title: Delivery of siRNA from lyophilized polymeric surfaces.
abstract: Standard in vitro gene silencing protocols are performed using aqueous formulations of transfection reagents and small interfering RNAs (siRNA) reconstituted immediately prior to use. In this study, we describe a method for producing gene silencing-active lyophilized cationic polymer (chitosan) or lipid (TransIT-TKO) siRNA formulations. We demonstrate specific and efficient knockdown of enhanced green fluorescent protein (EGFP) in H1299 human lung carcinoma cells transfected in plates pre-coated with both TransIT-TKO/siRNA ( approximately 85%) and a chitosan/siRNA formulation containing sucrose as lyoprotectant ( approximately 70%). This method removes the necessity for both siRNA reconstitution immediately prior to use and addition onto cells. Furthermore, silencing activity of the chitosan/siRNA formulation was shown over the period studied ( approximately 2 months) when stored at room temperature. Higher cell viability was observed using the chitosan system compared to the lipid formulation. Silencing of the proinflammatory cytokine tumour necrosis factor (TNF-alpha) was also demonstrated in the RAW macrophage cell line using the lyophilized chitosan/siRNA system suggesting that the coating can improve the biocompatibility of medical implants. This work describes an efficient gene silencing methodology using freeze-dried formulations with potential applications as a high throughput screening tool for gene function, biocompatible medical implant components and longer shelf-life therapeutics.
authors.affiliation: Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, 8000 Aarhus C, Denmark.
authors.forename: Morten Ø
authors.initials: MØ
authors.lastname: Andersen
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Howard
authors.forename: Søren R
authors.initials: SR
authors.lastname: Paludan
authors.forename: Flemming
authors.initials: F
authors.lastname: Besenbacher
authors.forename: Jørgen
authors.initials: J
authors.lastname: Kjems
mesh_terms: D000818:Animals; D002460:Cell Line; D048271:Chitosan / Q000737:chemistry* / Q000633:toxicity; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D053758:Nanoparticles / Q000737:chemistry; D034741:RNA, Small Interfering / Q000235:genetics*; D013499:Surface Properties; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism; D014867:Water / Q000737:chemistry
chemical_list: D008055:Lipids; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; C485184:enhanced green fluorescent protein; D014867:Water; D049452:Green Fluorescent Proteins; D048271:Chitosan