Effective Non-Viral Delivery of siRNA to Acute Myeloid Leukemia Cells with Lipid-Substituted Polyethylenimines

et al. (2012) Effective Non-Viral Delivery of siRNA to Acute Myeloid Leukemia Cells with Lipid- Substituted Polyethylenimines. PLoS ONE 7(8): e44197. doi:10.1371/journal.pone.0044197 Effective Non-Viral Delivery of siRNA to Acute Myeloid Leukemia Cells with Lipid-Substituted Polyethylenimines Breanne Landry 0 Hamidreza Montazeri Aliabadi 0 Anuja Samuel 0 Hilal Gu l-Uludag 0 Xiaoyan Jiang 0 Olaf Kutsch 0 Hasan Uludag 0 Partha Mukhopadhyay, National Institutes of Health, United States of America 0 1 Department of Chemical & Materials Engineering, Faculty of Engineering, University of Alberta , Edmonton, Alberta , Canada , 2 Department of Biomedical Engineering, Faculty of Medicine & Dentistry, University of Alberta , Edmonton, Alberta , Canada , 3 Terry Fox Laboratories , BC Cancer Agency , Vancouver, British Columbia , Canada , 4 Centre for AIDS Research, University of Alabama at Birmingham , Birmingham , Alabama, United States of America, 5 Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta , Edmonton, Alberta , Canada Use of small interfering RNA (siRNA) is a promising approach for AML treatment as the siRNA molecule can be designed to specifically target proteins that contribute to aberrant cell proliferation in this disease. However, a clinical-relevant means of delivering siRNA molecules must be developed, as the cellular delivery of siRNA is problematic. Here, we report amphiphilic carriers combining a cationic polymer (2 kDa polyethyleneimine, PEI2) with lipophilic moieties to facilitate intracellular delivery of siRNA to AML cell lines. Complete binding of siRNA by the designed carriers was achieved at a polymer:siRNA ratio of ,0.5 and led to siRNA/polymer complexes of ,100 nm size. While the native PEI2 did not display cytotoxicity on AML cell lines THP-1, KG-1 and HL-60, lipid-modification on PEI2 slightly increased the cytotoxicity, which was consistent with increased interaction of polymers with cell membranes. Cellular delivery of siRNA was dependent on the nature of lipid substituent and the extent of lipid substitution, and varied among the three AML cell lines used. Linoleic acid-substituted polymers performed best among the prepared polymers and gave a siRNA delivery equivalent to better performing commercial reagents. Using THP-1 cells and a reporter (GFP) and an endogenous (CXCR4) target, effective silencing of the chosen targets was achieved with 25 to 50 nM of siRNA concentrations, and without adversely affecting subsequent cell growth. We conclude that lipid-substituted PEI2 can serve as an effective delivery of siRNA to leukemic cells and could be employed in molecular therapy of leukemia. - Funding: This project was financially supported by operating grants from Alberta Innovates Health Sciences (AIHS), Canadian Institute of Health Research (CIHR) and Natural Science and Engineering Research Council of Canada (NSERC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Acute myeloid leukemia (AML) is the most common acute leukemia in adults, with an estimated .13,000 new cases yearly and a mortality rate of ,10,000 in the US alone [1]. Development of novel AML therapies is urgently needed due to poor prognosis of the disease with a five-year survival rate of 30% for younger adults and ,15% for elderly patients [2]. Only in childhood AML, ,60% of patients can be cured of AML with very intensive chemotherapy [3]. The chemotherapy remains the front-line treatment, but alternative therapeutic approaches are required due to high relapse rates and limited treatment options for patients that cannot bear the toxic side-effects of chemotherapy [4]. Chemotherapy also leads to long-term undesired consequences; ,66% of survivors have either a chronic or late-effect due to cancer treatment and ,33% of these effects are considered major, serious or life threatening [1]. With better understanding of molecular changes in malignant transformations, treatments that target tumor-specific changes will lead to more effective therapies as the normal cells transform into malignant cells. Towards this end, a highly specific leukemia therapy can be developed by exploiting the RNA interference (RNAi) to silence the aberrant protein(s) responsible for the disease [5,6]. There are two main approaches for RNAi, using either a plasmid encoding for short hairpin RNA (shRNA) or delivering small interfering RNA (siRNA) where the shRNA transcription and processing steps can be omitted [7]. The use of siRNA is a more practical approach bypassing the need to express the shRNA at sufficient quantities in hard-to-transfect primary cells. In cytosol, the siRNA duplexes assemble into a pre-RISC (RNA-induced silencing complex) containing specific proteins, including argonaute proteins (AGO1, 3 or 4) [8,9]. The siRNA duplexes become unwound in AGOs, where the guiding strand directs the matureRISC to target desired mRNA based on complementary base pairing [8]. Endonucleoyltic cleavage and/or translational repression of the mRNA [8,9] subsequently silences the desired protein target. Delivery systems, however, are an absolute necessity for effective use of siRNA since the molecules are highly sensitive to serum nucleases and their large (,13 kDa) and anionic nature (due to its phosphodiesterase backbone) prevents siRNA to traverse cellular membranes. Cationic biomolecules capable of binding and neutralizing the anionic charges of siRNA and packaging the siRNA into nano-sized complexes can serve as effective siRNA carriers [10]. The utility of cationic carries for siRNA therapy in AML has been explored as early as 2003, where Raf-1 and Bcl-2 proteins were suppressed in AML cells by using the synthetic carrier OligofectamineTM. However, the resulting apoptotic response required 400 nM siRNA [5], a concentration too high for practical applications. It was evident that a more efficient delivery system was required to advance siRNA therapy for AML. Recent RNAi delivery attempts in leukemia cells have employed a variety of commercial carriers, which included; (i) LipofectamineTM 2000 in chronic myeloid leukemia (CML) K562 cells, and AML cells (KG-1/HL-60/U937/primary) [1116], (ii) RNAiMAXTM in K562 [17], (iii) HiPerFectTM in K562 and TALL (Jurkat) cells [18,19], (iv) DOTAP in Bcr-abl positive CML cells (2Dp210-modified/patient samples) [20], (v) Lipofectin in myeloid neoplasm cells (HMC-1) [21] (vi) and OligofectamineTM in T-cell lympoblastic leukemia cells (CCRF-CEM) [21]. Other carriers used for siRNA delivery were cell penetrating peptides (TatLK15 peptide in K562 cells) [16], CADY peptide in THP-1 cells [23], and functionalized carbon nanotubes in K562 cells [24]. Many of the above studies focused on discovery of possible targets for silencing and/or mechanisms of drug action, without pursuing siRNA delivery (...truncated)