Systemic Delivery of Sirna-Based Therapeutics Using Single-Walled Carbon Nanotubes

Abstract

Background: Carbon nanotubes (CNTs) are potential candidates for drug, antigen and nucleic acid delivery vehicle in nanomedicine. The large surface of CNTs provides structural advantages and allows loading of functional groups or therapeutics such as nucleic acid, drugs and proteins. Aim: Our study is to deliver siRNA to cells using single walled carbon nanotube (SWNT) to achieve gene silencing effect. Methods: SWNT was functionalized by dissolving 1 mg of HiPCo SWNTs and 5 mg of PL-PEG-NH or PL-PEG-maleimide in 5 mL of water, sonicated for 60 min at room temperature and centrifuged for 6 h. The supernatant were collected and measured their concentration at 808 nm by ultraviolet-VIS-NIR spectrometer. The resulted noncovalent functionalized SWNTs were further conjugated with a 5′-thiolated siRNA against GFP (siGFP) and RFP (siRFP). In vitro silencing of GFP and RFP expression by SWNT-siRNA were evaluated in stable expression cell lines by fluorescence spectroscopy. Results: A range of 50%–80% GFP expression knocked down was observed in H1299, HeLa, MCF-7 and 293T cells by SWNT-siGFP. SWNT conjugated with both siGFP and siRFP were shown knocking down both GFP and RFP simultaneously in H1299 stable coexpression cell line. Also, gene silencing was observed despite incubation with inhibitors on different cellular internalization pathways. They are chlorpromazine for clathrin-mediated endocytosis inhibitor, genistein for caveolae-mediated endocytosis inhibitor and sodium azide for energy depletion agent. We also found that the internalization of SWNT by the nonphagocytic cells (H1299) did not solely depend on single cellular entry pathway to achieve the gene silencing effect. Conclusion: The successful knockdown of GFP expression in different cell lines indicated that siRNA were released from the conjugated SWNT-siRNA in the cytoplasm and silent the gene expression. It is also indicated that 2 different types of siRNA targets could be conjugated with SWNT and achieved 2 different gene silencing effects simultaneously.