The impact of reduced graphene oxide on corneal epithelial and fibroblast cells in vitro

Abstract

Purpose : Reduced graphene oxide (RGO) nanoparticles (NPs) have been utilized in wide applications in solar cells, biosensing, disease diagnosis and antiviral materials. Direct contact of eyes with RGO-NPs in the environment can lead to ocular damage. However, the impact of RGO-NPs on corneal wound healing is understudied. Thus, the purpose of this study was to evaluate the in vitro effects of RGO-NPs on corneal epithelial cell viability and migration, as well as fibroblast viability and myofibroblast differentiation.

Methods : Immortalized human corneal epithelial (hTCEpi) cells from passages 36-50 and primary rabbit fibroblasts (RCFs) from passages 3-7 were individually cultured for 24 h with RGO-NPs (400 nm; 0.05-50 µg/mL) and cell viability was assessed using MTT and Calcein-AM assays. Gold nanoparticle (15 nm; 5 µg/mL), 0.1% saponin and distilled water (DW) were used as negative, positive and vehicle controls, respectively. A round wound healing assay with a monolayer of hTCEpi cells was performed using RGO-NPs (0.05-7.5 µg/mL). RCFs were seeded in 6-well plates and treated for 24 h with DW or RGO-NPs (5 or 12.5 µg/mL) in the absence or presence of 10 ng/mL transforming growth factor-β1 (TGF-β1). RNA was then extracted and quantitative PCR was performed to determine mRNA expression of alpha smooth muscle actin (αSMA) as a myofibroblast phenotypic marker. Data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s test, or two-way ANOVA followed by Tukey’s test.

Results : Cell viability using either the MTT or Calcein-AM assay was significantly (P < 0.01) decreased at concentrations ≥2.5 µg/mL in hTCEpi cells and ≥12.5 µg/mL in RCFs. Migration of the hTCEpi cells was significantly inhibited (P < 0.05) following incubation with RGO-NPs at ≥ 5 µg/mL. Treatment with RGO-NPs at 5 and 12.5 µg/mL significantly increased (5.0- and 6.7-fold, respectively) αSMA mRNA expression in the presence of TGF-β1 (P < 0.001).

Conclusions : RGO-NPs decreased the viability of corneal epithelial cells and fibroblasts as well as epithelial cell migration in a concentration-dependent manner. RGO-NPs increased TGF-β1-induced αSMA mRNA expression. These in vitrostudies strongly indicate an impact of RGO-NPs on corneal epithelial cells and stromal fibroblasts that should be further studied in wound healing in vivo.

This is a 2020 ARVO Annual Meeting abstract.