Amelioration of systemic antitumor immune responses in cocktail therapy by immunomodulatory nanozymes.

Nanozymes that mimic natural enzyme-like activities have gradually emerged in cancer therapy. To overcome the bottlenecks of single-mode nanozymes, including "off-target" toxicity and ineffectiveness toward metastatic cancers, we designed magnetic nanoparticle-based multifunctional visualized immunomodulatory nanozymes. Besides the partial initiation of the prime immune response by intrinsic immunogenicity, as a smart drug delivery system with a temperature- and pH-sensitive dual response to the tumor microenvironment, these nanozymes released immune agonists to boost enhanced systemic immune response, eventually ameliorating the cancer immune microenvironment through many aspects: activating dendritic cells, improving the function of CD8+ T cells, and decreasing the population of myeloid-derived suppressor cells, which inhibited both primary and metastatic cancers. Mechanistically, these nanozymes regulated the reactive oxygen species-related Akt signaling pathway and consequently activated cell apoptosis-related signaling pathways, which provided a deeper understanding of the synergistic mechanism of multifunctional nanozymes. Our findings offer a promising imaging-guided cocktail therapy strategy through immunomodulatory nanozymes.

INTRODUCTION

Nanozymes, defined as “nanomaterials with enzyme-like characteristics,” can break through the limitations of low stability, high cost, and difficult storage of natural enzymes and therefore have been gradually applied in various fields, from biomedical sensing and theranostics to environmental protection (). In 2007, Fe3O4 nanoparticles (NPs) were first reported to have peroxidase-like activity (), which proved that magnetic NPs were powerful candidates for the preparation of nanozymes. Inspired by the pioneering work, magnetic NP–based peroxidase mimics were widely explored and studied, such as Fe3O4 (magnetite) (), Fe2O3 (hematite) (), and doped ferrites (). Subsequently, tailored to the specific characteristics of the tumor microenvironment (TME) (), including the excessive production of acid () and hydrogen peroxide (H2O2) overproduction () and low catalase activity (), the magnetic NPs with pH-dependent catalase-like activity were designed to produce reactive oxygen species (ROS) through Fenton reactions, resulting in the elimination of cancer cells (). This strategy has been extensively applied in chemodynamic therapy (CDT) of cancer (), which has been gradually booming into a new field called catalytic nanomedicine (). However, such a single-treatment mode mediated by nanozymes in vivo is facing inevitable challenges. Nanozymes need to be precisely controlled to target tumor sites for effective side effect avoidance. For advanced cancers, nanozymes also need to be devised to inhibit both primary and metastatic tumors through activating the acceptable systemic response. Hence, coordinated with the unique physical and chemical properties of magnetic NPs, the design of multifunctional nanozymes is of great significance for cancer therapy optimization.

Cancer immunotherapy is a systemic response to dynamically activate immune cells with the aim of attacking cancer cells or preventing them from escaping, involving monoclonal antibody therapy (), cytokine therapy (), cancer vaccines (), immune checkpoint blockade (ICB) (), and adoptive cell transfer (ACT) (). Conventional cancer immunotherapy has achieved admirable therapeutic effects, while some bottlenecks still exist in preclinical and clinical trials. For instance, ICB-derived adverse events bring about immune balance breaking or immune response silencing (); some cancer vaccines are prone to immunosuppression due to the potential instability (, ); ACTs suffer from both the unpredictable cytokine storm and economic pressure produced by engineering T cells (). Conforming to favorable trends, magnetic NPs have made a breakthrough in cancer immunotherapy owing to their stable structure, ultrasmall size, controllable surface modification potential, and unique biological characteristics (–). For instance, Fe3O4 could enhance the antigen presentation function of dendritic cells (DCs) (–); Fe2O3 could regulate the polarization of tumor-associated macrophages (TAMs) (). However, research on magnetic NPs in the field of cancer immunotherapy is still in its infancy, and the effect of some unique characteristics of magnetic NPs such as immunogenicity and enzyme-like activity needs to be further investigated in cancer immunotherapy. Hence, the combination of nanozyme technology and cancer immunotherapy can not only break through the bottleneck of nanozymes with the single mode but also further deepen the understanding of nanotechnology in cancer immunotherapy.

As described above, here, we designed a kind of immunomodulatory nanozyme based on Cu@Fe2C@mSiO2-R848-ICG-AS1411 for real-time visualization and synergistic cancer therapy. As the multifunctional candidate, iron carbide NPs are a type of biocompatible NP with excellent magnetic properties, photothermal conversion properties, and peroxidase-like activity, which can act as a magnetic resonance imaging (MRI) contrast agent (), a photothermal therapy (PTT) (, ) and CDT agent (). Copper ions can further enhance the photothermal conversion performance and peroxidase-like activity of iron carbide NPs for optimizing the therapeutic effect (). Meanwhile, synergistic cancer therapy can provide abundant cancer cell fragments as antigens and subsequently activate the immune system through antigen presentation for cancer immunotherapy. To further improve the effect of cancer immunotherapy, mesoporous silica is selected as a smart drug delivery system because of its good biocompatibility in vivo and affluent mesostructure (), ultimately achieving the effective loading and pH/temperature-controlled release of immune agonists (R848) () in cooperation with phase change materials [polyethylene glycol (PEG)/lauric acid (LA)]. Indocyanine green (ICG) can achieve near-infrared region (NIR-II) fluorescence imaging of deep tumors with a high spatial resolution (). In addition, nucleolin-specific aptamer AS1411 is attached to the surface of the immunomodulatory nanozymes to implement active targeting to tumor sites. In conclusion, we provided a strategy based on Cu@Fe2C@mSiO2-R848-ICG-AS1411 to achieve the synergistic treatment of PTT/CDT/immunotherapy guided by MRI/NIR-II dual-mode imaging (Fig. 1A). We mechanistically elucidated the enhanced antitumor synergistic effect mediated by this immunomodulatory nanozyme system, including the cooperativeness of immune responses induced by intrinsic immunogenicity and loaded immune agonists and the intrinsic regulation of molecular signaling pathway of cancer cells mediated by peroxidase-like activity, which would provide important therapeutic strategies and theoretical basis for clinical medicine (Fig. 1B).

Fig. 1.

Schematic illustration of the synthesis process and mechanism of the antitumor effect of Cu@Fe2C@mSiO2-PEG/LA-R848-ICG-AS1411 nanozymes.

(A) Schematic illustration of the sequential synthesis of Cu@Fe2C@mSiO2-PEG/LA-R848-ICG-AS1411 nanozymes. (B) Schematic illustration of the mechanism of the antitumor effect of Cu@Fe2C@mSiO2-PEG/LA-R848-ICG-AS1411 nanozymes.

RESULTS

Synthesis and characterization of Cu@Fe2C@mSiO2

A schematic illustration of the sequential synthesis of Cu@Fe2C@mSiO2-PEG/LA-R848-ICG-AS1411 is shown in Fig. 1A. First, monodispersed Cu@Fe2C NPs were synthesized by thermal decomposition in the oil phase and were successively integrated into the mesoporous silica NPs (MSNs), designated as Cu@Fe2C@mSiO2 nanospheres (NSs) (Fig. 2A). Immune agonists (R848) were loaded into the mesostructure by vacuum impregnation and sealed with phase change materials (PEG/LA) to prevent leakage. Furthermore, nucleolin-specific aptamer AS1411 and fluorescent molecule ICG were attached to the surface of Cu@Fe2C@mSiO2-PEG/LA-R848 NSs by a chemical bonding reaction. Representative transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) images of the as-synthesized Cu@Fe2C NPs (Fig. 2, B and C) and Cu@Fe2C@mSiO2 NSs (Fig. 2, D and E) were clearly revealed. Cu@Fe2C NPs with the core-shell structure consisted of a core of Cu NPs (~4 nm diameter) with a shell of Fe2C NPs (~20 nm thick); subsequently, the size of the coated mesoporous silica reached ~55 nm in diameter. Cu@Fe2C@mSiO2 NSs dispersed in an aqueous solution showed a larger hydrodynamic diameter of 299.9 nm measured by dynamic light scattering (DLS) (fig. S1). X-ray powder diffraction (XRD) analysis and energy-dispersive spectrometer (EDS) mapping results confirmed again that the phases were Cu@Fe2C and Cu@Fe2C@mSiO2 (figs. S2 and S3). Furthermore, x-ray photoelectron spectroscopy (XPS) results demonstrated that the valence states of copper and iron exerted no obvious change before and after mesoporous silica coating (fig. S4). To verify the rationality of mesostructure as the drug delivery system, the results of Brunauer-Emmett-Teller (BET) measurements indicated that the specific surface area was 230.909 m2 g−1, and the pore average diameter was 4.252 nm (fig. S5), which provided sufficient space for drug loading. The results of ultraviolet-visible (UV-vis) absorption spectra also indicated that mesoporous silicon may not notably affect the absorption peak (fig. S6).

Fig. 2.

Synthesis and characterization of Cu@Fe2C@mSiO2.

(A) Schematic illustration of the sequential synthesis of Cu@Fe2C@mSiO2 NSs. (B) TEM and (C) HRTEM images of Cu@Fe2C NPs. (D) TEM and (E) HRTEM images of Cu@Fe2C@mSiO2 NSs.

ROS generation ability and photothermal killing ability of Cu@Fe2C@mSiO2 in vitro

To explore the effect of Cu@Fe2C@mSiO2 NSs in vitro more objectively, first, the safe concentration of Cu@Fe2C@mSiO2 NSs that influenced the mouse embryonic fibroblast cell line NIH 3T3, the mouse breast cancer cell line 4T1, and the mouse melanoma cell line B16F10 was determined through the cell counting kit-8 (CCK8) assay. The results provided a reasonable concentration of Cu@Fe2C@mSiO2 NSs to incubate different cancer cells, which also proved that Cu@Fe2C@mSiO2 NSs were more sensitive to cancer cells 4T1 and B16F10 (fig. S7). After incubating with the cancer cells 4T1 and B16F10 for 6 hours, Cu@Fe2C@mSiO2 NSs were effectively phagocytized and mainly accumulated in lysosomes as indicated by cellular TEM images (Fig. 3A). Cu@Fe2C@mSiO2 NSs were conjugated with ICG, lysosomes were stained with LysoTracker Green, and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence confocal images also showed that Cu@Fe2C@mSiO2 NSs exhibited good colocalization with lysosomes, which were distributed in the cytoplasm (Fig. 3B). By transferring the absorbed energy into heat, iron carbide NPs have great potential for PTT (, , ). Expectedly, Cu@Fe2C@mSiO2 NSs also exhibited outstanding photothermal conversion performance in a dose- and power-dependent manner (fig. S8). The photothermal killing ability of Cu@Fe2C@mSiO2 NSs on cancer cells was evaluated by double staining of live/dead cells [calcein-AM (calcein acetoxymethyl ester) stained for live cells and propidium iodide (PI) stained for dead cells]. As shown in Fig. 3C, the group of Cu@Fe2C@mSiO2 NSs under the irradiation of an 808-nm laser showed the lowest proportion of live cells and the highest proportion of dead cells compared to the control group, the laser group, and the Cu@Fe2C@mSiO2 NS group. On the other hand, our previous study has proven the ferrous ion release of iron carbide NPs (Fe5C2) in an acidic environment, leading to •OH generation and eventual ROS production (). Here, considering the photothermal conversion properties of Cu@Fe2C@mSiO2 NSs, we selected 2′,7′-dichlorofluorescin diacetate (DCFH-DA) as an intracellular ROS indicator to further focus on the intrinsic ROS production ability of Cu@Fe2C@mSiO2 NSs without or with the 808-nm laser irradiation. As shown in Fig. 3D, two cancer cell lines (4T1 and B16F10) were used to demonstrate the universality of Cu@Fe2C@mSiO2 NSs as nanozymes. Green fluorescence intensity was notably increased in the Cu@Fe2C@mSiO2 NS group compared to the control group and the laser group. Green fluorescence intensity was further enhanced with Cu@Fe2C@mSiO2 NS administration and the 808-nm laser irradiation. It was speculated that the combination of Cu@Fe2C@mSiO2 NSs and the 808-nm laser could produce a more intense stress response on cancer cells, resulting in a more obvious up-regulation of ROS. The results of flow cytometry were also consistent with fluorescence images (Fig. 3E and fig. S9). To clarify the role of copper ion, we further made the simultaneous assessment of the ROS production capacity of Fe2C and Cu@Fe2C in 4T1 and B16F10 cells. Comparatively, the peak of flow cytometry shifted obviously to the right in the Cu@Fe2C group with the comparison of the Fe2C group (fig. S10), implying that copper ion could improve the Fenton reaction for more massive intracellular ROS production. All these results indicated Cu@Fe2C@mSiO2 NSs as the eligible PTT/CDT agents.

Fig. 3.

ROS generation ability and photothermal killing ability of Cu@Fe2C@mSiO2 in vitro.

(A) Bio-TEM images of 4T1 and B16F10 cells incubated with Cu@Fe2C@mSiO2 NSs for 6 hours. The red dashed boxes indicate the location of Cu@Fe2C@mSiO2 NSs. (B) Fluorescent images of 4T1 and B16F10 cells treated with NSs-PEG/LA-ICG for 6 hours. Lysosomes were stained with LysoTracker Green, and nuclei were stained with DAPI. Scale bars, 5 μm. (C) Bright-field and fluorescent images of 4T1 and B16F10 cells stained with calcein-AM (live cells, green fluorescence) and PI (dead cells, purple fluorescence) after treatment with PBS only, laser only, NSs only, and NSs + laser. Scale bars, 50 μm. (D) Bright-field and fluorescent images of 4T1 and B16F10 cells incubated with DCFH-DA after treatment with PBS only, laser only, NSs only, and NSs + laser. Scale bars, 50 μm. (E) Statistical analysis of flow cytometry of 4T1 and B16F10 cells incubated with DCFH-DA after treatment with PBS only, laser only, NSs only, and NSs + laser (n = 3).

Drug delivery ability of Cu@Fe2C@mSiO2 in vitro

Resiquimod (R848), as the Toll-like receptor 7/8 agonist, is an imidazoquinoline-based small-molecule compound, which can regulate various immune cells (, ). R848 can promote the activation and maturation of antigen-presenting cells (APCs) and subsequently induce the secretion of proinflammatory cytokines, type I interferons (IFNs), and chemokines (, ). In addition to the immunostimulatory function, R848 can also modulate immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) and M2-like macrophages. R848 can transform MDSCs into APCs such as DCs and macrophages and polarize TAMs from the M2-like phenotype into the M1-like phenotype (, ). Hence, we chose R848 as the immunostimulant to activate the systemic immune response. An ideal drug delivery system (, ) is usually provided with good biocompatibility and biodegradability (), controlled drug loading and release capacity (), and accurate targeting ability (). MSNs are regarded as one of the most favorable drug delivery candidates because of their stable biosafety in vitro and in vivo (, ). To further optimize the controllability of the MSN-based drug delivery system, we introduced temperature-sensitive phase change materials (PEG/LA), while using the weak acid environment of TME to construct dual-response (temperature and pH) smart drug delivery cargos. Taking the normal body temperature of mice (37°C) into account, we designed and adjusted the phase change temperature point of PEG/LA to 38.6°C by changing the proportion of PEG1000, PEG2000, and LA (Fig. 4A and fig. S11). The results of thermogravimetric analysis indicated that Cu@Fe2C@mSiO2 NSs had good heat resistance. When the temperature was above 200°C, the mass of Cu@Fe2C@mSiO2 NSs only decreased slightly, while the mass of other materials including PEG1000, PEG2000, LA, and R848 was substantially reduced. On the other hand, within the reasonable temperature range of PTT in vivo (below 60°C), all these materials maintained their original mass, ensuring stability and effectiveness during the treatment process in vivo (Fig. 4B). As the strategy indicated, R848 was loaded to full advantage into the mesostructure via vacuum impregnation and sealed with PEG/LA, with no change in the morphology of Cu@Fe2C@mSiO2 NSs (Fig. 4C). To further explore the effect of the acidic environment on the degradation of Cu@Fe2C@mSiO2 NSs for effective drug release, we designed the conditions with three different pH values (pH 7.4, 6.5, and 5.4) to simulate the normal physiological environment and TME. As shown in Fig. 4D, Cu@Fe2C@mSiO2 NSs still maintained complete morphology in the normal physiological environment (pH 7.4) even on day 9 but were gradually degraded with the extension of time in the weak acid environment (pH 6.5 and 5.4). These results indicated that the release of R848 could be controlled via acid stimulation of TME only when Cu@Fe2C@mSiO2 NSs reached the tumor sites, while the sustained stability of Cu@Fe2C@mSiO2 NSs under physiological conditions guaranteed the biosafety of drugs in vivo (Fig. 4E). We evaluated the effect of temperature and pH on the release efficiency of R848 after 24-, 48-, and 72-hour treatment through UV-vis absorption spectra (fig. S12). At 25° or 37°C, the release efficiency of R848 increased slightly at pH 5.4 compared to that at pH 7.4. At 45°C, the release efficiency of R848 was notably improved at both pH 5.4 and pH 7.4. The highest release ratio reached 85.06% [45°C (pH 5.4) for 72 hours] (Fig. 4F). It could be seen that the controlled release of R848 was more sensitive to temperature than to pH value, with the speculation that PEG/LA converted from the solid state to the liquid state at a temperature higher than the phase change temperature point, leading to the release of R848 from the mesostructure without sealing. All these results demonstrated that both temperature and pH value as a dual response achieved an efficient and controllable drug release.

Fig. 4.

Drug delivery ability of Cu@Fe2C@mSiO2 in vitro.

(A) Differential scanning calorimetry analysis of PEG1000/2000-LA, R848, Cu@Fe2C@mSiO2, and Cu@Fe2C@mSiO2-PEG/LA-R848. (B) Thermogravimetric analysis of PEG1000, PEG2000, LA, R848, Cu@Fe2C@mSiO2, and Cu@Fe2C@mSiO2-PEG/LA-R848. (C) TEM images of Cu@Fe2C@mSiO2 and Cu@Fe2C@mSiO2-PEG/LA-R848. (D) TEM images of Cu@Fe2C@mSiO2 after degradation with different pH values at different time points. Scale bars, 50 nm. (E) Schematic illustration of the degradation and drug release of the nanozymes with the drug delivery system based on Cu@Fe2C@mSiO2-PEG/LA-R848-ICG-AS1411. (F) R848 release efficiency curve of Cu@Fe2C@mSiO2-PEG/LA-R848 at different pH values and different temperatures.

Imaging and biosafety in vivo

ICG as an NIR-II fluorescence imaging agent was attached to the surface of Cu@Fe2C@mSiO2 NSs for imaging of deep tumors. Nucleolin has been proven to be commonly overexpressed in various kinds of cancer cells, including breast cancer cells () and melanoma cancer cells (). To better achieve active targeting of tumor sites, Cu@Fe2C@mSiO2 NSs were designed to be attached with the nucleolin-specific aptamer AS1411 that was specifically bound to nucleolin (Fig. 5A). In preclinical (, ) and clinical () trials, AS1411 has also been used for targeted cancer therapy. As expected, the results of fluorescence confocal images confirmed the excellent binding affinity of AS1411 targeting to 4T1 and B16F10 cells. AS1411 was conjugated to a fluorescein isothiocyanate (FITC) fluorescent molecule and was incubated with cancer cells for 24 hours. The colocalization of fluorescence indicated that most of AS1411 was bound to nucleolin that was usually located in the nucleus (Fig. 5B and movies S1 for 4T1 cells and S2 for B16F10 cells). In detail, the UV-vis absorption spectra (fig. S13) and fluorescence spectra (fig. S14) proved that Cu@Fe2C@mSiO2-PEG/LA-ICG (simplified as NSs-PEG/LA-ICG) and Cu@Fe2C@mSiO2-PEG/LA-ICG-AS1411 (simplified as NSs-PEG/LA-ICG-AS1411) had the characteristic peak of ICG, which suggested the attachment between ICG and NSs-PEG/LA. The formation of NSs-PEG/LA-ICG and NSs-PEG/LA-ICG-AS1411 nanozymes via chemical bonding reaction was further confirmed by a Fourier transform infrared spectrometer (fig. S15). With the sequential linking of ICG and AS1411 on the surface of Cu@Fe2C@mSiO2-PEG/LA (simplified as NSs-PEG/LA), the zeta potential change in NSs-PEG/LA, NSs-PEG/LA-ICG, and NSs-PEG/LA-ICG-AS1411 successively exerted an upward trend (fig. S16). All these various methods confirmed that ICG and AS1411 were successfully linked to the surface of NSs-PEG/LA. NSs-PEG/LA-ICG and NSs-PEG/LA-ICG-AS1411 were coincubated with 4T1 cells. The results of fluorescence imaging indicated that NSs-PEG/LA-ICG-AS1411 was more effectively enriched in 4T1 cells than NSs-PEG/LA-ICG (fig. S17). Subsequently, both NSs-PEG/LA and NSs-PEG/LA-AS1411 were linked to an FITC fluorescent molecule, and a cancer cell phagocytosis experiment measured by flow cytometry demonstrated that more NSs-PEG/LA-AS1411 were absorbed by 4T1 cells than NSs-PEG/LA (fig. S18). These results proved the good targeting performance of AS1411 in vitro. NIR-II fluorescence imaging was carried out with an excitation wavelength of 808 nm to track the in vivo behaviors of NSs-PEG/LA-ICG and NSs-PEG/LA-ICG-AS1411 after intravenous injection in 4T1 cell–bearing mice. The tumor sites of the NSs-PEG/LA-ICG-AS1411 group showed a strong fluorescent signal 12 hours after injection, which was maintained until 48 hours postinjection. In contrast, no obvious fluorescent signal appeared at the tumor sites for NSs-PEG/LA-ICG even 48 hours after injection. Moreover, the fluorescent signal of main tissues (heart, liver, spleen, lung, and kidney) was also detected ex vivo after intravenous injection for 48 hours, and the liver showed the most apparent fluorescent signal (Fig. 5C). Cu@Fe2C@mSiO2 NSs also had the potential to be agents for T2-weighted MRI, with an r2 value of 57.5 mM−1 s−1 in an aqueous solution (Fig. 5D and fig. S19). The results of Fig. 5E clearly indicated the enhancement of the negative signal at the tumor sites of mice treated with NSs-PEG/LA-AS1411 for 12, 24, and 48 hours postinjection. Conversely, the dropping signal change in the NSs-PEG/LA group was not evident even 48 hours after injection. Both MRI and fluorescence imaging results demonstrated that the accumulation of NSs-PEG/LA-AS1411 at tumor sites was more than that of NSs-PEG/LA, revealing the effectiveness of active targeting of AS1411. In addition, we also systematically evaluated the biosafety, tissue distribution, and metabolic pathways of NSs-PEG/LA and NSs-PEG/LA-AS1411 in vivo. NSs-PEG/LA and NSs-PEG/LA-AS1411 were visualized in vivo through NIR-II fluorescence imaging. The results showed that most NSs mainly accumulated in the liver 1 day postinjection; subsequently, the fluorescent signal of the liver gradually weakened from 3 to 14 days after intravenous injection, indicating that NSs were excreted from the body (Fig. 5F). To further explore the specific metabolic pathway of NSs, we collected the urine and feces at different time points after intravenous injection of NSs to quantitatively detect the content of copper by inductively coupled plasma optical emission spectrometry. The results showed that with the extension of time, the content of copper in urine and feces decreased gradually; in particular, ~90% of injected NSs were excreted from the body 14 days after injection. The enrichment of NSs in feces was evidently observed compared to that in urine, revealing that NSs were mainly metabolized out of the body through the liver (Fig. 5G). This result was also consistent with Fig. 5H, which indicated the highest concentration of NSs in the liver 24 hours postinjection. Moreover, NSs accumulated more at the tumor sites of the NSs-PEG/LA-AS1411 group than at the tumor sites of the NSs-PEG/LA group owing to the active targeting of AS1411. This adequate excretion could promote the clinical translation potential of Cu@Fe2C@mSiO2 NSs. In addition, the weight of mice with the administration of Cu@Fe2C@mSiO2 NSs also showed inconspicuous difference compared to the control group, suggesting the good biosafety of Cu@Fe2C@mSiO2 NSs in vivo (Fig. 5I).

Fig. 5.

Imaging and biosafety in vivo.

(A) Schematic illustration of the strategy of imaging in vivo based on different Cu@Fe2C@mSiO2 NSs. (B) Fluorescent images of 4T1 and B16F10 cells incubated with AS1411-FITC. Red fluorescence indicates nucleolin protein, green fluorescence indicates AS1411, and DAPI indicates nuclei. Scale bars, 10 μm. (C) Real-time NIR-II fluorescent images of the 4T1 cell–bearing mice after intravenous injection of NSs-PEG/LA-ICG and NSs-PEG/LA-ICG-AS1411. Ex vivo fluorescent images of heart (1), lung (2), spleen (3), liver (4), kidney (5), and tumor (6) that were obtained 48 hours after injection (left). Statistical analysis of fluorescence intensity of tumors after intravenous injection of NSs-PEG/LA-ICG and NSs-PEG/LA-ICG-AS1411, respectively (n ≥ 3) (right). (D) T2 relaxation rate (1/T2) as a function of Fe concentration for NSs-PEG/LA. (E) Real-time MRI of the 4T1 cell–bearing mice after intravenous injection of NSs-PEG/LA and NSs-PEG/LA-AS1411 (left). Statistical analysis of the relative MRI signal intensity of tumors after intravenous injection of NSs-PEG/LA-ICG and NSs-PEG/LA-ICG-AS1411, respectively (n = 4) (right). (F) Real-time NIR-II fluorescent images of the 4T1 cell–bearing mice after intravenous injection of NSs-PEG/LA-ICG and NSs-PEG/LA-ICG-AS1411 at 0, 1, 3, 5, 7, 10, and 14 days. Ex vivo fluorescent images of heart (1), lung (2), tumor (3), liver (4), kidney (5), and spleen (6) that were obtained 14 days after injection. (G) Cu relative content of urine and feces of the 4T1 cell–bearing mice after intravenous injection of NSs-PEG/LA and NSs-PEG/LA-AS1411 at 1, 7, 10, 14, 21, and 28 days (n = 5). (H) Cu relative content of main tissues (heart, liver, spleen, lung, kidney, and tumor) of the 4T1 cell–bearing mice after intravenous injection of NSs-PEG/LA and NSs-PEG/LA-AS1411 for 24 hours (n ≥ 5). (I) Body weight curve of the 4T1 cell–bearing mice after intravenous injection of PBS and Cu@Fe2C@mSiO2 NSs, respectively (n = 3). ns, not significant.

Synergetic therapy in vivo

Synergistic therapy strategy (i.e., PTT/CDT/immunotherapy) based on Cu@Fe2C@mSiO2-PEG/LA-R848-ICG-AS1411 (simplified as NSs-PEG/LA-R848-ICG-AS1411) for the treatment of 4T1 cell– or B16F10 cell–bearing mice was implemented according to the schematic illustration in Fig. 6A. According to previous results, NSs were enriched at the tumor sites to reach the maximum amount 24 hours after injection. Therefore, laser irradiation was applied to the mice with the treatment of NSs-PEG/LA-R848-ICG-AS1411 24 hours after injection, and the local temperature of the tumor site rapidly increased from 29.1° to 44.8°C, while the temperature was only changed from 31.9° to 38.3°C in mice without treatment of NSs-PEG/LA-R848-ICG-AS1411 (Fig. 6B). On early-phase tumor suppression assessments, compared with the other five groups, NSs-PEG/LA-R848-ICG-AS1411 combined with the 808-nm laser irradiation presented the most satisfactory suppression effects on subcutaneous tumor cells of 4T1 (Fig. 6, C and D) with tumors that almost disappeared, and the survival of this treated group was also notably prolonged (Fig. 6E), indicating the highly effective antitumor effect of synergistic therapy (PTT/CDT/immunotherapy) induced by NSs-PEG/LA-R848-ICG-AS1411 nanozymes. Perhaps, because of the more complex immunosuppressive TME, NSs-PEG/LA-R848-ICG-AS1411 nanozymes with the 808-nm laser irradiation also showed an imperfect inhibitory effect on the advanced-phase tumors (fig. S20). The histochemical analysis of hematoxylin and eosin (H&E) staining in the tumor of each group suggested that extensive necrosis appeared with severe cell shrinkage and loss of contact in the NSs-PEG/LA-R848-ICG-AS1411+Laser group, whereas fewer necrotic areas were observed in the mice treated with NSs-PEG/LA-R848-ICG or NSs-PEG/LA-R848-ICG-AS1411. No obvious necrosis was observed in the other three groups (Fig. 6F). To highlight the deep penetration ability of NIR-II PTT and the systemic effect of immunotherapy, metastatic cancer mouse models of 4T1 and B16F10 cells were established. Analogously, the most evident tumor inhibition effect was observed in the NSs-PEG/LA-R848-ICG-AS1411+Laser group, followed by the NSs-PEG/LA-R848-ICG-AS1411 group and the NSs-PEG/LA-R848-ICG group, while the other three groups had almost no effect (Fig. 6G and fig. S21). In vivo biosafety of these nanozymes was also evaluated during the treatment process. Injected NSs made no contribution to the weight of mice in these six groups (fig. S22), as well as the morphology of pivotal tissues (heart, liver, spleen, lung, and kidney) (fig. S23), revealing the good biocompatibility of NSs in vivo.

Fig. 6.

Synergetic therapy in vivo.

(A) Schematic illustration of the strategy of synergetic therapy in vivo based on different Cu@Fe2C@mSiO2 NSs for different tumor models. (B) Real-time thermal infrared images of the 4T1 cell–bearing mice after intravenous injection of PBS and NSs-PEG/LA-R848-ICG-AS1411 for 24 hours under 808-nm laser irradiation (0.3 W/cm2, 5 min). (C) Representative images of tumors of mice in six groups for the early-phase subcutaneous tumor treatment model. (D) Tumor volume curve of mice in six groups for the early-phase subcutaneous tumor treatment model (n ≥ 3). (E) Survival curve of mice in six groups for the early-phase subcutaneous tumor treatment model (n = 6). (F) H&E-stained images of tumors of mice in six groups for the early-phase subcutaneous tumor treatment model at the end of the treatment. (G) Statistical analysis of tumor numbers of mice in six groups for the metastatic tumor treatment model of 4T1 and B16F10 cells (n = 6).

Immunomodulatory effect in vivo

To further explore the immunomodulatory effect of the NS-mediated nanozyme system on mice, we made a comprehensive and systematic evaluation. First, the intrinsic immunogenicity of NSs was roughly proved by the increased number and proportion of total spleen cells, CD45+ lymphocytes, and CD8+ T cells. R848 was released continuously in vivo by NSs as a smart drug delivery system, allowing the total spleen cells, CD45+ lymphocytes, and CD8+ T cells to be further activated and proliferated (fig. S24). Meanwhile, considering the regulatory effect of NS-mediated nanozymes on cancer cells and different immune cells, the uptake of the nanozymes by cancer cells and different immune cells should be evaluated (). With the increased incubation time of the nanozymes with cancer cells, the peak of flow cytometry shifted gradually to the right, implying the time-dependent uptake of nanozymes by cancer cells (fig. S25). Subsequently, the uptake of nanozymes by different immune cells was also detected by flow cytometry. In both subcutaneous tumor models (Fig. 7A and fig. S26A) and metastatic tumor models (Fig. 7B and fig. S26B), as DCs and macrophages were potent phagocytic cells, the nanozymes were most effectively taken up by these cells in a time-dependent manner; however, MDSCs had the least uptake of the nanozymes. In addition, these immune cells around subcutaneous or metastatic tumor tissues also exerted a similar uptake ability to the nanozymes as shown in fig. S27. For a successful induction of immune responses, the maturation and activation of DCs are the prerequisites (). The results of Fig. 7 (C and D) demonstrate that NSs-PEG/LA-ICG slightly up-regulated the costimulatory molecules CD80 and CD86 expressed in DCs, while loading R8484 could obviously enhance the up-regulation effect. Among the six groups, the NSs-PEG/LA-R848-ICG-AS1411+Laser group exhibited the most mature and active state of DCs owing to the enhanced antigen presentation induced by synergistic therapy. Similarly, the DCs around tumor tissues in the NSs-PEG/LA-R848-ICG-AS1411+Laser group showed the highest expression of CD80 and CD86, which suggested the potential initiation of the strongest immune response (fig. S28). MDSCs as inhibitory immune cells are a population of myeloid cells generated during a large array of pathologic conditions including various types of cancers. In general, the studies have demonstrated that MDSCs contribute to tumor cell proliferation by impairing T cell function (). The results of Fig. 7E and fig. S29 indicate that NSs-PEG/LA-R848-ICG-AS1411 nanozymes substantially down-regulated the proportion of MDSCs to alleviate the immunosuppression of TME. On the other hand, CD8+ T cells have served as the primary immune cells for cancer cell elimination (), which greatly aroused our interest. T-bet () and Eomesodermin (Eomes) (), as the key transcription effectors, intrinsically regulate CD8+ cell proliferation and activation at the transcriptional level. In the NSs-PEG/LA-R848-ICG-AS1411+Laser group, CD8+ T cells exhibited the highest expression of T-bet and Eomes, indicating the greatest degree of CD8+ T cell activation. CD8+ T cells of mice treated with NSs-PEG/LA-ICG also showed a higher expression of T-bet and Eomes than the control group, further proving the intrinsic immunogenicity of NSs (Fig. 7F and fig. S30). Subsequently, we explored the CD8+ T cell responsiveness to 4T1 cells. Splenic CD8+ T cells were first coincubated with 4T1 cells for 4 hours, while phosphate-buffered saline (PBS) was the negative control, and phorbol 12-myristate 13-acetate (PMA) + ionomycin (P + I) was the positive control. Cytokines such as IFN-γ and tumor necrosis factor–α (TNF-α) act as markers of CD8+ T cell function. The CD8+ T cells in the NSs-PEG/LA-R848-ICG-AS1411+Laser group exhibited a notable increment in the production of IFN-γ and TNF-α, and the expression of CD107a, a marker of CD8+ T cell degranulation, in response to 4T1 cell stimuli, indicated the strongest antitumor ability of CD8+ T cells in the NSs-PEG/LA-R848-ICG-AS1411+Laser group. Consistently, similar results were found in the PBS group and the P + I group (Fig. 7G and fig. S31). Furthermore, we isolated lymphocytes from the tumor tissues in these six groups. The number and proportion of immune cells (total spleen cells, CD45+ lymphocytes, and CD8+ T cells), the activation (T-bet and Eomes) and the function (IFN-γ, TNF-α, and CD107a) of CD8+ T cells, and the population of MDSCs were also detected through the same methods. The results showed that besides the immunogenicity of NSs, the targeting performance of AS1411 could also appropriately improve the activation and function of CD8+ T cells and reduced the population of MDSCs, because more nanozymes were accumulated at tumor sites for easier immune response improvement (fig. S32). The systemic immune activation of R848 also had an identical effect (fig. S33). All these results strongly confirmed the multifaceted immune response regulation of our nanozyme system to cancer (Fig. 1B).

Fig. 7.

Immunomodulatory effect in vivo.

(A) Statistical analysis of flow cytometry of the uptake of NS-mediated nanozymes in different immune cells of the spleen in the subcutaneous tumor model (n = 3). (B) Statistical analysis of flow cytometry of the uptake of NS-mediated nanozymes in different immune cells of the spleen in the metastatic tumor model (n = 3). (C) Flow cytometry and corresponding statistical analysis of CD11c+MHC II+CD80+ splenocytes of mice in six groups for the early-phase subcutaneous tumor treatment model (n = 3). (D) Flow cytometry and corresponding statistical analysis of CD11c+MHC II+CD86+ splenocytes of mice in six groups for the early-phase subcutaneous tumor treatment model (n = 3). (E) Statistical analysis of flow cytometry of CD11b+Gr-1+ splenocytes of mice in six groups for the early-phase subcutaneous tumor treatment model (n = 3). (F) Statistical analysis of flow cytometry of CD3+CD8+T-bet+ and CD3+CD8+Eomes+ splenocytes of mice in six groups for the early-phase subcutaneous tumor treatment model (n = 3). (G) Statistical analysis of flow cytometry of CD3+CD8+CD107a+, CD3+CD8+IFN-γ+, and CD3+CD8+TNF-α+ splenocytes of mice in six groups with different stimulations for the early-phase subcutaneous tumor treatment model (n = 3).

Effect of Cu@Fe2C@mSiO2 on signaling pathways

The physiological proliferation and apoptosis of cells are commonly regulated by intrinsic molecular signaling pathways. The dysregulation of these signaling pathways usually leads to pathological changes in cells. For instance, excessive ROS can regulate various signaling pathways [e.g., nuclear factor κB (NF-κB)], resulting in functional consequences, which successively mediate pathological processes (). The different properties of NPs for the antitumor effect can induce the changes in different molecular signaling pathways of cancer cells, eventually leading to the death of cancer cells. However, research in this field has been in its infancy, which has greatly aroused our research interest. Hence, we selected 4T1 and B16F10 cells without or with the treatment of Cu@Fe2C@mSiO2 NSs to explore the change in mRNA level via RNA sequencing (RNA-seq)–based global gene expression profiling analysis. A great number of genes were up- or down-regulated in both 4T1 and B16F10 cells (Fig. 8A). In detail, Cu@Fe2C@mSiO2 NSs induced the up- and down-regulation of 2.4% of the genes in 4T1 cells and 1.4% of the genes in B16F10 cells, respectively (Fig. 8B). By quantitative analysis, the top 20 genes with the most up-regulation or down-regulation induced by Cu@Fe2C@mSiO2 NSs in 4T1 and B16F10 are found in the heatmap in Fig. 8C and the volcano plot in fig. S34. Although the changed genes were slightly different in 4T1 and B16F10 cells, most of these genes have been proven to be related to normal cell physiological behaviors, such as cell development, cell migration and invasion, vesicle maturation, and neurotransmitter release, as well as to some diseases such as autism spectrum disorder and various kinds of cancers. To further find clearer and more specific molecular signaling pathways, gene ontology enrichment analysis was carried out. The phosphatidylinositol 3-kinase (PI3K)–Akt pathway and the cell cycle–related pathway stood out in 4T1 and B16F10 cells, respectively. Some conservative signaling pathways were also involved, including the DNA replication–related signaling pathway, the cell apoptosis–related signaling pathway, the hypoxia-inducible factor 1 (HIF-1) signaling pathway, and the p53 signaling pathway (Fig. 8D). The PI3K-Akt signaling pathway happened to be regulated by ROS (), which strongly suggested the intrinsic regulation of the peroxidase-like activity of our nanozymes at the molecular level. To further verify the RNA-seq results at the protein level, some key effectors of the PI3K-Akt signaling pathway and cell apoptosis–related protein were detected in 4T1 and B16F10 cells treated with Cu@Fe2C@mSiO2 NSs. The PI3K-Akt signaling pathway is crucial to many aspects of cell growth and survival, as well as in physiological and pathological conditions (e.g., cancer). The treatment of Cu@Fe2C@mSiO2 NSs up-regulated the expression of Akt phosphorylation at serine-473, which suggested the activation of Akt. Similarly, the key downstream kinase of the PI3K-Akt pathway, IκBα, was also activated via the increased expression of IκBα phosphorylation at serine-32. Moreover, the expression of NF-κB(p65), cleaved caspase 3, and cleaved poly(adenosine 5′-diphosphate–ribose) polymerase (PARP) was also up-regulated by Cu@Fe2C@mSiO2 NSs (Fig. 8E). Mechanistically, these kinases (Akt and IκBα) activated by Cu@Fe2C@mSiO2 NSs further increased the expression of NF-κB(p65) as the transcription factor and consequently up-regulated the expression of cell apoptosis–related proteins (cleaved caspase 3 and cleaved PARP), leading to the cell apoptosis of 4T1 and B16F10 cells (Fig. 1B). All these results suggested that Cu@Fe2C@mSiO2 NSs induced cell apoptosis of 4T1 and B16F10 cells via the Akt–IκBα–NF-κB pathway and the cell apoptosis–related signaling pathway. The cell proliferation–related (Ki-67) and apoptosis-related (Bcl-2 and Bax) signaling pathways regulated by Cu@Fe2C@mSiO2 NSs were also proved by immunohistochemistry in vivo. As shown in Fig. 8F, the expression of Ki-67 and Bcl-2 was the highest in the control group and the laser group. With the gradual increase in treatment intensity, the expression of Ki-67 and Bcl-2 was down-regulated, which was lowest in the NSs-PEG/LA-R848-ICG-AS1411+Laser group. These results indicated that the synergistic therapy based on our nanozymes could notably inhibit cell proliferation and promote cell apoptosis in vivo. Furthermore, the ROS level in vivo was also evaluated, and the administration of Cu@Fe2C@mSiO2 NSs in mice prominently increased ROS generation at the tumor sites (Fig. 8G).

Fig. 8.

Effect of Cu@Fe2C@mSiO2 on signaling pathways.

(A) Heatmap of RNA-seq analysis of 4T1 and B16F10 cells incubated without or with Cu@Fe2C@mSiO2 NSs for 24 hours. (B) The proportion analysis of up- and down-regulated genes in 4T1 and B16F10 cells incubated with Cu@Fe2C@mSiO2 NSs for 24 hours. (C) The top 20 up- and down-regulated genes in 4T1 and B16F10 cells incubated with Cu@Fe2C@mSiO2 NSs for 24 hours. (D) Gene ontology enrichment analysis of 4T1 and B16F10 cells incubated with Cu@Fe2C@mSiO2 NSs for 24 hours. (E) Western blotting of 4T1 and B16F10 cells incubated with Cu@Fe2C@mSiO2 NSs for 24 hours with antibodies against Akt, Pho-Akt(Ser473), IκBα, Pho-IκBα(Ser32), NF-κB(p65), cleaved caspase 3, and cleaved PARP. β-Actin was used as a loading control. (F) Immunohistochemistry staining of Ki-67, Bcl-2, and Bax in tumors of mice in six groups for the early-phase subcutaneous tumor treatment model at the end of the treatment. (G) DHE and DAPI staining in tumors of mice treated without or with NSs for 48 hours.

DISCUSSION

For a long time, people have had great interest in improving the intrinsic antitumor ability of nanozymes, as well as the synergetic enhancement strategy via multifunctionalization of nanozymes. To break through the potential therapeutic bottleneck of nanozymes, we designed a type of visualized immunomodulatory nanozyme for ameliorating the complex immunosuppressive TME to eliminate both primary and metastatic cancers. First, the composite-phase NPs (copper ion) enhanced Fenton reaction compared to that induced by single-phase NPs (iron carbide NPs), resulting in higher ROS production. Meanwhile, these nanozymes, as a smart drug delivery system, made immune agonist release more efficient and controllable through the dual domination of temperature and pH. The active targeting effect also enabled the nanozymes to be more effectively enriched at tumor sites, avoiding damaging normal tissues. However, NPs with a composite phase usually require a larger size; thus, their metabolic efficiency in vivo needs further improvement while ensuring their excellent performance.

Intrinsic immunogenicity of nanozymes combined with the effect of immune agonist boosted systemic antitumor immune response; simultaneously, adverse immune side effects were avoided because of the specific accumulation of nanozymes at the tumor sites by active targeting. These nanozymes promoted the maturation of DCs, improved the activation and function of CD8+ T cells, and inhibited the proportion of MDSCs, maintaining the good immune dynamic balance of TME for killing cancer cells. Of course, the systemic immune response is a very complex physiological process, and the changes in some immune cells often involve a variety of reasons. In our study, the reduction of the MDSC population aroused our concern. Considering the uptake of the nanozymes by MDSCs, we can assume that MDSCs may be directly killed by either PTT or CDT induced by the nanozymes. Unfortunately, the uptake of nanozymes by MDSCs is very limited (Fig. 7, A and B, and figs. S26 and S27), especially when compared with DCs or macrophages. Hence, the decrement of MDSCs may be caused by the change of the TME. Previous studies have proven that R848 can influence the differentiation of MDSCs into DCs or macrophages (). Meanwhile, with the appearance of the therapeutic effect of nanozymes, smaller tumor tissues are always accompanied by fewer MDSCs. TAMs, one of the most abundant immune cells in the TME, are divided into M1- or M2-like phenotypes. In general, M1-like macrophages have a phagocytic effect on cancer cells, while M2-like macrophages promote cancer cell growth through the secretion of some cytokines (). To further enrich our evaluation of the immunomodulatory effect induced by our nanozymes, the polarization of macrophages was also detected. As shown in fig. S35, our nanozymes could polarize M2-like macrophages to M1-like macrophages to a certain extent. In general, the regulatory effect of multifunctional nanozymes on the immune system is worthy of further research in the future.

In addition to the immunomodulatory effect, mechanistically, these nanozymes also activated the Akt-IκBα signaling pathway of cancer cells, subsequently up-regulated the expression of the transcription factor NF-κB, and eventually promoted cancer cell apoptosis. Moreover, many signaling pathways have been reported to be associated with ROS (). Our subsequent results preliminarily suggested that the Cu@Fe2C@mSiO2-based nanozymes could up-regulate the phosphorylation of signal transducer and activator of transcription 3 (STAT3); however, more experiments are needed to further clarify the relationship between the therapeutic effect of nanozymes and the STAT3 signaling pathway. Exploring the effect of nanozymes on tumor cells in the gene level and the transcription level is still in its infancy, and a great amount of research is needed to improve the theoretical basis. In summary, the nanozyme-based comprehensive analysis of immune activation function and influence on endogenous gene and protein levels in cancer cells provided more theoretical support for clinical translation of nanozymes.

MATERIALS AND METHODS

Synthesis of monodisperse Cu@Fe2C NPs and Cu@Fe2C@mSiO2 NSs

Cu@Fe2C NPs were synthesized by a facile seedmediated growth method. In the typical synthesis, copper acetylacetonate [Cu(acac)2] (1 mmol; J & K), 1octadecene (46.875 mmol; Alfa Aesar), and oleamine (15 mmol; J & K) were mixed under a gentle N2 flow for 30 min in a fournecked flask. Then, the solution was heated to 110°C and kept for 30 min to remove the organic impurities. Fe(CO)5 (5 mmol) was injected into the reaction system when the temperature reached 180°C and kept for 10 min. Subsequently, the solution temperature was increased up to 265°C for 2 hours. At room temperature, the solution was washed three times with acetone and hexane, and the products were kept in trichloromethane.

For the synthesis of Cu@Fe2C@mSiO2 NSs, triethylamine (0.18 g; Xilong Chemicals), N-hexadecyl trimethyl ammonium chloride (CTAC) (10.56 g; Sigma-Aldrich), and Cu@Fe2C NPs (1 mmol) were mixed in a deionized water solution (36 ml) at 60°C for 3 hours. Then, tetraethyl orthosilicate (0.94 g, 28%; Xilong Chemicals) dissolved in hexane (20 ml, 5%, v/v) was added into the reaction system and kept at 60°C for 48 hours. The products were washed three times with ethanol and kept in ethanol.

Synthesis of NSs-PEG/LA-R848-ICG-AS1411

First, composite phase change materials (PEG/LA) were synthesized via the mass ratio of PEG1000 (Xilong Chemicals) and PEG2000 (Xilong Chemicals) as 1:1 and simultaneously allocated with LA (Alfa Aesar) with a mass ratio of 10%. For the preparation of R848 (MC0662, Immunoway) loading, Cu@Fe2C@mSiO2 NSs were washed in 1% (w/w) NaCl of methanol solution for 36 hours to remove CTAC from the reaction system and then dried into a powder state in a vacuum.

For the synthesis of NSs-PEG/LA-R848, Cu@Fe2C@mSiO2 NSs (10 mg) and R848 (2 mg) were mixed in PEG/LA (250 mg) via efficient stirring at 50°C for 30 min and then kept for another 30 min. The excess PEG/LA was removed by magnetic enrichment. NSs-PEG/LA-R848 was kept at −20°C. Moreover, for the cellular and animal experiment, NSs-PEG/LA-R848 was dispersed in ethanol by ultrasound, cleaned three times by magnetic enrichment, and lastly dispersed in an aqueous solution. The excess ethanol was removed by vacuum for 1 hour.

For the synthesis of NSs-PEG/LA-R848-ICG-AS1411, 1-ethyl-3-(3-(dimethylamino)propyl) urea hydrochloride (1.9 mg, 99%; Sigma-Aldrich) and N-hydroxysuccinimide (2.2 mg, 98%; Sigma-Aldrich) were mixed with NSs-PEG/LA-R848 (20 mg) in an aqueous solution for 4 to 8 hours. Subsequently, ICG (0.2 mg) and AS1411 (60 μg) were added into the reaction system for 24 hours. The products were washed three times and kept in an aqueous solution.

Characterization

TEM images were obtained from the FEI Tecnai T20 microscope. HRTEM images were obtained from the FEI Tecnai F30 microscope. A reinforced carbon membrane support grid was used to obtain the EDS mapping. XPS measurements were performed on an imaging x-ray photoelectron spectrometer using Al Kα radiation (Axis Ultra DLD, Kratos Analytical Ltd.). All the collected spectra were calibrated with the contaminated C 1s peak at 284.8 eV and were analyzed using CasaXPS software (2.3.12 Dev7). XRD patterns were carried out using a Rigaku DMAX-2400 x-ray diffractometer equipped with Cu Kα (λ = 0.15405 nm) radiation. Magnetization was measured by Physical Property Measurement System (PPMS-9, Quantum Design, USA). DLS was measured using a particle size analyzer (Zetasizer Nano ZS-90, Malvern, England). The concentrations of Fe and Au were quantified using an inductively coupled plasma–atomic emission spectrometer (Prodigy 7, Leeman, USA). UV-vis–NIR absorption spectra were measured on a UV-vis spectrophotometer (UV-2550, SHIMADZU, Japan). BET was measured by an Autosorb-iQ2 apparatus (Quantachrome, USA).

Photothermal effect of Cu@Fe2C@mSiO2 NSs

A total of 350 μl of Cu@Fe2C@mSiO2 dispersions with different concentrations (0, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mM) was irradiated with a laser (808 nm, 0.4 W/cm2) for 5 min, and their temperature in solution was recorded by an online-type thermocouple thermometer. Similarly, to study the influence of optical density on photothermal conversion, 350 μl of 0.4 mM Cu@Fe2C@mSiO2 dispersions was irradiated with an 808-nm laser with different power densities (0.2, 0.4, 0.6, 0.8, and 1.0 W/cm2) for 5 min. The change of temperature in solution was recorded by an online-type thermocouple thermometer. The photostability of Cu@Fe2C@mSiO2 dispersions (0.4 mM) was evaluated in a quartz cuvette under laser irradiation (808 nm, 0.4 W/cm2) for 5 min, and then the dispersions were cooled down to room temperature without irradiation. The photostability was tested by repeating such processes three times.

Cell culture

NIH 3T3, 4T1, and B16F10 cell lines were obtained from the Cancer Institute and Hospital of the Chinese Academy of Medical Science. NIH 3T3, 4T1, and B16F10 cell lines were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified environment at 37°C with 5% CO2. All the reagents for cell culture were purchased from Invitrogen.

In vitro cytotoxicity assay

In vitro cytotoxicity of Cu@Fe2C@mSiO2 NSs was evaluated by the CCK8 from Dojindo Laboratories (Tokyo, Japan). NIH 3T3, 4T1, and B16F10 cells (5 × 103 cells per well) were seeded into a 96-well culture plate. When the cell density reached 70 to 80%, all the cells were incubated with Cu@Fe2C@mSiO2 NSs at different Fe concentrations (0, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6, and 51.2 μM) for 24 hours. The relative cell viabilities were determined as directed by the instructions. The absorbance of each well was measured with a luminescence microplate reader (Bio-Rad 680) at 450 nm. According to the results, 4T1 and B16F10 cells for all the cell experiments were treated with Cu@Fe2C@mSiO2 NSs at an Fe concentration of 51.2 μM.

Bio-TEM characterization

Bio-TEM was used to determine the subcellular distribution of the Cu@Fe2C@mSiO2 NSs in 4T1 and B16F10 cells. 4T1 and B16F10 cells were seeded into a six-well culture plate (105 cells per well). When the cell density reached 80 to 90%, the cells were then incubated with Cu@Fe2C@mSiO2 NSs for 6 hours. The treated cells were washed three times with PBS, fixed using paraformaldehyde and osmium tetraoxide, and then dehydrated with ethanol. Eventually, the treated cells were embedded in Spurr resin and sectioned to 70 nm in thickness.

Colocalization of lysosomes and NSs assay

The as-prepared Cu@Fe2C@mSiO2 NSs were labeled with ICG, defined as NSs-ICG. 4T1 and B16F10 cells (5 × 104 cells per well) were seeded into a glass-bottom cell culture dish (20 mm). When the cell density reached 80 to 90%, the cells were then incubated with Cu@Fe2C@mSiO2 NSs for 6 hours. Subsequently, 4T1 and B16F10 cells were counterstained with LysoTracker Green (L7526, Life Technologies, USA) and DAPI (C0060, Solarbio, Beijing, China) for 15 min. Last, the cells were washed three times with PBS for NIR-II fluorescence imaging observation through a multidimensional confocal microfluorescence imaging system (FLIM+confocal+AFM, Q2, ISS-USA).

Double staining of the live/dead cell assay

Double staining of the live/dead cell assay was carried out using the Calcein-AM/PI Double Stain Kit (40747ES76, Yeasen, Shanghai, China). 4T1 and B16F10 cells were seeded into a 24-well culture plate (2 × 104 cells per well). When the cell density reached 70%, the cells were then incubated with Cu@Fe2C@mSiO2 NSs for 24 hours. After washing out the free NPs with PBS, the fresh culture medium was added. Laser (808 nm, 0.3 W/cm2) was then used to irradiate the cells for 5 min. The staining method was directed by the instructions. The cells were lastly visualized using an inverted microscope (Olympus IX71).

Intracellular ROS assay

Intracellular ROS assay was measured by the ROS Assay Kit (S0033, Beyotime, China). 4T1 and B16F10 cells were seeded into a 24-well culture plate (2 × 104 cells per well). When the cell density reached 70%, the cells were then incubated with Cu@Fe2C@mSiO2 NSs for 24 hours. After washing out the free NPs with PBS, the fresh culture medium was added. Laser (808 nm, 0.3 W/cm2) was then used to irradiate the cells for 5 min. The staining method was directed by the instructions. The cells were lastly visualized using an inverted microscope (Olympus IX71) or detected through flow cytometry using a BD Fortessa flow cytometer (BD Biosciences).

Immunofluorescence

AS1411 was labeled with FITC. 4T1 and B16F10 cells (5 × 104 cells per well) were seeded into a glass-bottom cell culture dish (20 mm). When the cell density reached 70%, the cells were transfected with AS1411 (800 ng per well) using Lipo3000 (Invitrogen, USA) for 24 hours according to the instructions. The treated cells were fixed in 4% paraformaldehyde for 15 min at room temperature. Immunofluorescence was performed as described previously (). Antibodies used were anti-nucleolin (1:100; Abcam, ab129200) and Dylight 594 goat anti-rabbit immunoglobulin G (1:500; sc-362281, Santa Cruz Biotechnology). The images and videos were taken using a confocal microscope (Zeiss LSM 880, Germany).

Animals and tumor models

Balb/c mice and C57BL/6J mice (female; 20 g) were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). For the subcutaneous tumor model, 4T1 cells (2 × 106 cells in 0.1 ml of saline) were injected subcutaneously into Balb/c mice at the root of the right hind legs. For the metastatic tumor model, 4T1 and B16F10 cells (5 × 105 cells in 0.1 ml of saline) were injected intravenously into Balb/c mice and C57BL/6J mice, respectively. All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Peking University, Beijing, China.

In vivo MRI

The 4T1 cell–bearing mice were intravenously injected with NSs-PEG/LA and NSs-PEG/LA-AS1411 (20 mg/kg, 200 μl). After the injection, T2 images were obtained at 0, 6, 12, 24, and 48 hours by a clinic 3-T MRI scanner [Philips; TR (Repetition time) = 1200 ms, TE (Echo time) = 30.2 ms, and slice thickness = 2.5 mm]. The intensity of the MRI signal before injection was used as the control.

In vivo fluorescence imaging

The 4T1 cell–bearing mice were intravenously injected with NSs-PEG/LA and NSs-PEG/LA-AS1411 (20 mg/kg, 200 μl). After the injection, fluorescent images were obtained at 3, 6, 12, 24, and 48 hours by the self-assembled NIR-II fluorescence imaging system. To confirm the in vivo distribution of NSs-PEG/LA and NSs-PEG/LA-AS1411, mice were euthanized 48 hours after injection. The main organs (heart, liver, spleen, lung, kidney, and tumor) were collected for fluorescence imaging.

In vivo treatment experiments

The subcutaneous tumor–bearing mice (4T1 cells) and the metastatic tumor–bearing mice (4T1 and B16F10 cells) were randomly divided into six groups: (i) control (PBS), (ii) laser (PBS + laser), (iii) NSs-PEG/LA-ICG, (iv) NSs-PEG/LA-R848-ICG, (v) NSs-PEG/LA-R848-ICG-AS1411, and (vi) NSs-PEG/LA-R848-ICG-AS1411+Laser. Different NSs (20 mg/kg, 200 μl) were intravenously injected into mice according to the time point of the schematic diagram (Fig. 5A). For 24 hours postinjection, the tumors of mice were exposed to 808-nm laser irradiation (0.3 W/cm2) for 5 min. An infrared thermal imaging instrument (FLIR A325SC camera) was used to record the temperature detection and thermal image. An 808-nm high-power multimode pump laser (Shanghai Connect iber Optics Co.) was used for NIR laser.

Histological evaluation

Mice in different groups were euthanized at the end of the treatment, and the tissues (heart, liver, spleen, lung, kidney, and tumor) were selected, followed by fixing with 4% paraformaldehyde. The tissues were embedded in paraffin and sectioned at 5 mm; H&E staining was performed for histological evaluation. The slides were observed under an optical microscope.

In vivo ROS assay

The frozen sections of tumor tissues in mice were prepared after the administration of NSs for 48 hours. Then, the sections were incubated with DHE (dihydroethidium) and eventually stained with DAPI. The slides were observed under a fluorescence microscope.

Immunohistochemistry

The paraffin sections of tumor tissues in mice were prepared at the end of the treatment. Immunohistochemistry was performed as described previously (). Primary antibodies were used including anti–Ki-67 (1:500; Cell Signaling Technology, 9449), anti–Bcl-2 (1:200; Cell Signaling Technology, 15071), and anti-Bax (1:50; Absin, 130057).

Flow cytometry

Flow cytometry was performed using a BD Fortessa flow cytometer (BD Biosciences), and data were analyzed using FlowJo 10 software (TreeStar). The following monoclonal antibodies against mouse were purchased from BioLegend: CD45 (1:200; 103111), CD3 (1:200; 100335), CD8 (1:200; 100733), Gr-1 (1:200; 108405), CD11b (1:200; 101215), T-bet (1:200; 644809), Eomes (1:200; 157703), CD107a (1:100; 121613), IFN-γ (1:200; 113603), TNF-α (1:200; 506323), CD80 (1:200; 104707), CD86 (1:200; 105027), major histocompatibility complex (MHC) II (1:200; 107605), F4/80 (1:200; 123107), CD206 (1:200; 141705), and CD11c (1:200; 117317). The following monoclonal antibodies against mouse were purchased from Thermo Fisher Scientific: MHC II (1:200; 12-5321-81) and F4/80 (1:200; 12-4801-80).

Uptake assay of NSs by different immune cells

According to the different color matching of antibodies marking the different immune cells, NSs were linked with the appropriate fluorescence molecules (FITC or DAPI fluorescence molecules) for subsequent flow cytometry analysis. Splenocytes or lymphocytes around tumors were cocultured with NSs (Fe concentration, 51.2 μM) at 37°C for different times. The specific experimental steps were performed as described previously (), and lastly, the uptake of NSs by different immune cells was analyzed by flow cytometry.

CD8+ T cell function assay

Splenocytes (2 × 106) or lymphocytes around tumors were cocultured with target cells (4T1, 2 × 106). BD GolgiStop reagent (BD Biosciences) was used to inhibit intracellular protein transport; meanwhile, allophycocyanin-conjugated anti-CD107a antibody or the respective control isotypes were added at the beginning of incubation. Splenocytes stimulated with PMA (50 ng/ml) plus ionomycin (1 μM) were used as a positive control. PBS was used as a negative control. After coculturing for 4 hours, cells were harvested and stained with the indicated antibodies, fixed, permeabilized with Cytofix/Cytoperm Buffer (BD Biosciences), and then stained with CD107a, IFN-γ, and TNF-α antibody.

RNA-seq analysis

For RNA-seq analysis, 4T1 and B16F10 cells were seeded into a six-well culture plate (105 cells per well). When the cell density reached 80 to 90%, the cells were then incubated with Cu@Fe2C@mSiO2 NSs for 24 hours. The treated cells were washed three times by PBS and then harvested for total RNA extraction with a total RNA purification kit (GeneMarkbio), and RNA was converted into RNA-seq quantification libraries (low-input library; 200 ng). RNA-seq libraries were sequenced with the paired-end option using an Illumina-HiSeq-PE150 Sequencer at Novogene (Beijing, China) as per the manufacturer’s recommended protocol.

Western blotting

For Western blotting, 4T1 and B16F10 cells were seeded into a six-well culture plate (105 cells per well). When the cell density reached 80 to 90%, the cells were then incubated with Cu@Fe2C@mSiO2 NSs for 24 hours. Subsequently, cells were harvested and lysed in radioimmunoprecipitation assay buffer (Cell Signaling Technology). Western blot analysis was performed with the use of conventional protocols as described previously (). The following primary antibodies were purchased from Cell Signaling Technology: Akt (1:1000; 4691T), Pho-Akt(Ser473) (1:1000; 4060T), IκBα (1:1000; 4814T), Pho-IκBα(Ser32) (1:1000; 2859T), NF-κB(p65) (1:1000; 8242T), cleaved caspase 3 (1:1000; 9664T), cleaved PARP (1:1000; 5625T), and β-actin (1:5000; 3700T).

Statistical analysis

Results were presented as means ± SD. Student’s two-tailed nonpaired t test was used to determine significance between treatment and control groups in all experiments. P < 0.05 was considered statistically significant; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.