Delivery of Molecules into Human Corneal Endothelial Cells by Carbon Nanoparticles Activated by Femtosecond Laser

PLoS One

Zhiguo He¹  Philippe Gain¹  Clotilde Jumelle¹  Michel Peoc’h¹  Fabien Forest¹  Gilles Thuret²  Sophie Acquart²  Julien Houzet³  Cyril Mauclair⁴  Aurélien Bernard⁴ 

[1] Corneal Graft Biology, Engineering and Imaging Laboratory, EA2521, SFR143, Faculty of Medicine, Saint-Etienne, France;Department of Pathology, University Hospital, Saint-Etienne, France;GIE Manutech-Ultrafast Surfacing Design, Saint-Etienne, France;Hubert Curien Laboratory, UMR-CNRS 5516, Pôle Optique Rhône-Alpes, Saint-Etienne, France

关键词: Cornea;    Lasers;    Nanoparticles;    Endothelial cells;    Toxicity;    Cell membranes;    Flow cytometry;    Laser beams;   

DOI  :  10.1371/journal.pone.0132023

学科分类：医学（综合）

来源: Public Library of Science

【 摘 要 】

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.

【 授权许可】

CC BY