July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Targeted Gene Therapy of the Corneal Endothelium using Functionalized Nanoparticle Assisted Femtosecond Laser Technology
Author Affiliations & Notes
  • Rami Darwich
    Department of Ophthalmology, Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada
    Faculty of Medicine, McGill University, Montreal, Quebec, Canada
  • Hasitha de Alwis Weerasekera
    Department of Ophthalmology, Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada
    Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario, Canada
  • Sergiy Patskovsky
    Department of Engineering Physics, Polytechnique, Motreal, Quebec, Canada
  • Marilyse Piche
    Department of Ophthalmology, Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada
  • Joseph Roman Casey
    Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada
    Department of Ophthalmology, Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada
  • Ariel Wilson
    Department of Engineering Physics, Polytechnique, Motreal, Quebec, Canada
  • Santiago Costantino
    Department of Ophthalmology, Université de Montréal, Montreal, Quebec, Canada
  • Przemyslaw Mike Sapieha
    Department of Ophthalmology, Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada
    Department of Ophthalmology, Université de Montréal, Montreal, Quebec, Canada
  • Michel Meunier
    Department of Engineering Physics, Polytechnique, Motreal, Quebec, Canada
  • Isabelle Brunette
    Department of Ophthalmology, Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada
    Department of Ophthalmology, Université de Montréal, Montreal, Quebec, Canada
  • Footnotes
    Commercial Relationships   Rami Darwich, None; Hasitha de Alwis Weerasekera, None; Sergiy Patskovsky, None; Marilyse Piche, None; Joseph Casey, None; Ariel Wilson, None; Santiago Costantino, None; Przemyslaw Mike Sapieha, None; Michel Meunier, None; Isabelle Brunette, None
  • Footnotes
    Support  Fonds de recherche en ophtalmologie de l'Université de Montréal, Canadian Institutes for Health research (CIHR) and FRQS Vision Health Research Network.
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 4553. doi:
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      Rami Darwich, Hasitha de Alwis Weerasekera, Sergiy Patskovsky, Marilyse Piche, Joseph Roman Casey, Ariel Wilson, Santiago Costantino, Przemyslaw Mike Sapieha, Michel Meunier, Isabelle Brunette; Targeted Gene Therapy of the Corneal Endothelium using Functionalized Nanoparticle Assisted Femtosecond Laser Technology. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4553.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Gold-nanoparticle (AuNP) mediated femtosecond laser optoporation transiently permeabilizes the cellular membrane, allowing the introduction of membrane-impermeable molecules, such as DNA plasmids, into targeted cells. Maximizing the specificity of AuNP towards target cells is necessary to enhance the safety of the technique for clinical use. The goal of this study was to assess the specificity and efficacy of antibody (Ab) conjugated AuNP and its effect on optoporating corneal endothelial cells.

Methods : Thiol mediated antibody (Ab) conjugation of AuNP was confirmed by dynamic light scattering (DLS) and UV-Vis spectroscopy. Ab-AuNP specificity was assessed by quantifying specific adherence of AuNP on human corneal endothelial cells (HCEC) and murine cornea (in vivo) using immunofluorescence and backscattering microscopy. HCEC viability was tested at a range of Ab-AuNP concentrations (7.00 – 0.01 µg/mL) and laser fluences (60 – 140 mJ/cm2). Optoporation of HCEC with GFP plasmid was conducted using the off-resonance excitation of cell membrane bound Ab-AuNP using a femtosecond laser (Ti:Sapphire laser, λ = 800 nm).

Results : Ab-AuNP surface functionalization was confirmed by the larger hydrodynamic diameter (DLS) and by the change in the AuNP dielectric microenvironment (UV-Vis). In vivo results depicted a sixteen-fold increase in the specific cellular adherence of Anti-Slc4a4-AuNP (98±27 AuNP/cell) on the endothelium, outperforming the PEG-AuNP control (6±1 AuNP/cell). A concentration of 3.05 µg/mL of Ab-AuNP and <80 mJ/cm2 of laser fluence were found to be optimal in maintaining HCEC viability (>99% viability in comparison to control). Successful internalization and transient expression of GFP in HCEC using Ab-AuNP assisted optoporation was documented.

Conclusions : We report a significant improvement on the specificity of the targeted AuNP by antibody conjugation. In addition, this is the first report that demonstrates the successful plasmid optoporation of corneal endothelial cells. Further optimization of the laser parameters is needed to increase optoporation efficacy. Corneal is an ideal model for applying this new technology and with further optimization this research will provide a novel therapeutic gene transfer toolset and will have a significant impact in ophthalmology.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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