(M1430-03-13) Combination eEF2K Gene and Drug Delivery for Treatment of Breast Cancer

Purpose: Combination treatment strategies have been investigated for enhanced anti-cancer activity. Specifically, the idea of silencing a gene causing abnormality in combination with chemotherapy has been investigated in recent years. Eukaryotic elongation factor 2 kinase (eEF2K) is one of the promising molecular targets for effective breast cancer treatment. eEF2K regulates elongation phase of protein synthesis under stress conditions and has a role in cancer cell survival and progression [1]. siRNAs are the main tools used for inhibition of gene expression. Although gene regulation is a powerful strategy, delivery of siRNAs into cells is challenging. To improve the current landscape of treatment strategies, the purpose of this study was combination therapy using eEF2K silencing (eEF2K siRNA) and chemotherapy (Doxorubicin (DOX)) to treat ERα positive breast cancer cells and using gold nanoparticles (AuNPs) as the delivery vehicle. We tested various siRNA: Drug ratios for effective cancer cell proliferation inhibition. Two different methods were used for siRNA and chemotherapy drug delivery. The two therapeutics were loaded on the same NP (1) and separate NPs in a mixture (2). eEF2K siRNAs and DOX were delivered to the ERα positive breast cancer cells to test their synergistic growth inhibition effect.

Methods: 13 nm AuNPs were synthesized using citrate reduction method. siRNAs were conjugated to the surface of AuNPs via thiol-gold linkage and DOX was loaded through intercalation between siRNA base pairs. Bare and modified NPs were characterized using dynamic light scattering (DLS) and UV-Vis spectroscopy. The loading of the therapeutics was evaluated with UV-Vis and fluorescence spectroscopy. The intracellular uptake of the DOX was measured using flow cytometry. Gene silencing effect of siRNAs was determined with Western blotting. The proliferation rates of the breast cancer cells (MCF7) were investigated using Alamar blue viability test, live/dead, and colony formation assays.

Results: The size of bare AuNPs was 13 nm. Approximately 33±5 siRNAs were loaded per NP and the DOX loading was tunable. The size of the AuNPs increased to approximately 21 nm after siRNA loading. Free DOX was internalized by almost 100% of the cells within 2h, while DOX/siRNA loaded NPs were taken up by 80% of cells even after 24h incubation. siRNAs demonstrated 50% silencing efficiency (p < 0.01). 1:2.25 siRNA: DOX ratio showed synergistic growth inhibition on cancer cells. Delivery of the therapeutics delivered on separate NPs showed higher synergistic growth inhibition (combination index: 0.74 ±0.04) on cancer cells than co-delivery on the same nanoparticle (combination index: 0.87 ±0.06).

Conclusion: In this study, we demonstrated successful design and construction of a simple, cost effective, and potent gene silencing and chemotherapy drug co-delivery platform based on AuNPs. The drug molecules were delivered to breast cancer cells simultaneously using two different methods. eEF2K expression was downregulated effectively. It was determined that the siRNA/DOX ratio as well as delivery method play key roles for success of combination therapy. Future work involves evaluation of the delivery strategy in in vivo models.

References: 1. Karakas, D. and B.J.J.o.M.M. Ozpolat, Eukaryotic elongation factor-2 kinase (eEF2K) signaling in tumor and microenvironment as a novel molecular target. 2020. 98(6): p. 775-787.

Acknowledgements: C.U.T. recognizes the financial support of Scientific and Technological Research Council of Turkey (TÜBİTAK).


Figure 1: Schematic representation of AuNPs delivery methods for combination therapy


Figure 2: Proliferation inhibition rates of MCF7 cells after eEF2K silencing and DOX delivery.