BMP7 Gene Transfer via Gold Nanoparticles into Stroma Inhibits Corneal Fibrosis In Vivo

Abstract

This study examined the effects of BMP7 gene transfer on corneal wound healing and fibrosis inhibition in vivo using a rabbit model. Corneal haze in rabbits was produced with the excimer laser performing -9 diopters photorefractive keratectomy. BMP7 gene was introduced into rabbit keratocytes by polyethylimine-conjugated gold nanoparticles (PEI2-GNPs) transfection solution single 5-minute topical application on the eye. Corneal haze and ocular health in live animals was gauged with stereo- and slit-lamp biomicroscopy. The levels of fibrosis [α-smooth muscle actin (αSMA), F-actin and fibronectin], immune reaction (CD11b and F4/80), keratocyte apoptosis (TUNEL), calcification (alizarin red, vonKossa and osteocalcin), and delivered-BMP7 gene expression in corneal tissues were quantified with immunofluorescence, western blotting and/or real-time PCR. Human corneal fibroblasts (HCF) and in vitro experiments were used to characterize the molecular mechanism mediating BMP7’s anti-fibrosis effects. PEI2-GNPs showed substantial BMP7 gene delivery into rabbit keratocytes in vivo (2×104 gene copies/ug DNA). Localized BMP7 gene therapy showed a significant corneal haze decrease (1.68±0.31 compared to 3.2±0.43 in control corneas; p<0.05) in Fantes grading scale. Immunostaining and immunoblot analyses detected significantly reduced levels of αSMA (46±5% p<0.001) and fibronectin proteins (48±5% p<0.01). TUNEL, CD11b, and F4/80 assays revealed that BMP7 gene therapy is nonimmunogenic and nontoxic for the cornea. Furthermore, alizarin red, vonKossa and osteocalcin analyses revealed that localized PEI2-GNP-mediated BMP7 gene transfer in rabbit cornea does not cause calcification or osteoblast recruitment. Immunofluorescence of BMP7-transefected HCFs showed significantly increased pSmad-1/5/8 nuclear localization (>88%; p<0.0001), and immunoblotting of BMP7-transefected HCFs grown in the presence of TGFβ demonstrated significantly enhanced pSmad-1/5/8 (95%; p<0.001) and Smad6 (53%, p<0.001), and decreased αSMA (78%; p<0.001) protein levels. These results suggest that localized BMP7 gene delivery in rabbit cornea modulates wound healing and inhibits fibrosis in vivo by counter balancing TGFβ1-mediated profibrotic Smad signaling.

The Effect of Silica Nanoparticles on Human Corneal Epithelial Cells

“…Recently, it was suggested that this long challenging problem could be overcome with the use of nano-based drug carriers56. Several recent reports of successful intraocular drugs or gene deliveries by various nanoparticles further increased such expectations18192021222324. The major advantage of nanoparticles is enhanced cellular uptake due to their small size.…”

Targeted AAV5-Smad7 gene therapy inhibits corneal scarring in vivo

“…Haze scoring was performed according to the Fantes grading scale [29, 30]. Briefly, grade 0 was a completely clear cornea; grade 0.5 had trace haze visualized with careful tangential illumination on slit-lamp biomicroscopy; grade 1 was more prominent haze not interfering with the visibility of fine iris details; grade 2 represented sufficient haze as to cause mild obscuration of iris details; grade 3 was moderate obscuration of the iris and lens; and grade 4 was complete opacification of the cornea in the area of ablation.…”

Wounding the cornea to learn how it heals

“…Currently, the rabbit cornea is being used to study gene transfer procedures (Mohan et al, 2013). A recent study showed that gold nanoparticles coated with BMP7 can reduce corneal fibrosis in the rabbit (Tandon et al, 2013). Disadvantages of using rabbits include higher cost for purchase and maintenance than rats, restricted availability of polyclonal antibodies against various proteins of interest, and reduced availability of genetically diverse strains and transgenic animals.…”