Abstract Metastatic lung cancer remains a leading cause of cancer-related mortality worldwide, underscoring the urgent need for early detection and intervention strategies. Current diagnostic and therapeutic methods often fall short in sensitivity and efficacy, particularly for early-stage disease. In this study, we develop CaCO[3]@quercetin-Mn nanoparticles (CQM NPs) integrating dual-stage macrophage reprogramming with T[1]-weighted magnetic resonance imaging (MRI) enhancement for theranostic management of pulmonary metastasis. CQM NPs exploit dynamic macrophage polarization: initial M2 polarization promotes nanoparticles accumulation in metastatic lesions, enabling detection of lesions as small as 0.11 mm, while subsequent acid/glutathione (GSH)-triggered degradation reprograms tumor-associated macrophages to antitumor M1 phenotype, suppressing metastatic growth by 81.64% and preventing circulating tumor cells (CTCs) from colonizing the lungs by 78.08%. These findings demonstrate a significant improvement in MRI sensitivity and an integrated approach to therapy, contrasting with traditional methods that separate these challenges. This “detect-to-treat” paradigm bridges the critical gap between imaging sensitivity and immunomodulatory therapy, offering a blueprint for precision nanomedicine in metastatic cancers. Graphical abstract [46]graphic file with name 12951_2025_3749_Figa_HTML.jpg Supplementary Information The online version contains supplementary material available at 10.1186/s12951-025-03749-5. Keywords: Pulmonary metastasis, Magnetic resonance imaging, Macrophage polarization, Nanoplatform, Theranostic Introduction According to the latest USA cancer statistics, lung cancer accounts for substantially more deaths than any other malignancy, with a five-year relative survival rate of just 27% [[47]1]. Metastasis is the leading cause of lung cancer-related mortality, with metastatic lung cancer standing out for its particularly poor prognosis. Statistics from 2011 to 2017 reveal a dismal 5-year relative survival rate of only 6% for patients with metastatic lung cancer in the United States [[48]2, [49]3]. Although early detection is critical, reliable identification of submillimeter metastatic lesions remains challenging [[50]4–[51]6], delaying treatment and limiting therapeutic efficacy. Molecular imaging, particularly MRI, has advanced the noninvasive detection of pulmonary metastases [[52]6–[53]11]. The benefits of MRI, including its free of ionizing radiation, deep tissue penetration, and high spatial resolution, are enhanced by contrast agents (CAs) [[54]12–[55]15]. However, conventional CAs struggle to detect ultra-small lesions and lack therapeutic capacity, underscoring the need for multifunctional agents that can both diagnose and treat metastases [[56]5, [57]16]. Nanoparticles (NPs) have emerged as promising theranostic tools [[58]17–[59]21], yet systemic administration is hindered by rapid clearance and nonspecific macrophage uptake [[60]22, [61]23]. In the tumor microenvironment (TME), macrophages play dual roles: M1 macrophages suppress tumor growth, while M2 macrophages and tumor-associated macrophages (TAMs) promote metastasis [[62]24–[63]26]. Current evidence suggests that NP phagocytosis can modulate macrophage polarization, but the direction of polarization (M1 vs. M2) is highly NP-dependent [[64]27–[65]29]. An optimal nanoplatform should enable programmable polarization: first exploiting M2 macrophages for targeted lesion accumulation, then reprogramming them to M1 for therapeutic benefit [[66]30–[67]32]. To address these challenges, we designed a core-shell nanoparticle (CQM NP) composed of a quercetin-Mn (QM) shell and CaCO[3] core. The QM shell functions as a T[1]-weighted MRI CA [[68]33, [69]34], and induces M2 polarization to guide metastatic targeting [[70]35, [71]36]. Following vesicular transport to tumor sites, the acidic TME and elevated glutathione levels trigger degradation of both the QM complexes and CaCO[3] nanoparticles (CNPs), releasing bioactive components (manganese ions, calcium ions, quercetin, and CO[2]) that reprogram TAMs to the tumor-suppressive M1 phenotype. This innovative design enables the CQM NPs to achieve three critical functions: (1) MRI detection of submillimeter metastatic lesions through enhanced contrast, (2) inhibition of metastatic colony formation via targeted accumulation, and (3) therapeutic intervention through TME modulation (Scheme [72]1). By integrating early diagnostic capability with timely therapeutic intervention, this multifunctional nanoplatform represents a significant advancement in the proactive management of metastatic lung cancer, offering new possibilities for combating this lethal disease. Scheme 1. [73]Scheme 1 [74]Open in a new tab Schematic illustration of programmable macrophage-polarizing CQM NPs for MRI-guided early detection and treatment of pulmonary metastases Materials and methods Materials MnCl[2]·4H[2]O and CaCl[2]·2H[2]O were purchased from Shanghai Titan Scientific Co., Ltd. NH[4]HCO[3] was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. Quercetin was bought from Shanghai Macklin Biochemical Co., Ltd. Ethanol, NaOH, NaH[2]PO[4]·2H[2]O, and Na[2]HPO[4]·12H[2]O were purchased from Sinopharm Chemical Reagent Co., Ltd. Reduced GSH was bought from Sigma-Aldrich Trading Co., Ltd. 3,3′,5,5′-tetramethylbenzidine (TMB) was purchased from Aladdin Bio-Chem Technology Co., Ltd. 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) was purchased from Bide Pharmatech Ltd. Sulfo-Cyanine5 (Cy5) was obtained from MedChemExpress (USA). D-Luciferin potassium salt was bought from Solarbio Science & Technology Co., Ltd. Phosphate-buffered saline (PBS) was obtained from HyClone (USA). Trypsin, RPMI Medium 1640 basic (1X), Dulbecco’s Modified Eagle Medium (DMEM), and fetal bovine serum (FBS) were purchased from Thermo Fisher Biochemical Products Co., Ltd. 4% neutral paraformaldehyde solution and penicillin-streptomycin (Pen-Strep) were obtained from Beijing Labgic Technology Co., Ltd. Puromycin was bought from Solarbio Science & Technology Co., Ltd. BCECF-AM and DNA Damage Assay Kit by γH2AX Immunofluorescence were obtained from Beyotime Biotechnology Co., Ltd. CellTrace^™ Violet (Invitrogen, [75]C34557) and CellTrace^™ CFSE (Invitrogen, [76]C34554) were purchased from Thermo Fisher Scientific (USA). Anti-Mo F4/80 (eFluor^™ 450), Anti-Mo CD86 (PE-Cyanine7), and Anti-Mo CD206 (APC) were obtained from Thermo Fisher Scientific (Ann Arbor, MI, USA). 4’,6-diamidino-2-phenylindole (DAPI) and Cell Counting Kit-8 (CCK-8) Assay Kit were purchased from Beyotime Biotechnology Co., Ltd. Antifade solution was obtained from Boster Biological Technology Co., Ltd. Synthesis of CNPs 0.6 g of CaCl[2]·2H[2]O was dissolved in 400 mL of ethanol in a beaker covered with aluminum foil pierced with pores. The beaker was placed in a vacuum environment containing 10 g of NH[4]HCO[3] for 24 h at 30 °C. After the reaction was completed, the resulting CNPs were centrifuged at 8000 rpm and washed three times with ethanol before being redispersed in ethanol for further use. Synthesis of CQM NPs CNPs (7 mg·mL^−1, 1 mL) were dispersed in 197 mL of ethanol. The reaction solution was continuously stirred at 800 rpm while 1 mL of quercetin solution (8 mM in ethanol) was added at a controlled rate of 0.01–0.05 mL/s. Under real-time monitoring with a pH meter, the pH was adjusted to 8.0 by dropwise addition of NaOH solution (0.1 M). Subsequently, 1 mL of MnCl[2]·4H[2]O solution (40 mM in ethanol) was introduced at the same rate (0.01–0.05 mL/s). The reaction was adjusted to 8.0 by NaOH solution (0.1 M) again and stirred overnight at room temperature with continuous stirring at 800 rpm. The resulting suspension was then centrifuged at 15,000 rpm for 20 min, followed by repeated washing with deionized water until the supernatant became clear. The resulting CQM NPs were redispersed in deionized water and stored at −4 °C for future use. Elemental mapping of CQM NPs using energy-dispersive X-ray spectroscopy (EDS) coupled with transmission electron microscopy (TEM) Elemental mapping of CQM NPs was conducted using energy-dispersive X-ray spectroscopy (EDS) coupled with TEM. The CQM NPs were ultrasonically dispersed in absolute ethanol (5 min, 40 kHz) and deposited onto copper grids. After vacuum drying at 25 °C overnight, the grids were loaded into a field emission transmission electron microscope (FETEM) equipped with an energy-dispersive X-ray spectrometer (EDS) detector to visualize the elemental distribution of C, O, Ca, and Mn in CQM NPs. Prior to mapping, the microscope was calibrated using a copper standard (Cu Kα at 8.04 keV) and operated at 200 kV with probe current stabilized at 0.5 nA to balance spatial resolution and elemental sensitivity. HAADF-STEM imaging initially identified individual CQM NPs, whereafter EDS spectral maps were acquired. For quantitative analysis, characteristic X-ray lines were selected as follows: C Kα (277 eV), O Kα (525 eV), Ca Lα (341 eV), and Mn Lα (637 eV) with respective energy windows set to ± 10 eV to exclude overlapping signals. The final elemental distribution profiles were displayed in ‌false color‌. Post-processing included background subtraction using Cliff-Lorimer correction and quantitative elemental distribution analysis via Bruker Esprit software (v2.1). The overlapping signals between Ca Lα and Mn Lα peaks were deconvoluted through Gaussian fitting, and the atomic percentages were normalized to the sum of detected elements excluding copper from the grid. Measurements of Quercetin in CQM NPs Ethanol solutions containing quercetin at different concentrations were prepared, and their absorbance at 256 nm was measured. A standard curve of absorbance versus concentration was established using these measurements. The content of quercetin in CQM NPs was then calculated based on this standard curve. Measurements of manganese and calcium contents in CQM NPs by inductively coupled plasma mass spectrometer (ICP-MS) 200 µL of CQM NPs solution (1 mg·mL^−1) was fully dissociated in 5 mL of concentrated nitric acid (HNO[3]) at 70 °C for 24 h. Subsequently, the mixture solution was volatilized at 150 °C to remove HNO[3]. Finally, 5 mL of deionized water was added, and the manganese and calcium contents were determined by ICP-MS. The peroxidase (POD)-like activity of CQM NPs under different pH conditions The POD-like activity of CQM NPs under different pH conditions was evaluated using TMB as the substrate in the presence of H[2]O[2]. In detail, 10 µL of CQM NPs solution (0.8 mg·mL^−1) was added into 3 mL of 0.2 M buffer solution (pH 7.4, 6.5, and 5.5) containing 100 µL of TMB (2 mg·mL^−1 in dimethyl sulfoxide) and 900 µL of H[2]O[2] (3%). The ultraviolet-visible (UV-vis) absorption spectra of oxidized TMB (oxTMB) was recorded by UV-vis spectrophotometer. The glutathione peroxidase (GPx)-like activity of CQM NPs To evaluate GPx-like activity, CQM NPs (C[Mn]: 0.1, 0.2, 0.3, 0.4, and 0.5 mM) were respectively added to GSH solutions (10 mM, pH 6.5), followed by the addition of DTNB (6 mM, 100 µL). Then the reaction solution was diluted 100 times, and the UV-vis absorption spectra of different reaction solutions was recorded by UV-vis spectrophotometer. Cell lines and culture 4T1 and RAW264.7 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). 4T1-Luc cells were purchased from Xiamen Immocell Biotechnology Co., Ltd. (Xiamen, China). 4T1 and 4T1-Luc cells were cultured in RPMI Medium 1640 basic (1X) supplemented with 10% FBS and 1% Pen-Strep, with the exception that 4T1-Luc cells were subjected to additional puromycin (0.5 µg·mL^−1) for cell screening. RAW264.7 cells were cultured in DMEM containing 10% FBS and 1% Pen-Strep. All cells were cultured under stable conditions of 37 °C and 5% CO[2]. Animal model Female Balb/c mice (5–6 weeks of age) were purchased from Hubei Beiente Biotechnology Co., Ltd. and fed under specific pathogen-free (SPF) conditions. To establish pulmonary metastasis model, 1 × 10^6 4T1-Luc cells were injected intravenously into the tail vein of each mouse. To confirm pulmonary metastases, mice were intraperitoneally injected with D-luciferin potassium salt solution (15 mg·mL^−1, 200 µL) 10 min before bioluminescence imaging (BLI). After in vivo imaging or therapeutic interventions, the metastatic lung tumors were further visualized and quantitatively analyzed by hematoxylin and eosin (H&E) staining. The disintegration behavior of CNPs and CQM NPs under different pH conditions CNPs and CQM NPs were dispersed in buffer solutions at various pH values (7.4, 6.5, and 5.5) and incubated at 37 °C under continuous shaking (150 rpm). Morphological and size changes of CNPs and CQM NPs were analyzed by transmission electron microscopy (TEM) at predetermined time points. The disintegration behavior of QM and CQM NPs in GSH solutions at various concentrations QM and CQM NPs were individually incubated with GSH solutions at concentrations of 0, 1.25, and 2.50 mM at 37 °C under continuous shaking (150 rpm). Morphological and size changes of QM and CQM NPs were analyzed by TEM at predetermined time points. Mn ion release profiles of CQM NPs under varying pH or GSH conditions CQM NPs were incubated with buffer solutions at varying pH values (5.5 and 6.5) or GSH solution (2.50 mM). At specific time points, 1 mL of the solution was extracted for subsequent analysis, and 1 mL of fresh buffer solution was added. The concentration of Mn ions in the extracted solution was determined by ICP-MS. Bio-transmission electron microscopy (Bio-TEM) of CQM NPs in 4T1 cells 4T1 cells were incubated with CQM NPs (C[Mn] = 0.1 mM) for 3 h. After incubation, 4T1 cells were washed three times with PBS, followed by being harvested via gentle scraping in fresh PBS containing protease inhibitors. The pelleted cells were fixed with 2.5% glutaraldehyde for 24 h at 4 °C. Finally, the as-prepared samples were observed by Bio-TEM. The quantification of CQM NPs uptake by RAW264.7 cells RAW264.7 cells were seeded in 6-well plates and cultured overnight. The next day, the medium was replaced with 1 mL of fresh DMEM containing CQM NPs, polystyrene@quercetin-Mn nanoparticles (PQM NPs), or CaCO[3]@dopamine-Mn nanoparticles (CDM NPs) (C[Mn] = 0.1 mM). Cells were incubated with NPs for varying durations (0, 10, 20, 30, 60, 120, and 180 min). At each time point, cells were washed three times with PBS and harvested for manganese content analysis by ICP-MS. Cytotoxicity assessment 4T1 cells and RAW264.7 cells were separately seeded into two 96-well cell culture plates (5 × 10^3 cells per well) and cultured overnight. The next day, 4T1 cells and RAW264.7 cells were incubated with varying concentrations of CQM NPs for 24 h. After incubation, the cell viability was measured using a CCK-8 assay kit. Assessments of RAW264.7 cells polarization RAW264.7 cells were seeded in 6-well plates (5 × 10^5 cells per well) and cultured overnight. Next day, RAW264.7 cells were treated with CQM NPs, PQM NPs, or CDM NPs (C[Mn] = 0.1 mM). At specified time points (0, 1, 3, and 24 h), the cells were washed, collected, and subsequently stained with Anti-Mo F4/80 (eFluor^™ 450), Anti-Mo CD86 (PE-Cyanine7), and Anti-Mo CD206 (APC) for flow cytometry (FCM) analysis. FCM analysis was performed in triplicate for each sample. Tumor-targeting delivery of CQM NPs via macrophage hitchhiking RAW264.7 cells and 4T1 cells were separately plated into 6-well plates and cultured overnight. Next day, RAW264.7 cells and 4T1 cells were pre-stained with Dulbecco’s phosphate-buffered saline (DPBS) containing CellTrace^™ Violet (5µM) and CellTrace^™ CFSE (5µM) at 37 °C away from light for 30 min, respectively. Then, RAW264.7 cells were incubated with Sulfo-Cyanine 5@CQM NPs (Cy5@CQM NPs) (C[Mn] = 0.1 mM) for 1 h. After aspirating the supernatant, RAW264.7 cells were washed three times with DPBS and replenished with complete medium. Cells were harvested using a cell scraper, gently pipetted to homogeneity, then co-cultured with pre-stained 4T1 cells in 6-well plates for defined durations (0.5, 1, 2, 3, and 4 h). After coincubation, cells underwent DPBS rinsing followed by 15-min fixation with 4% paraformaldehyde at room temperature. Washed coverslips were mounted on glass slides for confocal imaging. RAW264.7 cells and 4T1 cells were respectively plated into 6-well plates and cultured overnight. RAW264.7 cells were then divided into two groups: one group was left untreated, and the other group was pre-treated with 2-deoxy-D-glucose (2-DG) (8 mM) for 12 h to suppress phagocytosis. Both groups were incubated with Cy5@CQM NPs for 1 h, followed by triple PBS washing to remove uninternalized Cy5@CQM NPs. The harvested RAW 264.7 cells were co-cultured with 4T1 cells for specified durations (0, 0.5, 1, 2, 3, and 6 h). After carefully removing RAW264.7 cells, the remaining 4T1 cells were fixed and stained with Actin-Tracker Green (cytoskeleton) and DAPI (nucleus) for confocal laser scanning microscopy (CLSM) imaging. Lysosomal escape assay 4T1 cells were co-cultured with RAW264.7 cells pre-loaded with Cy5@CQM NPs through 1-hour incubation. After co-incubation for varying durations (1, 2, 3, and 4 h), RAW264.7 cells were removed carefully. The remaining 4T1 cells were washed three times with PBS, followed by staining with Lyso-Tracker Green probe (50 nM, 37 °C for 15 min) and DAPI (5 min) for CLSM imaging. Intracellular pH measurements 4T1 cells were seeded into confocal dishes at a density of 5 × 10^4 cells per dish and cultured overnight. Next day, 4T1 cells were incubated with CQM NPs (C[Mn] = 0.1 mM) for varying durations (1, 2, 3, 4, and 5 h) prior to being stained with BCECF-AM pH-sensitive fluorescent probe (37 °C, 30 min). Finally, the intracellular pH level of 4T1 cells was observed by CLSM. Intracellular DNA damage assay 4T1 cells were seeded into 6-well plate (1 × 10^5 cells per well) and cultured overnight. Next day, 4T1 cells were incubated with CQM NPs (C[Mn] = 0.1 mM) for different durations (0, 2, 4, 6, and 8 h) prior to γH2AX immunofluorescence staining. Finally, the intracellular DNA damage level of the 4T1 cells was observed by CLSM. MRI contrast performance of CQM NPs in vitro The longitudinal relaxation time (T[1]) of CQM NPs samples under different pH conditions (7.4, 6.5, and 5.5) or GSH conditions (1.25, 2.50, and 5.00 mM) was measured by a 7 T MRI scanner. The specific parameters were set as follows: RARE T[1] + T[2] map sequence, matrix size = 256 × 256, slice thickness = 1 mm. The r[1] relaxivity was determined through linear fitting of 1/T[1] as a function of Mn concentration. Additionally, the T[1]-weighted MR images of the corresponding CQM NPs samples were captured on a 7 T MRI scanner. The parameters for these images were set as follows: RARE-T[1] sequence, Repetition Time‌ (TR) = 50.0 ms. Echo Time (TE) = 9.0 ms, rare factor = 1, number of average = 8, slice thickness = 1.0 mm, and matrix size = 256 × 256. Biosafety test of CQM NPs To assess the biosafety of CQM NPs, healthy Balb/c mice (female, 6 weeks old) were randomly divided into three groups. Mice of one group were left without any treatment. The mice of the other two groups were treated with CQM NPs (C[Mn] = 0.5 mM) via intratracheal (i.t.) administration and intravenous (i.v.) injection, respectively. At 24 h post-treatment, these mice were euthanized, and blood samples were collected. Some of the blood samples were stabilized with an anti-clotting agent for blood routine analysis, while the rest were centrifuged at 3000 rpm for 10 min to obtain serum for blood biochemical analysis. In vivo MR imaging of pulmonary metastases After successfully establishing the pulmonary metastasis model, Balb/c mice with lung metastases were divided into three groups and intravenously injected with 200 µL of CQM NPs solution, PQM NPs solution or CDM NPs solution (C[Mn] = 0.5 mM). They were then scanned by a 7 T MRI scanner at 2 h post-injection. The acquisition parameters of T[1]-weighted MRI images were as follows: RARE-T[1] sequence, TR = 400 ms, TE = 10 ms, number of average = 4, rare factor = 1, matrix size = 256 × 256, FOV = 3 cm × 2.5 cm, slice thickness = 0.8 mm without gap, and acquisition time = 10 min 14 s 400 ms. Spatial transcriptomic analysis of dynamic macrophage polarization profiles (1) Spatially barcoded nuclei preparation. Fresh-frozen mouse lungs were cryosectioned at 10–20 μm thickness using a Leica cryostat at −20 °C. Sections were processed with the SeekSpace Single Cell Spatial Transcriptome‑seq Kit ([77]K02501‑08) to release nuclei and incorporate spatial barcodes. After two rounds of gentle washing and filtration through a 40 μm strainer, nuclei were stained with AO/PI and counted on a Seekgene Fluorescence Cell Analyzer (M002B). The final nuclei suspension was kept on ice until library preparation. (2) Library construction and sequencing. We generated both single‑cell RNA and spatial‑barcode libraries following the manufacturer’ s instructions (SeekSpace Kit [78]K02501‑08). Libraries were purified with VAHTS DNA Clean Beads (Vazyme N411), quantified on a Qubit Fluorometer (Thermo Fisher Q33226), and quality‑checked on a Bio‑Fragment Analyzer (Bioptic Qsep400). Paired‑end (2 × 150 bp) sequencing was performed on an Illumina NovaSeq6000. (3) Data processing. We trimmed adapters and filtered low‑quality bases with Fastp (v0.20.1), and assessed read quality metrics. Clean reads were processed by the SeekSpace Tool (v1.0.0) to assign spatial barcodes and evaluate library complexity. We aligned reads to the mouse reference genome (mm10, refdata‑gex‑mm10‑2020‑A) using STAR (v2.7.3a). UMI counts were extracted and imported into Seurat (v4.0.5) as a spatial object, retaining spatial coordinates in the reductions slot. (4) Clustering and annotation. We normalized gene counts by log‑normalization, identified 2,000 highly variable genes with FindVariableFeatures, and scaled all genes for dimensionality reduction. Principal component analysis (PCA) used the top 30 PCs for clustering via a graph‑based approach (FindClusters). We visualized clusters with t‑distributed stochastic neighbor embedding (t‑SNE) and detected marker genes using FindAllMarkers (min.pct = 0.25). We annotated cell types with SingleR (v1.6.1) against standard reference datasets and custom SeekGene references.