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

et al. (2013) BMP7 Gene Transfer via Gold Nanoparticles into Stroma Inhibits Corneal Fibrosis In Vivo. PLoS ONE 8(6): e66434. doi:10.1371/journal.pone.0066434 BMP7 Gene Transfer via Gold Nanoparticles into Stroma Inhibits Corneal Fibrosis In Vivo Ashish Tandon 0 Ajay Sharma 0 Jason T. Rodier 0 Alexander M. Klibanov 0 Frank G. Rieger 0 Rajiv R. Mohan 0 Che John Connon, University of Reading, United Kingdom 0 1 Harry S. Truman Memorial Veterans' Hospital , Columbia , Missouri, United States of America, 2 Mason Eye Institute, School of Medicine, University of Missouri , Columbia , Missouri, United States of America, 3 Departments of Chemistry and Biological Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts , United States of America, 4 College of Veterinary Medicine, University of Missouri , Columbia, Missouri , United States of America 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 (PEI2GNPs) 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 [a-smooth muscle actin (aSMA), 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 (26104 gene copies/ug DNA). Localized BMP7 gene therapy showed a significant corneal haze decrease (1.6860.31 compared to 3.260.43 in control corneas; p,0.05) in Fantes grading scale. Immunostaining and immunoblot analyses detected significantly reduced levels of aSMA (4665% p,0.001) and fibronectin proteins (4865% 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 TGFb demonstrated significantly enhanced pSmad-1/5/8 (95%; p,0.001) and Smad6 (53%, p,0.001), and decreased aSMA (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 TGFb1-mediated profibrotic Smad signaling. - Funding: The work was supported by 1I01BX000357-01Veteran Health Affairs Merit (RRM), RO1EY17294 National Eye Institute, National Institutes of Health (RRM), RO1EB000244 National Institutes of Health (AMK), and Research to Prevent Blindness Unrestricted (Mason Eye Institute) grants. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have the following interests. Rajiv R. Mohan is a PLOS ONE Editorial Board member. There are no patents, products in development or marketed products to declare. This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. Trauma, infection, or chemical/surgical injury to the cornea can cause fibrosis/scarring resulting in loss of corneal transparency. Though corneal scarring is the third leading cause of blindness worldwide and affects over one million Americans every year [1], no effective therapy is yet available to treat corneal scarring. Steroids have been used, but their effectiveness remains controversial and they have significant side effects [2,3]. Mitomycin C is commonly used in clinic to treat laser surgery-induced corneal scarring [46]. However, its use has been associated with serious side effects both in preclinical and clinical studies [711]. Due to the lack of effective and safe drugs, many cases of corneal scarring require corneal transplantation. Despite its high success rate, corneal transplantation poses the challenges of postsurgical complications and the limited availability of high quality donor corneas. Thus, there is a need for newer and effective treatments for corneal scarring. Innovations in nanotechnology have generated nanoparticles that can potentially be used as gene delivery vectors, largely due to their ability to carry therapeutic molecules with high efficiency and low toxicity into targeted cells/tissues. A variety of nanoparticles recently have been tested for their potential as a gene therapy vectors for various cell types [1215]. Some of these nanoparticles such as gold, albumin, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and poly(lactic-co-glycolic acid) (PLGA) have been tested for gene therapy to treat corneal diseases including fibrosis or neovascularization [1619]. Recently, we identified that polyethylenimine-conjugated gold nanoparticles (PEI2-GNPs) are capable of delivering genes into corneal cells both in vitro and in vivo with high potency and low-moderate toxicity [20]. The PEI2-GNPs are a promising non-viral vector for ocular gene therapy due to their many unique features such as ease of synthesis, high plasmid binding capacity, biocompatibility and low immunogenicity. These characteristics and high transfection efficiency prompted us to hypothesize that PEI2-GNPs-mediated targeted gene delivery into keratocytes of rabbit stroma can provide required levels of therapeutic genes to treat corneal diseases. Bone morphogenetic proteins (BMP) are a large family of growth factors with more than 10 members [21,22]. The cornea has been reported to express several BMPs and their receptors [2327]. We have previously reported that BMP2 and BMP4 modulate keratocyte proliferation and apoptosis in the human cornea [23]. Among other BMP family members, BMP7 has been shown to play a pivotal role in eye development during embryogenesis, and BMP7- knockout mice have anophthalmia [28,29]. In adult animals, endogenous BMP7 levels in the eye and other organs decline but exogenous BMP7 administration has been demonstrated to attenuate fibrosis [30,31]. In the cornea, therapeutic effects of BMP7 have been examined by topical application of recombinant protein or adenovirus-mediated gene deli (...truncated)