Breast cancer targeted therapy using ZnO nanostructures

Abstract

There are an estimated 14.1 million cancer cases around the world every year, with this number expected to increase to 24 million by 2035. Breast cancer is the most common cancer in the UK, with 50,000 new cases per year. Due to the limitations and side effects of most available anticancer therapy, there is an urgent need to develop new anticancer agents. There has been significant progress in utilizing nanotechnology in several areas of cancer care, including in vitro diagnostics, imaging, and therapy. The first generation of novel nanomaterials (NMS) as anticancer agents has successfully entered a widespread use. Recent studies demonstrate that zinc oxide (ZnO) NMS hold a considerable promise as potential anticancer agents. In addition to the novel size and shape properties, ZnO NMS exhibit a pH-sensitive dissolution. These properties of ZnO NMS could potentially allow increased cancer selectivity, changes in pharmacokinetics and amplification of cytotoxic effects. The selectivity of ZnO NMS can be further induced by attaching of cancer target reactive-ligands. The aim of this study is to test the hypothesis that zinc oxide nanoparticles (ZnO NPs), grafted with RGD peptide, would target tumours that up- regulate integrin alpha v beta 3 (αvβ3) receptors, and that targeting using this peptide would further enhance the antitumor efficacy of ZnO. Therefore, the toxicity and uptake of bare zinc oxide nanostructures and their RGD-targeted counterparts to a panel of malignant breast cancer cell lines was investigated. Two breast cancer cell lines of differing metastatic and invasive capacities were used: MDA-MB-231 cell line that is oestrogen receptor-negative and highly invasive; and, MCF-7 cell line that is oestrogen receptor-positive and non-metastatic. Healthy epithelial MCF-10-2A cell line was used as a control. To test the toxicity of the ZnO NPs (bare and RGD targeted), a combination of cell viability assays including; Alamar Blue, LDH, and ATP assays was used. Flow Cytometry was used to detect necrosis and apoptosis. The intracellular uptake of the ZnO NPs (bare and RGD targeted) was investigated and compared using different correlative microscopy techniques; Transmission electron microscopy (TEM), scanning electron microscopy (SEM), and confocal microscopy. Furthermore, the protein corona formed around the particles was analysed using mass spectroscopy (MS). The bare and RGD targeted ZnO NPs exhibited dose and time dependent toxicity to the MCF-7 and the MDA-MB-231 cell lines in a concentration range of 10-100 µg/ml. Significant differences in the responses of the MCF-7 and MDA-MB-231 cells after exposure to the bare ZnO NPs were observed in the LDH assay and the Alamar Blue assays. Cell death had features of necrosis and apoptosis with mitochondrial structural changes. RGD-targeting of the ZnO NPs was efficient and successfully increased the toxicity of the ZnO NPs to breast cancer cells at lower doses, while preserving the viability of the healthy cells at the same dose and exposure time indicating that a potential therapeutic dose window exists. In the Alamar Blue viability assay, for example, the IC50 values of the bare and targeted ZnO NPs after 24 h exposure to the MDA-MB-231 cells were 30 and 22 μg/ml, respectively. In cell free experiments, the bare ZnO NPs dissolved very rapidly in simulated body fluid of lysosomal pH 5.2, whereas they were more stable at pH 7.4. Confocal and transmission electron microscopy confirmed uptake of both classes of ZnO NPs and a rise in intracellular Zn2+ concentration prior to cell death after exposure to ZnO NPs. RGD-grafted ZnO NPs were more associated to MDA-MB-231 cells possibly by binding to integrin αvβ3 compared to the bare ZnO. The kinetics of intracellular dissolution of the RGD-targeted and bare ZnO NPs showed differences. The bare ZnO NPs, showed a clear time dependent increase in intracellular dissolution while the RGD-targeted ZnO NPs showed very low intracellular dissolution with time and then a sudden burst of ionic zinc. Preliminary studies were also performed on one dimensional ZnO nanowires and data are in broad agreement with the NP study, showing dose and time dependent toxicity in the MDA-MB-231 cells. This research establishes a successful NP-based platform for cancer targeting and therapy.