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

Jumelle C, Mauclair C, Houzet J, Bernard A, He Z, Forest F, Peoc'h M, Acquart S, Gain P, Thuret G - PLoS ONE (2015)

Bottom Line: For clinical applications, physical methods have a more favorable individual and general benefit/risk ratio than most biological vectors, but are often less efficient.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.

View Article: PubMed Central - PubMed

Affiliation: Corneal Graft Biology, Engineering and Imaging Laboratory, EA2521, SFR143, Faculty of Medicine, Saint-Etienne, France.

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.

No MeSH data available.


Related in: MedlinePlus

Static laser beam: Effect of fluence and exposure time on calcein delivery into adherent corneal endothelial cells.(A) constant fluence at 10 mJ/cm² and increasing exposure times; (B) constant exposure time at 10 min and increasing fluences. All images were obtained with a x10 objective and merged phase contrast/FITC. Green fluorescence (FITC+) indicated uptake of calcein. Scale bar 200 μm.
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pone.0132023.g003: Static laser beam: Effect of fluence and exposure time on calcein delivery into adherent corneal endothelial cells.(A) constant fluence at 10 mJ/cm² and increasing exposure times; (B) constant exposure time at 10 min and increasing fluences. All images were obtained with a x10 objective and merged phase contrast/FITC. Green fluorescence (FITC+) indicated uptake of calcein. Scale bar 200 μm.

Mentions: No spontaneous calcein uptake was observed in the absence of either FsL irradiation or CNPs. With constant fluence of 10 mJ/cm², calcein+ CECs were visible even for 3 min exposure, but their number seemed to increase with exposure time. The area of the cell culture where uptake of calcein was present was strictly restricted to the area irradiated by the FsL (1 mm in diameter), showing that the reaction was very localized. With a constant exposure of 10 min, calcein+ CECs were visible only from 8 mJ/cm² and increased with fluence (Fig 3). The static irradiation proved the concept of delivery of a small molecule by CNPs activated by a FsL in a monolayer of adherent cells, and of the possibility to modulate efficiency with the laser parameters (fluence and exposure time).


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

Jumelle C, Mauclair C, Houzet J, Bernard A, He Z, Forest F, Peoc'h M, Acquart S, Gain P, Thuret G - PLoS ONE (2015)

Static laser beam: Effect of fluence and exposure time on calcein delivery into adherent corneal endothelial cells.(A) constant fluence at 10 mJ/cm² and increasing exposure times; (B) constant exposure time at 10 min and increasing fluences. All images were obtained with a x10 objective and merged phase contrast/FITC. Green fluorescence (FITC+) indicated uptake of calcein. Scale bar 200 μm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4489806&req=5

pone.0132023.g003: Static laser beam: Effect of fluence and exposure time on calcein delivery into adherent corneal endothelial cells.(A) constant fluence at 10 mJ/cm² and increasing exposure times; (B) constant exposure time at 10 min and increasing fluences. All images were obtained with a x10 objective and merged phase contrast/FITC. Green fluorescence (FITC+) indicated uptake of calcein. Scale bar 200 μm.
Mentions: No spontaneous calcein uptake was observed in the absence of either FsL irradiation or CNPs. With constant fluence of 10 mJ/cm², calcein+ CECs were visible even for 3 min exposure, but their number seemed to increase with exposure time. The area of the cell culture where uptake of calcein was present was strictly restricted to the area irradiated by the FsL (1 mm in diameter), showing that the reaction was very localized. With a constant exposure of 10 min, calcein+ CECs were visible only from 8 mJ/cm² and increased with fluence (Fig 3). The static irradiation proved the concept of delivery of a small molecule by CNPs activated by a FsL in a monolayer of adherent cells, and of the possibility to modulate efficiency with the laser parameters (fluence and exposure time).

Bottom Line: For clinical applications, physical methods have a more favorable individual and general benefit/risk ratio than most biological vectors, but are often less efficient.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.

View Article: PubMed Central - PubMed

Affiliation: Corneal Graft Biology, Engineering and Imaging Laboratory, EA2521, SFR143, Faculty of Medicine, Saint-Etienne, France.

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.

No MeSH data available.


Related in: MedlinePlus