title: Surface-mediated delivery of siRNA from fibrin hydrogels for knockdown of the BMP-2 binding antagonist noggin.
abstract: Antagonists and inhibitory molecules responsible for maintaining tissue homeostasis can present a significant barrier to healing when tissue engineering/regenerative medicine strategies are employed. One example of this situation is the up-regulation of antagonists such as noggin in response to increasing concentrations of bone morphogenetic protein-2 (BMP-2) present from endogenous bone repair processes or delivered exogenously from biomaterials (synthetic bone grafts). While recombinant human (rh)BMP-2 delivered from synthetic bone grafts has been shown to be an effective alternative to autografts and allografts, the supraphysiological doses of rhBMP-2 have led to clinically-adverse side effects. The high rhBMP-2 dosage may be required, in part, to overcome the presence of antagonists such as noggin. Small interfering RNA (siRNA) is an appealing approach to overcome this problem because it can knock-down antagonists or inhibitory molecules in a temporary manner. Here, we conducted fundamental studies on the delivery of siRNA from material surfaces as a means to knock-down antagonists like noggin. Non-viral cationic lipid (Lipofectamine)-siRNA complexes were delivered from a fibrin hydrogel surface to MC3T3-E1 preosteoblasts that were treated with a supraphysiological dose of rhBMP-2 to achieve noggin mRNA expression levels higher than cells naïve to rhBMP-2. Confocal microscopy and flow cytometry showed intracellular uptake of siRNA in over 98% of MC3T3-E1 cells after 48 h. Doses of 0.5 and 1 μg noggin siRNA were able to significantly reduce noggin mRNA to levels equivalent to those in MC3T3-E1 cells not exposed to rhBMP-2 with no effects on cell viability.
  
  
  CONCLUSIONS
  Small interfering RNA (siRNA) has been considered for treatment of diseases ranging from Alzheimer's to cancer. However, the ability to use siRNA in conjunction with biomaterials to direct tissue regeneration processes has received relatively little attention. Using the bone morphogenetic protein 2 antagonist, noggin, as a model, this research describes an approach to knock-down molecules that are inhibitory to desired regenerative pathways at the mRNA level via siRNA delivery from a hydrogel surface. Interactions between the material (fibrin) surface and polycation-siRNA complexes, release of the siRNA from the material surface, high levels of cellular uptake/internalization of siRNA, and significant knockdown of the targeting (noggin) mRNA are demonstrated. Broader future applications include those to nerve regeneration, cardiovascular tissue engineering, directing (stem) cell behavior, and mitigating inflammatory responses to materials.
authors.affiliation: School of Biomedical Engineering and Sciences, Virginia Tech-Wake Forest University, Medical Center Blvd, Winston-Salem, NC 27157, United States; Department of Chemical, Paper and Biomedical Engineering, Miami University, 650 High St, Oxford, OH 45056, United States.
authors.forename: Christine J
authors.initials: CJ
authors.lastname: Kowalczewski
authors.affiliation: Department of Chemical, Paper and Biomedical Engineering, Miami University, 650 High St, Oxford, OH 45056, United States. Electronic address: sauljm@miamioh.edu.
authors.forename: Justin M
authors.initials: JM
authors.lastname: Saul
mesh_terms: D000327:Adsorption; D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D005337:Fibrin / Q000737:chemistry* / Q000648:ultrastructure; D005434:Flow Cytometry; D055785:Gene Knockdown Techniques; D006801:Humans; D020100:Hydrogels / Q000737:chemistry*; D007700:Kinetics; D051379:Mice; D018613:Microscopy, Confocal; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D000099176:Noggin Protein
chemical_list: D055396:Bone Morphogenetic Protein 2; D002352:Carrier Proteins; D005337:Fibrin; D020100:Hydrogels; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D000099176:Noggin Protein; C499469:recombinant human bone morphogenetic protein-2
keywords: Natural polymer; Regenerative medicine; Tissue engineering; Transfection; mRNA