title: Gene expression and internalization following vector adsorption to immobilized proteins: dependence on protein identity and density.
abstract: BACKGROUND
  Gene delivery by non-specific adsorption of non-viral vectors to protein-coated surfaces can reduce the amount of DNA required, and also increase transgene expression and the number of cells expressing the transgene. The protein on the surface mediates cell adhesion and vector immobilization, and functions to colocalize the two to enhance gene delivery. This report investigates the mechanism and specificity by which the protein coating enhances gene transfer, and determines if the protein coating targets the vector for internalization by a specific pathway.
  
  
  METHODS
  Proteins (FBS, BSA, fibronectin, collagen I, and laminin) were dried onto culture dishes, followed by PEI/DNA complex adsorption for surface delivery. Reporter genes were employed to characterize transfection as a function of the protein identity and density. Vector immobilization was measured using radiolabeled plasmid, and internalization was quantified in the presence and absence of the endocytosis inhibitors chlorpromazine and genistein.
  
  
  RESULTS
  Fibronectin coating yielded the greatest expression for PEI/DNA polyplexes, with maximal expression at intermediate protein densities. Expression in control studies with bolus delivery was independent of the protein identity. Substrate binding was independent of the protein identity; however, internalization was greatest on surfaces coated with fibronectin and collagen I. Inhibition of caveolae-mediated endocytosis reduced gene expression more than clathrin-mediated endocytosis. Similarly, inhibition of caveolae-mediated endocytosis significantly reduced the intracellular levels of DNA.
  
  
  CONCLUSIONS
  Fibronectin at intermediate densities mediated the highest levels of transgene expression, potentially by targeting internalization through caveolae-mediated endocytosis. Substrate modifications, such as the identity and density of proteins, provide an opportunity for modification of biomaterials for enhancing gene expression.
authors.affiliation: Department of Interdepartmental Biological Sciences, Northwestern University, 2145 Sheridan Rd E156, Evanston, IL 60208-3120, USA.
authors.forename: Zain
authors.initials: Z
authors.lastname: Bengali
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Rea
authors.forename: Lonnie D
authors.initials: LD
authors.lastname: Shea
mesh_terms: D000327:Adsorption; D000818:Animals; D020099:Coated Materials, Biocompatible; D024042:Collagen Type I / Q000378:metabolism; D003587:Cytomegalovirus / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D005353:Fibronectins / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007797:Laminin / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D011094:Polyethyleneimine / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014162:Transfection
chemical_list: D020099:Coated Materials, Biocompatible; D024042:Collagen Type I; D005353:Fibronectins; D007797:Laminin; D049452:Green Fluorescent Proteins; D012710:Serum Albumin, Bovine; D011094:Polyethyleneimine; D004247:DNA; D008156:Luciferases