B. Landry, J. Valencia-Serna, H. Uludag
University of Alberta,
Keywords: polymeric biomaterials, siRNA delivery, anti-cancer therapy
Summary:Leukemias are a group of heterogeneous disorders characterized by abnormal proliferation of blood cells. Molecular underpinnings of leukemia development are now quickly elucidated by high-throughput sequencing efforts, making it possible to rely on molecular therapies rather than broad spectrum chemotherapy. Although chemotherapy yields high remission rates in most leukemias, the majority of patients eventually relapse due to the proliferation of drug-resistant leukemic blasts . Current therapies also display immediate toxic side-effect, patient incompatibility with high-dose treatments, and late adverse effects especially in pediatric patients. A new approach to cancer therapy has been emerging based on RNA Interference (RNAi), where endogenous RNAs regulate the expression of mRNAs. The pharmacological mediator of RNAi, short interfering RNA (siRNA), is currently explored as a highly specific therapeutic agent in several cancers. Unfortunately, siRNA is an incompetent agent on its own and a functional delivery system is needed for intracellular uptake. To explore the potential of siRNA in leukemia, we are investigating the utility of lipid-modified, cationic polymers to deliver siRNA and silence desired proteins in leukemic cells. Lipid-substituted polymers were synthesized by modifying polyethylenimines with various lipids, including caprylic, palmitic and linoleic acids. The carriers were self-assembled in aqueous buffers with siRNAs to form complexes at ratios above the minimum required for complete siRNA binding (0.5). While the native PEI2 did not display cytotoxicity on leukemic cells, lipid-grafting on PEI2 slightly increased the cytotoxicity, which was consistent with increased interaction of polymers with cell membranes. Cellular delivery was investigated by using FAM-labeled siRNA; siRNA delivery was dependent on the nature and extent of lipid substitution. In acute myeloid leukemia cells (AML), caprylic and linoleic acid-substituted polymers performed best among the prepared polymers  and gave a siRNA delivery equivalent to better performing commercial reagents. With chronic myeloid leukemia (CML) cells, the same polymers were not effective and we found a particular palmitic acid-substituted PEI to be most effective in siRNA delivery . Initial silencing studies were performed using cells modified with a reporter (GFP) gene, which was effectively silencing on AML and CML cells at 25-100 nM siRNA concentration. The aberrant Bcl-Abl expression was silencing in CML cells, resulting in increased apoptosis induction. In AML cells, an effective suppression of CXCR4 protein was obtained, leading to decreased cell survival, which was attributed to a decrease in proliferation. The decrease in cell numbers due to CXCR4 silencing occurred both in the absence and presence of human bone marrow stromal cells, suggesting that siRNA therapy could be effective in the protective bone marrow environment. The combination of silencing CXCR4 and the drug (cytarabine) treatment resulted in more effective drug toxicity when the cells were co-incubated with bone marrow cells. We conclude that amphiphilic polymers created by grafting lipid moieties onto cationic PEIs can deliver siRNA effectively into hard-to-transfect leukemic cells. The delivery efficiency was influenced by the structural features of polymers and functional (therapeutic) outcomes was obtained in two types of leukemias.