Delivery of macromolecules into the endothelium of whole ex vivo human cornea by femtosecond laser-activated carbon nanoparticles

Background The targeted delivery of drugs or genes into corneal endothelial cells (ECs) during eye banking could help improve graft quality and quantity. Physical methods raising less safety concerns than viral ones, we previously adapted, for in vitro ECs, a recent innovative technique of drug delivery based on the activation of carbon nanoparticles (CNPs) by a femtosecond laser (fsL). The aim of the present pilot study was to adapt this method to enable molecule delivery into the intact endothelium of ex vivo human corneas. Methods ECs from 40 organ-cultured corneas were perforated by photoacoustic reaction induced by irradiation of CNPs by a fsL. This enabled intracellular delivery of Alexa Fluor 488 dextran, a 4000 Da fluorescent macromolecule. The influence of increasing laser fluences (15, 20, 30 and 40 mJ/cm 2 ) and of protective additives (ROCK inhibitor and poloxamer 407) on delivery and mortality rates was quantified using ImageJ. Results No dextran was delivered with a fluence lower than 20 mJ/cm 2 . Dextran was delivered into 3% (range 0%–7%) of cells at 20 mJ/cm 2 , 7% (range 2%–12%) at 30 mJ/cm 2 and reaching a median 13% (range 3%–24%) for 40 mJ/cm 2 , showing that dextran uptake by ECs increased significantly with fluence. Induced mortality varied from 0% to 53% irrespective of fluence, but likely to be related with the endothelial status (EC density and morphometry, donor age, storage duration and presence of Descemet9s folds). ROCK inhibitor slightly increased uptake efficiency, unlike poloxamer. However, none of them decreased the mortality induced by laser. Conclusions This study shows that a macromolecule can be delivered specifically into ECs of a whole organ-cultured human cornea, using fsL-activated CNPs. The delivery rate was relatively high for a non-viral method. Further optimisation is required to understand and reduce variability in cell mortality.