title: Nanoparticle-mediated delivery of shRNA.VEGF-a plasmids regresses corneal neovascularization.
abstract: OBJECTIVE
  To determine the efficacy of a plasmid containing a small hairpin RNA expression cassette (pSEC.shRNA) against VEGF-A-loaded poly(lactic co-glycolic acid) nanoparticles (PLGA NPs) in the sustained regression of murine corneal neovascularization.
  
  
  METHODS
  PLGA nanoparticles were loaded with pSEC.shRNA.VEGF-A plasmids using the double emulsion-solvent evaporation method. KNV was induced in BALB/c mice by mechanical-alkali injury. Four weeks after induction of KNV, the mice were randomly divided to receive one of four treatments intrastromally: pSEC.shRNA.VEGF-A PLGA NPs (2 μg plasmid); naked pSEC.shRNA.VEGF-A plasmid only (2 μg plasmid); control blank PLGA NPs (equivalent dry weight of NPs); and vehicle. Two and five days after intervention, corneas were harvested to determine VEGF-A gene and protein expression using reverse transcriptase polymerase chain reaction and ELISA, respectively. Four weeks after intervention, corneas were photographed, mice sacrificed, and the corneal whole mounts were immunostained for CD31 (panendothelial cell marker). Immunofluorescence microscopy was performed and the neovascular area was quantitated.
  
  
  RESULTS
  VEGF-A mRNA (49.6 ± 12.4 vs. 82.9 ± 6.0%, P < 0.01) and protein (4.0 ± 5.2 vs. 20.0 ± 7.5 ρg VEGF-A/mg total protein, P < 0.05) expression were significantly reduced in pSEC.shRNA.VEGF-A PLGA NP-treated corneas as compared with control blank NP. The pSEC.shRNA.VEGF-A PLGA NP-treated corneas showed significant regression in the mean fractional areas of KNV (0.125 ± 0.042; 12.5%, P <0.01) compared with both naked plasmid only (0.283 ± 0.004; 28.3%) and control (blank NPs = 0.555 ± 0.072, 55.5%) at 4 weeks post-treatment.
  
  
  CONCLUSIONS
  The pSEC.shRNA.VEGF-A-loaded PLGA NPs are an effective, nonviral, nontoxic, and sustainable form of gene therapy for the regression of murine KNV.
authors.affiliation: John A. Moran Eye Center, Salt Lake City, UT 84132, USA.
authors.forename: Yureeda
authors.initials: Y
authors.lastname: Qazi
authors.forename: Brian
authors.initials: B
authors.lastname: Stagg
authors.forename: Nirbhai
authors.initials: N
authors.lastname: Singh
authors.forename: Swita
authors.initials: S
authors.lastname: Singh
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Zhang
authors.forename: Ling
authors.initials: L
authors.lastname: Luo
authors.forename: Jacquelyn
authors.initials: J
authors.lastname: Simonis
authors.forename: Uday B
authors.initials: UB
authors.lastname: Kompella
authors.forename: Balamurali K
authors.initials: BK
authors.lastname: Ambati
mesh_terms: D000818:Animals; D016510:Corneal Neovascularization / Q000378:metabolism / Q000517:prevention & control*; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D020868:Gene Silencing; D051379:Mice; D008807:Mice, Inbred BALB C; D053758:Nanoparticles; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A