Improvement of delivery of molecules into corneal endothelium using nanoparticles activated by femtosecond laser pulses

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Abstract

Purpose

NanoFemtoTransfection (NFT) is an innovative and promising technique of gene transfer (Nature nanotechnology 2011). It consists in permeabilizing cell membrane with nanoparticles (NP) of black carbon activated by Ti-Sapphire femtosecond laser (FsL) in order to safely introduce molecule of interest. Initially described for cell suspension, we adapted during last year NFT to human corneal endothelial cells (EC) Aim: to further optimize NFT of EC in vitro

Methods

The model of NFT of calcein (a small molecule) in an EC line (B4G12, DMSZ, Germany) was used to optimize exposition times, fluence and Ti-Saphir FsL beam movements in order to determine the best efficiency/toxicity ratio. Calcein transfer was observed with fluorescent microscopy (IX81, Olympus) and quantified by flow cytometry (FACSCalibur, BD, CA). Viability of EC was assessed by propidium iodide staining. Staining of actine with phalloidin and immunostaining of ZO-1 was performed to study cell alteration caused by NFT

Results

With a static laser beam increasing FsL fluence and exposure time increased transfection efficiency but also EC mortality. The use of mobile FsL beam (with a XY micrometric stage, cheaper than a scanner), permitted an irradiation of the entire area of EC culture resulting in a noticeably increased efficiency that reached 38% of transfected EC and a limited toxicity (3%). Calcein positive EC presented the same phalloidin and ZO-1 patterns as control EC

Conclusion

Optimized NFT parameters are currently being tested in our laboratory to transfect plasmids. Grants: Fondation pour la Recherche Médicale, Fondation des Aveugles de France

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