Abstract
Purpose :
When people age, liquefaction of the vitreous can result in Posterior Vitreous Detachment (PVD), routinely treated by a vitrectomy. Unfortunately, this procedure is invasive and risky since it might lead to complications such as retinal tears. As an alternative, we explored enzymatic vitreolysis both as a replacement or pre-treatment to vitrectomy. To this end, we aimed to immobilize enzymes on the surface of nanoparticles (NPs) to minimize enzyme-induced toxicity caused by retinal penetration and thus generate safe and effective vitreous liquefaction.
Methods :
NPs and enzyme were incubated for 24 hours at room temperature under continuous stirring. Next, multiple centrifugation cycles were performed in order to remove the free enzyme. The activity of the enzyme-modified NPs in vitro was evaluated by measuring substrate-FITC-fluorescence with a plate-reader. The fluorescence intensity of the FITC-labeled substrate increased upon degradation by the enzyme. Additionally, ex vivo activity experiments in bovine vitreous were performed using single particle tracking (SPT). SPT measurements allow mobility tracking of fluorescent particles. We used fluorescent beads which are immobile in vitreous but are able to diffuse upon vitreous liquefaction. Finally, potential penetration of the enzyme-modified NPs into the retina upon injection in bovine vitreo-retinal explants was determined with confocal microscopy. Necessary controls with non-modified NPs and/or free enzyme were performed in every experimental set-up.
Results :
We repeatedly succeeded in generating stable 300 nm NPs with an in vitro activity of 170 units/100 µL sample. SPT experiments validated that we could generate sufficient vitreous liquefaction. Upon treatment of the bovine vitreous with enzyme-modified NPs, the mobility of injected fluorescent beads increased enormously. As expected, microscopy imaging showed clear alignment of the enzyme-modified NPs at the inner limiting membrane (ILM) while no penetration to the retina could be observed. Finally, we could not see significant structural retinal changes in the vitreo-retinal explants upon treatment with the enzyme-modified NPs.
Conclusions :
This study showed that enzyme-immobilization is a promising technique for effective vitreous liquefaction. Future goals include extensive toxicity studies both ex vivo and in vivo to provide more information about the safety of this therapy.
This is a 2021 ARVO Annual Meeting abstract.