Photodisruption of the ILM to enhance retinal drug delivery after intravitreal injection

Abstract

Purpose : The development of many therapies involving retinal delivery of biologics or cells by intravitreal injection is greatly hampered by obstruction of the therapeutics at the inner limiting membrane (ILM). Therefore, we explore photodisruption of the ILM to generate therapeutic entryways that can greatly enhance retinal delivery. To this end, the photothermal dye indocyanine green (ICG) is applied to the ILM and irradiated with pulsed laser light in order to create vapor nanobubbles (VNBs) which upon their collapse can disrupt the ILM (Fig 1).

Methods : To perforate the ILM, we applied varying concentrations of ICG (0,1 mg – 1mg/ml) on top of bovine retinal explants followed by scanning of the laser beam at lower and higher laser energies (0,24 or 0,4 J/cm²). For the latter, an Nd:YLF laser was applied to generate 800 nm single laser pulses of a 2 picosecond pulse width and frequency of 1 kHz. Propidium iodide staining was applied to assess retinal cell death right after treatment and after 24 hours in retinal cryosections (n=3). To assess the impact on delivery, 120nm-sized Lipid Nanoparticles (LNPs) loaded with luciferase-mRNA were dripped on top of explants (according with a dose of 1 µg of mRNA) prior to their culture for 24 hours after which retinal luciferase expression was measured in intact explants using an IVIS sytem (n=4). During these experiments, untreated, ICG-only and laser-only controls were added. Finally, dark-field microscopy was applied to image VNB formation upon laser pulse irradiation in patient-derived isolated human ILM (n=2).

Results : The strongest conditions (1 mg/ml ICG; 0,43 J/cm2) elicited significant cell death, especially in the GCL and INL layer, yet, milder conditions (0,1 mg/ml ICG; 0,43 J/cm2) did not affect retinal viability, even after 24 hours. Strikingly, the latter conditions elicited a nearly 5-fold increase (4.7 ± 0.4 SD, p<0001) in retinal luciferase expression when compared to explants treated with LNPs only. Dark-field microscopy on the human ILM confirmed that this effect is based on pore formation owing to VNB generation.

Conclusions : This ex vivo study reveals that ICG-mediated photodisruption of the ILM can greatly enhance delivery of nanoparticles into the retina in a safe manner. Driven by this positive outcome, future plans include assessment of the safety and efficacy of our technology in vivo.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.