Electromagnetic wireless remote control of mammalian transgene expression

Fabrication of CBCFO

CFO nanoparticles were synthesized according to the literature with modifications³³. To prepare CFO nanoparticles, iron(III) chloride hexahydrate (0.995 g) and cobalt(II) chloride (0.239 g) were mixed in deionized water (35 ml) containing hexadecyltrimethylammonium bromide (2.041 g). Sodium hydroxide solution (6 M) was then added dropwise to the mixture under continuous stirring to achieve a final pH of 11.0. After ultrasound stimulation for 30 min, additional hydrothermal treatment was applied to the mixture at 180 °C for 24 h in a 50-ml Teflon-lined stainless-steel autoclave. The resulting black precipitates were washed with deionized water and ethanol several times after cooling to room temperature.

To synthesize BCFO magnetoelectric nanoparticles, a sol–gel treatment was applied to the as-prepared CFO nanoparticles³³. Briefly, CFO nanoparticles (50 mg) were dispersed into 30 ml ethylene glycol (catalogue number 324588, Sigma-Aldrich) containing bismuth(III) nitrate pentahydrate (0.160 g) and iron(III) nitrate nonahydrate (0.121 g). After 2 h of sonication, the sol mixture was moved to a vacuum oven and dried for 24 h. Next, the resulting gel-state mixture was preheated at 400 °C for 30 min to eliminate organic compounds and successively calcined at 500 °C for 90 min. The resulting BCFO nanoparticles were washed several times with deionized water and ethanol on a nylon membrane and collected with a neodymium permanent magnet after ultrasound treatment.

Chitosan (catalogue number 448877-50 G, Sigma-Aldrich) was first dissolved in 0.1-M NaCl to form a 0.1% solution after acidification with 1% acetic acid. Rhodamine B isothiocyanate (RITC)-labelled chitosan was prepared by dissolving RITC (40 µM, catalogue number CAY20653-100 mg, Cayman) in methanol and mixing it 1:1 with a 10 mg ml⁻¹ chitosan solution under nitrogen protection, followed by dialysis against 0.1-M NaCl. The prepared BCFO nanoparticles were then dispersed and mixed in the chitosan solution (5 mg ml⁻¹) by sonication for 1 h. The CBCFO nanoparticles were collected by centrifugation and washed with water three times. RITC-CBCFO nanoparticles were fabricated by mixing BCFO nanoparticles with RITC-labelled chitosan.

For cellular uptake, all nanoparticles were sonicated at 35 kHz for 30 min (Bandelin Electronic, RK100H) and filtered through a 0.22-µm filter (catalogue number P668.1, Carl Roth).

Characterization of CBCFO

The morphology of the obtained CFO, BCFO and CBCFO nanoparticles was examined by TEM (FEI F30) and STEM (JEM-F200). The distribution of elements along the nanoparticles was studied by STEM EDX mapping (JEM-F200). The crystallographic structure of the nanostructures was analysed by XRD on a Bruker AXS D8 Advance 1 X-ray diffractometer, equipped with a copper target at a wavelength of 1.542 Å. The magnetic properties were evaluated by scanning probe microscopy (Bruker Dimension ICON) according to the magnetic force model. The zeta potential and the hydrodynamic size of samples were measured by a dynamic light scattering Zetasizer (Malvern, ZEN3600) in DPBS (0.01 M, pH 7.4). Relative charge separation and ROS induction from nanoparticles were evaluated by TA assay (3 mM, λ_ex/λ_em = 310/430 nm) and MB assay (5 mM, λ_abs = 664 nm), respectively, using a plate reader (Tecan, Spark Reader). For TA and MB assays, an aqueous solution (400 μl) containing different nanoparticles was exposed to a magnetic field under constant agitation, and 100-μl aliquots of the supernatant were transferred to 96-well plates for colorimetric or fluorometric measurement.

Magnetic field stimulation

Electromagnet-containing 3D-printed holders (Supplementary Figs. 5a and 8c) were designed to minimize the thermal effect on biological systems. Samples were exposed to a uniform EMF by placing them in the central area (5.8 cm × 5.8 cm) of a Helmholtz-coil-based device. The circuits (Supplementary Fig. 5c) for magnetic field stimulation were powered by custom-designed electrical drivers. The field strength generated by the Helmholtz-coil device was 9–21 mT and that generated by the single-coil device was 20–22 mT at a plane of 0.3–0.5 cm from the coil, with the frequency fixed at 1 kHz (sinusoidal). The amplitude of the applied alternating magnetic field was confirmed by a gaussmeter.

Cell culture and engineering

Cell culture

Human embryonic kidney cells (HEK-293, ATCC, CRL-11268), human telomerase-immortalized mesenchymal stem cells (hMSC-TERT, RRID: CVCL_Z015), human liver cancer cell line (HepG2, ATCC, CRL-11997), Chinese hamster ovary cells (CHO-K1, ATCC, CCL-61), baby hamster kidney cells (BHK-21, ATCC, CCL-10) and mouse pituitary tumour cells (AtT-20, ATCC, CCL-89), were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, catalogue number 52100-39, Thermo Fisher Scientific) supplemented with 100 mM proline (CHO-K1 only), 10% fetal bovine serum (FBS, catalogue number F7524, Sigma-Aldrich) and 1% (v/v) streptomycin/penicillin (catalogue number L0022, Biowest) at 37 °C in a humidified atmosphere containing 5% CO₂.

Cell transfection

For transfection, 10⁴ cells (CellDrop BF Brightfield Cell Counter, DeNovix) were seeded per well in a 96-well plate (catalogue number 3599, Corning Life Sciences) 24 h before transfection by addition of 20 µl of a mixture containing 0.3 µg polyethyleneimine (PEI MAX, mol. wt 40,000, 1 μg μl⁻¹ in double-distilled H₂O, catalogue number 24765-2, Polysciences) and 0.1 µg plasmid DNA (equimolar concentrations for plasmid mixtures) per well. After 8 h, the mixture was replaced with a standard cultivation medium or nanoparticle medium suspension (100 µl) for further characterization.

Monoclonal cell line construction

HEK-293 cells (1.5 × 10⁵) were cotransfected with pJH1101 (ITR-P_hCMV-NRF2-pA: P_RPBSA-ECFP-P2A-PuroR-pA-ITR) (200 ng), pJH1054 (ITR-P_hCMV-KEAP1-P2A-BlastR-pA-ITR) (550 ng), pJH1096 (ITR-P_ARE-NLuc-P2A-mINS:P_mPGK-ZeoR-pA-ITR) (400 ng) and pJH42 (P_hCMV-SB100X-pA) encoding constitutive expression of a hyperactive Sleeping Beauty (SB) transposase (200 ng)⁵⁸. After selection for two passages in culture medium supplemented with 2.5 μg ml⁻¹ puromycin, 300 μg ml⁻¹ blasticidin and 300 μg ml⁻¹ zeocin, the resistant polyclonal population was divided by ECFP-based FACS-mediated single-cell sorting into 48 monoclonal cell lines. Twelve monoclonal cell lines with the highest ECFP-based fluorescence intensity were loaded with CBCFO nanoparticles (50 μg per 10⁶ cells) and stimulated by EMF (1 kHz, 21 mT, 3 min). HEK_EMPOWER (clone number 3), showing best-in-class EMF-stimulated transgene-fold induction, was chosen for further studies (Extended Data Fig. 7a).

Microencapsulation and implantation of HEK_EMPOWER cells

To protect HEK_EMPOWER cells from the mouse immune system while permitting the exchange of nutrients and release of therapeutic proteins, we used a clinical trial-validated alginate-based encapsulation technology⁴⁸. HEK_EMPOWER cells were encapsulated in alginate/poly(l-lysine)/alginate microcapsules with a diameter of 400 µm by treating a mixture of 9.0 × 10⁷ cells with 18 ml alginate (w/v, 1.6%; Na-alginate, catalogue number 71238, Sigma-Aldrich) in an encapsulator (Inotech Encapsulator IE-50R, EncapBiosystems) equipped with a 200-μm nozzle. A 20-ml syringe was operated at a flow rate of 20 ml min⁻¹ with a vibration frequency of 1.2 kHz and 1.2 kV voltage for bead dispersion. A 100-ml poly(l-lysine) 2000 (w/v, 0.05%; catalogue number 25988-63-0, Alamanda Polymers) solution and a 100-ml 0.03% alginate solution were sequentially used to form the microcapsules. For delivery, 2.5 × 10⁶ encapsulated cells in 0.5 ml serum-free DMEM were subcutaneously implanted through a 3-ml syringe (catalogue number 9400038, Becton Dickinson) with a 0.7-mm × 30-mm needle (catalogue number 30382903009009, Becton Dickinson).

Animal experiments

Preparation of experimental mouse models

C57BL/6JRJ mice were kept and monitored in groups (n = 5) in an environment controlled at 21 ± 2 °C and 55 ± 10% humidity and maintained under a 12-h reverse light–dark cycle, with free access to standard diet and water. All procedures were performed in compliance with Swiss animal welfare regulations, approved by the Veterinary Office of the Canton Basel-Stadt, Switzerland (license number 2996_34477), the French Republic (project number DR2018-40v5 and APAFIS number 16753) and the People’s Republic of China (Institutional Animal Care and Use Committee of Westlake University, protocol ID20-009-XMQ). The experiments were conducted by P.G.R. (license number LTK 5507), G. Charpin-El Hamri (number 69266309; University of Lyon, Institut Universitaire de Technologie) or by S. Xue (Westlake University). Two groups of mice were utilized: WT and experimentally induced T1D mice. To induce the T1D condition, male WT mice (8–9 weeks old, 18–23 g) were intraperitoneally injected with streptozotocin (STZ; 75 mg kg⁻¹, 0.2 M citrate buffer, pH 4.2; Sigma-Aldrich, catalogue number S0130) for 4 consecutive days following a 6-h fasting period⁵⁹. Control WT mice from Janvier Labs (18–23 g) received identical injections without STZ. At 10 days after the final injection of STZ, fasting blood-glucose levels were measured using ContourNext test strips and a ContourNext ONE reader (Ascensia Diabetes Care; catalogue numbers 84191451 and 85659367) to confirm persistent hyperglycaemia and T1D status in the STZ-treated group.

Experimental procedure

Microencapsulated HEK_EMPOWER cells with CBCFO nanoparticles (50 μg per 10⁶ cells) were subcutaneously implanted in the experimental and control groups. The hair on the dorsoventral side of the mice was completely shaved, and the animals were anaesthetized with 4% isoflurane and maintained under 2% isoflurane during surgery. Microencapsulated HEK_EMPOWER cells were injected subcutaneously (0.5 ml DMEM, 5 × 10⁶ cells) on the dorsoventral side using a 5-ml syringe with a 21-gauge needle to reduce the risk of aseptic loosening. After a 24-h stabilization period, the HEK_EMPOWER cells were wirelessly stimulated using a portable (single-coil-based) device (Fig. 4b) for 3 min once every 24 h in the EMFS (+) group. For the rest of each day, treated animals were not restrained. The single-coil devices (n = 5) were fitted into a 3D-printed holder (Supplementary Fig. 8d) and a rectangular tunnel (with five parallel holes, Supplementary Fig. 8b) was used to maximize efficiency and facilitate parallel experiments. The animals were fasted for 6 h before measuring blood-glucose and insulin levels. For the GTT experiment, treated animals were intraperitoneally injected with 1.5 g kg⁻¹ glucose and glycaemia was recorded at regular intervals over 2 h. Real-time blood-glucose monitoring was performed at regular time points over a period of 4 weeks after a fasting period of 6 h. Alongside glycaemic levels, the corresponding blood insulin levels were also measured and compared with those of untreated WT and T1D groups.

Blood collection

The level of blood glucose was monitored periodically using ContourNext test strips and a ContourNext ONE reader (catalogue numbers 84191451 and 85659367, Ascensia Diabetes Care)⁶⁰. Blood insulin levels were assessed in serum samples collected in Microtainer serum separator tubes (centrifuged at 6,000g for 10 min at 4 °C; catalogue number 365967, Becton Dickinson) with an ultrasensitive ELISA assay (catalogue number 10-1247-01, Mercordia).

Histology

Microencapsulated HEK_EMPOWER and surrounding tissue were explanted from EMF-stimulated and unstimulated mice and fixed overnight in 10% buffered formalin (100 ml 40% formalin, 900 ml double-distilled H₂O, 4 g l⁻¹ NaH₂PO₄, 6.5 g l⁻¹ Na₂HPO₄, pH 7). The tissue samples were trimmed, dehydrated in increasing concentrations of ethanol, cleared with xylene, embedded in paraffin wax, processed into 5-µm slices using an EXAKT 300 CP system (EXAKT Technologies) and stained with haematoxylin and eosin. The tissue sections were analysed by light microscopy (Olympus CKX53) and images were acquired with an Olympus DP75 camera.

Statistics and reproducibility

The data presentation, sample size of biological replicates (n), statistical analysis and significance of differences are shown in the figure legends. All in vitro experiments were repeated at least twice unless otherwise stated. For the mouse experiments, biological replicates (n = 5 mice per group) were randomly assigned to different experimental groups. The details are described in each figure legend. To determine the statistical significance of differences in the case of multiple comparisons we used GraphPad Prism 10 (v.10.1.0, GraphPad Software) and a two-tailed, unpaired, Student’s t-test and one-way or two-way analysis of variance (ANOVA). No statistical methods were used to prespecify sample sizes, but our sample sizes are the same as previously reported^12,14. Data distribution was assumed to be normal, but was not formally tested. All investigators involved in this study were blinded to group allocation during data collection and analysis. No animals or data points were excluded from the analyses for any reason.

Reporting summary

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