BCA protein assay kit (#23225) and Isopropylthio-β-D-thiogalactopyranoside (IPTG, #R0392) were purchased from Thermo Fisher Scientific (USA). β-actin (#ab 8226), HSP 70 (#ab 5439), Bid (#ab 10640), Cyt C (#ab 133504), VEGF (#ab 32152), and Caspase-3 (#ab 13847) antibodies were purchased from Abcam (USA). Bax (#YM 3619) and HIF-1α (#YT 2133) antibodies were purchased from ImmunoWay Biotechnology, Inc (USA). Calcein-AM and propidium iodide assay kit (#04511) and MTT (#M2128) were obtained from Sigma-Aldrich (USA).

Expression and purification of recombinant encapsulinABC

Gene sequence of encA (MXAN_3556), encB (MXAN_3557), and encC (MXAN_4464) were chosen from GenBank, and constructed into vector pRSFDuet-1 (encA) or pCDFDuet-1 (encB and encC), respectively, by Sangon Biotech (Shanghai), Co., Ltd, China. A single colony of E. coli BL 21 (DE 3) cells, transformed with protein expression plasmid, was transferred into 5 mL LB medium, supplemented with 100 ng/μL antibiotic, and incubated overnight at 37 °C with 180 rpm shaking. The overnight preculture was then inoculated into 500 mL LB medium and incubated at 37 °C with 180 rpm shaking. Recombinant protein production was induced at OD600 = 0.6 by the addition of 1 mM IPTG for another 4-h incubation. Cells were pelleted by centrifugation at 7000 × g for 10 min at 4 °C, and resuspended in tenfold (volume per gram of cell pellet) in lysis buffer for sonication on ice with 2-s burst of sonication at 40% amplitude and 4-s of cooling, the total time of sonication is 7 min. Cell lysate clarified by centrifugation at 10,000 × g, 10 min, 4 °C, and then polyethylene glycol added to the supernatant on ice and mixed for 45 min, after which precipitated proteins were pelleted. The pellets were resuspended in PBS (pH 7.0) on ice for 1 h, and then centrifuged at 10,000 × g, 10 min, 4 °C to remove the sediment. The supernatant was filtrated using 0.22-μm syringe filter (Millipore, UK) for molecular sieve (AKTA Purifier, GE). The collected proteins were verified by SDS-PAGE, dynamic light scatter (DLS), and cryo-electron microscope (Cryo-EM), and then stored at 4 °C.

Preparation of eMIONs and fMIONs

First, a radius flask (50 mL) with a stirrer was filled with Argon gas, then added a degassed NaCl solution (pH 8.5, 8.0 mL, 100 mM) and encABC (2 mg) with stirring of 1000 rpm and 50 °C. Next, Fe2+ (125 mM, FeSO4·6H2O) and fresh H2O2 (41.7 mM) were slowly added into the vessel (5 μL/min) by syringe simultaneously. In total, 50 mM NaOH was titrated into a vessel to keep pH to 8.5. For the fMIONs, ferritin (2 mg) was added into an Argon gas-filled radius flask, followed by the addition of 8 mL degassed 100 mM NaCl solution to the flask. Next, Fe2+ (12.5 mM, FeCl2·4H2O) and fresh H2O2 (4.17 mM) were injected into the vessel (31.3 μL/min) by syringe simultaneously. In all, 50 mM NaOH was titrated into a vessel to keep pH to 8.5. The reacted temperature was kept at 65 °C, and with 1000 rpm stirring for 1 h. After the reaction, the solution purified via ultrafiltration (30 KDa, 2000 rpm/30 min), and then resuspended in 2 mL PBS.

Characterization of eMIONs and fMIONs

Then, the collected eMIONs and fMIONs were characterized by DLS (Malvern Instruments, Malvern, UK), TEM (TecnaiG2 Spirit, FEI), BCA kit (Thermo Fisher), Inductively Coupled Plasma Mass Spectrometer (ICP-MS). The crystallinity and composition of samples were measured by X-ray diffraction (XRD, Rigaku Ultima IV, Japan) and X-ray photoelectron spectroscopy (XPS). Magnetic characterizations of the samples were represented by a vibrating-sample magnetometer (VSM) option under a magnetic field up to 20 kOe at 5 K. The T2-weighted MR imaging ability was performed with a 9.4-T MRI scanner (Bruker, USA).

Specific absorption rate (SAR) measurement

The magnetic-to-thermal conversion capacity of eMIONs was measured by SAR-based time-dependent calorimetric measurements under AMF. eMIONs with a series of concentration (0.1, 0.2, 0.5 mM) were positioned at a circular copper coil (10-turn copper coil with an 8 cm outer diameter). Magnetic field was produced by an AMF generator (SPG-10-II, Shenzhen Double-Power Technology Co. Ltd. China) and with different powers (10, 15, 20 KA/m, 300 kHz). The temperature changes of the samples were measured by a thermocouple thermometer (TES-1310, Taiwan). Finally, the SAR value was calculated using the following Eq. (1)42:

$${\mathrm{SAR}} = {\mathrm{C}}_{\mathrm{P}}\frac{{{\mathrm{{\Delta}}}T}}{{{\mathrm{{\Delta}}}t}} \times \frac{{m_V}}{{m_{NP}}},$$


where Cp is the heat capacity of the medium, ΔTt is the experimentally observed heating rate, mV is the mass of the suspension, mNP is the iron content of the suspension.

Study of catalase-like ability of eMIONs

The oxygen generation and its generated rate in aqueous solutions were used to represent the catalase-like ability of eMIONs. For the catalysis ability detection, the fresh H2O2 (8.8 M) was diluted to 100 mM by deoxygenated water with different pH (5.5, 6.5, and 7.4), and then 30 mL prepared H2O2 was injected in a 50 mL of closed three-neck flask with oxygen electrode probe of portable dissolved oxygen meter (Rex, JPBJ-608, China) to measure the concentration of oxygen in solution real time. The eMIONs and fMIONs with different concentrations (0.1, 0.5, 1.0, 2.0, and 5 μg/mL) were injected into the flask by a syringe. Then, the device was placed in a water bath with different temperatures (25, 37, and 43 °C) and AMF to detect the effect of temperature and magnetic field on the catalytic efficiency of eMIONs. Finally, the Michaelis–Menten kinetic curves of eMIONs and fMIONs were gained by plotting the respective initial velocities, and the maximal velocities were calculated by the Lineweaver–Burk plotting with Prism 7.0.

Cellular experiments

To evaluate the magneto-catalytic therapeutic ability, A549, LM3, U87, 4T1, HUEVC, MSC, LO2, and 293T cell lines were used as cell models and cultured in DMEM (with 10% FBS) at 37 °C with 5% CO2. Cells (1 × 104) were seeded on a 96-well plate overnight, then added eMIONs or fMIONs with different concentrations (1.0, 2.5, 5.0, 10.0, 20.0 μg/mL), and closed by aseptic paraffin to culture another 6 h. The medium was replaced with a fresh one and closed by aseptic paraffin, and the hyperthermia groups to receive MHT (15 KA/m, 300 kHz, 10 min), then incubated for another 12 h. After that, a standard MTT assay was utilized to evaluate the cell viabilities (n = 5).

For AM/PI staining, calcein-AM and propidium iodide solutions (4 μM) were added into A549 cell lines (3 × 105/well, six-well plate) after different treatments, and then incubated for 30 min, after that, washed three times with PBS carefully for fluorescence image by inverted microscopes (Nikon DS-Ti2).

Western blotting experiments to verify the protein expression of the apoptosis signal path, HSP 70, HIF-1α, and VEGF. A549 cell lines were seeded on a six-well plate (3 × 105/well) to incubate overnight. Then the samples were received different treatments as mentioned above. The cells were harvested for western blotting, and the abundance of proteins was calculated via Image J2x (, Rawak Software, Inc).

In vivo MR imaging

Animal experiments were approved by the Animal Care and Use Committee (CC/ACUCC) of Xiamen University. Male BALB/c nude mice (6 weeks, 20 g) were obtained from Beijing Vital River Laboratory Animal Technology Co. Ltd. For the subcutaneous xenograft tumor model, 106 A549 cells were injected into the right flank of nude mice, and housing conditions of mice is the specific pathogen-free (SPF) laboratory animal center, the humidity keeps at 40–60% (26–28 °C), and keeps at 10 h of light and 14 h of dark cyclic condition. The mice were used when their tumor volumes reached ~100 mm3. The eMIONs (CFe 2 mg/kg) were injected via tail vein, and MR imaging was executed on a 9.4-T scanner (Bruker, USA) at the scheduled time (0, 3, 6, 12, and 24 h), respectively. T2-weighted MR images were obtained with the following parameters: TR = 2500, TE = 33 ms, field of view (FOV) = 4 × 4 cm, slice thickness = 1 mm, and 19 contiguous slices.

In vivo magneto-catalytic therapy assisted by eMIONs

For in vivo investigations, when the tumors reach ~100 mm3, the mice were randomly divided into six groups (n = 5). Then the mice treated with CFe 5 mg/kg of various formulations at day 1, 4, and 7 through the tail vein. In groups only AMF, fMIONs with AMF, and eMIONs with AMF, the tumors were received with AMF (15 KA/m, 300 kHz, 10 min) at 9 h after injection. For the orthotopic model, we established orthotopic HCC, as described previously. Briefly, a laparotomy executed in the anesthetized nude mouse, and then 25 μL fLuc-LM3 cells (107) were injected into the right liver lobe. After 12 days, bioluminescence imaging by IVIS Lumia II was utilized to screening out tumor-bearing mice, and randomly divided into three groups (n = 5). Then the mice treated with CFe 11.2 μg/per mouse by ultrasound imaging-guided intratumor injection, and monitoring the delivery via MRI at days 1, 4, and 7. After that, in group eMIONs with AMF, the tumors were received with AMF (15 KA/m, 300 kHz) for 10 min. On day 14, we collected about 200 μL blood of the mice from the orbit, and randomly sacrificed a mouse from each group for further H&E staining and immunofluorescence staining.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.