Abstract
Purpose :
Treatment of rare corneal diseases besides transplantation are sparse. Delivery of biologics such as siRNA and mRNA could provide effective treatments however it still remains challenging due to the numerous ocular barriers.
Irradiation of light absorbing nanoparticles (NPs) leads to an ultrafast heating causing evaporation of the surrounding medium and the formation of vapor nanobubbles (VNB) which when collapsed porate cell membranes allowing intracellular delivery of macromolecules. This method, photoporation, has been studied in the past years to deliver different cargos. In this project we investigate bioinspired poly-dopamine (PDA) NPs to improve biologics delivery in the corneal epithelium.
Methods :
Freshly enucleated bovine eyes were used to isolate and culture primary bovine corneal epithelial cells (pBCEC) or used intact as an ex vivo model to test different PDA NP sizes (90 nm vs. 260 nm) and concentrations (8.0-160x107 nps/ml). These NPs were irradiated by a nanosecond pulsed laser (3 ns at 532 nm with fluences of 0.9-2.7 J/cm2) to allow VNB formation. These NPs were investigated on their ability to form pores after laser treatment allowing to transfect pBCEC with FITC-dextrans of different molecular weights (10 kDa, 150 kDa and 500 kDa).
Results :
We observed 10 kDa dextrans were more efficiently delivered than 500 kDa dextrans (80% vs 40% respectively) likely due to their smaller molecular weight. Comparing 90 nm and 260 nm sized NPs in optimized conditions did not show a difference in delivery yield and only a slight change in relative mean fluorescent intensity (up to 1.3 times higher for 500 kDa dextrans). The 260 nm NPs did however show an increased toxicity at equal concentrations.
Intact bovine eyes were treated with 90 nm PDA NPs and 10 kDa dextrans. Optimal concentrations determined in vitro however did not show increased uptake compared to dextrans alone. Increasing the PDA concentration (500 fold) however showed clearly increased uptake as visualized through full cornea microscopy and corneal sections.
Conclusions :
In this work we have successfully synthesized 90 and 260 nm biocompatible PDA NPs. We have shown these NPs were able to create VNB and porate the cell membrane as observed with the increase in delivery efficiency in vitro. In an intact bovine eye, we have been able to efficiently deliver dextrans to the corneal epithelial layer.
This is a 2021 ARVO Annual Meeting abstract.