Premium
Pore size assessment during corneal endothelial cell permeabilization by femtosecond laser‐activated carbon nanoparticles
Author(s) -
Jumelle C.,
Mauclair C.,
Houzet J.,
Bernard A.,
He Z.,
Piselli S.,
Perrache C.,
Gain P.,
Thuret G.
Publication year - 2015
Publication title -
acta ophthalmologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.534
H-Index - 87
eISSN - 1755-3768
pISSN - 1755-375X
DOI - 10.1111/j.1755-3768.2015.0595
Subject(s) - calcein , dextran , viability assay , biophysics , flow cytometry , membrane , materials science , femtosecond , chemistry , nanotechnology , cell , laser , chromatography , microbiology and biotechnology , biochemistry , biology , optics , physics
Purpose Therapeutic molecules delivery represents a promising solution to maintain human corneal endothelial cells ( HCEC s) 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 ( CNP s) 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, HCEC s (B4G12 cell line) were put in contact with CNP s and irradiated with a 500 µm diameter Ti:Sa FsL focalized spot. Four sizes of fluorescent reporter molecules were delivered into HCEC s 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 2 MD a (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 HCEC s, FITC ‐Dextran 4 kDa in 42 ± 4%, FITC ‐Dextran 70 kD a in 22 ± 3% and finally FITC ‐Dextran 2 MD a 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 CNP s 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 HCEC s. GRANT : Fondation des Aveugles de France, Fondation de l'Avenir, Fondation Visaudio ( ET 1‐638).