A novel high-density-lipoprotein-like nanoparticle (HDL NP) mitigates conjunctivalization and neovascularization in mustard agent -induced corneal injury

Abstract

Purpose : Ocular exposure to sulfur mustard (SM), a chemical warfare agent, results in acute and delayed phases of corneal disruption. Corneal conjunctivalization, neovascularization, and persistent inflammation are most problematic, and can result in blindness. Available therapies for mustard keratopathy(MK) are inadequate and often have adverse side effects. We developed a novel optically transparent HDL NP synthesized using pentaerythritol tetraoleate (PE-O4) as a molecular scaffold to support the self-assembly of apolipoprotein A-I and phospholipids into oc-HDL NP. We now report on the in vivo efficacy of this oc-HDL NP (oc = organic core) in treating MK.

Methods : MALDI mass-spectrometric imaging (MSI) verified the penetration of HDL NPs topically applied to the intact ocular surface. Nitrogen mustard (NM) is an SM analog that replicates many corneal pathologies caused by SM. Mouse corneas were exposed to 0.5% NM for 1 min. Two hrs post exposure, corneas were topically treated with the PE-O4 oc-HDL NP (2µM) or PBS (control) daily for 3 days (acute phase) and 14 days (delayed phase). The corneas were imaged for haze scoring and epithelial fluorescein penetration. RT-qPCR examined the expression of pro-inflammatory genes. H&E-stained mouse eye sections assessed corneal epithelial integrity, inflammation, and epithelial conjunctivalization. Corneal whole mounts were stained with CD31 to assess stromal blood vessels.

Results : Four hrs after topical application of the HDL NPs to the mouse corneal surface, MALDI MSI detected the spatial distribution of the phospholipids making up the HDL NPs in the corneal epithelium and stroma. In the acute phase, PE-O4 oc-HDL NPs reduced haze by 32% and pro-inflammatory genes by at least 40%. H&E staining revealed that the HDL NPs restored epithelial thickness by 80% and dramatically reduced inflammation by 58%. In the delayed phase, the HDL NPs reduced haze by 20%. Importantly, there was a 65% reduction in corneal epithelial goblet cells, and a 40% reduction in corneal stromal blood vessels. These reductions indicate that PE-O4 oc-HDL NPs diminish NM-induced conjunctivalization and angiogenesis, hallmarks of a limbal stem cell deficiency (LSCD).

Conclusions : As an eyedrop formulation developed for corneal MK, this novel oc-HDL NP has the potential to correct LSCD, a sight-threatening condition.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.