The AAPS Journal

, Volume 6, Issue 3, pp 72–80

Neural retina limits the nonviral gene transfer to retinal pigment epithelium in an in vitro bovine eye model

Authors

    • Department of PharmaceuticsUniversity of Kuopio
    • Department of OphthalmologyKuopio University Hospital
  • Jukka Pelkonen
    • Department of Clinical MicrobiologyUniversity of Kuopio
    • Department of Clinical MicrobiologyKuopio University Hospital
  • Marika Ruponen
    • Department of PharmaceuticsUniversity of Kuopio
  • Seppo Rönkkö
    • Department of PharmaceuticsUniversity of Kuopio
  • Arto Urtti
    • Department of PharmaceuticsUniversity of Kuopio
Article

DOI: 10.1208/aapsj060325

Cite this article as:
Pitkänen, L., Pelkonen, J., Ruponen, M. et al. AAPS J (2004) 6: 72. doi:10.1208/aapsj060325

Abstract

We investigated the permeation of liposomal and polymeric gene delivery systems through neural retina into retinal pigment epithelium (RPE) and determined the roles of various factors in permeation and subsequent uptake of the delivery systems by RPE. Anterior parts and vitreous of fresh bovine eyes were removed. Retina was left intact or peeled away. Complexes of ethidium monoazide (EMA)-labeled plasmid DNA and cationic carriers (polyethyleneimine, poly-L-lysine, DOTAP liposomes) were pipetted on the retina or RPE. Two hours later the neural retina was removed, if present, and the RPE cells were detached. Contaminants were removed by sucrose centrifugation, and the RPE cells were analyzed for DNA uptake by flow cytometry. Cellular uptake of FITC-dextrans (molecular weight [mw] 20 000, 500 000 and 2 000 000), FITC-poly-L-lysine (mw 20 000), FITC-labeled oligonucleotide (15-mer), and naked EMA-labeled plasmid DNA was determined after pipetting the solutions on the RPE or neural retina. Location of the fluorescent materials in the retina was visualized with fluorescence microscopy. Neural retina decreased the cellular uptake of DNA complexes by an order of magnitude, the uptake of FITC-dextrans slightly, whereas delivery of polycationic FITC-poly-L-lysine to RPE was almost completely inhibibited. Neural retina decreased the cellular uptake of FITC-oligonucleotides, while the uptake of uncomplexed plasmid was always negligible. conclusions from FACS and fluorescence microscopy were similar: delivery of polymeric and liposomal DNA complexes into RPE are limited by the neural retina. This is due to the size and positive charge of the complexes.

Keywords

gene deliveryintravitrealretinaliposomepolymer

Copyright information

© American Association of Pharmaceutical Scientists 2004