title: Corneal epithelium as a platform for secretion of transgene products after transfection with liposomal gene eyedrops.
abstract: BACKGROUND
  The first objective of the study was to evaluate the transfection of corneal epithelium with non-viral vectors to secrete transgene products into the tear fluid and aqueous humor. The second goal was to evaluate the differentiated corneal epithelial cell culture for transfection studies.
  
  
  METHODS
  The human corneal epithelial (HCE) cell line was cultured to different stages of differentiation and transfected with complexes of pCMV-SEAP2 with DOTAP/DOPE, DOTAP/DOPE/protamine sulfate (PS) and polyethylenimine (PEI). The complexes of DOTAP/DOPE with plasmid (CMV-SEAP2 or pCMV-Luc4) were subsequently applied topically to the rabbit eyes. Secreted alkaline phosphatase (SEAP) was analyzed using chemiluminescent assay. Luciferase (Luc) was detected at the mRNA level in cornea and conjunctiva using a qRT-PCR.
  
  
  RESULTS
  The transfection levels decreased with differentiation of HCE cells. PEI was effective in transfecting both the dividing and partly differentiated cells, but ineffective in differentiated cells. DOTAP/DOPE showed high activity in differentiated cell cultures, while added PS did not improve transfection. Significant SEAP expression was observed for three days after in vivo transfection in the tear fluid and aqueous humor. The luciferase mRNA was found both in the cornea and conjunctiva. The rates of SEAP secretion from both the basolateral side of differentiated HCE cells and cornea in vivo were within the same range.
  
  
  CONCLUSIONS
  Corneal epithelium can be transfected topically to secrete gene products to the tear fluid and aqueous humor. The differentiated HCE model is a useful tool in the evaluation of non-viral carriers for corneal transfection.
authors.affiliation: Department of Pharmaceutics, University of Kuopio, P.O. Box 1627, FI-70211 Kuopio, Finland. elisa.toropainen@uku.fi
authors.forename: Elisa
authors.initials: E
authors.lastname: Toropainen
authors.forename: Margit
authors.initials: M
authors.lastname: Hornof
authors.forename: Kai
authors.initials: K
authors.lastname: Kaarniranta
authors.forename: Pinja
authors.initials: P
authors.lastname: Johansson
authors.forename: Arto
authors.initials: A
authors.lastname: Urtti
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D019573:Epithelium, Corneal / Q000737:chemistry / Q000378:metabolism*; D005123:Eye / Q000378:metabolism; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D006801:Humans; D008081:Liposomes / Q000008:administration & dosage*; D008156:Luciferases / Q000235:genetics; D009883:Ophthalmic Solutions; D010539:Permeability; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010957:Plasmids / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D011817:Rabbits; D013666:Tears / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D019076:Transgenes
chemical_list: C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D005229:Fatty Acids, Monounsaturated; D008081:Liposomes; D009883:Ophthalmic Solutions; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D012333:RNA, Messenger; D008156:Luciferases; D000469:Alkaline Phosphatase; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane