Transcorneal delivery of topically applied silver nanoparticles does not delay epithelial wound healing

Abstract

Silver nanoparticles (AgNPs) are a common antimicrobial additive for a variety of applications, including wound care. However, AgNPs often undergo dissolution resulting in release of silver ions, with subsequent toxicity to mammalian cells. The cornea is a primary exposure site to topically administered AgNPs in and around the eye but their impact on corneal wound healing is understudied. Thus, the purpose of this study was to determine in vitro toxicity of AgNPs on corneal epithelial cells and fibroblasts as well as their effects on corneal epithelial wound healing utilizing an in vivo rabbit model. Non-coated 20 nm sized AgNP (AgNP-20) as well as 1% and 10% silver silica NPs (AgSiO₂NPs) were tested at concentrations ranging from 0.05-250 μg/mL. Immortalized human corneal epithelial (hTCEpi) cells and primary rabbit corneal fibroblasts (RCFs) were incubated for 24 h with AgNPs and cell viability was tested. Additionally, a round wound healing assay was performed to determine hTCEpi cell migration. Quantitative real-time PCR and western blot analysis was performed to determine α-smooth muscle actin (α-SMA, a myofibroblast marker) mRNA and protein expression, respectively, in RCFs treated with 50 μg/mL of AgNPs. Corneal epithelial wound healing was evaluated with 1%-AgSiO₂NPs (10 and 250 μg/mL) using an in vivo rabbit model. Rabbits were subsequently euthanized, and histologic sections of the enucleated globes were used to determine corneal penetration of 1%-AgSiO₂NPs with autometallography and hyperspectral darkfield microscopy. Cell viability of both the hTCEpi cells and fibroblasts was significantly decreased by the three AgNPs in a dose dependent manner. Migration of hTCEpi cells was significantly inhibited by the three AgNPs. Alpha-SMA mRNA expression was significantly inhibited with three AgNPs, but only the 1%-AgSiO₂NPs inhibited protein expression of α-SMA. In vivo epithelial wound closure did not significantly differ between groups treated with 10 or 250 μg/mL of 1%-AgSiO₂NPs or vehicle control. The 1%-AgSiO₂NPs penetrated throughout all corneal layers and into the anterior chamber in all treated eyes with no histopathological changes observed. In conclusion, the 1%-AgSiO₂NPs are safe and have potential therapeutic applications through its efficacy of the corneal penetration and reduced scar formation during corneal wound healing.

Keywords: Corneal epithelial wound healing; Keratocyte-fibroblast-myofibroblast transformation; Silver nanoparticles; Silver silica nanoparticles; Transcorneal penetration.