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

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-Llysine, 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), FITClabeled 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 inhibited. Neural retina decreased the cellular uptake of FITColigonucleotides, 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.

Can Ultrasound Solve the Transport Barrier of the Neural Retina?

“…Unfortunately, in contrast to the situation in vitreous, we show in this study that a high pegylation degree of DOTAP DOPE lipoplexes does not improve their permeation through the neural retina. Our results confirm previous experiments by Pitkänen et al (33) who found that neural retina completely blocks nonpegylated lipoplexes. Their data indicated that the cationic charge of the penetrating substance is even more important than its size.…”

“…The eyes were kept at +9°C. The eyes were treated as previously described by Pitkänen et al (33) Briefly, after the eyes were cleaned of extra-ocular tissue and dipped in a 0.9% NaCl solution containing 1% penicillin/streptomycin (Gibco, Merelbeke, Belgium), they were opened circumferentially ±8 mm behind the limbus. The anterior tissues and the vitreous were separated gently from the neural retina.…”

“…The IPRM, located between the outer limiting membrane of the retina and apical border of the RPE, also contains GAGs (32) and may thus also form a barrier for the penetrating gene complexes. Previously Pitkänen et al (33) demonstrated that the passage of polyethyleneimine polyplexes, poly-L-lysine polyplexes and DOTAP liposomes through the neural retina was restricted by the ILM.…”

Dendrimers for gene delivery – a potential approach for ocular therapy?

“…[68] Moreover, the inner and outer limiting membranes at the back of the eye present a tight barrier for gene delivery to the retinal tissues. [69] Pitkanen et al [72] demonstrated that the diffusion of PEI and PLL polyplexes through the neural retina was restricted by the inner limiting membrane, which contains laminin, collagen and several proteoglycans. Thus, additional surface modifications such a PEGylation may be required to overcome this barrier.…”

New Techniques for Drug Delivery to the Posterior Eye Segment

“…Cationic lipids and polymers are frequently used owing to binding and condensing of DNA and RNA to small particulates in the range of 100 nm. Despite their small size, the nanoparticulate systems have limited diffusion in the tissues, possibly due to the barriers placed by the vitreous and retina (74,75). This may be due to the steric hindrance and electrostatic interactions with ocular polyanions, such as hyaluronic acid and chondroitin sulfate.…”