Targeted delivery of siRNA to hepatocytes and hepatic stellate cells by bioconjugation.
Previously, we successfully conjugated galactosylated poly(ethylene glycol) (Gal-PEG) to oligonucleotides (ODNs) via an acid labile ester linker (Zhu et al., Bioconjugate Chem. 2008, 19, 290-8). In this study, sense strands of siRNA were conjugated to Gal-PEG and mannose 6-phosphate poly(ethylene glycol) (M6P-PEG) for targeted delivery of siRNAs to hepatocytes and hepatic stellate cells (HSCs), respectively. These siRNA conjugates were purified by ion exchange chromatography and verified by gel retardation assay. To evaluate their RNAi functions, the validated siRNA duplexes targeting firefly luciferase and transforming growth factor beta 1 (TGF-β1) mRNA were conjugated to Gal-PEG and M6P-PEG, and their gene silencing efficiencies were determined after transfection into HepG2 and HSC-T6 cells. The disulfide bond between PEG and siRNA was cleaved by dithiothreitol, leading to the release of intact siRNA. Both Gal-PEG-siRNA and M6P-PEG-siRNA conjugates could silence luciferase gene expression by about 40% without any transfection reagents, while the gene silencing effects reached more than 98% with the help of cationic liposomes at the same dose. Conjugation of TGF-β1 siRNA with Gal-PEG and M6P-PEG could silence endogenous TGF-β1 gene expression as well. In conclusion, these siRNA conjugates have the potential for targeted delivery of siRNAs to hepatocytes and hepatic stellate cells for efficient gene silencing in vivo.
Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, Tennessee 38103, United States.
Lin
L
Zhu
Ram I
RI
Mahato
D045744:Cell Line, Tumor; D005690:Galactose / Q000737:chemistry; D018014:Gene Transfer Techniques; D055166:Hepatic Stellate Cells / Q000201:enzymology / Q000378:metabolism*; D022781:Hepatocytes / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008360:Mannosephosphates / Q000737:chemistry; D015394:Molecular Structure; D011092:Polyethylene Glycols / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism
D008360:Mannosephosphates; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D053773:Transforming Growth Factor beta1; C027693:mannose-6-phosphate; D011092:Polyethylene Glycols; D008156:Luciferases; D005690:Galactose