title: Nonviral vector loaded collagen sponges for sustained gene delivery in vitro and in vivo.
abstract: BACKGROUND
  Naked DNA and standard vectors have previously been used for gene delivery from implantable carrier matrices with great potential for gene therapeutic assistance of wound healing or tissue engineering. We have previously developed copolymer-protected gene vectors which are inert towards opsonization. Here we examine their potency in carrier-mediated gene delivery in comparison to standard vectors using a vector-loaded collagen sponge model.
  
  
  METHODS
  Equine collagen type I sponges were loaded by a lyophilization method with naked DNA, polyethylenimine (PEI)-DNA, DOTAP/cholesterol-DNA and copolymer-protected PEI-DNA. These preparations were characterized in terms of vector-release, cell growth on the matrices and reporter gene expression by cells colonizing the sponges in vitro and in vivo. Subcutaneous implantation of sponges in rats served as an in vivo model.
  
  
  RESULTS
  At the chosen low vector dose, the loading efficiency was at least 86%. Naked DNA-loaded collagen matrices lost 77% of the DNA dose in an initial burst in aqueous buffer in vitro. The other preparations examined displayed a sustained vector release. There was no difference in cell growth and invasion of the sponges between vector-loaded and untreated collagen grafts. Reporter gene expression from cells colonizing the sponges in vitro was observed for not more than 7 days with naked DNA, whereas the lipoplex and polyplex preparations yielded long-term expression throughout the experimental period of up to 56 days. The highest expression levels were achieved with the PEI-DNA-PROCOP (protective copolymer) formulation. Upon subcutaneous implantation in rats, no luciferase expression was detected with naked DNA preparations. DOTAP/cholesterol-DNA and PEI-DNA-loaded implants lead to reporter gene expression for at least 3 days, but with poor reproducibility. PEI-DNA-PROCOP collagen matrices yielded consistently the highest reporter gene expression levels for at least 7 days with good reproducibility.
  
  
  CONCLUSIONS
  With the preparation method chosen, lipoplex- and polyplex-loaded collagen sponges are superior in mediating sustained gene delivery in vitro and local transfection in vivo as compared to naked DNA-loaded sponges. Protective copolymers are particularly advantageous in promoting the tranfection capacity of polyplex-loaded sponges upon subcutaneous implantation, likely due to their stabilizing and opsonization-inhibiting properties.
authors.affiliation: Institute of Experimental Oncology, Technische Universität München, Ismaninger Str. 22, 81675 Munich, Germany.
authors.forename: Franz
authors.initials: F
authors.lastname: Scherer
authors.forename: Ulrike
authors.initials: U
authors.lastname: Schillinger
authors.forename: Ursula
authors.initials: U
authors.lastname: Putz
authors.forename: Axel
authors.initials: A
authors.lastname: Stemberger
authors.forename: Christian
authors.initials: C
authors.lastname: Plank
mesh_terms: D016475:3T3 Cells; D000818:Animals; D003094:Collagen; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D008156:Luciferases / Q000235:genetics; D051379:Mice; D051381:Rats
chemical_list: D003094:Collagen; D008156:Luciferases