Reprogramming Dysfunctional Dendritic Cells by a Versatile Catalytic Dual Oxide Antigen-Captured Nanosponge for Remotely Enhancing Lung Metastasis Immunotherapy

Abstract

Dendritic cells (DCs) play a crucial role in initiating antitumor immune responses. However, in the tumor environment, dendritic cells often exhibit impaired antigen presentation and adopt an immunosuppressive phenotype, which hinders their function and reduces their ability to efficiently present antigens. Here, a dual catalytic oxide nanosponge (DON) doubling as a remotely boosted catalyst and an inducer of programming DCs to program immune therapy is reported. Intravenous delivery of DON enhances tumor accumulation via the marginated target. At the tumor site, DON incorporates cerium oxide nanozyme (CeO₂)-coated iron oxide nanocubes as a peroxide mimicry in cancer cells, promoting sustained ROS generation and depleting intracellular glutathione, i.e., chemodynamic therapy (CDT). Upon high-frequency magnetic field (HFMF) irradiation, CDT accelerates the decomposition of H₂O₂ and the subsequent production of more reactive oxygen species, known as Kelvin's force laws, which promote the cycle between Fe³⁺/Fe²⁺ and Ce³⁺/Ce⁴⁺ in a sustainable active surface. HFMF-boosted catalytic DON promotes tumors to release tumor-associated antigens, including neoantigens and damage-associated molecular patterns. Then, the porous DON acts as an antigen transporter to deliver autologous tumor-associated antigens to program DCs, resulting in sustained immune stimulation. Catalytic DON combined with the immune checkpoint inhibitor (anti-PD1) in lung metastases suppresses tumors and improves survival over 40 days.

Keywords: T cell infiltration; antigen capture; immunotherapy; lung metastasis; nanozymes.

Figure 1

Schematic illustration of dual catalytic oxide nanosponge (DON) a dual catalyst and an inducer of T cell infiltration to program immune therapy. (a) DON consisting of cerium oxide nanozyme (CeO₂), iron based-MOF, and iron oxide nanocubes (IO) as agents to promote the cycle between Fe³⁺/Fe²⁺ and Ce³+/Ce⁴⁺ as well as to decompose H₂O₂ upon a high-frequency magnetic fields (HFMF) irradiation. (b) The hyperthermia and chemodynamic therapy (CDT) via redox reactions promoted cancer cell apoptosis and release TAAs. Porous DON enhances the retention of antigen release, facilitating sustained immune stimulation and inhibiting tumor metastasis.

Figure 2

Synthesis and characterizations of PB, PC, and DON. (a) Schematic representation showing the synthesis of DON for HFMF-enhanced catalytic therapy. SEM and TEM images of (b,c) PB, (d,e) PC, (f,g) PBA, and (h,i) DON. (j,k) Element mapping analysis of PBA and DON.

Figure 3

Physicochemical characterization of PB, PC, PBA, and DON. (a) Size distributions and (b) surface charges of PB, PC, PBA, and DON. (c) Thermogravimetric analysis curves and (d) field-dependent magnetization curves of PB, PC, PBA, and DON. (e) XPS spectrum of C 1s, O 1s, and Fe 2p of PBA and DON. (f) XRD spectrum of PB, PC, PBA, and DON. (g) BET analysis of N₂ adsorption–desorption isotherms of DON.

Figure 4

(a) Degradation patterns of rhodamine B (RhB) under an HFMF with the magnetic strengths of 0, 20, and 40 mT. RhB concentration: 10 mg/L; H₂O₂ concentration: 5 mM; pH: 7.4. Statistical significance was assessed using one-way ANOVA. Data represent mean ± SEM, n = 5. (b) Relationship between magnetic strength and reaction kinetic rate. Statistical significance was assessed using one-way ANOVA. Data represent mean ± SEM, n = 5. (c) Comparing the observed reaction kinetic rates (kobs) in the presence and absence of an HFMF across different pH values, while maintaining a constant dosage of 5 mL of H₂O₂. Statistical significance was assessed using one-way ANOVA. Data represent mean ± SEM, n = 5. (d) Monitoring the change in reaction kinetic rate upon activation and deactivation of the HFMF. Statistical significance was assessed using one-way ANOVA. Data represent mean ± SEM, n = 6. (e) The peroxidase mimic catalytic activity of PBA and DON. UV–vis absorption spectra of catalyzed oxidation of OPD (ox OPD) in an acid environment. (f) The GSH depletion of PBA and DON. (g) Mechanism of peroxidase mimic catalytic process and GSH depletion test of nanoparticles. (h) CLSM images of B16F10 cells incubated with PBA and DON. Blue, green and red represents nucleus stained with DAPI, cytoskeleton with F-Actin, and particles stained with QD, respectively. (i) CLSM images of B16F10 cells incubated with DON to evaluate the lysosomal escape effect of DON after HFMF irradiation. (j) Flow cytometry analysis of PBA and DON.

Figure 5

(a,b) Cell viability of B16F10 treated with PB, PC, PBA, and DON at various concentrations with and without subjecting to HFMF. Statistical significance was assessed using one-way ANOVA. Data represent mean ± SEM, n = 6. *p < 0.05, **p < 0.01, ***p < 0.005. (c) CLSM images of in vitro assessments of the intracellular hydroxyl radical ROS generation and anti-CRT via the catalysis of PBA and DON nanoparticles. (d) CLSM images of autophagy activation through the LC3B protein expression of B16F10 cells incubated with PBA, DON, PBA+HFMF, and DON+HFMF. Purple fluorescence indicates LC3B expression, serving as a measure of autophagosome abundance. (e) Quantitative levels of ROS and anti-CRT are expressed as the percentage of hydroxyl identified in control cells. Statistical significance was assessed using one-way ANOVA. Data represent mean ± SEM, n = 6. **p < 0.01, ****p < 0.001. (f) CLSM images of DC 2.4 cells treated with antigen@DON for 24 h.

Figure 6

In vivo study. (a) Animal treatment schedule. (b) In vivo IVIS organ biodistribution images of control, PBA, DON, and DON+HFMF-treated mice at 24 h post-treatment. (c) CLSM images of mice bearing GFP-B16F10 lung metastases after treatment with PBA, DON, and DON+HFMF, respectively, after 24 h of treatment. (d) The number of foci in dissected lung metastases treated with PBS (control), PBA, PBA+HFMF, DON, and DON+HFMF intravenously at 14 days postinjection was quantified using ImageJ. Statistical significance was assessed using one-way ANOVA. Data represent mean ± SEM, n = 5. ****p < 0.001. (e) Biochemical indices of liver and kidney after 24 h of treatment. Statistical significance was assessed using one-way ANOVA. Data represent mean ± SEM, n = 3. (f) Survival patterns of animals with lung metastases following treatment with PBS (control), PBA+HFMF, DON, DON+HFMF, and DON+HFMF+αPD-1 (n = 6).

Figure 7

T cell infiltration in metastasis. (a) CLSM images of the lungs 24 h postintravenous injection of particles, along with measurements of CD4⁺ and CD8⁺ T cells. (b) Flow cytometry gating strategy for T cells. The lymphocyte population was selected based on SSC and FSC properties. Fluorescence gating for CD3, CD45, CD4, and CD8 was performed using fluorescence minus one (FMO) controls and single-staining compensation. The CD3⁺ population within the lymphocytes represents mature T lymphocytes, and further gating on CD45⁺ cells identify the cytotoxic T and T-helper cells. (c) Quantification of in vivo CD4⁺ and CD8⁺ T cells via flow cytometry analysis 24 h after control, PBA, DON, and DON+HFMF treatments.

Figure 8

In vivo study of particle treatment in mice bearing B16F10 lung metastases. (a) The scheme of mechanism by which DON captures antigen and delivers it to antigen-presenting cells (APCs) such as dendritic cells (DCs). DCs then transport the antigen to lymph nodes, where they activate the immune system to induce a T-cell immune response. (b) Comparison of the percentage of antigen captured by PBA and DON, respectively. (c) CLSM image of dissected lymph node tissue 24 after injection. White, green, and red fluorescence represent nuclei stained with DAPI, DCs labeled with CD86, and nanoparticles labeled with QDs, respectively. (d) In vivo flow cytometry analysis of LN tissue dissected after 24 h post-treatment by PBA, DON, and DON+HFMF.

Figure 9

Antigen release after PC, DON, DON+HFMF, and chemodrug-treated cells.