Transfection efficiency of lipoplexes for site-directed delivery.
Targeted gene delivery is a promising strategy to cure disease on its basic level at the site of interest. The ultrastructure, internalization, and transfection efficiency of lipoplexes was investigated. We found that at a charge ratio (rho) of 4.0 lipoplexes had optimum characteristics for gene delivery in vitro. To decrease the size of lipoplexes, we used a method of continuous-flow microfluidics. PEGylation of lipoplexes did not hinder internalization, but was found to hamper transfection. To discriminate between uptake and transfection efficiency of lipoplexes, we used fluorescence-based approaches: microscopy and FACS. To this end, GFP plasmid was labeled with Alexa 594, and, in parallel experiments, GFP plasmid was combined with rhodamine-labeled lipid. Our studies confirm that cellular uptake does not imply transfection efficiency, and that hurdles in cellular processing have to be taken before targeted gene delivery becomes an established therapeutic option.
Jellema
Reint K
RK
Department of Pathology, Electron Microscopy Unit, Maastricht University, Maastricht, The Netherlands.
Bomans
Paul
P
Deckers
Niko
N
Ungethum
Liset
L
Reutelingsperger
Chris P M
CP
Hofstra
Leo
L
Frederik
Peter M
PM
D020285:Cryoelectron Microscopy; D004247:DNA / Q000008:administration & dosage; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D005434:Flow Cytometry; D018390:Gene Targeting / Q000379:methods*; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000008:administration & dosage* / Q000737:chemistry; D044085:Microfluidics; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009997:Osmotic Pressure; D011092:Polyethylene Glycols / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D014162:Transfection / Q000379:methods*
D005229:Fatty Acids, Monounsaturated; D008055:Lipids; D008081:Liposomes; D000644:Quaternary Ammonium Compounds; D049452:Green Fluorescent Proteins; D011092:Polyethylene Glycols; D004247:DNA; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane