Functionalized Hybrid Iron Oxide–Gold Nanoparticles Targeting Membrane Hsp70 Radiosensitize Triple-Negative Breast Cancer Cells by ROS-Mediated Apoptosis

SIMPLE SUMMARY: Breast cancer is the most common cancer in women worldwide, and triple-negative breast cancer (TNBC) is the malignancy with the worst prognosis. Although the vast majority of TNBC patients are treated with multimodal therapies, including ionizing irradiation (IR), as a standard of care, radiation-resistant tumor cells and off-target toxicities preclude an advantageous clinical outcome. We studied the radiosensitizing effect of novel Hsp70-specific, hybrid iron oxide–gold (Fe(3)O(4)-Au) nanoparticles (NPs) functionalized with the Hsp70 peptide TPP via a PEG4 linker (TPP-PEG4) to target tumor-specific membrane Hsp70 (mHsp70) on TNBCs. TPP can increase the affinity and uptake of hybrid Fe(3)O(4)-Au nanoparticles (FeAuNPs) into TNBC cells. TPP-PEG4-FeAuNPs, but not control hybrid FeAuNPs, significantly sensitize TNBC cells against radiation by activating a G2/M checkpoint arrest and elevating the production of reactive oxygen species (ROS), which induce DNA double-strand breaks in TNBC. ABSTRACT: Triple-negative breast cancer (TNBC) a highly aggressive tumor entity with an unfavorable prognosis, is treated by multimodal therapies, including ionizing radiation (IR). Radiation-resistant tumor cells, as well as induced normal tissue toxicity, contribute to the poor clinical outcome of the disease. In this study, we investigated the potential of novel hybrid iron oxide (Fe(3)O(4))-gold (Au) nanoparticles (FeAuNPs) functionalized with the heat shock protein 70 (Hsp70) tumor-penetrating peptide (TPP) and coupled via a PEG4 linker (TPP-PEG4-FeAuNPs) to improve tumor targeting and uptake of NPs and to break radioresistance in TNBC cell lines 4T1 and MDA-MB-231. Hsp70 is overexpressed in the cytosol and abundantly presented on the cell membrane (mHsp70) of highly aggressive tumor cells, including TNBCs, but not on corresponding normal cells, thus providing a tumor-specific target. The Fe(3)O(4) core of the NPs can serve as a contrast agent enabling magnetic resonance imaging (MRI) of the tumor, and the nanogold shell radiosensitizes tumor cells by the release of secondary electrons (Auger electrons) upon X-ray irradiation. We demonstrated that the accumulation of TPP-PEG4-FeAuNPs into mHsp70-positive TNBC cells was superior to that of non-conjugated FeAuNPs and FeAuNPs functionalized with a non-specific, scrambled peptide (NGL). After a 24 h co-incubation period of 4T1 and MDA-MB-231 cells with TPP-PEG4-FeAuNPs, but not with control hybrid NPs, ionizing irradiation (IR) causes a cell cycle arrest at G2/M and induces DNA double-strand breaks, thus triggering apoptotic cell death. Since the radiosensitizing effect was completely abolished in the presence of the ROS inhibitor N-acetyl-L-cysteine (NAC), we assume that the TPP-PEG4-FeAuNP-induced apoptosis is mediated via an increased production of ROS.