title: Nerve growth factor expression by PLG-mediated lipofection.
abstract: Biomaterials capable of efficient gene delivery provide a fundamental tool for basic and applied research models, such as promoting neural regeneration. We developed a system for the encapsulation and sustained release of plasmid DNA complexed with a cationic lipid and investigated their efficacy using in vitro models of neurite outgrowth. Sustained lipoplex release was obtained for up to 50 days, with rates controlled by the fabrication conditions. Released lipoplexes retained their activity, transfecting 48.2+/-8.3% of NIH3T3 cells with luciferase activity of 3.97x10(7)RLU/mg. Expression of nerve growth factor (NGF) was employed in two models of neurite outgrowth: PC12 and primary dorsal root ganglia (DRG) co-culture. Polymer-mediated lipofection of PC12 produced bioactive NGF, eliciting robust neurite outgrowth. An EGFP/NGF dual-expression vector identified transfected cells (GFP-positive) while neurite outgrowth verified NGF secretion. A co-culture model examined the ability of NGF secretion by an accessory cell population to stimulate DRG neurite outgrowth. Polymer-mediated transfection of HEK293T with an NGF-encoding plasmid induced outgrowth by DRG neurons. This system could be fabricated as implants or nerve guidance conduits to support cellular and tissue regeneration. Combining this physical support with the ability to locally express neurotrophic factors will potentiate regeneration in nerve injury and disease models.
authors.affiliation: Interdepartmental Biological Sciences Program, Northwestern University, Evanston, IL 60208, USA.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Whittlesey
authors.forename: Lonnie D
authors.initials: LD
authors.lastname: Shea
mesh_terms: D000818:Animals; D001672:Biocompatible Materials; D002460:Cell Line; D018920:Coculture Techniques; D004274:DNA, Recombinant / Q000008:administration & dosage* / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D019344:Lactic Acid; D008081:Liposomes; D008422:Materials Testing; D051379:Mice; D008854:Microscopy, Electron; D041681:NIH 3T3 Cells; D020932:Nerve Growth Factor / Q000235:genetics*; D009416:Nerve Regeneration; D016501:Neurites / Q000502:physiology / Q000648:ultrastructure; D016716:PC12 Cells; D011100:Polyglycolic Acid; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011108:Polymers; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics; D014162:Transfection
chemical_list: D001672:Biocompatible Materials; D004274:DNA, Recombinant; D008081:Liposomes; D011108:Polymers; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; D020932:Nerve Growth Factor