Abstract
Purpose :
The inner limiting membrane (ILM) represents a major hurdle for therapeutics to efficiently reach the retina after intravitreal injection. To tackle this issue, we explore indocyanine green (ICG) mediated photodisruption to locally perforate the ILM. In this concept, ICG is delivered to the ILM followed by pulsed laser treatment generating vapor nanobubbles. Upon their collapse, mechanical effects disrupt the ILM paving the way for therapeutics to enter the retina. Given the increasing interest in mRNA therapy to treat retinal diseases, the goal of this ex vivo study is to characterize the enhancement of mRNA lipid nanoparticle (LNP) delivery after photodisruption.
Methods :
Luciferase encoding mRNA LNPs (C12-200;DSPC;Cholesterol;DMG-PEG), fluorescently labeled with DiD dye, were synthesized via microfluidic mixing. Physicochemical characterization was performed by use of Dynamic Light Scattering to determine the size and zeta potential. To disrupt the ILM, ICG was applied on top of bovine retinal explants followed by pulsed laser treatment (2ps, 800nm). After photodisruption, explants were cultured and transfected with LNPs (1µg mRNA/explant) for 24h. Finally, cryosections were prepared followed by immunostaining with collagen IV antibodies to investigate ILM integrity as well as retinal penetration of LNPs with confocal microscopy. To evaluate mRNA expression, the bioluminescent signal -resulting from luciferase expression- was measured after 24h of transfection by use of the IVIS equipment.
Results :
Confocal microscopy revealed that LNPs (107±8nm, 2.4±1.7mV) were not able to cross the intact ILM since limited retinal penetration was observed in untreated samples. On the contrary, ILM damage provoked by the photodisruption treatment resulted in substantial penetration of LNPs into the inner retinal layers. Strikingly, this enhanced LNP entry furthermore boosted their efficacy since a 5.8±0.9 fold increase in luciferase expression was obtained compared to explants without photodisruption treatment.
Conclusions :
This study indicates that the delivery of LNPs can be substantially enhanced by ILM photodisruption. Additionally, the improved retinal LNP penetration led to a significant increase in mRNA expression. Considering these promising results, future experiments will focus on finding the optimal efficacy safety balance as well as exploring realistic target cells for LNP delivery.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.