Femtosecond Laser and Gold Nanoparticles-Mediated mRNA Delivery in Corneal Endothelial Cells Ex Vivo

Endothelial diseases impair the vision of approximately 246 million people worldwide. The only approved treatment is corneal transplantation, which is invasive, costly, and limited by donor shortages. We present a femtosecond (fs) laser-based method for gene delivery to corneal endothelial cells (CECs) via transcorneal irradiation of gold nanoparticles (AuNPs). Upon irradiation, the plasmonic response of AuNPs transiently permeabilizes CEC membranes (optoporation), enabling the delivery of exogenous molecules such as mRNA. Mouse and rabbit corneas are used ex vivo to evaluate mRNA transfection and nanoparticle uptake, respectively. Mice receive intracameral injections of AuNPs and Cy3 EGFP mRNA, while rabbit corneas are incubated with AuNPs and fluorophores. After 1 h incubation, a 45 fs laser (λ = 800 nm, 1 kHz) is focused on the CEC layer. Fluorescence and bright-field microscopy assess optoporation, viability, and specificity. Lipofectamine-mRNA serves as a transfection control. Results show successful optoporation, with CEC viability above 70% at 48 h, 49% mRNA transfection in mice, and 95% fluorophore uptake in rabbits. This targeted, noninvasive approach offers a promising alternative for gene and drug delivery to CECs.