Efficient delivery of a Bcl-2-specific antisense oligodeoxyribonucleotide (G3139) via transferrin receptor-targeted liposomes Shih-Jiuan Chiu a,b , Shujun Liu c , Danilo Perrotti d , Guido Marcucci c,d,e, ⁎ , Robert J. Lee a,b,c, ⁎ a Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH, USA b NSF Nanoscale Science and Engineering Center (NSEC), The Ohio State University, Columbus, OH, USA c NCI Comprehensive Cancer Center (CCC), The Ohio State University, Columbus, OH, USA d Division of Human Cancer Genetics, Department of Internal Medicine, The Ohio State University, Columbus, OH, USA e Division of Hematology and Oncology, Department of Internal Medicine, The Ohio State University, Columbus, OH, USA Received 24 October 2005; accepted 15 February 2006 Available online 6 March 2006 Abstract A novel transferrin receptor (TfR)-targeted liposomal formulation was synthesized and evaluated for the delivery of a phosphorothioate antisense oligodeoxyribonucleotide (ODN) (G3139, oblimerson sodium, or Genasense™) to Bcl-2 in K562 leukemia cells. Liposomes composed of DC-Chol/egg PC/PEG–DSPE (25:73.5:1.5, mol/mol/mol) were loaded with G3139 with high efficiency (70–80%). To prepare targeted liposomes, transferrin was first coupled to PEG–DSPE and then incorporated into the bilayer by post-insertion. The liposomes had a mean diameter of 100 to 150 nm and exhibited colloidal stability for up to 8 weeks. Uptake of Tf-conjugated G3139-containing liposomes in TfR positive K562 cells was found to be more efficient than that of the non-targeted control formulation and could be blocked by excess free Tf. Treatment with Tf-conjugated liposomes resulted in Bcl-2 protein downregulation in K562 cells that was approximately 2-fold greater than with non-targeted liposomes (p <0.05) and 10-fold greater than with free G3139. Treatment with 2 μM G3139 in Tf-conjugated liposomes resulted in > 80% reduction in Bcl-2 transcript. In addition, Tf-conjugated liposomal G3139-sensitized K562 cells to daunorubicin, lowering IC 50 from 1.8 μM to 0.18 μM. In conclusion, Tf-conjugated liposomes are effective delivery vehicles for G3139 antisense oligos in TfR positive K562 cells and warrant further investigation as an in vivo oligo delivery vehicle. © 2006 Elsevier B.V. All rights reserved. Keywords: Antisense oligonucleotide; Transferrin; Bcl-2; Liposomes; Leukemia 1. Introduction Antisense oligodeoxyribonucleotides (ODNs) are currently being investigated as therapeutic agents for the treatment of solid tumors and hematological malignancies [1–3]. Since ODNs have relatively rapid plasma clearance, they are usually administered via continuous intravenous infusion over an extended period of time in order to maintain therapeutic levels in plasma [4]. Novel delivery strategies for ODNs have been investigated in a number of recent studies [5], including the use of polymeric carriers [6,7], micelles [8], nanoparticles [9–11], and conjugation to a targeting ligand [12]. Tumor cell targeted liposomes can potentially improve the therapeutic efficacy of ODNs by achieving sustained plasma concentration, enhanced accumulation in tumor tissues, as well as increased rate of internalization by tumor cells [5,13–15]. G3139 (Genasense™ or oblimerson sodium) [16,17] is an 18-mer ODN (5′–TCT CCC AGC GTG CGC CAT–3′) designed to specifically bind the first six codons of the human bcl-2 mRNA and downregulate bcl-2 expression [18–20]. Bcl-2 is an antiapoptotic protein frequently overexpressed in tumor cells and is associated with resistance to chemotherapy. Bcl-2 downregulation, therefore, is being evaluated as a potential strategy to reverse chemoresistance [16,20,21]. We have Journal of Controlled Release 112 (2006) 199 – 207 www.elsevier.com/locate/jconrel ⁎ Corresponding authors. Guido Marcucci is to be contacted at 310 10th Ave., Starling Loving Hall, Division of Hematology and Oncology, The Ohio State University, Columbus, OH, 43210, United States. Tel.: +1 614 293 9868; fax: +1 614 293 7526. Robert J. Lee, 500 W 12th Ave., L. M. Parks Hall, College of Pharmacy, The Ohio State University, Columbus, OH 43210, United States. Tel.: +1 614 292 4172; fax: +1 614 292 7766. E-mail addresses: marcucci-1@medctr.osu.edu (G. Marcucci), lee.1339@osu.edu (R.J. Lee). 0168-3659/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2006.02.011