ICG-mediated photoporation of the ILM to enhance retinal drug delivery after intravitreal injection

Abstract

Purpose : The inner limiting membrane (ILM) is the primary barrier hindering effective drug delivery to the retina after intravitreal injection. Therefore, we aim to create therapeutic entryways into the retina by puncturing the ILM using photoporation. To explore our concept, we applied indocyanine green (ICG) at the ILM surface of bovine and human retinal explants followed by laser scanning of the retina with 800 nm laser light (Figure).

Methods : To perforate the ILM, we applied varying concentrations of ICG (0,1 mg – 1mg/ml) on top of bovine and human retinal explants followed by scanning of the laser beam at lower and higher laser energies (0,24 and 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. Following laser treatment, 100 nm sized PEGylated nanoparticles (NPs) were applied on top of the explants prior to their culture for 24 hours. Finally, retinal cryosections were prepared and immunostained for Collagen IV to check for ILM integrity as well as NP entry into the retina with confocal microscopy.

Results : Bovine retinal explants treated with laser only (0,4 J/cm²) showed an intact ILM. In explants treated with both ICG and laser light, however, the ILM was clearly affected: high ICG concentration and laser energy led to full ILM ablation, while lower conditions led to partial ILM destruction. As anticipated, 100 nm sized NPs were largely unable of crossing the intact ILM of untreated and laser-treated explants. By contrast, complete treatment (ICG and laser) resulted in a massive entry of NPs into the retina for all conditions. Additionally, we effectively ablated the ILM of human explants of patients with varying age (27 – 70 years) which indicates that our approach is also effective for treating the thicker and more complex human ILM.

Conclusions : We have successfully ablated the ILM of bovine and human explants by means of ICG-mediated photoporation. In addition, this ILM damage resulted in extensive entry of NPs into the retina. Future plans include fine-tuning of our approach in accordance with the limitations set by toxicity evaluations ex vivo and in vivo.

This is a 2021 ARVO Annual Meeting abstract.

 

A vapor nanobubble (VNB) emerges around an accumulation of ICG at the ILM by absorption of a short laser pulse. The collapse of the VNBs induces local perforation of the ILM, allowing therapeutics to enter the retina.