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Poly(ethylene glycol)-Protected, pH-Dependent Liposomes Enhance RNAi Delivery

Debra T. Auguste1, Kay Furman2, Andrew D. Wong1, Jason Fuller3, Steven P. Armes4, Timothy Deming5, and Robert S. Langer3. (1) School of Engineering and Applied Sciences, Harvard University, 29 Oxford St., Cambridge, MA 02138, (2) Department of Material Science and Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, (3) Department of Chemical Engineering, Massachusetts Institute of Technology, 45 Carleton St, E25-342, Cambridge, MA 02142, (4) Department of Chemistry, The University of Sheffield, Dainton Building, Sheffield, United Kingdom, (5) Department of Bioengineering, UCLA, Bolter Hall, Los Angeles, CA 90095

Short interfering RNA (siRNA) has potential therapeutic applications in regulating gene expression; however, its use is limited by inefficient delivery. We can enhance the transfection of siRNA using pH dependent liposomes coated with poly(ethylene glycol) (PEG)-b-polycation polymers. The PEG coating enables protection from immune recognition during circulation (pH 7.4) and triggering the release of the PEG layer within the cellular endosome (pH ~5.5) allows subsequent intracellular delivery of siRNA. Polycationic blocks, poly[2-(dimethylamino) ethyl methacrylate] (DMA) (31 or 62 monomers) or polylysine (K) (21 monomers), act as anchors for a PEG (113 monomer) protective block on the lipsomes. Incorporation of 1,2-dioleoyl-3-dimethylammonium-propane (DAP), a titratable lipid (pKa ~ 6.7), allows the liposome's net charge to increase as the pH shifts from 7.4 in the bloodstream to 5.5 within the endosome. The net increase in liposome cationicity repels the polycationic blocks, which decreases the PEG polymer adsorption, and allows the liposome to fuse with the endosomal membrane; thus, releasing the siRNA into the cytoplasm. Our pH dependent (PD) liposomes encapsulate siRNA that demonstrate green fluorescent protein (GFP) silencing in genetically-modified, GFP expressing HeLa cells and GAPD knockdown in human umbilical vein endothelial cells (HUVEC). The liposomes coated with PEG113–DMA31 and PEG113–DMA62 exhibited a 0.32 ± 0.3 and 0.33 ± 0.03 fraction of GFP knockdown, whereas direct siRNA administration and Oligofectamine complexed siRNA only reduced GFP expression by 0.06 ± 0.02 and 0.14 ± 0.02. Our in vitro data indicates that polymer desorption from PD liposomes may enhance siRNA-mediated gene knockdown, contrary to our membrane fusion results which show greater fusion with uncoated PD liposomes.