Femtosecond laser activated carbon nanoparticles for corneal gene therapy
Résumé
Cell permeabilization allows us to deliver all sorts of molecules, especially genetic material able to modify cell behavior, into cells. A new cell therapy method published in Nature Nanotechnology in 2011 [1] consists in ephemerally permeabilizing cell membranes using a photo-acoustic reaction produced by carbon nanoparticles and femtosecond laser. Authors introduced a small molecule called calcein into cells in suspension whose membrane is normally impermeable with a good efficiency (until 90%) and a very low toxicity (<10%). The aim of this work is to adapt this innovative and promoting technique on a confluent monolayer of human corneal endothelial cells (HCEC). Our experimentations were inspired from teams working on the same technique but with the use of gold [2] and polystyrene [3] nanoparticles. 15 nm diameter carbon black nanoparticles (CNPs) were put in contact with HCEC and calcein and irradiated on the entire cell surface with a 500 μm diameter Ti:Sa FsL focalized spot (Bright, Thales, Paris, France). Many parameters were tested to obtain the best efficiency of calcein penetration in the cells while limiting toxicity: beam fluence, velocity and pitch, CNPs concentration and volume. After irradiation, cells were dissociated, stained by propidium iodide (IP) to highlight died or dying cells and counted by flow cytometry to precisely determine the percentage of cells with calcein (efficiency) and of dead cells (toxicity). Preliminary studies showed a real concordance between the FsL beam energetic profile and the geometry of cell area with calcein uptake showing a very space limited reaction. The greater the energy of the FsL beam arriving on CNPs, the higher the efficiency and the toxicity or/and cell detachment. Calcein was inhomogeneously uptaken by cells making a fluorescence gradient. After several optimizations, the best efficiency obtained was around 38% for a very low toxicity of around 3 %. Compared to the others papers [1, 2], our permeabilization efficiency are lower. However, considering the cell density used for our experiments in order to be as close to an ex vivo model as possible, cell permeabilization by photo-acoustic reaction already presents excellent results on confluent HCEC monolayer which are promising for gene transfer. [1] P. Chakravarty, Qian W, El-Sayed MA, Praunitz MR, Delivery of molecules into cells using carbon nanoparticles activated by femtosecond laser, Nat. Nanotech., vol.5, pp. 605-11, (2010). [2] J. Baumgart, L. Humbert, E. Boulais, R. Lachaine, JJ. Lebrun, M. Meunier, Off-resonance plasmonic enhanced femtosecond laser optoporation and transfection of cancer cells, Biomaterials, vol. 33, pp. 2345-50, (2012). [3] M. Terakawa, Y. Tsunoi, T. Mitsuhashi, In vitro perforation of human epithelial carcinoma cell with antibody-conjugated biodegradable microspheres illuminated by a single 80 femtosecond near-infrared laser pulse, Int. J. Nanomedicine, vol. 7, pp. 2653-60, (2012).