Published: March 01, 2011 r2011 American Chemical Society 429 dx.doi.org/10.1021/bc1004697 | Bioconjugate Chem. 2011, 22, 429–435 ARTICLE pubs.acs.org/bc Dicetyl Phosphate-Tetraethylenepentamine-Based Liposomes for Systemic siRNA Delivery Tomohiro Asai, †, * Saori Matsushita, † Eriya Kenjo, † Takuma Tsuzuku, † Norihito Yonenaga, † Hiroyuki Koide, † Kentaro Hatanaka, † Takehisa Dewa, ‡ Mamoru Nango, ‡ Noriyuki Maeda, § Hiroshi Kikuchi, ^ and Naoto Oku † † Department of Medical Biochemistry and Global COE, University of Shizuoka Graduate School of Pharmaceutical Sciences, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan ‡ Department of Life and Materials Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan § Nippon Fine Chemical Co. Ltd., 5-1-1 Umei, Takasago, Hyogo 676-0074, Japan ^ DDS Research, Formulation Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, 300-2635, Japan b S Supporting Information ’ INTRODUCTION RNA interference (RNAi) refers to post-transcriptional gene silencing induced by double-stranded RNA. 1,2 Small RNAs such as small interfering RNA (siRNA) and microRNA (miRNA) are expected to become novel drug therapeutics for intractable diseases such as cancer. 3 Compared with local injection of siRNA drug candidates, systemic injection of them is quite limited at present because of a serious problem of siRNA delivery; 4,5 i.e., naked RNA is readily degraded by nucleases and poorly pene- trates plasma membrane of target cells. Thus, establishment of an siRNA delivery system is a major challenge for the development of RNAi-based drugs. 5 In recent studies, a variety of drug delivery system (DDS) technologies such as liposomes, 6 micelles, 7 other nanoparticles, 8,9 and cholesterol-conjugation 10 of siRNA have been investigated for siRNA delivery. Also, useful nonviral vectors enabling both potent gene silencing in target cells and in vivo delivery to target cells have been awaited to achieve siRNA therapy via systemic administration. We reported earlier that polycation liposomes (PCL), one of the nonviral vectors, possess the advantages of both liposomes and polycations for the delivery of plasmid DNA and siRNA. 11-17 Previously, we synthesized cetylated polyethylenimine- [1800] (cetyl-PEI[1800]) and polycation (spermidine, spermine or PEI[1800])-dialkyl phosphate conjugates to prepare various types of PCLs. 11-13 PCLs are simply prepared with these conjugates through anchoring via the hydrophobic portion of Received: October 25, 2010 Revised: January 26, 2011 ABSTRACT: Dicetyl phosphate-tetraethylenepentamine (DCP-TEPA) conjugate was newly synthesized and formed into liposomes for efficient siRNA delivery. Formulation of DCP-TEPA-based polycation liposomes (TEPA-PCL) com- plexed with siRNA was examined by performing knockdown experiments using stable EGFP-transfected HT1080 human fibrosarcoma cells and siRNA for GFP. An adequate amount of DCP-TEPA in TEPA-PCL and N/P ratio of TEPA-PCL/ siRNA complexes were determined based on the knockdown efficiency. Then, the biodistribution of TEPA-PCL modified with poly(ethylene glycol) (PEG) was examined in BALB/c mice. As a result, TEPA-PCL modified with PEG6000 avoided reticuloendothelial system uptake and showed long circulation in the bloodstream. On the other hand, PEGylation of TEPA-PCL/ siRNA complexes caused dissociation of a portion of the siRNA from the liposomes. However, we found that the use of cholesterol- conjugated siRNA improved the interaction between TEPA-PCL and siRNA, which allowed PEGylation of TEPA-PCL/siRNA complexes without siRNA dissociation. In addition, TEPA-PCL complexed with cholesterol-conjugated siRNA showed potent knockdown efficiency in stable luciferase-transfected B16-F10 murine melanoma cells. Finally, the biodistribution of cholesterol- conjugated siRNA formulated in PEGylated TEPA-PCL was examined by performing near-infrared fluorescence imaging in Colon26 NL-17 murine carcinoma-bearing mice. Our results showed that tumor targeting with siRNA via systemic administration was achieved by using PEGylated TEPA-PCL combined with active targeting with Ala-Pro-Arg-Pro-Gly, a peptide used for targeting angiogenic endothelium.