L Prasmickaite1, A Høgset, T E Tjelle, V M Olsen, K Berg. 1. Department of Biophysics, Institute for Cancer Research, The Norwegian Radium Hospital, Montebellow, Oslo. lina.prasmickaite@labmed.uio.no
Abstract
BACKGROUND: Most non-viral gene therapy vectors deliver transgenes into cells through the endocytic pathway. Lack of escape from endocytic vesicles in many cases constitutes a major barrier for delivery of the functional gene. We have developed a new technology named photochemical internalisation (PCI) to achieve light-inducible cytosolic delivery of the transgene. The technology is based on a photochemical treatment employing photosensitisers localised in endocytic vesicles. In this work mechanisms involved in PCI-mediated transfection (photochemical transfection) were studied. METHODS: Human melanoma or colon carcinoma cells were pre-incubated with the photosensitiser aluminium phthalocyanine disulfonate (AlPcS2a) followed by treatment with plasmid encoding enhanced green fluorescent protein (EGFP) complexed with poly-L-lysine, N-(1-(2,3-dioleoxyloxy)propyl)-N,N,N,-trimethylammonium-methyl-sulfate (DOTAP) or polyethylenimine (PEI) and light exposure. The expression of the EGFP-gene was scored by fluorescence microscopy and flow cytometry. RESULTS: The photochemical treatment using light doses corresponding to D50 substantially improves the efficiency of transfection mediated by poly-L-lysine and PEI, but not by DOTAP. The treatment does not enhance the delivery of the plasmid complex across the plasma membrane, since the amount of internalised plasmid is similar for irradiated and non-irradiated cells. Light-inducible transfection occurs only under temperature conditions allowing endocytic uptake and is not improved by chloroquine or ammonium chloride, but is inhibited by bafilomycin A1 (agents that increase vesicular pH and interfere with the endocytic transport). CONCLUSIONS: Photochemical transfection occurs through endocytosis, followed by cytosolic release of the transfecting DNA from photochemically permeabilised endocytic vesicles. Release of plasmid from early endosomes seems to be of importance in photochemical transfection, although a role of later endocytic vesicles can, however, not be ruled out.
BACKGROUND: Most non-viral gene therapy vectors deliver transgenes into cells through the endocytic pathway. Lack of escape from endocytic vesicles in many cases constitutes a major barrier for delivery of the functional gene. We have developed a new technology named photochemical internalisation (PCI) to achieve light-inducible cytosolic delivery of the transgene. The technology is based on a photochemical treatment employing photosensitisers localised in endocytic vesicles. In this work mechanisms involved in PCI-mediated transfection (photochemical transfection) were studied. METHODS:Humanmelanoma or colon carcinoma cells were pre-incubated with the photosensitiser aluminium phthalocyanine disulfonate (AlPcS2a) followed by treatment with plasmid encoding enhanced green fluorescent protein (EGFP) complexed with poly-L-lysine, N-(1-(2,3-dioleoxyloxy)propyl)-N,N,N,-trimethylammonium-methyl-sulfate (DOTAP) or polyethylenimine (PEI) and light exposure. The expression of the EGFP-gene was scored by fluorescence microscopy and flow cytometry. RESULTS: The photochemical treatment using light doses corresponding to D50 substantially improves the efficiency of transfection mediated by poly-L-lysine and PEI, but not by DOTAP. The treatment does not enhance the delivery of the plasmid complex across the plasma membrane, since the amount of internalised plasmid is similar for irradiated and non-irradiated cells. Light-inducible transfection occurs only under temperature conditions allowing endocytic uptake and is not improved by chloroquine or ammonium chloride, but is inhibited by bafilomycin A1 (agents that increase vesicular pH and interfere with the endocytic transport). CONCLUSIONS: Photochemical transfection occurs through endocytosis, followed by cytosolic release of the transfecting DNA from photochemically permeabilised endocytic vesicles. Release of plasmid from early endosomes seems to be of importance in photochemical transfection, although a role of later endocytic vesicles can, however, not be ruled out.
Authors: Caitriona O'Rourke; Colin Hopper; Alexander J MacRobert; James B Phillips; Josephine H Woodhams Journal: Int J Pharm Date: 2017-06-01 Impact factor: 5.875