Pore size assessment during corneal endothelial cell permeabilization by femtosecond laser-activated carbon nanoparticles

Purpose Therapeutic molecules delivery represents a promising solution to maintain human corneal endothelial cells (HCECs) viability, but transport across cell membrane must be facilitated. A new delivery method consists in ephemerally permeabilizing cell membranes using a photo-acoustic reaction produced by carbon nanoparticles (CNPs) and femtosecond laser (FsL). The aim of this work is to investigate the size of pores formed at cell membrane by this technique. Methods To induce cell permeabilization, HCECs (B4G12 cell line) were put in contact with CNPs and irradiated with a 500 µm diameter Ti:Sa FsL focalized spot. Four sizes of fluorescent reporter molecules were delivered into HCECs to investigate pore sizes: calcein (1.2 nm), FITC-Dextran 4 kDa (2.8 nm) and FITC-Dextran 70 kDa (12 nm) and FITC-Dextran 2MDa (50 nm). Uptake of each molecule was assessed by flow cytometry immediately after irradiation. Results The delivery rate was dependent of their size. Calcein was delivered in 56 ± 8% of HCECs, FITC-Dextran 4 kDa in 42 ± 4%, FITC-Dextran 70 kDa in 22 ± 3% and finally FITC-Dextran 2MDa in 13 ± 2%, suggesting that a large number of pores in the size ranging from 1.2 to 2.8 nm were formed. However, 12 nm and larger pores were almost half more infrequent. Conclusions Pore sizes formed at cell membrane by the technique of cell permeabilization by FsL activated CNPs were investigated for the first time. This innovative non-viral method is characterized by pore sizes large enough for the efficient delivery of small, medium and big therapeutic molecules on HCECs. GRANT: Fondation des Aveugles de France, Fondation de l'Avenir, Fondation Visaudio (ET1-638).