Delivery of siRNA from lyophilized polymeric surfaces.
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.
Andersen
Morten Ø
MØ
Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, 8000 Aarhus C, Denmark.
Howard
Kenneth A
KA
Paludan
Søren R
SR
Besenbacher
Flemming
F
Kjems
Jørgen
J
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
D008055:Lipids; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; C485184:enhanced green fluorescent protein; D014867:Water; D049452:Green Fluorescent Proteins; D048271:Chitosan