Efficiency and toxicity of liposome-mediated gene transfer to corneal endothelial cells.
Gene transfer to corneal endothelial cells could be an important advance to modulate functions of these critical cells and is a field of current investigations. The development of gene transfer methods is a prerequisite for gene therapy to realize its full potential. We attempted to investigate and optimize the efficacy and safety of cationic liposome mediated gene transfer into corneal endothelial cells using different lipid formulations. Mono- and polycationic lipids and the neutral helper lipid dioleolphosphotidyl-ethanolamine (DOPE) were used for preparation of cationic liposomes. Six liposomal formulations containing DAC/DOPE 30/70 (DAC 30), DOSGA/DOPE 30/70 (DOSGA 30), DOSGA 100, DMRIE/DOPE 50/50 (DMRIE 50) and SP/DOPE 20/80 (SP 20) were complexed with the pUT 651-plasmid, encoding the E. coli beta-galactosidase gene. Subconfluent primary and passaged bovine corneal endothelial cells (BCEC) were transfected with different amounts of liposomes and DNA or uncomplexed free DNA as control. Quantitative expression of beta-galactosidase was measured using a colorimetric assay. In order to assess the effects on cell viability and growth, a modified acidic phosphatase assay was employed. Differences were detected using these various liposome preparations. Transfection experiments demonstrated the highest gene expression using SP 20> DMRIE 50 ranging at approximately 3 mU per beta-gal per well. Low expression of beta-galactosidase was achieved using DAC 30, DOSGA 30 and DOSGA 100. No beta-galactosidase expression was found in control dishes. There was no difference seen following transfection of primary or subsequent passages of BCEC. As indicated by the acid phosphatase assay, no significant toxicity was detected for the most efficient lipids used. Of the preparations studied, SP 20 appeared as the optimal vehicle for plasmid-mediated transfection of BCEC. The ability to deliver genes to BCEC via liposomes could be valuable, since the use of other vectors for transfection may be limited by undesired effects.
Pleyer
U
U
Department of Ophthalmology, Charité, Campus Virchow, Humboldt University, Augustenburger Platz 1, D-13353 Berlin, Germany. uwe.pleyer@charite.de
Groth
D
D
Hinz
B
B
Keil
O
O
Bertelmann
E
E
Rieck
P
P
Reszka
R
R
D000818:Animals; D002417:Cattle; D002455:Cell Division; D002470:Cell Survival; D003124:Colorimetry; D004728:Endothelium, Corneal / Q000166:cytology / Q000502:physiology*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D008055:Lipids / Q000737:chemistry; D008081:Liposomes; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
D008055:Lipids; D008081:Liposomes; D010744:Phosphoric Monoester Hydrolases; D001616:beta-Galactosidase