Application of Enzyme-Loaded Microspheres to Disrupt the Internal Limiting Membrane for optimization of Retinal Ganglion Cell Engraftment in a Rodent Model

Abstract

Purpose : Cell therapy is a promising treatment for replacing lost retinal ganglion cells (RGCs) with healthy cells in diseases caused by RGC loss (e.g., glaucoma and Neurofibromatosis Type 1). The internal limiting membrane (ILM) represents a significant barrier hindering the success of cell-based therapies aimed at replacing inner retinal neurons. Herein, we propose the use of enzyme-loaded microspheres (MSs) to disrupt the ILM without causing damage to the retina for improving engraftment of intravitreally-delivered RGCs.

Methods : The degradation capabilities of MSs loaded with Vitamin-E (VitE) and collagenase or pronase-E were determined via ELISA and immunocytochemistry. Additionally, adult C57BL/6J mice were intravitreally injected with volumes ranging from 1-2mL of either formulation (0.1mgMSs/mL). Free collagenase and pronase-E were used for comparison. MSs labelled Nile Red were used as negative control for co-localization. Retinal sections and flat mounts were analyzed via immunohistochemistry (H&E) and immunofluorescence with antibodies directed against laminin and Collagen Type IV (Col4) to evaluate the integrity of the ILM.

Results : Pronase-E MSs showed complete degradation of Col4 in coated plates compared to collagenase MSs, which showed partial degradation. Our ELISA profile evidenced maximum Col4 degradation rate in the first hour upon addition of MSs and the degradation was sustained up to 2 h. In vivo data showed Col4 degradation while preserving laminin structure upon injection of collagenase/VitE-loaded MSs. While pronase-E/VitE MSs degraded laminin within the ILM in a dose-dependent fashion. Intravitreal delivery of MSs caused no obvious abnormalities in the retina while injection of free enzymes was associated with intraretinal hemorrhage and cataract.

Conclusions : Our data indicates that both enzyme-loaded MSs could serve to circumvent the ILM. With further optimization, we aim at improving the survival and integration rate of RGCs after transplantation with the pre-treatment of MSs to further restore vision upon RGC loss.

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