Abstract
Purpose: :
TGFβ produces myofibroblasts and scarring in the cornea. Modulation of TGFβ biological activity is a novel approach to prevent fibrosis. Soluble TGFβRII receptor (sTGFβRII) can sequester TGFβ prior to signaling and fibrotic response. We tested the hypothesis that sTGFβRII gene delivered to human corneal fibroblasts (HCF) with linearized PEI nanoparticles (LPEI) could inhibit fibrosis in the cornea using an in vitro model.
Methods: :
The sTGFβRII containing TGFβRII extracellular domain bound to human immunoglobulin Fc chain was cloned into mammalian pcDNA3.1 expression vector (pcDNA3.1-sTGFβRII). Restriction enzyme and DNA sequencing confirmed nucleotide sequence of generated construct. A pcDNA3.1-gfp construct was used for optimization. HCF generated from donor human corneas using MEM medium supplemented with 10% serum. At 60-70% confluence, cDNA3.1-sTGFβRII or pcDNA3.1-gfp construct was delivered into HCF with LPEI (nitrogen/phosphate ratios 8, 30, 60, 120/2ml medium) or Lipofectamine-2000 (5µl/2ml medium). Tranfected/untransfected cultures were exposed to TGFβ (1ng/mL) for 5 days under serum-free conditions. Supernatant was used for ELISA to quantify sTGFβRII. Cells were used to analyze fibrosis markers [α-smooth muscle actin (SMA), collagen, f-actin] with real-time PCR, western blotting and immunocytochemistry. Toxicity of LPEI was assessed with phase-contrast microscopy and MTT assay.
Results: :
Gene delivery efficacy and toxicity of LPEI depended on N/P ratio. N/P ratio of 8 and 30 showed low toxicity and high gene delivery comparable to Lipofectamine-2000, a high-efficiency commercial vector, among tested N/P ratios. ELISA quantification of lipofectamine or nanoparticle delivered sTGFβRII in HCF ranged from 1620-1636 pg/mL suggesting high transgene delivery by either vehicle. Immunocytochemistry quantification of SMA in TGFβ-treated non-transfected HCF showed 17±5 SMA+ cells/40x field (90%) whereas sTGFβRII delivered with Lipofectamine or LPEI nanoparticles reduced SMA+ cells by 60-65% (6±4 cells/40x field; p<0.01). Similar level of SMA inhibition was detected with qRT-PCR (63%, p<0.05) and western blotting (65%; p<0.001).
Conclusions: :
LPEI nanoparticles (up to N/P 30) are an efficient, low toxicity vector for corneal gene therapy. LPEI-nanoparticle-mediated sTGFβRII gene therapy could be used to inhibit fibrotic response in the cornea.
Keywords: gene transfer/gene therapy • cornea: stroma and keratocytes • wound healing