DOI: 10.1002/cbic.200800540 Self-Assembling Polyethylenimine Derivatives Mediate Efficient siRNA Delivery in Mammalian Cells Gaelle Creusat and Guy Zuber* [a] Synthetic 21–22-nt-long RNA duplexes (siRNAs) can trigger specific mRNA degradation and selectively control gene ex- pression, and because of this they have potential therapeutic applications. [1] Unfortunately, the intracellular localization of siRNA targets and the inability of oligonucleotides to diffuse across cellular membranes requires siRNA conjugation or for- mulation with synthetic delivery vectors. [2] Interestingly, poly- ACHTUNGTRENNUNGethylenimine (PEI), a popular gene transfection agent [3] that showed promise for cancer gene therapy, [4] appears inefficient for siRNA delivery in vitro. [5] The poor performance of PEI could be linked with the length of the siRNA anionic segment, which is too short to maintain electrostatic cohesion with the soluble cationic poly- mer. Consequently, siRNA polyplexes break apart too readily upon contact with polyanions present at cell surfaces. [6] We reasoned that the transformation of water-soluble PEI into a molecule with self-assembly properties should stabilize siRNA polyplexes and hence favor overall siRNA delivery. A water-soluble polymer could acquire new aggregating properties through the addition of hydrophobic domains. We avoided using alkyl chains, which, by mixing with the cell lipid bilayer, might act as a detergent and cause cell death. Our preference was instead for natural a-amino acids with decreas- ing hydropathy indices, [7] such as leucine (L), phenylalanine (F), tryptophan (W), and tyrosine (Y). Indeed, these amino acids constitute the cores of globular proteins and help maintain protein structures. They also can be coupled to PEI amines without destroying nucleic acid binding properties, because over the course of the reaction a cationic amine replaces the reacted one. Commercially available 25 kDa branched PEI contains pri- mary, secondary, and tertiary amines in a ratio of 1:1:1. [8] The primary amines were allowed to react fully with the succini- midyl esters of butyloxycarbonyl-protected amino-acids (Boc- aa-OSu) (Scheme 1). Removal of the Boc groups with trifluoro- acetic acid (TFA) and subsequent dialysis in aqueous HCl gave the desired products, with a-amino acid contents of 30 % per ethylenimine, in 30–60 % overall yields. The aggregating properties of the polymers were evaluated by measurement of dynamic light scattering (Table 1). All poly- mers, as hydrochloride salts, were soluble in water, even at a concentration of 0.5 m. Upon dilution in RPMI cell culture medium, no light scattering signals were detected in the cases of PEI and PEIL. On the other hand, PEIF, PEIW, and PEIY self-as- sembled into particles with apparent diameters between 0.3– 0.7 mm. Addition of siRNA permitted PEI and PEIL to form com- plexes and led to slight increases in the sizes of the self-assem- bling polymers in a range that had previously been observed to be effective for PEI-mediated gene delivery. [9] Transmission electron micrographs showed that PEIY (Figure 1 A) forms fiber-like structures of fused irregular spheroids roughly 20 nm in diameter. Addition of siRNA (Figure 1B) led to a twofold ACHTUNGTRENNUNGdiminution in the fiber diameters and to further clustering. Nanoparticles, including viruses, are too large to diffuse freely across membranes and thus need to divert the cell ma- chinery for translocation. They first anchor to receptors on the cellular surface and are then actively dragged into cells within membrane-coated vesicles (often endosomes). [10] Evidence sug- gests that the high buffer capacity of PEI enables rupture of the endosome membranes and hence effective nucleic acid translocation. [11] We therefore examined whether amino acid modification impacted this important function by acid–base ACHTUNGTRENNUNGtitration (Figure 1). Profiles of modified PEIs superposed almost perfectly in the pH 6.0–8.0 range and showed that the poly- mers have higher buffering capacities than PEI, probably due to the electron-withdrawing effect of carboxamide on the a- terminal amines. This greater “proton sponge” ability should Scheme 1. Synthesis. a) Boc-aa-OSu. b) TFA. Table 1. Sizes [nm] of polymer self-assemblies in RPMI medium. Polymer [a] Alone + siRNA [b] PEI – 155 Æ 31 nm PEIL – 175 Æ 13 nm PEIF 700 Æ 34 nm 880 Æ 66 nm PEIW 475 Æ 11 nm 620 Æ 41 nm PEIY 325 Æ 28 nm 570 Æ 33 nm [a] 120 mm in ethylenimine. [b] 2.4 mm in phosphate. [a] G. Creusat, Dr. G. Zuber Laboratoire de Chimie GØnØtique, ULP CNRS UMR 7175 FacultØ de Pharmacie 74, Route du Rhin, 67400 Illkirch (France) Fax: (+ 33) 3-9024-4306 E-mail : zuber@bioorga.u-strasbg.fr Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author: protocols for polymer synthesis and delivery experiments. ChemBioChem 2008, 9, 2787 – 2789  2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 2787