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Marta Czugala, Olga Mykhaylyk, Bernhard B Singer, Sebastian Wesselborg, Christian Plank, Friedrich E Kruse, Thomas Armin Fuchsluger; Magnetolipofection - promising tool for targeted gene therapy of corneal endothelium. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1176.
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© ARVO (1962-2015); The Authors (2016-present)
Gene therapy of corneal endothelium (CE) faces the challenge of specific targeting. Topical or intracameral application does not allow to target the CE precisely. Association of delivery vectors with magnetic nanoparticles (MNP) can overcome this problem by delivery to sites of gradient magnetic field application, thus minimizing the risk of uncontrolled transfection. In this study, we performed plasmid DNA delivery to CE cells (CEC) in complexes with MNP and liposomal enhancers upon application of a gradient magnetic field (magnetolipofection) and evaluated resulting transfection efficiency, immune compatibility and effect on endothelial function.
Transfection efficiency and viability in CEC were assessed after magnetolipofection with three types of MNP and transfection enhancers. Expression of CD25 and CD69 activation markers, cell viability and proliferation in human PBMCs were studied using flow cytometry. Cell viability was determined by PI or AnnexinV/PI staining. Transfection efficiency in human corneas was examined by confocal microscopy.
Magnetolipofection in CEC has proven to be up to four times more efficient than standard lipofection-based methods. MNP increased efficiency for XTremeGeneHP by 60% (p=0.0002). Already with very low dose of pDNA (3pg/cell) and low iron-to-pDNA w/w ratio (0.25), 39.5±2,7% of cells were successfully transfected while only 4.6±1.1% of cells were non-viable. For a dose of 6pg/cell, efficiency was only 30.6±4.0% with 9.8±3.7% of non-viable cells. In human donor corneas 9.9% transfected cells were observed for a dose of 360ng pDNA/ml (approx. 2.5pg/CE cell). Biocompatibility studies with human PBMCs from healthy donors have shown no significant effects on cell viability or activation of T cells after magnetolipofection.
In this study, magnetic nanoparticles enabled high transfection efficiency in CEC at low dose of nucleic acid per cell, resulting in low toxicity. Our results indicate that the method can be suitable for in vitro cornea therapy. The data also show that magnetolipofection itself does not impair endothelial function or trigger immune response in human PBMCs, thus indicating good biocompatibility. In addition, the magnetic features of the nanoparticles enable to control their distribution in vivo. We conclude, that magnetolipofection provides a promising tool for targeted gene therapy of corneal endothelial pathologies.
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