A Lysosome‐Targeted Magnetic Nanotorquer Mechanically Triggers Ferroptosis for Breast Cancer Treatment

Abstract Targeting ferroptosis has attracted exponential attention to eradicate cancer cells with high iron‐dependent growth. Increasing the level of intracellular labile iron pool via small molecules and iron‐containing nanomaterials is an effective approach to induce ferroptosis but often faces insufficient efficacy due to the fast drug metabolism and toxicity issues on normal tissues. Therefore, developing a long‐acting and selective approach to regulate ferroptosis is highly demanded in cancer treatment. Herein, a lysosome‐targeted magnetic nanotorquer (T7‐MNT) is proposed as the mechanical tool to dynamically induce the endogenous Fe2+ pool outbreak for ferroptosis of breast cancer. T7‐MNTs target lysosomes via the transferrin receptor‐mediated endocytosis in breast cancer cells. Under the programmed rotating magnetic field, T7‐MNTs generate torques to trigger endogenous Fe2+ release by disrupting the lysosomal membrane. This magneto‐mechanical manipulation can induce oxidative damage and antioxidant defense imbalance to boost frequency‐ and time‐dependent lipid peroxidization. Importantly, in vivo studies show that T7‐MNTs can efficiently trigger ferroptosis under the magnetic field and play as a long‐acting physical inducer to boost ferrotherapy efficacy in combination with RSL3. It is anticipated that this dynamic targeted strategy can be coupled with current ferroptosis inducers to achieve enhanced efficacy and inspire the design of mechanical‐based ferroptosis inducers for cancer treatment.


Characterization of T7-MNTs. Transmission electron microscopy (TEM, JEOL JEM
2100F) and vibrating sample magnetometer (VSM, LakeShore7404, US) were performed for characterizing the morphological, structural, and magnetic features of T7-MNTs.T7 peptide modification was characterized by UV−vis spectrophotometer (Cary 60, Agilent).The hydrodynamic particle size and zeta potential of T7-MNTs and MNP@SiO2 were detected by ZETA SIZER Nano series (Nano-ZS90, Malvern Ltd.).MFG-100 equipment was used to record the magneto-mechanical properties of T7-MNTs.T7-MNTs were dispersed in cell culture medium with 25 µg/mL concentration of Fe and the MF with parameters of 40 mT, 0.5 Hz was applied.
Cells without any treatment were used as the control group.Cell viability was measured after different incubation time (t = 24 h, 72 h, 120 h, 168 h).

LC-MS Analysis of
Lipidomics. 1×10 7 MDA-MB-231 cells were seeded on 100 mm dish for 24 h."MF+" and "MF-" groups were incubated with T7-MNTs (50 μg/mL of Fe content) for 24 h.Afterwards, the "MF+" group was treated with MF application (260 mT, 15 Hz, 30 min).Then, cells were digested by trypsin and collected in centrifuge tubes.After centrifuging at 1000 rpm for 3 min, the cell clusters were collected to extract the lipids.All the samples were examined by liquid chromatography-mass spectrometry (LC-MS) analysis.LC-MS analysis was performed by using an UHPLC-Q Exactive HF-X systerm (equipping with a Accucore C30 column of 100 mm × 2.1 mm, 2.6 μm).50% of acetonitrile-water sollution (containing 0.1% of formic acid and 10 mmol/L ammonium acetate) was used as the mobile phase A. The mobile phase B consisted of 10% acetonitrile, 88% isopropanol and 2% water.
The washed gradient was as follows: 0 ~ 4 min, 65% ~ 40% A; 4 ~ 12 min, 40% ~ 15% A; 12 ~ 15 min, 15% ~ 0% A; 15 ~ 17 min, 0% A. The flow rate was 0.4 mL/min and the column temperature was maintain at 40 °C.The injection volume was 5 μL.All the samples were ionized by electrospray, and the mass spectrum signals were acquired by positive and negative modes (the spray voltage of 3.0 kV and -3.0 kV), respectively.The equipment conditions were set as following: scan type as 200-2000 (m/z), sheath gas flow rate as 60 psi, aux gas flow rate as 20 psi, aux gas heater temp as 370℃, normalized collision energy as 20-40-60 (v), respectively.The datas were analyzed by the Lipidsearch software (Thermo Fisher，USA).The Cytotoxicity of T7-MNTs Treatment in Cancer andNormal Cell Lines.6×10 3 A549, HepG2 and 3T3 cells were seeded on the 96-well plates, respectively.Then, the cells were incubated with T7-MNTs (50 μg/mL of Fe content) or RSL3 (20 μM) for 24 h.Afterwards, the "MF+" group was treated with MF stimulation (260 mT, 15 Hz, 30 min).Cells without any treatment were used as the control group.Cell viability of all groups was measured by the CCK-8 kit.

Figure S2 .
Figure S2.Calculation of torques generated by T7-MNTs.T7-MNTs dispersed in a cell culture medium and assembled under the magnetic field.As B0 = 40 mT, the single T7-MNT had a saturated magnetization of Ms = 40.61emu/g and a remanent magnetization of Mr = 0.26 emu/g.The torques generated by T7-MNTs were calculated using the following equation: [1]   Sin B Cos M M M mass N r r gle    − +   = 0 0 sin ) ) ( (

Figure S3 .
Figure S3.Transferrin receptors expression in normal and cancer cell lines.

Figure S9 .
Figure S9.Colocalization coefficient of intracellular iron with lysosomes in MCF-7 and MDA-MB-231 cells.Histograms of tMr (Manders' overlap coefficient for red signals in thresholded image) corresponding to the signals of FerroOrange (red) and Lyso-Tracker Green, respectively (n = 5, ***p < 0.001, five randomly selected images were analyzed by Image J).

Figure S10 .
Figure S10.Lipid metabolomics analysis of T7-MNTs treatment.(a) PCA analysis of the different expression lipids profile in MDA-MB-231 cells with (MF+) and without T7-MNTs treatment (MF-).Axes in the PCA plot are relative to the two principal components with the largest variance.(b) Volcano plot of the different expression lipids in the MF-and MF+ groups.

Figure S11 .
Figure S11.The expression level of SLC7A11 and ferritin with different treatment conditions in MCF-7 (a) and MDA-MB-231 cells (b).RSL3 group was treated withRSL3 (20 µM) for 24 h.The "MF+" group was treated with T7-MNTs and followed by MF application (260 mT, 15 Hz, 30 min)."MF+DFO" group were incubated with DFO (50 μM) for 6 h before MF application.The cells incubated with T7-MNTs were used as the "MF-" group, and untreated cells were used as the control group.

Figure S12 .
Figure S12.T7-MNTs-mediated cytotoxicity in A549 and HepG2 cells.Cell viability of A549 (a) and HepG2 cells (b) with different treatments (n = 6, ****p < 0.0001).The "MF+" group was treated with MF (260 mT, 15 Hz) for 30 min.20 µM of RSL3 was used as a positive control.The cells incubated with T7-MNTs were used as the "MF-" group, and untreated cells were used as the control group.Cell viability was measured by CCK-8 kit.

Figure S16 .
Figure S16.Lipid peroxidation in MDA-MB-231 cells with treatments under different frequencies of MF.The T7-MNTs concentration was 50 μg/mL, and the MF exposure time of 30 min.Scale bar: 100 µm.

Figure S17 .
Figure S17.Lipid peroxidation in MDA-MB-231 cells with MF treatment at different periods.Cells treated with T7-MNTs at a Fe dose of 50 μg/mL.The frequency