Delivery of molecules into corneal endothelium using nanoparticles activated by femtosecond laser pulses: proof of concept

Purpose NanoFemtoTransfection (NFT) is an innovative and promising non-viral technique to transfer molecules into cells (Chakravarty. Nature Nanotechnology 2010;5:607). It consists in temporarily permeabilizing cell membrane by a photoacoustic effect obtained by nanoparticles of black carbon activated by Ti-Saphir femtosecond laser (fsL) pulses. Calcein (622 Da), tagged bovine serum albumine (70 kDa) and one eGFP plasmid (5 MDa) were transfected into two non-adherent cell lines (DU145 prostate-cancer and GS-9L rat gliosarcoma). Our aim was to adapt the NFT to adherent human corneal endothelial cells (HCEC) Methods We tested the NFT of calcein in vitro on the HCEC line HCEC-12 seeded at 1500cells/mm2 in 6 wells plates and ex vivo on whole human organ cultured corneas. A matrix of experiments comprising 4 exposition times, 6 fluences and 2 fsL beam movements was performed in order to obtain transfection with minimal toxicity. After exposition to fsL, nuclei were counterstained with Hoechst33342 and transfection efficiency was determined by observation on a fluorescence inverted microscope (IX81, Olympus, Japan) and further quantified by flow cytometry (FACSCalibur, BD, CA). Viability was assessed by Trypan blue staining Results In HCEC-12, a fluence of 100 mJ/cm² and a laser beam movement of 3,5 mm/s gave a transfection of 17% and a viability of 97%. In whole corneas, with the same parameters, transfection was detectable in disseminated EC Conclusion We obtained the proof of concept of the NFT in HCEC. Further optimization is ongoing to increase the transfection rate while maintaining minimal toxicity, especially for bigger molecules, like plasmids