# Effects of a Novel Dexamethasone Hydrogel Drug Delivery System on Cytokine and Mucin Expression in a Three-Dimensional In Vitro Conjunctival Inflammation Model

## TL;DR

A new 3D model for conjunctival inflammation was developed to test a dexamethasone hydrogel system, showing reduced inflammation and increased mucin expression.

## Contribution

A novel dexamethasone hydrogel drug delivery system was tested in a 3D in vitro conjunctival inflammation model.

## Key findings

- Combined TNF-α and IL-1β stimulation significantly increased cytokine and mucin expression.
- Dex-formulations significantly reduced IL6 expression and further elevated mucin levels.
- The model provides a new in vitro platform for testing DED treatments.

## Abstract

Inflammation of the ocular surface is one of the key symptoms in dry eye disease (DED). Various eye drops are available to alleviate conjunctival inflammation but have limited effect and poor patient compliance. To facilitate testing of new treatments, we established a three-dimensional (3D) in vitro conjunctival inflammation model and tested a novel dexamethasone (Dex) hydrogel drug-delivery system on cytokine and mucin expression.

Primary human conjunctival fibroblasts were embedded in a compressed collagen matrix. Primary epithelial cells were seeded on top and cultured at the air-liquid-interface for 15 days. Inflammation was induced via TNF-α, IL-1β, or their combination. Pristine Dex, poly(2-oxazoline) (POx)–based Dex micelles and modified acrylic acid based hydrogel loaded with Dex micelles were applied to the inflammation model to verify the therapeutic efficacy. Pro-inflammatory cytokines were analyzed via real-time quantitative PCR (RT-qPCR) and ELISA. Conjunctival mucins were analyzed via RT-qPCR.

The expression of all tested cytokines (IL1A, IL1B, IL6, IL8, MMP9) was significantly increased after combined stimulation with TNF-α and IL-1β. Treatment with Dex-formulations significantly reduced IL6 expression. The expression of mucins was significantly increased after stimulation and was further elevated after treatment with Dex-formulations.

We established an inflammation model, in which the effects of novel treatment options on cytokine and mucin expression can be analyzed. This opens a new in vitro test platform for DED medication and enables deeper investigations into conjunctival mucin expression in inflamed tissue.