title: Vascular gene transfer from metallic stent surfaces using adenoviral vectors tethered through hydrolysable cross-linkers.
abstract: In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.
authors.affiliation: Department of Pediatrics, Division of Cardiology, The Children's Hospital of Philadelphia, University of Pennsylvania; fishbein@email.chop.edu.
authors.forename: Ilia
authors.initials: I
authors.lastname: Fishbein
authors.affiliation: Department of Pediatrics, Division of Cardiology, The Children's Hospital of Philadelphia, University of Pennsylvania.
authors.forename: Scott P
authors.initials: SP
authors.lastname: Forbes
authors.affiliation: Department of Pediatrics, Division of Cardiology, The Children's Hospital of Philadelphia, University of Pennsylvania.
authors.forename: Richard F
authors.initials: RF
authors.lastname: Adamo
authors.affiliation: Department of Pediatrics, Division of Cardiology, The Children's Hospital of Philadelphia, University of Pennsylvania.
authors.forename: Michael
authors.initials: M
authors.lastname: Chorny
authors.affiliation: Department of Pediatrics, Division of Cardiology, The Children's Hospital of Philadelphia, University of Pennsylvania.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Levy
authors.affiliation: Department of Pediatrics, Division of Cardiology, The Children's Hospital of Philadelphia, University of Pennsylvania.
authors.forename: Ivan S
authors.initials: IS
authors.lastname: Alferiev
mesh_terms: D000256:Adenoviridae / Q000737:chemistry / Q000235:genetics; D017130:Angioplasty; D000818:Animals; D002417:Cattle; D054855:Drug-Eluting Stents; D042783:Endothelial Cells / Q000502:physiology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics; D008297:Male; D032389:Myocytes, Smooth Muscle / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley