Abstract
Corneal keratocyte apoptosis triggered by cornel debridement is one mechanism of corneal disorders. In this study, the feasibility of cyclo-(d-Trp-Tyr) peptide nanotubes (PNTs) as carriers of caspase 3 silence shRNA delivery was assessed. A model of epithelial injury by epithelial debridement was applied to investigate the feasibility of PNTs as gene delivery carriers on corneal injury. First, the PNTs were found within 2 μm in length and 300 nm in width by an atomic force microscope and confocal laser microscope system. Plasmid DNAs were observed to be associated with PNTs by atomic force microscope and confocal laser scanning microscope. The plasmids were associated with tyrosine of PNTs with a binding constant of 2.7 × 108 M−1. The stability of plasmid DNA with PNTs against the DNase was found at 60 min. Using thioflavin T pre-stained PNTs on the corneal eye drop delivery, the distribution of PNTs was in the epithelial and stroma regions. After corneal debridement, the rhodamine-labeled plasmid DNA and thioflavin T pre-stained PNTs were also delivered and could be observed in the stroma of cornea. PNTs complexed with anti-apoptotic plasmid caspase 3 silencing shRNA eye drop delivery decreased 41% of caspase 3 activity after the first dose by caspase 3 activity and Western blot analysis.
Highlights
Cornea epithelial debridement often results from cornea surgery, such as laser-assisted in situ keratomileusis (LASIK) and photorefractive keratectomy (PRK) in the treatment of myopia, hyperopia or astigmatism [1,2,3]
In epithelial debridement-induced cornea injury with delayed wound healing, keratocyte apoptosis resulted in the anterior stroma, and subsequent myofibroblast formation determined final visual regression and stromal haze formation, which often occurred after mechanical epithelial scrape and PRK for myopia [1,11,12,13,14]
The tube-shaped peptide nanotubes (PNTs) composed of cyclo-(D-Trp-Tyr) appeared to be 1.8 ± 0.6 μm in length as observed by scanning electron microscope (SEM) (Figure 1A,B)
Summary
Cornea epithelial debridement often results from cornea surgery, such as laser-assisted in situ keratomileusis (LASIK) and photorefractive keratectomy (PRK) in the treatment of myopia, hyperopia or astigmatism [1,2,3]. In epithelial debridement-induced cornea injury with delayed wound healing, keratocyte apoptosis resulted in the anterior stroma, and subsequent myofibroblast formation determined final visual regression and stromal haze formation, which often occurred after mechanical epithelial scrape and PRK for myopia [1,11,12,13,14]. Using topical administration of pCMV-bcl-xL-eGFP DNA with sphere-type polymeric micelles (PM) after corneal epithelial debridement, bcl-xL-eGFP fusion protein was detected in wounded ocular tissues, and both DNA fragmentation and caspase-3 activity were significantly decreased [16]. We delivered the plasmid-encoding caspase 3 silencing shRNA with PNTs via eye drop to cornea with epithelial debridement and assessed its anti-apoptotic effect
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