Lipid Modified Polymers as BCR-ABL siRNA Carriers for Growth Arrest in Chronic Myeloid Leukemia Cells

Valencia-Serna, Juliana (University of Alberta)
Chan, Nicole (University of Alberta)
Yang, Xiaohong (University of Alberta)
Aliabadi, Hamid M (University of Alberta)
Parmar, Manoj B (University of Alberta)
Jiang, Xiaoyan (British Columbia Cancer Agency)
Uludagğ, Hasan (University of Alberta)

Introduction

Chronic Myeloid Leukemia (CML) is a disease initialized by hematopoietic stem cells after chromosomal translocation, which results in the BCR-ABL fusion oncogene. This fusion causes permanent activation of the ABL tyrosine kinase that leads to myeloid cell expansion and accumulation of immature blasts in bone marrow and bloodstream [1]. The shortcomings of current leukemia treatments such as acquired drug resistance and drug insensitivity [2] call for development of new treatments. To control the expression of BCR-ABL and other genes involved in the malfunctioning pathways, synthetic small interfering RNA (siRNA) can be delivered to diseased cells to interact with the target mRNA of aberrant genes and silence the protein expression. The aim of this project is to develop an effective polymeric carrier in order to deliver siRNA against BCR-ABL mRNA of CML cells. The polymer consists of low molecular weight (MW) polyethylenimine grafted with linoleic acid (PEI1.2-LA). This study evaluated the potential of the polymer in reduction of CML cell growth in vitro and in vivo.

Materials and Methods

To substitute PEI (1,2 kDa) with LA, PEI1.2 was dissolved in dichloromethane (DCM) and sufficient linoleyl chloride was added in DCM to get a PEI1.2:LA mol feed ratio of 1:4. Final product was analyzed by 1H-NMR (Bruker 600 MHz). Extent of lipid substitution on the polymer was 0.5 lipid/PEI. Complexes were prepared with siRNA and polymer at a polymer:siRNA ratio of 8:1 or 12:1 in RPMI medium (Sigma) and incubated for 30 min at room temperature. Comparison of the polymer was performed against commercial PEI25, Lipofectamine 2000 and, Turbofect, and a carrier developed in the authors lab, palmitic acid-substituted PEI1.2 (PEI1.2-PA) [3,4]. Green fluorescent Protein (GFP)-expressing CML cells (GFP-K562 cells) were used as cell model. Decrease in GFP fluorescence after siRNA delivery was assessed by flow cytometry as a measure of silencing. The MTT experiments were performed to account for any decrease of viability after BCR-ABL siRNA delivery. In vivo experiments were performed in accordance with the University of Alberta guidelines. For tumor formation, five-week old female nu-nu mice (Taconic Farms) were injected in the flank with 2 x 107 GFP-K562 cells. Tumors were injected in the vicinity of the tumor (subcutaneously) or intraperitoneally with PEI1.2-LA/siRNA complexes 3 times every 72 h (10 µg siRNA, ratio of 1:12 or 1:8). Tumor volume was measured every 3 d. Tumors were excised and processed for absolute quantification of BCR-ABL mRNA expression by digital PCR (dPCR).

Results

Based on flow cytometry analysis, PEI1.2 did not show any silencing while PEI25, Turbofect and Lipofectamine showed ~80% silencing (Fig 1). Lipid-modified polymers PEI1.2-PA and PEI1.2-LA showed a comparable silencing of ~62 and ~54%, respectively without inducing as much cell death as PEI25 and Turbofect (not shown). To evaluate therapeutic effect of PEI1.2-LA, GFP-K562 cells were transfected with GFP (negative control) or BCR-ABL siRNA. An initial toxicity soon after the transfection in the control group was seen; however, a slow cell recovery was also obtained. In contrast, BCR-ABL group showed a cell growth arrest, which reached a significant difference with control group on day 4 (p<0.01, Fig 2). Effect of BCR-ABL siRNA delivery was then evaluated in an animal model. For CML tumors injected with siRNA in their vicinity, GFP siRNA has a slight decrease in tumor volumes after day 10 suggesting some degree of toxicity due to complex injection. Treatment with BCR-ABL siRNA was more effective in reducing tumor volumes starting from day 3 (Fig 3A). dPCR supported this silencing effect by revealing a reduction of the BCR-ABL mRNA expression (Fig 3B). Similar results were found when tumors were injected with siRNA intraperitoneally (p<0.01, Fig 5C).

Discussion and Conclusion

With the aim of balancing out effective transfection with lower cytotoxicity, PEI1.2 was grafted with a lipid moiety and degree of modification. PEI1.2-LA polymer showed similar effect to PEI25 in terms of transfection but having a less toxic effect on cells. Although there is an initial toxicity due to complexes internalization, a recovery is seen over time. Decrease of BCR-ABL expression by RNAi mediated by siRNA delivery showed its functional effect in restraining cell proliferation. Although there was not a strong statistically significant difference among the groups, the tendency of tumors treated with BCR-ABL siRNA complexes was to show a retardation in tumor growth. All these results demonstrate the potential of PEI1.2-LA polymer to effectively deliver siRNA and therefore its potential for use in the treatment of CML.


Fig 1. GFP silencing in GFP-K562 cells. Decrease in mean GFP fluorescence was expressed as a percentage of control siRNA treated cells.


Fig 2. Cell viability expressed as a percentage of no treated cells


Fig 3. GFP-K562 xenografts in nu/nu mice treated in the vicinity of the tumor. Changes in tumor volumes expressed as the tumor volume at any day divided by the initial tumor volume (A). Absolute BCR-ABL mRNA quantified by dPCR (B). Xenografts treated with intraperitoneal injections (C)

Acknowledgements

Financial support was provided by Alberta Cancer Foundation (ACF), Natural Sciences and Engineering Research Council of Canada (NSERC) and, Canadian Institutes of Health Research (CIHR). J.V.S. was supported by graduate studentships from NSERC CREATE Program (provided to Dr. G. Laroche, Laval University)

References

[1] I. Sloma, et al, Leukemia, vol. 24, no. 11, pp. 1823–1833, Sep. 2010. [2] H. Zhang, et al, Protein and Cell, vol. 4, no. 3, pp. 186–196, Mar. 2013. [3] J. Valencia-Serna, et al, J Control Release, vol. 172, no. 2, pp. 495–503, Dec. 2013. [4] K. C. Remant Bahadur, et al, Acta Biomaterialia, vol. 7, no. 5, pp. 2209–2217, May 2011.

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