Attenuation of corneal myofibroblast development through nanoparticle-mediated soluble transforming growth factor-β type II receptor (sTGFβRII) gene transfer

To explore (i) the potential of polyethylenimine (PEI)-DNA nanoparticles as a vector for delivering genes into human corneal fibroblasts, and (ii) whether the nanoparticle-mediated soluble extracellular domain of the transforming growth factor-β type II receptor (sTGFβRII) gene therapy could be used to reduce myofibroblasts and fibrosis in the cornea using an in vitro model. PEI-DNA nanoparticles were prepared at a nitrogen-to-phosphate ratio of 30 by mixing linear PEI and a plasmid encoding sTGFβRII conjugated to the fragment crystallizable (Fc) portion of human immunoglobulin. The PEI-DNA polyplex formation was confirmed through gel retardation assay. Human corneal fibroblasts (HCFs) were generated from donor corneas; myofibroblasts and fibrosis were induced with TGFβ1 (1 ng/ml) stimulation employing serum-free conditions. The sTGFβRII conjugated to the Fc portion of human immunoglobulin gene was introduced into HCF using either PEI-DNA nanoparticles or Lipofectamine. Suitable negative and positive controls to compare selected nanoparticle and therapeutic gene efficiency were included. Delivered gene copies and mRNA (mRNA) expression were quantified with real-time quantitative PCR (qPCR) and protein with enzyme-linked immunosorbent assay (ELISA). The changes in fibrosis parameters were quantified by measuring fibrosis marker α-smooth muscle actin (SMA) mRNA and protein levels with qPCR, immunostaining, and immunoblotting. Cytotoxicity was determined using cellular viability, proliferation, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. PEI readily bound to plasmids to form nanoparticular polyplexes and exhibited much greater transfection efficiency (p