Cell lines and animals

Human myeloid mononuclear THP-1 cells (SCSP-567) were obtained from the Cell bank of Chinese Academy of Sciences (Shanghai, China). Genetically transfected ACE2-293T cells (41107ES03) were purchased from Yeasen Biotechnology (Shanghai, China). Human ACE2 transgenic mice (hACE2-mice) and C57 mice were used in the study. The mice were permitted to consume food and water at their discretion, and they were maintained in an ambient temperature range of 21–26 °C, with a humidity level of 50-60%, and a 12-h light/dark cycle. All animal procedures were performed in accordance with the Guidelines for the Institutional Animal Care and Use Committee (IACUC) and approved by the Animal Ethics Committee of Southern Medical University (Permit Number: SMUL-202311053).

Materials

Magnetotactic bacteria AMB-1 (ATCC 700264) was obtained from Biobw (Beijing, China). This wild-type, Gram-negative, facultatively anaerobic, magnetotactic bacterium possesses a fully sequenced genome and biomineralizes magnetosomes. All experiments were performed using the parental strain without further genetic modification. Cur and LPS was bought from Sigma-Aldrich (USA). DBCO-PEG₄-NHS ester was bought from Guangzhou Tanshtech Co., Ltd (Guangzhou, China). Ac₄GalNAz was purchased from Macklin (Shanghai, China). HSA was obtained from Bomeibio (Hefei, China).

Preparation of 2TM

THP-1 cells were cultivated in RPMI 1640 media, and ACE2-293T cells were cultured in high glucose DMEM media. All media contain 10% fetal bovine serum with 1% penicillin and streptomycin. Azido sugar Ac₄GalNAz (50 μM) was added into THP-1 cells and ACE2-293T cells respectively. After incubating for 48 h, activated azide (N₃)-labeling THP-1 cells (THP1-N₃) and ACE2-293T cells (ACE2-293T-N₃) were then obtained.

Cell membrane was obtained according to the previous report^54,55,56. Briefly, THP1-N₃ cells or ACE2-293T-N₃ cells were lysed, followed by mechanical membrane disruption (Huxi JY92-IIDN, China), and 1 h ice-cold incubation. The resulting solutions were then conducted with differential centrifugation (Beckman Optima XPN-100 ultracentrifuge, USA), and the THP1-N₃ cell membrane (TM) and ACE2-293T-N₃ cell membrane (2 M) were obtained and stored at −80 °C.

By adding 500 μL of ultrapure water to 2 M and TM respectively, the isolated cell membranes were re-suspended and mixed at a 1:1 protein mass ratio (2 M:TM). The hybrid membrane (2TM) was then prepared by sonication for 20 s followed by repeated extrusion through a 220 nm polycarbonate membrane.

Fabrication of CurNPs@2TM and ICGNPs@2TM

CurNPs were prepared by adding 200 μL of curcumin solution (2.5 mg mL⁻¹ in dimethyl sulfoxide) dropwise into 4 mL of HSA (2.5 mg mL⁻¹ in pure water) under sonication⁵⁷. CurNPs were then obtained by ultrafiltration with pure water for three times. And the mixture of 2TM and CurNPs were physically squeezed repeatedly through 220 nm polycarbonate membrane for the formation of CurNPs@2TM.

ICGNPs were prepared by slowly adding 200 μL of ICG solution (2.5 mg mL⁻¹ in dimethyl sulfoxide) into 4 mL of HSA solution (2.5 mg mL⁻¹ in water) under continuous sonication. ICGNPs were then obtained by ultrafiltration with pure water for three times. The 2TM and ICGNPs mixture was repeatedly extruded through a 220 nm polycarbonate membrane to generate ICGNPs@2TM.

Preparation of AR and ICGNPs@2TM-AMB-1

AMB-1 (10⁷ CFU mL⁻¹) were cultured in enriched magnetic spirillum growth medium (EMSGM) at 30 °C in the dark⁵⁸. AMB-1 were washed three times with pure water to remove residual medium and then resuspended in pure water. Then, 1 × 10⁸ of AMB-1 were incubated with 0.1 mg mL⁻¹ of DBCO-PEG₄-NHS for 1 h at room temperature to acquire DBCO-labeled AMB-1 (AMB-1-DBCO). To remove unreacted DBCO-PEG₄-NHS, AMB-1-DBCO were washed three times with pure water.

AMB-1-DBCO were incubated with azido-functionalized CurNPs@2TM at room temperature for 1 h. After 10 min centrifugation at 5,000 x g and washing with PBS for three times, the resulting CurNPs@2TM-AMB-1 (AR) were collected.

AMB-1-DBCO were incubated with azido-functionalized ICGNPs@2TM at room temperature for 1 h. After 10 min centrifugation at 5000 x g and washing with PBS for three times, the resulting ICGNPs@2TM-AMB-1 were collected.

Characterization of CurNPs@2TM and AR

The morphology of CurNPs, CurNPs@2TM, AMB-1, and AR was characterized using scanning electron microscopy (SEM, Quattro S, Thermo, USA), transmission electron microscopy (TEM, JEOL-1400, Japan), and fluorescence microscopy (Eclipse Ti2, Nikon, Japan). The absorption spectrum was recorded using a UV-vis spectrometer (Shimadzu UV-2600, Japan) and the fluorescence spectrum was obtained using a fluorescence spectrophotometer (Shimadzu RF-6000, Japan). The hydrodynamic diameters of CurNPs and CurNPs@2TM were measured by dynamic light scattering (DLS, Malvern Zetasizer Nano ZSE, UK). The zeta potential of CurNPs, 2TM, CurNPs@2TM, AMB-1 and AR were also measured by DLS. Room-temperature magnetic properties of AMB-1 and AR were measured using a physical property measurement system (PPMS-9; Quantum Design, USA).

THP-1 and ACE2-293T cells pretreated with or without Ac₄GalNAz (50 μM) were incubated with fluorescent DBCO-Cy5, and the fluorescence intensity was obtained by fluorescence microscope imaging and flow cytometric analysis (BD LSRFortessa X-20, USA).

SDS-PAGE was performed to analyze the membrane proteins of CurNPs@2TM. Membrane proteins were extracted from 2 M, TM, 2TM, and CurNPs@2TM using RIPA lysis buffer, and total protein concentration was quantified by BCA assay (Beyotime, China). Samples (100 μg protein per lane) were denatured at 100 °C for 10 min in SDS loading buffer, then separated on 7.5% SDS-PAGE gels using the Mini-PROTEAN Tetra System (Bio-Rad, USA). Proteins were transferred onto PVDF membranes at 240 mA for 2 h. Membranes were incubated overnight at 4 °C with primary antibodies against ACE2 (Santa Cruz Biotechnology, sc-390851), IL6ST (ABclonal, A18036), CD116 (ABclonal, A23001), and ATP1A1 (Proteintech, 14418-1-AP), followed by 1 h incubation at 37 °C with HRP-conjugated secondary antibodies (anti-rabbit IgG-Proteintech, SA00001-2; anti-mouse IgG-Proteintech, SA00001-1). Protein bands were visualized using the ChemiDoc MP imaging system (Bio-Rad).

Propidium iodide (PI) dye was used to mark dead AR 1 day after AMB-1 loaded with CurNPs@2TM. And we diluted AR and coated it on a EMSGM solid-state culture plate to detect the survival rate of AR. AR density was quantified by measuring optical density at 600 nm (OD₆₀₀) and referencing a standard curve.

Motion evaluation of AR under magnetic field

The controllable motion of AMB-1 and AR was actuated by rotating magnetic field generated by a homemade three-dimensional Helmholtz coil system. AMB-1 and AR with appropriate concentration were dispersed in simulated lung fluid (SLF) in a confocal dish. Samples were positioned at the center of a Helmholtz coil to acquire a uniform magnetic field and imaged using a Nikon Ti2-A microscope. Time-lapse images were recorded with ΔT of = 200 ms. These image sequences were analyzed by ImageJ software with manual tracking plugin. The motion of AMB-1 and AR was manipulated by changing the frequency and the phase of the rotating magnetic field.

In Vivo detection of AR in lung

In order to observe how magnetic motion enhances the penetration into the lung mucus, AR were labeled with Cy5. In short, Cy5-maleimide was conjugated to activated sulfhydryl groups on the surface of AMB-1 bacteria via Michael addition. Next, DBCO was labeled on the surface of Cy5 labeled AMB-1, and the resulting bacteria were connected with N₃ labeled CurNPs@2TM, ultimately forming Cy5 labeled AR. C57 mice were assigned to two groups randomly (AR group and AR + M group). The mice were intratracheally (i.t.) inhaled with Cy5 labeled AR. The mice from AR + M group were positioned in the center of the homemade triaxial Helmholtz coil for magnetic actuation for 30 min after 1 h of i.t. inhalation of AR. The mice were sacrificed after magnetic actuation. Lungs were harvested and fixed in 4% paraformaldehyde for frozen sectioning. Sections were stained with DAPI and imaged using DAPI and Cy5 filters.

C57 mice were assigned to three groups randomly (ICGNPs@2TM group, ICGNPs@2TM-AMB-1 group and ICGNPs@2TM-AMB-1 + M group). The mice were intratracheally (i.t.) inhaled with ICGNPs@2TM or ICGNPs@2TM-AMB-1 using micro-sprayer aerosolizer (YUYANBIO, Shanghai, China). The mice from ICGNPs@2TM-AMB-1 + M group were placed in the center of the homemade triaxial Helmholtz coil for magnetic actuation for 30 min after 1 h of inhalation. In vivo imaging system (IVIS) was used to record the retention of the samples in lung at different times after inhalation (3 h, 12 h, 24 h, 48 h, 72 h) (Ex: 710 nm, Em: 770 nm). The mice were sacrificed after 72 h of i.t. inhalation. The major organs were collected and sectioned for FL imaging to detect the retention of ICGNPs@2TM or ICGNPs@2TM-AMB-1.

C57 mice were assigned to three groups randomly (CurNPs@2TM group, AR group and AR + M group). The mice were intratracheally (i.t.) inhaled with CurNPs@2TM or AR. The mice from AR + M group were placed in the center of the homemade triaxial Helmholtz coil for magnetic actuation for 30 min after 1 h of i.t. inhalation of AR. The mice were sacrificed after 72 h of i.t. inhalation. Lungs were collected and fixed in 4% paraformaldehyde for frozen sectioning. Sections were stained with DAPI and imaged using DAPI and FITC filter sets.

Prepared of pseudovirus (PsV)

PsV were developed following the procedures below. 293T cells were co-transfected with pCDH-Luciferase, psPAX2, and pCAGGS-SARS-2-S-flag plasmids encoding viral glycoproteins. Pseudovirus (PsV) was harvested from the supernatant 48 h post-transfection and concentrated by ultracentrifugation at 100,000 × g through a 20% sucrose cushion for 2.5 h at 4 °C using an Optima XPN-100 ultracentrifuge (Beckman Coulter, USA). The resulting PsV pellet was resuspended in PBS and stored at −80 °C.

In vitro Neutralization of PsV and Inflammatory Cytokines

ACE2-293T cells were seeded in 24-well plates at 1 × 10⁵ cells per well and cultured for 24 h. Prior to infection, 5 μL of PsV was mixed with 500 μL of culture medium containing PBS, AMB-1, CurNPs@2TM and AR (the concentration of AMB-1 was 10⁷ cfu per mL, and Cur was 1 μg mL⁻¹) respectively. Then 500 μL of the mixture was added into ACE2-293T cells, and the culture media were replaced after 12 h. After an additional 48 h of incubation, the Luciferase Assay System (AAT Bioquest, USA) was employed to analyze the luciferase activity.

To determine the cytokine binding effect of AR, 100 μL of PBS containing AMB-1, CurNPs@2TM or AR were incubated with 100 μL of PBS containing IL-6 (2,000 pg mL⁻¹) or GM-CSF (500 pg mL⁻¹) for 30 min. Samples were then centrifuged at 15,000 × g for 15 min to remove nanoparticles, and cytokine levels in the supernatant were quantified using IL-6 or GM-CSF ELISA kits (BioLegend, USA).

In vivo neutralization of PsV and inflammatory cytokines

HACE2-mice pre-inhaled with PsV were randomly divided into six groups (PBS, AMB-1, AMB-1 + M, CurNPs@2TM, AR, AR + M). The mice were then i.t. inhaled with AMB-1, CurNPs@2TM or AR using micro-sprayer aerosolizer after 0.5 h of PsV inhalation. The mice from AMB-1 + M group and AR + M group were placed in the center of the homemade triaxial Helmholtz coil for magnetic actuation for 30 min after 1 h of i.t. inhalation of AMB-1 or AR.

After inhaling PsV at different times (0 h, 48 h, and 72 h), d-fluorescein (150 mg kg⁻¹) was intravenously injected for the quantification of the PsV infection in lung tissue using IVIS system.

The mice were sacrificed 72 h after PsV inhalation, and lungs were collected, fixed in 4% paraformaldehyde, and processed for frozen sectioning followed by H&E staining. Lung injury was scored using the Mikawa method^33,53 based on four parameters graded from 0 to 4: alveolar congestion, pulmonary hemorrhage, neutrophil infiltration, and septal thickening. The sum of these scores represented the overall lung injury.

72 h after PsV inhalation, mice were euthanized and positioned on an endotracheal intubation platform. The lungs were lavaged three times with 0.8 mL of ice-cold PBS via a tracheal catheter. Bronchoalveolar lavage fluid (BALF) was collected and centrifuged at 15,000 × g for 10 min at 4 °C, and the supernatant was harvested for subsequent analysis. The BALF levels of IL-6 and GM-CSF were measured by corresponding ELISA kits.

In vivo inhibition of acute pneumonia

C57 mice pre-inhaled with LPS (10 mg mL) were randomly divided into six groups (PBS, AMB-1, AMB-1 + M, CurNPs@2TM, AR, AR + M). The mice were then i.t. inhaled with AMB-1, CurNPs@2TM or AR using micro-sprayer aerosolizer after 3 h of LPS inhalation. The mice from AMB-1 + M group and AR + M group were placed in the center of the homemade triaxial Helmholtz coil for magnetic actuation for 30 min after 1 h of AMB-1 or AR inhalation.

To assess oxidative stress in LPS-induced acute pneumonia, a reactive oxygen species–sensitive probe (L-012; 25 mg kg⁻¹, Wako Chemicals, Japan) was administered via intravenous injection 24 h after LPS exposure. Fluorescence (FL) signals were monitored using an IVIS imaging system over a 30 min period.

The mice were sacrificed after 24 h of LPS inhalation and the lungs of each group were collected and fixed in 4% PFA and then frozen sectioned for H&E staining. Lung injury scores were also evaluated by Mikawa method^33,53.

The mice were sacrificed after 24 h of LPS inhalation and the lungs of each group were collected. Harvested lung tissues were weighed immediately to obtain wet weight, then dehydrated at 65 °C for 72 h to determine dry weight. The lung wet-to-dry (W/D) weight ratio was subsequently calculated.

24 h after LPS inhalation, mice were euthanized and positioned on an endotracheal intubation platform. The lungs were lavaged three times with 0.8 mL of ice-cold PBS via a tracheal catheter. Bronchoalveolar lavage fluid (BALF) was collected and centrifuged at 15,000 × g for 10 min at 4 °C, and the supernatant was harvested for downstream analysis. The BALF levels of IL-6 and GM-CSF were measured by corresponding ELISA kits.

Analysis of M1 Type and M2 Type alveolar macrophages

Mice with acute pneumonia were sacrificed after 24 h of LPS inhalation and the lungs of each group were excised and fixed with 4% PFA. The tissues were embedded in paraffin, followed by cutting into slices with 4 μm thickness. Alveolar M1 and M2 macrophages were identified in tissue sections by co-staining with Cy3-conjugated anti-F4/80 and Cy5-conjugated anti-CD11c antibodies (for M1) or Cy3-conjugated anti-F4/80 and Cy5-conjugated anti-CD206 antibodies (for M2). Stained sections were imaged using fluorescence microscopy.

The collected lung tissues were homogenized through a nylon mesh to obtain single-cell suspensions, which were stained with PerCP-Cy5.5–anti-F4/80 (Biolegend, 123128), BV605–anti-CD11b (BD, 563015), PE–anti-CD11c (BD, 557401), and APC–anti-CD206 (Thermo, 17-2061-80) antibodies. After washing, cells were resuspended in PBS for flow cytometry analysis. M1 macrophages were defined as F4/80⁺CD11b⁺CD11c⁺ cells, while M2 macrophages were identified as F4/80⁺CD11b⁺CD206⁺ cells.

In Vivo and in vitro biosafety detection

The biosafety of AR on ACE2-293T cells was evaluated by CCK8 assay (GLPBIO, USA). ACE2-293T cells were seeded in 96-well plates at a density of 8 ×  10³ cells per well and cultured for 24 h. The medium was then replaced with fresh medium containing AMB-1 (10⁸ CFU mL⁻¹), CurNPs@2TM, or AR (Cur concentration, 10 μg mL⁻¹). After 48 h of incubation, the supernatant was removed, and 100 μL of fresh medium containing 10% CCK-8 solution was added. Following incubation for 0.5–2 h, absorbance was measured using a multifunctional microplate reader (Spark, TECAN, Switzerland).

For in vivo toxicity evaluation, serum was collected after treatment and analyzed for liver (ALT/AST) and kidney (BUN/CREA) function using corresponding activity assay kits. Major organs including the heart, liver, spleen, lungs, and kidneys were harvested, fixed in 4% paraformaldehyde, sectioned, and subjected to H&E staining for histological analysis under a microscope.

To further ensure the safety, C57 mice were i.t. inhaled with AR (the concentration of AMB-1 was 10⁸ cfu mL⁻¹, and Cur was 10 μg mL⁻¹). And then, the mice were placed in the center of the homemade triaxial Helmholtz coil for magnetic actuation for 30 min after 1 h of AR inhalation. The major organs of the mice were extracted, weighed, and homogenized in EMSGM medium on day 1, 2, 3 and 4 after lung administration of AR with RMF. These samples were diluted (10 times) and coated on EMSGM solid-state culture plate. After incubation, the colonies of the tissues in each group were quantified.

Statistical analysis

All statistical analyses were analyzed by Microsoft Excel 2019, Graphpad Prism 8.0, origin 85, and Image J (1.52a). Flow cytometry was analyzed using Flowjo_V10 software. Statistical analysis was conducted using one-way ANOVA analysis followed by Tukey’s post-test, and the date represent mean ± SD. The asterisk denoted statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Reporting summary

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