Delivery of siRNA from lyophilized polymeric surfaces

• Published in: Biomaterials 2008-02, Vol.29 (4), p.506-512
• Main Authors: Andersen, Morten Ø, Howard, Kenneth A, Paludan, Søren R, Besenbacher, Flemming, Kjems, Jørgen
• Format: Article
• Language: English
• Subjects: Advanced Basic Science; Animals; Cell Line; Chitosan; Chitosan - chemistry; Chitosan - toxicity; Dentistry; Drug delivery; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Humans; Lipids - chemistry; Lyophilization; Macrophages; Macrophages - drug effects; Macrophages - metabolism; Mice; Nanoparticles - chemistry; RNA interference; RNA, Small Interfering - genetics; SiRNA; Surface Properties; Tumor Necrosis Factor-alpha - genetics; Tumor Necrosis Factor-alpha - metabolism; Water - chemistry
• ISSN: 0142-9612; 1878-5905
• DOI: 10.1016/j.biomaterials.2007.10.003

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 ( Trans IT - 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 Trans IT - TKO/siRNA (∼85%) and a chitosan/siRNA formulation containing sucrose as lyoprotectant (∼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 (∼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- α ) 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.