Transfer of molecules into the endothelial cells of whole human corneas using carbon nanoparticles activated by femtosecond laser
Abstract
Purpose
The targeted delivery of drugs and genes represents a promising solution to modify human corneal endothelial cells (HCECs), however, it remains limited by the difficulty to efficiently cross cell membranes without altering their integrity. Aim: to adapt an innovative technique of macromolecules delivery in corneal endothelium using carbon nanoparticles (CNPs) activated by femtosecond laser (FsL).
Methods
HCECs of 35 whole human corneas stored in organ culture were permeabilized by photo-acoustic reaction induced by irradiation of CNPs by FsL beam. The uptake of dextran AlexaFluor 488, a 3000-Da fluorescent macromolecule, was assessed using fluorescent microscopy. Three fluences (20, 30 and 40 mJ/cm2) and protective additives (ROCK inh and Poloxamer 407) were tested in order to increase uptake efficiency while limiting cell mortality (ethidium +). Dextan uptake and cell mortality rates were quantified using the CorneaJ plugin for ImageJ.
Results
Dextran delivery significantly increased with fluence, reaching a mean of 13% of HCECs for 40 mJ/cm2. Induced mortality varied from 0 to 53% and was more dependent of the cornea itself than on fluence. Treatment by either protective additive did significantly enhance the delivery rate, nor HCECs viability.
Conclusions
For the first time, ex vivo HCECs of whole corneas were permeabilized by mediated-nanoparticles laser perforation. Even if the delivery rate was lower than for viral vectors, it remained high for a non-viral technique. Mortality was variable according to corneas tested even with the use of protective additives. GRANT: Fondation des Aveugles de France, Fondation de l'Avenir, Fondation Visaudio (ET1-638).
