Vitreous is a barrier in nonviral gene transfer by cationic lipids and polymers.
OBJECTIVE
To investigate the role of vitreous in nonviral gene delivery into retinal pigment epithelial (RPE) cells.


METHODS
Human RPE cell line D407 was cultured in six-well plates. Bovine vitreous, hyaluronan, or DMEM was added on the cells. Complexes of DNA and cationic carriers (polyethyleneimine, poly-L-lysine, DOTAP liposomes) were pipetted onto the vitreous, hyaluronan, or DMEM. Cellular uptake of DNA was studied with ethidium monoazide DNA and gene expression with GFP-plasmid complexes. FITC-dextrans and FITC-polylysines were used to probe the effects of the size and cationic charge on permeation in the vitreous in a similar experimental setup. Fluorescent cells were analyzed by flow cytometry.


RESULTS
Vitreous decreased the cellular uptake of DNA complexes 2-30 times, and GFP expression was also impaired. In hyaluronan solutions the cellular uptake of the complexes was also decreased significantly in most cases. In vitreous, cellular uptake of all FITC-dextrans decreased slightly, and uptake of poly-L-lysines was decreased substantially, whereas in hyaluronan solutions the effects were mild or nonexistent.


CONCLUSIONS
Polymeric and liposomal gene delivery is substantially limited by the vitreous. This is probably because of the size and charge of the retinal gene delivery after intravitreal injections.
Department of Pharmaceutics, University of Kuopio, Kuopio, Finland.
Leena
L
Pitkänen
Marika
M
Ruponen
Jenni
J
Nieminen
Arto
A
Urtti
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D005229:Fatty Acids, Monounsaturated / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D018014:Gene Transfer Techniques; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism; D008055:Lipids / Q000737:chemistry / Q000493:pharmacokinetics*; D010857:Pigment Epithelium of Eye / Q000378:metabolism / Q000502:physiology; D011094:Polyethyleneimine / Q000378:metabolism; D011107:Polylysine / Q000378:metabolism; D011108:Polymers / Q000737:chemistry / Q000493:pharmacokinetics*; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D014822:Vitreous Body / Q000737:chemistry* / Q000502:physiology*
D005229:Fatty Acids, Monounsaturated; D008055:Lipids; D011108:Polymers; D000644:Quaternary Ammonium Compounds; D011107:Polylysine; D011094:Polyethyleneimine; D006820:Hyaluronic Acid; D016650:Fluorescein-5-isothiocyanate; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane