for the estimation of iron from the patient's tears, cells were incubated with free serum medium and healthy tears (1:1) and different FeSO₄ concentrations. Epithelial cells incubated with reflex tears of KC patients showed increased mortality (27.7 ± 3.9%, p = 0.0003, for KC1 and 17.6 ± 0.95%, p = 0.014, for KC2) compared to epithelial cells maintained in control healthy tears (8.6 ± 1.2%). This difference in mortality was correlated with tear iron concentration, which was estimated at 4.58 μg/mL for the healthy subjects, at 56.28 μg/mL for KC1, and at 8.7 μg/mL for KC2 patient. Application of LF-CLs counteracted KC tear cytotoxicity restoring viability obtained in the presence of control tears. Therapeutic contact lenses obtained by LF loading can reduce oxidative stress induced by patients' tears and might represent an efficient device to arrest the progression of keratoconus.