Metal Oxide Nanoparticles Induce Stiffening of Rabbit Corneal Fibroblasts in vitro

Abstract

Purpose : Metal oxide nanoparticles (NPs) zinc oxide (ZnO) and titanium dioxide (TiO₂) are routinely applied near the eye via cosmetics and sunscreens. Despite the high probability of exposure there remains a paucity of research as to the ocular consequences. The biomechanics of the corneal stroma are altered during wound repair, indicating a need to examine mechanics in ocular toxicity. We hypothesize that ZnO and TiO₂ NPs increase the mechanical stiffness of corneal fibroblast cells, and thus contribute to keratocyte-fibroblast-myofibroblast transformation.

Methods : Rabbit Corneal Fibroblasts (RCFs) at passage 6 were cultured with and without 10 ng/mL Transforming Growth Factor Beta 1 (TGFβ-1) for 48 h at which time phosphate buffered saline (PBS-control), 10 µg/mL ZnO or 250 µg/mL TiO₂ NPs were added, and cells incubated for an additional 24 hrs. After incubation cells were then gently washed 3x with PBS and live cell stiffness measured by Atomic Force Microscopy (AFM). For each experimental condition, 5 force curves per cell were collected from 10 individual cells. Force curves were taken with PNP-TR-50 silicon nitride cantilevers that were calibrated for the deflection inverse optical lever sensitivity by indentation in PBS on glass and the spring constant determined using the Asylum Research software at a scan velocity of 1.98 µm/s. Data were analyzed to determine elastic modulus and were obtained by mathematically fitting the indentation curves to the Hertz model for spherical geometry and then subjected to a one-way analysis of variance (ANOVA) followed by Tukey’s test.

Results : RCFs exposed to ZnO and TiO₂ NPs had a significantly (p<0.001) greater elastic modulus than control cells to a degree that approximated the effect seen with TGFβ-1 alone. For ZnO, the degree of stiffening was not augmented by the presence of TGFβ-1. RCFs treated with TiO₂ NPs were stiffer and in contrast to results obtained with ZnO NPs, the effect was significantly augmented by TGFβ-1.

Conclusions : ZnO as well as TiO₂ NPs increased stiffness of RCFs. The degree of stiffening induced by ZnO NPs was unaffected by the presence or absence of TGFβ-1. For TiO₂, TGFβ-1 further increased the stiffness of cells. These results suggest that ZnO and TiO₂ NPs alter cytoskeletal dynamics and further studies of the NPs on KFM transformation during stromal wound healing are warrranted.

This is a 2021 ARVO Annual Meeting abstract.