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Vitreous is a barrier in nonviral gene transfer by cationic lipids and polymers
Leena Pitkänen1, Marika Ruponen, Jenni Nieminen
1Department of Pharmaceutics, University of Kuopio, Kuopio, Finland.
Purpose:
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.