Rapid And Substantial Gene Delivery Into Cornea In Vivo And In Vitro With Linearized Polyethyleneimine Nanoparticles

Abstract

Purpose: : Polyethyleneimine is biocompatible, biodegradable and FDA approved agent for human medical applications. The aims of this study were to test the toxicity and gene transfer efficacy of linearized PEI nanoparticles (PEI-NP) for the mouse cornea in vivo and human cornea in vitro.

Methods: : Female black C57 mice for in vivo and human stromal fibroblast (HSF) for in vitro studies were used. Transfection solution was prepared by incubating PEI-NP with plasmid (pTRUF11 expressing green fluorescent protein) in 10% glucose at 37 oC for 30 minutes. Two microliters of vehicle, PEI-NP, or transfection solution was topically applied once to the cornea of anesthetized animal for two minutes using defined hair dryer-based application technique. Mouse eyes were collected 1d, 3d, 7d, and 14d after vector application. HSF cultures were exposed to vehicle, PEI-NP, or PEI-NP-plasmid (10µl/ml medium) for 6h for in vitro investigations. Visual eye exam, and stereo- and slit-lamp biomicroscopy were used to monitor corneal health and fluorescent gene delivery in the eyes of live animals. Immunocytochemistry, western blotting, and real-time PCR techniques tested delivered transgene expression. Phase-contrast microscopy, H&E staining, trypan blue, and TUNEL assays evaluated phenotype, cellular viability, and toxicity.

Results: : Trypan blue and TUNEL assays demonstrated ≥95% cellular viability. No phonotypic changes in HSF were detected with phase-contrast microscopy. A significantly high (30-37%; p<0.01) transgene delivery was noted in HSF exposed to PEI-NP transfection solution for 6h. Slit-lamp biomicroscopy revealed no significant inflammation, redness, or opacity in PEI-NP treated corneas. However, moderate corneal haze and delayed epithelial healing were observed in few animals. PEI-NP treated mouse corneas showed substantial transgene expression in a time-dependent manner. Mouse corneal tissue section of 14d time point showed significantly higher fluorescent gene expression compared to 1d time point. Additional real-time PCR, immunocytocychemistry, western blotting, and quantification analyses are underway.

Conclusions: : Tested PEI-NP has potential for developing non-viral gene transfer approaches.

Keywords: gene transfer/gene therapy • cornea: basic science • cornea: stroma and keratocytes