Linear Polyethylenimine–Based DNA Nanoparticle Delivery Into Mouse Retinas

Abstract

Purpose: : The cationic linear–polyethylenimine (LPEI) has recently been explored as a gene transfer vector in various cell types. In this study, we evaluated the LPEI–based DNA nanoparticles for gene transfer into retinal and periocular mouse tissues.

Methods: : Complexed as nanoparticles with linear, 22 kDa polyethylenimine, three different plasmid DNAs (pMOPS500–GFP, pCBA–GFP pCMV–GFP) were used. A homogeneous population of LPEI/DNA nanoparticles was produced with mean a diameter of approximately 50 nm. Before injection, we confirmed the resistance of LPEI/DNA complexes to DNase I digestion. Then, the LPEI/DNA complexes were subretinally injected into both eyes of adult C57Bl6 mice at various DNA concentrations and different N/P ratios (PEI nitrogen/DNA phosphate). As controls, naked plasmid DNA, rAAV5 vector with same promoters and GFP cDNA and nanoparticles without DNA were subretinally injected. At 2, 3, 4, 5, 6, 7, 9, 12, 14 and 21 days after injection, the right eyes of each mouse were enucleated and briefly fixed in 4% paraformaldehyde. The retinas were then dissected free from the choroid and sclera, whole mounted and examined viewed by fluorescence microscopy. The left eyes were processed as frozen sections and stained with GFP antibody. At 2 and 3 weeks post–injection, ERGs were recorded to evaluate any toxicity of LPEI/DNA nanoparticles on retinal function.

Results: : LPEI/DNA nanoparticle–mediated GFP transduction is efficient in the mouse retina. High levels of GFP expression were found in photoreceptors two days after subretinal injection with nanoparticles having a N/P ratio of 9 nitrogen equivalents per DNA phosphate. Fewer cells of the inner nuclear layer, RPE, choroid and ganglion cell layer were also positive. Reporter gene expression was dose– and N/P ratio–dependent in all tissues examined. High amounts of concentration of plasmid DNA (>0.5 ug/ul in a 1ul subretinal injection) or a high N/P ratio (>9) damaged the retina. Both the morphology and ERG data showed that the optimal concentration of plasmid DNA in a nanoparticle is about 0.2 ug/ul for a 1ul njection and the best N/P ratio is 6. Time course data showed that LPEI nanoparticle expression initiated before 2 days and lasted longer than 21 days after injection.

Conclusions: : Linear polyethylenimine–based nanoparticle transfection was found to be effective for in vivo gene transfer into the mouse retina by subretinal administration.

Keywords: gene transfer/gene therapy • gene/expression • retina