title: Delivery of Molecules into Human Corneal Endothelial Cells by Carbon Nanoparticles Activated by Femtosecond Laser.
abstract: Corneal endothelial cells (CECs) form a monolayer at the innermost face of the cornea and are the engine of corneal transparency. Nevertheless, they are a vulnerable population incapable of regeneration in humans, and their diseases are responsible for one third of corneal grafts performed worldwide. Donor corneas are stored in eye banks for security and quality controls, then delivered to surgeons. This period could allow specific interventions to modify the characteristics of CECs in order to increase their proliferative capacity, increase their resistance to apoptosis, or release immunosuppressive molecules. Delivery of molecules specifically into CECs during storage would therefore open up new therapeutic perspectives. For clinical applications, physical methods have a more favorable individual and general benefit/risk ratio than most biological vectors, but are often less efficient. The delivery of molecules into cells by carbon nanoparticles activated by femtosecond laser pulses is a promising recent technique developed on non-adherent cells. The nanoparticles are partly consummated by the reaction releasing CO and H2 gas bubbles responsible for the shockwave at the origin of cell transient permeation. Our aim was to develop an experimental setting to deliver a small molecule (calcein) into the monolayer of adherent CECs. We confirmed that increased laser fluence and time exposure increased uptake efficiency while keeping cell mortality below 5%. We optimized the area covered by the laser beam by using a motorized stage allowing homogeneous scanning of the cell culture surface using a spiral path. Calcein uptake reached median efficiency of 54.5% (range 50.3-57.3) of CECs with low mortality (0.5%, range (0.55-1.0)). After sorting by flow cytometry, CECs having uptaken calcein remained viable and presented normal morphological characteristics. Delivery of molecules into CECs by carbon nanoparticles activated by femtosecond laser could prove useful for future cell or tissue therapy.
authors.affiliation: Corneal Graft Biology, Engineering and Imaging Laboratory, EA2521, SFR143, Faculty of Medicine, Saint-Etienne, France.
authors.forename: Clotilde
authors.initials: C
authors.lastname: Jumelle
authors.affiliation: Hubert Curien Laboratory, UMR-CNRS 5516, Pôle Optique Rhône-Alpes, Saint-Etienne, France; GIE Manutech-Ultrafast Surfacing Design, Saint-Etienne, France.
authors.forename: Cyril
authors.initials: C
authors.lastname: Mauclair
authors.affiliation: Hubert Curien Laboratory, UMR-CNRS 5516, Pôle Optique Rhône-Alpes, Saint-Etienne, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Houzet
authors.affiliation: Corneal Graft Biology, Engineering and Imaging Laboratory, EA2521, SFR143, Faculty of Medicine, Saint-Etienne, France.
authors.forename: Aurélien
authors.initials: A
authors.lastname: Bernard
authors.affiliation: Corneal Graft Biology, Engineering and Imaging Laboratory, EA2521, SFR143, Faculty of Medicine, Saint-Etienne, France.
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: He
authors.affiliation: Corneal Graft Biology, Engineering and Imaging Laboratory, EA2521, SFR143, Faculty of Medicine, Saint-Etienne, France; Department of Pathology, University Hospital, Saint-Etienne, France.
authors.forename: Fabien
authors.initials: F
authors.lastname: Forest
authors.affiliation: Corneal Graft Biology, Engineering and Imaging Laboratory, EA2521, SFR143, Faculty of Medicine, Saint-Etienne, France; Department of Pathology, University Hospital, Saint-Etienne, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Peoc'h
authors.affiliation: Eye Bank, Etablissement Français du Sang Loire/Auvergne, Saint-Etienne, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Acquart
authors.affiliation: Corneal Graft Biology, Engineering and Imaging Laboratory, EA2521, SFR143, Faculty of Medicine, Saint-Etienne, France; Department of Ophthalmology, University Hospital, Saint-Etienne, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Gain
authors.affiliation: Corneal Graft Biology, Engineering and Imaging Laboratory, EA2521, SFR143, Faculty of Medicine, Saint-Etienne, France; Department of Ophthalmology, University Hospital, Saint-Etienne, France; Institut Universitaire de France, Paris, France.
authors.forename: Gilles
authors.initials: G
authors.lastname: Thuret
mesh_terms: D002244:Carbon / Q000008:administration & dosage / Q000528:radiation effects*; D002448:Cell Adhesion; D002461:Cell Line, Transformed; D002463:Cell Membrane Permeability; D004333:Drug Administration Routes; D042783:Endothelial Cells / Q000187:drug effects*; D004728:Endothelium, Corneal / Q000166:cytology*; D005452:Fluoresceins / Q000008:administration & dosage* / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000008:administration & dosage*; D019220:High-Energy Shock Waves; D006801:Humans; D007834:Lasers; D045423:Microbubbles; D053758:Nanoparticles / Q000008:administration & dosage* / Q000528:radiation effects; D013997:Time Factors
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D002244:Carbon; C007740:fluorexon