Zinc–selenium synergistic nanoplatform for augmented cancer immunotherapy via trace-elements-mediated immunomodulation

Abstract

Essential trace elements (ETEs) are crucial nutrients in maintaining the immune function of the body. Designing nanomedicines based on ETEs has become an emerging strategy for enhancing immunotherapy by utilizing the metabolism of constituent ETEs and their immunomodulatory functions. However, their medical applications are challenged by the dosage-dependent balance between therapeutic necessity and toxicity. The narrow safety zones of ETEs pose great challenges for high efficacy without exceeding strict safety thresholds. Nanomedicinal strategies based on multiple ETEs hold promising potentials for exerting safe and effective immunomodulation functions of ETEs with an expanded therapeutic window. Herein, ultrasmall ZnS/Se/BSA nanoclusters (ZSB NCs) were synthesized via a biomineralization approach, acting as a synergetic lymph nodes (LNs)-targeting nanoplatform integrating the immunomodulatory effects of ETEs (Zn and Se) and the advantages of albumins for cancer immunotherapy. ZSB NCs could remarkably target LNs after subcutaneous injection, where the released zinc ions and transformed selenoproteins stimulated the cyclic guanosine monophosphate-adenosine monophosphate synthase-interferon gene (cGAS-STING) pathway. Subsequently, ZSB NCs effectively induced the activation and maturation of dendritic cells (DCs) and activated T cells to secrete inflammatory factors for enhancing immunomodulatory effects. The cancer immunotherapy efficacy and biosafety of ZSB NCs were validated in a orthotopic breast cancer model, where tumor growth was significantly suppressed. Our findings indicate that ZSB NCs can act as a promising candidate for improved synergetic cancer immunotherapy.

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Chunying Chen

Congratulations to Nanoscale Horizons on this impressive 10-year milestone! We celebrate its consistent publication of outstanding research and its vital role in advancing nanoscience. It has been a privilege to serve on the Nanoscale Horizons Advisory Board and to witness the journal's growth over the last ten years. Our group has published and will continue to report our significant research in Nanoscale Horizons, benefiting from the journal's broad reach and high impact. I extend my best wishes for the journal's continued success in pioneering the future of nanoscience.

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New concepts

Recent advancements in essential trace element (ETE)-mediated immunotherapy highlight their substantial potential for immunomodulation. However, their healthcare applications still face the challenge of dosage-dependent necessity/toxicity balance. The narrow safety zones bring great difficulties in achieving high efficacy without exceeding strict safety thresholds. To overcome this limitation, we have developed ZnS/Se/BSA nanoclusters (ZSB NCs) as a synergistic multi-ETE platform capable of targeted co-delivery of Zn and Se to lymph nodes and enhanced immunomodulation at safe dosages. The designed ZSB NCs integrate the synergistic immune effects of Zn and Se, as well as the LNs-targeting capability of BSA, leveraging the biotransformed zinc ions and selenoproteins to activate the cGAS-STING pathway. Compared with zinc- or selenium-based nanoparticles, ZSB NCs exhibit superior immune activation effects both in vitro and in vivo, resulting in significant tumor regression in the orthotopic breast cancer model at decreased administrated dosage. This work establishes a research paradigm based on synergistic ETE networks, achieving robust immunomodulation within an expanded therapeutic window that surpasses the limitations of the individual elements.

Introduction

The in vivo distribution and metabolic behavior of nanomedicines determine their therapeutic efficacy, safety profiles, and translational potential.^1–3 The targeted delivery and controlled metabolic behavior of nanomedicines in biological systems represent a key research Frontier for deciphering the underlying mechanism of improved therapeutic efficacy and biosafety.^4–6 Lymphatic targeting is of paramount importance to achieve the desired immunomodulation effects. The targeting efficiency and accumulation in lymphoid tissues can be precisely improved via structural engineering of nanomaterials, which enables immune crosstalk and induces a pro-immunogenic niche.^7,8 Notably, programmed lymph node-targeting strategies have demonstrated the capacity to orchestrate coordinated anti-tumor immunity,⁹ as evidenced by the robust suppression of 4T1 mammary tumor growth in mouse models.¹⁰ The in vivo metabolism of nanoparticles is largely governed by their elemental composition, particularly in case of essential trace element (ETE)-based nanoparticles, such as selenium,¹¹ molybdenum,¹² iron,¹³ copper,^14,15 zinc,¹⁶ and manganese.^17,18 ETE-based nanoparticles undergo biotransformation into bioactive species including ions, enzyme cofactor and biomolecule conjugates and are bioavailable and involved in diverse physiological activities.^19,20

The unique bioavailability and bioactivity of ETE-based nanoparticles demonstrate the intrinsic advantages of ETE-based nanotherapeutics.²¹ The development of nanomedicines incorporating ETEs has emerged as a novel strategy to enhance immunotherapy, leveraging the intrinsic metabolic pathways and immunoregulatory properties of ETEs to optimize therapeutic outcomes. Zinc- and selenium-based nanomaterials exhibit great potential in immunotherapy.^22–25 Zinc ion-mediated immunity is the key antitumor mechanism of zinc-based nanomedicines: (1) Zn ions regulate Toll-like receptor 4-mediated signaling pathways, promoting the secretion of TNF-α, IL-1β, IFN-γ, and other inflammatory factors;²⁶ (2) Zn²⁺ can also enhance the cGAS enzyme activity and assist in activating the cGAS-STING pathway by promoting the phase separation of cGAS-DNA,²⁷ stimulating the maturation of dendritic cells and antigen presentation. Zn-based nanoparticles have been widely studied as cancer metallo-immunotherapy nanoplatforms.^28–30 Se-based nanomaterials have also made substantial progress in the development of COVID-19 vaccines and tumor immunotherapy, while the underlying mechanisms have not been fully understandood.^25,31–33 One possibility is that the metabolized selenocystine participates in the synthesis of various selenoproteins and exerts immunomodulatory functions.^34–36 Therefore, ETE-based nanotherapeutics exert their immunomodulatory functions mainly via the metabolized ETE-mediated immunity.

Nevertheless, ETE-based nano-immunomodulators still face several critical challenges, including narrow zones of safety and adequate intakes of ETEs, suboptimal efficacy of nano-systems containing an individual element, as well as the insufficient elucidation of the relationship between immunomodulatory effects and metabolic behavior. Therefore, we proposed a synergistic strategy based on multiple ETEs to develop multi-elemental nano-immunomodulators with lymphatic targeting, adequate uptake, good biocompatibility and amplified immunity. In this study, we designed a lymph node-targeting zinc–selenium synergistic nanoplatform by leveraging dual-ETE-mediated immunity potentiation and albumin-facilitated targeted delivery. Ultrasmall ZnS/Se/BSA nanoclusters (ZSB NCs) were synthesized via biomineralization approach in which bovine serum albumin (BSA), as a natural biomolecule with prominent intrinsic affinity for lymphatic vessels, could effectively deliver nanoclusters to lymph nodes, promote the internalization by antigen-presenting cells (APCs) and improve the biocompatibility.^37,38 The released zinc ions and transformed selenoproteins activated the cGAS-STING pathway, effectively inducing the maturation and activation of dendritic cells (DCs). T cells were further activated, promoting the secretion of the inflammatory cytokines and inducing a cascade of immunomodulatory effects (Scheme 1). Given the importance of immunotherapy in breast cancer treatment, particularly for triple-negative breast cancer, we validated the immunotherapeutic efficacy of ZSB NCs using a BALB/c mouse 4T1 breast cancer orthotopic model. In summary, we developed an efficient nano-immunomodulator capable of inducing immune activation effects via dual ETE-mediated immunity, demonstrating its significant therapeutic potential in cancer treatment.

Scheme 1 Schematic of the ZSB NCs for synergistically augmented cancer immunotherapy via trace elements-mediated immunomodulation. (a) Illustration of the ZSB NCs preparation. ZSB NCs exerting anti-breast cancer immunotherapy by activating immunity in draining lymph nodes (b) and tumor (c). Sec: selenocysteine; SelK: selenoprotein K; and GPX4: glutathione peroxidase 4.

Results and discussion

Preparation and characterization of ZSB NCs

ZSB NCs were synthesized using a biomimetic mineralization and self-assembly method. The synthesis process involved the redox reaction and chemical binding among Zn²⁺, BSA, sodium sulfide and sodium selenite, where Zn²⁺ is bound to the BSA network, sulfides, selenides and selenites (Scheme 1a). Transmission electron microscopy (TEM) images revealed that the ZSB NCs had an average size of 3.1 nm and exhibited excellent dispersion and stability (Fig. 1a and d and Fig. S1). Next, we analyzed the composition of ZSB NCs using elemental mapping performed by field-emission transmission electron microscopy (FETEM) (Fig. 1b). Zn, Se, and S elements were spread evenly on the ZSB NCs, with these three elements being almost completely co-localized, indicating stable integration. Scanning electron microscopy (SEM) was employed to investigate the microstructure of ZSB NCs, revealing their extremely small spherical morphology (Fig. 1c). The chemical composition of ZSB NCs was further characterized with X-ray photoelectron spectroscopy (XPS) and synchrotron radiation-X-ray absorption near edge structure spectroscopy (SR-XANES). XPS analysis provided insights into the surface chemical composition of ZSB NCs, confirming the presence and valence states of Zn, Se, and S elements (Fig. 1e). Specifically, Zn was primarily represented by a pair of Zn 2p peaks at 1021.7 eV and 1045 eV, corresponding to the 2p_1/2 and 2p_3/2 spin–orbitals, respectively (Fig. 1f). XPS confirmed the presence of Se through its characteristic Se 3d_3/2 and Se 3d_5/2 peaks at 56.87 eV and 55.81 eV, respectively. In addition, a fraction of Se(IV) is observed, which is associated with the Se 3d_3/2 and Se 3d_5/2 peaks at 59.14 eV and 61.00 eV, respectively (Fig. 1g), indicating that Se is present as a mixture of Se²⁻ and SeO₃²⁻. Additionally, S was mainly represented by the S 2p orbital peak at 161.96 eV, confirming the presence of S²⁻ (Fig. 1h). As determined by SR-XANES, Zn is mainly present as Zn–cysteine (67.8%), which originated from the binding between zinc ions and the cysteine residues in BSA, and the other Zn species were sulfides (13.0%), selenides (5.8%) and selenites (13.3%). Meanwhile, Se existed in the chemical forms of selenide (31.5) and selenite (68.5) (Fig. 1i).

Fig. 1 Characterization of ZSB NCs. (a) Transmission electron microscopy (TEM) images of the ZSB NCs. Scale bar: 50 nm. (b) Field emission transmission electron microscope (FETEM) image and energy dispersive X-ray spectroscopy (EDX) elemental mapping images of the ZSB NCs. Scale bar: 100 nm. (c) Scanning electron microscopy (SEM) image of the ZSB NCs. Scale bar: 500 nm. (d) Size distribution histogram of the ZSB NCs obtained from TEM images. (e) XPS spectra of ZSB NCs. High-resolution XPS spectra of (f) Zn 2p, (g) Se 3d and (h) S 2p. (i) Zn and Se K-edge XANES (solid lines) and fitted curves (dashed lines) of the ZSB NCs.

Biodistribution and lymph nodes-targeting of ZSB NCs

To elucidate the in vivo transportation pathway and mechanism of ZSB NCs for immunomodulation, we subcutaneously injected Cy5-labeled ZSB NCs (ZSB NCs-Cy5) into the posterior neck of BALB/c mice bearing orthotopic breast cancer. To eliminate the interference from free NHS-Cy5, we validated the stability of the Cy5 fluorescence dye-labeled ZSB NCs in vitro and in vivo. ZSB NCs-Cy5 was subjected to ultrafiltration centrifugation after incubation in the systems of PBS buffer supplemented with FBS for different time periods. Fluorescence spectra of both the filtrate and the collected material were measured (Fig. S2). The results demonstrated the basically unchanged fluorescence signal of ZSB NCs-Cy5. Moreover, the fluorescence intensity of the filtrate was very weak, showing a signal that was two orders of magnitude lower than that of the nanomaterials. Meanwhile, free NHS-Cy5 was subcutaneously injected into mice, and in vivo fluorescence imaging was performed at different time points. At the same injection dose to ZSB NCs-Cy5, the biodistribution of free NHS-Cy5 showed obviously different patterns, which further confirmed the stability of ZSB NCs-Cy5 (Fig. S3). Then, the biodistribution of ZSB NCs in the mice was tracked by detecting the Cy5 signal with a small-animal optical 3D in vivo imaging system (IVIS-Spectrum), as shown in Fig. 2a. The fluorescence intensity at the injection site gradually decreased over time, revealing the translocation of ZSB NCs from the injection site. The Cy5 signal in the axillary lymph nodes appeared detectable at 2 h post-injection, reaching maximum intensity at 12 h, and subsequently weakened with time. This demonstrated the LNs-targeting delivery of ZSB NCs, which was endowed by intrinsic affinity of BSA for lymphatic vessels. Imaging from the abdominal side revealed a slight accumulation in the major organs, with a distribution trend of peaking at 1 d and a gradual reduction and almost complete disappearance at 7 d post-injection. The high accumulation in the intestine at 1 d and subsequent decrease indicated the excretion from body. The in vivo fluorescence imaging demonstrated the lymph node-targeting capability and the excretion via the intestinal system.

Fig. 2 Biodistribution of the ZSB NCs in orthotopic 4T1 tumor-bearing mice after subcutaneous injection. (a) The in vivo fluorescent imaging at different time points (0 h, 2 h, 6 h, 12 h, 1 d, 3 d, and 7 d) after subcutaneous injection with ZSB NCs-Cy5. The quantitative biodistribution of Zn (b) and Se (c) of the ZSB NCs in different organs measured by ICP-MS.

To further evaluate the lymph nodes-targeting capability, as well as their biodistribution and metabolism process, we collected and digested the major organs, tumors, lymph nodes, and skin at the injection sites from BALB/c mice with orthotopic breast cancer at 6 h, 12 h, 1 d, 3 d, and 7 d post-injection, and quantified the accumulation of Zn and Se by ICP-MS (Fig. 2b and c). After subcutaneous injection, ZSB NCs primarily accumulated at the injection site (subcutaneous tissue and muscle) and gradually decreased over time. No significant accumulation was observed in major organs such as the heart, lungs, or tumors compared to the control group at various time points. A slight accumulation was noted in the major metabolic organs, including the liver and kidney. As evidenced by ICP-MS data and fluorescence imaging (Fig. 2), ZSB NCs initially accumulated in the liver and intestine, then the amount in the hepatobiliary and intestinal system decreased, indicating clearance via the hepatobiliary pathway, which was consistent with the previous studies.^39–41 The intense and substantial accumulation in the lymph nodes, peaking at 12 h, also demonstrated the lymph node-targeting capability of ZSB NCs, in line with the in vivo fluorescence imaging. It is noteworthy that the different distribution patterns of Zn and Se at the same tissues implied the disassembly of ZSB NCs. These findings indicated that subcutaneously administered ZSB NCs achieved lymphatic-specific targeting delivery with desired body clearance and biosafety.

Spatial distribution of ZSB NCs in lymph nodes

To observe the spatial distribution of ZSB NCs in the lymph nodes, synchrotron radiation X-ray fluorescence (SR-XRF) imaging was conducted to detect the signal of Zn and Se elements in the lymph nodes collected at the indicated time points after subcutaneous injection (Fig. 3). The spatial distribution of ZSB NCs in the axillary lymph nodes (AN) and inguinal lymph nodes (IN) showed slightly different patterns, which may be attributed to the different distances of AN and IN from the injection site (subcutaneous tissue of the posterior neck). Similarly, the difference in distance also resulted in a slight variation in the accumulation amount of ZSB NCs in AN and IN measured by ICP-MS (Fig. 2b and c). The XRF images revealed that Zn and Se elements at 12 h were predominantly localized at the edge of the lymph nodes, specifically in the cortical region, which is primarily composed of antigen-presenting cells and B lymphocytes, as shown in Fig. S4. Since the observed Zn and Se signals were non-uniformly distributed in the cortical region and present in the medullary region inside the lymph nodes at 1 d, we hypothesize that ZSB NCs are mainly present in the afferent lymphatics of the lymph nodes. This spatial-temporal pattern aligns with the established migratory pathway of dendritic cells (DCs), which predominantly enter lymph nodes via afferent lymphatics during immune surveillance processes. Therefore, we speculated that ZSB NCs can be internalized by dendritic cells and subsequently drained into the lymph nodes after subcutaneous injection, where they interact with immune cells (B cells and T cells). Lymph nodes serve as critical regional command centers in the immune system and play a pivotal role in regulating immune responses. The accumulation of ZSB NCs in lymph nodes and their interaction with immune cells disclose their potential applications in immunomodulation.

Fig. 3 SR-XRF imaging of the distribution of the ZSB NCs in the lymph node at indicated time points (0 h, 6 h, 12 h, 1 d, and 7 d) after subcutaneous injection. Zn and Se in axillary lymph nodes (a) and inguinal lymph nodes (c). The color bars represent the X-ray fluorescence intensity of Se and Zn. (b and d) The bright-field images of the lymph node sections corresponding to the images in (a) and (c).

ZSB NCs in vitro immune-activating effect

Based on the aforementioned results, to verify the immunomodulatory effects of ZSB NCs, we evaluated the internalization by antigen-presenting cells (APCs) and subsequent activation of the downstream immune cells. Firstly, we assessed the cytotoxicity of ZSB NCs by co-incubating different concentrations of ZSB NCs with BMDCs and DC2.4 cells for 24 h, and analyzing the cell viability using the MTT assay (Fig. S5 and S6). The results demonstrated that the ZSB NCs exhibited no obvious cytotoxicity to both BMDCs and DC2.4 cells at concentrations of less than 0.8 µg mL⁻¹ (in terms of Zn) and had minimal impact on the cell viability at 0.5 µg mL⁻¹ of ZSB NCs. Consequently, a concentration of 0.5 µg mL⁻¹ ZSB NCs was selected for subsequent experiments.

We firstly co-incubated ZSB NCs-Cy5 with cells to observe the cellular internalization. After 12 h of incubation, the uptake of ZSB NCs by the DC2.4 cells was visualized using laser scanning confocal microscopy (LSCM). As shown in Fig. 4a, ZSB NCs-Cy5 were co-localized within lysosomes in DC2.4 cells. Additionally, flow cytometry revealed that approximately 87.6% of DC2.4 cells were detected with the signal of LysoTracker-green and NHS-Cy5, further validating the endocytosis of ZSB NCs by DC2.4 cells (Fig. 4b).

Fig. 4 Endocytosis of ZSB NCs in antigen-presenting cells (APC) and their immune activation effects in vitro. (a) LSCM image of DC2.4 after incubation with ZSB NCs-Cy5 for 12 h. Scale bar: 10 µm; (blue: nucleus, green: lysosome, and red: ZSB NCs). (b) Flow cytometry results indicating the internalization of ZSB NCs in lysosomes of DCs. (c) Flow cytometry results of DC2.4 incubated with ZB NPs, SeB NPs, ZB NPs + SeB NPs, and ZSB NCs for 24 h, indicating the activation and maturation of DCs. (d) Western blot results of DC2.4 incubated with different concentrations of ZSB NCs for 24 h and the quantification of the band intensities (e). (f) Western blot results of DC2.4 incubated with ZB NPs, SeB NPs, ZB NPs + SeB NPs and ZSB NCs for 24 h and the quantification of the band intensities. (g) Molecular weight labels on the left denote the position of the protein ladders. p-TBK1: phosphorylated TBK1 and p-IRF3: phosphorylated IRF3.

Subsequently, we investigated the immune activation effects onto DCs. DC2.4 cells were incubated with ZB NPs, SeB NPs, ZB NPs + SeB NPs and ZSB NCs (ZB NPs: ZnS/BSA NPs, SeB NPs: Se/BSA NPs, ZB NPs + SeB NPs: mixture of ZnS/BSA and Se/BSA NPs, ZSB NCs: ZnS/Se/BSA nanoclusters) for 24 hours. Flow cytometry results revealed that the ZSB NCs treatment significantly enhanced the expression of CD80 and CD86 on DC2.4 cells. (Fig. 4c), indicating the most activation and maturation of DC2.4 by the ZSB NCs. Furthermore, we examined the expression of MHCII in BMDCs after incubation with ZSB NCs, as MHCII plays a critical role in the antigen presentation. The results showed that ZSB NCs significantly upregulated the expression of MHCII in BMDCs (Fig. S7).

To elucidate the underlying mechanisms behind the activation of BMDCs by ZSB NCs, we conducted western blot analysis to investigate the role of the cGAS-STING pathway during the immunomodulatory process. As shown in Fig. 4d–g (raw data are shown in Fig. S8 and S9), the expression levels of downstream proteins TBK1, IRF3, p-TBK1 and p-IRF3 in the cGAS-STING pathway, as well as selenoproteins (Selenoprotein K (SelK) and GPX4), were significantly upregulated after the treatment with ZSB NCs. Previous studies have demonstrated the contributions of zinc ions and selenoproteins to the activation of the cGAS-STING pathway. Zn²⁺ stabilizes catalytic cGAS-DNA condensates through enhanced liquid–liquid phase separation, boosting cGAS activity and amplifying cGAMP production.²⁷ SelK promotes STING oligomerization and COPII-mediated ER-to-Golgi transport in the endoplasmic reticulum, essential for signalosome assembly.³⁴ Glutathione peroxidase 4 (GPX4) scavenges lipid peroxides to prevent STING cysteine carbonylation, ensuring functional complex trafficking.³⁵ We speculated that the immune activation effects could result from the biotransformed chemical species of ZSB NCs.

Furthermore, SR-XANES was performed to reveal the chemical biotransformation of ZSB NCs in dendritic cells. After incubation with dendritic cells for 24 h, sulfides, selenides and selenites disappeared and transformed to Zn₃(PO₄)₂-, Zn–N-, Zn–O–CO- and Zn–Cys-like species, which could be attributed to the rich cellular phosphate, imidazole, carboxyl groups and cysteine residues (Fig. 5a, c). Meanwhile, the Zn–cysteine species increased to 74.7% due to the strong chelating between Zn²⁺ and cysteine-rich proteins.⁴² The biotransformation of Zn in ZSB NCs is consistent with previous reported results.⁴³ In ZSB NCs, Se exists in the chemical form of selenide and selenite (Fig. 5b), which is consistent with the analysis results of the Se valence state by XPS (Fig. 1g). In dendritic cells, the contents of selenides and selenites decreased and metabolized into selenocystine, an essential component in the biosynthesis of selenoproteins, which is in line with previous findings about the metabolism of selenium nanoparticles.^25,44 The biotransformation of Se in ZSB NCs promoted the upregulated expression of SelK and GPX4 in dendritic cells (Fig. 4d). The biodegraded Zn and Se significantly contribute to the immune activation effects of ZSB NCs.

Fig. 5 Chemical biotransformation of the ZSB NCs in DCs. Zn (a) and Se (b) K-edge XANES (solid lines) and fitted curves (dashed lines) of ZSB NCs and the DCs incubated with ZSB NCs. (c) Chemical forms and ratio of the Zn and Se species in the ZSB NCs and the DCs incubated with ZSB NCs, fitted from the XANES spectra.

ZSB NCs in vivo tumor immunotherapy efficacy

Breast cancer is the most prevalent type of cancer in women, with triple-negative breast cancer (TNBC) being particularly challenging due to the negative expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Immunotherapy has recently attracted growing attention and significance in addressing TNBC. An orthotopic breast cancer mouse model was utilized to evaluate immunotherapy effects of ZSB NCs. This model exhibits invasive characteristics, poor immunogenicity, and spontaneous metastasis to distant organs.

At the third, sixth, and ninth day after cancer cells inoculation, the mice were administered with ZSB NCs, ZnS/BSA NPs (ZB NPs), Se/BSA NPs (SeB NPs), or PBS via subcutaneous injections (with the same dosage in terms of Zn or Se; Zn: 4.0 mg kg⁻¹, Se: 0.27 mg kg⁻¹) (Fig. 6a). The combination of Zn with Se sharply reduced the dosage of Se by 50–80% compared to the dosage of Se NPs used in previously published studies (0.5–1 mg kg⁻¹),^25,45 beneficial for the improved biosafety. The body weight and tumor volume were continuously monitored throughout the time period. There is no significant reduction in the body weight compared with the normal group, indicating acceptable biosafety of all three nanoparticles (Fig. 6b). The tumor growth curve revealed that the ZSB NCs group significantly inhibited tumor growth, while the other three groups showed no significant suppressive effect. At the end of the experiment, tumors were excised, photographed, and weighed, with the tumor weight and visual size consistent with the tumor volume monitoring results (Fig. 6c–i). Subsequently, hematoxylin and eosin (H&E) staining and Ki67 immunohistochemistry of the tumor demonstrated that the ZSB NCs group induced the highest levels of tumor cell death (Fig. 6j) and significantly inhibited tumor cell proliferation compared to other groups (Fig. 6k). Overall, subcutaneous injection of ZSB NCs exhibited notable immunotherapeutic effects in the 4T1 orthotopic breast cancer mouse model. Leveraging the synergistic effect allows for a dose reduction while maintaining biosafety and efficacy simultaneously, thereby improving the overall risk-benefit profile.

Fig. 6 Therapeutic efficacy of ZSB NCs against orthotopic 4T1 tumor-bearing mice. (a) Schematic of the treatment process of tumor-bearing mice. (b) Body weight and (c) tumor volumes after different treatments. Data are represented as mean ± S.D., n = 5. (d) Photographs and weight of dissected tumors of different groups. Data are represented as mean ± S.D., n = 5. (f–j) Tumor growth curves of different groups. (j) H&E staining and (k) Ki67 staining of tumor tissues after different treatment. Scale bar: 500 µm. Statistical comparisons were tested using ordinary one-way analysis of variance (ANOVA) with Tukey's multiple-comparisons test. ****P < 0.0001, ***P < 0.001, and **P < 0.01.

ZSB NCs tumor immunotherapy mechanism study

It has been validated that ZSB NCs efficiently activate the BMDCs and DC2.4, but the underlying mechanism for the anti-tumor immune response in vivo also needs to be clarified. We firstly investigated whether ZSB NCs could promote the maturation and activation of DCs in lymph nodes. As shown in Fig. 7a, the proportion of MHCII⁺ DCs in the ZSB NCs group was significantly higher than that in the ZB NPs, SeB NPs and PBS, suggesting that ZSB NCs remarkably enhanced the antigen-presenting capability of DCs in lymph nodes. Additionally, as shown in Fig. 7b, the surface markers CD80 and CD86 on DCs in the lymph nodes of mice treated with ZSB NCs were significantly upregulated compared to other groups. These findings demonstrate that ZSB NCs exhibit superior efficacy compared to ZB NPs and SeB NPs in inducing dendritic cell (DC) maturation and activation in vivo, thereby initiating downstream adaptive immune responses. Subsequently, we further assessed the impact of ZSB NCs on local or systemic immune activation by analyzing the proportions of CD4⁺ and CD8⁺ T cells in lymph nodes. Flow cytometric analysis revealed that ZSB NCs induced marked elevation in CD4⁺ and CD8⁺ T-cell ratios compared to other groups (Fig. 7c and d), indicating that ZSB NCs exhibit potentiated immunocompetence in driving T-lymphocyte maturation and proliferation in vivo.

Fig. 7 Immune response of the ZSB NCs in lymph nodes determined by flow cytometry. (a and b) Flow cytometry analysis and quantitative result of the percentages of maturated DCs in the LNs of different groups. Flow cytometry analysis and quantitative result of the percentages of (c) CD4⁺ T lymphocytes and (d) CD8⁺ T lymphocytes in mice with different treatments (n = 3). Data are represented as mean ± S.D., n = 3. Statistical comparisons were tested using ordinary one-way analysis of variance (ANOVA) with Tukey's multiple-comparisons test. ****P < 0.0001, ***P <0.001, and **P < 0.01.

To further verify the inhibitory effect of ZSB NCs on tumor growth, we performed immunohistochemistry (IHC) staining of tumor sections to detect CD8⁺ T cells and CD4⁺ T cells (Fig. 8a). Additionally, flow cytometry was used to analyze the proportions of CD4⁺ T cells and CD8⁺ T cells in the tumor. The results demonstrated that after ZSB NCs exerted an immune-activating effect in the lymph nodes, activated T cells were recruited to the tumor site, leading to a significant increase of CD4⁺ T cells and CD8⁺ T cells in the tumor, which is useful for effectively inhibiting tumor growth (Fig. 8b and c). Compared with other groups, increased proportions of CD4⁺ T cells and CD8⁺ T cells were observed in tumors after treatment with ZSB NCs. Notably, this infiltration was more pronounced for CD4⁺ T cells in tumor tissues than in lymph nodes. Given this distinct recruitment, we measured the serum chemokine levels and found that ZSB NCs induced the most potent CXCL10 expression (Fig. 8d), indicating that ZSB NCs enhance T cell infiltration in tumors by promoting CXCL10-mediated recruitment. The increased proportion of CD45⁺ cells at the tumor site also demonstrated robust immune cell recruitment, which in turn suggested that the tumor immune activation is induced by ZSB NCs (Fig. S11).

Fig. 8 Immune response of ZSB NCs in the tumor. (a) Immunohistochemical images of tumor sections in different groups. Flow cytometry analysis and quantitative result of the percentages of (b) CD4⁺ T lymphocytes and (c) CD8⁺ T lymphocytes in tumors of different groups (n = 3). (d–g) CXCL10, IFN-γ, IL-1β and TNF-α in serum measured by ELISA. Statistical comparisons in (b–g) were tested using ordinary one-way analysis of variance (ANOVA) with Tukey's multiple-comparisons test. ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05.

The inhibitory effect of T lymphocytes on tumor growth is closely associated with the secretion of anti-tumor cytokines such as TNF-α, IFN-γ, and IL-1β, which are indicators of the body's anti-tumor immunity. Therefore, we measured the levels of TNF-α, IFN-γ, and IL-1β in the serum of mice from different treatment groups using ELISA. As shown in Fig. 8e–g and Fig. S10, in the ZB NPs, SeB NPs, and ZSB NCs groups, the levels of TNF-α, IFN-γ, and IL-1β were simultaneously higher than those in the PBS group. Among them, the ZSB NCs-treated group exhibited the highest levels of TNF-α, IFN-γ, and IL-1β expression in the serum, which also validates the more efficient tumor suppression effect in the ZSB NCs group than other nanoparticles.

Biosafety assessment

We subsequently evaluated the in vivo and in vitro biosafety of ZSB NCs. We firstly evaluated the cytotoxicity of ZSB NCs to skin cells (HSF cells) and 4T1 tumor cells. After incubation with HSF cells and 4T1 cells for 1 day, ZSB NCs showed no obvious cytotoxicity to HSF cells and 4T1 cells at the experimental concentration (0.5 µg mL⁻¹) (Fig. S12 and S13). Therefore, tumor growth inhibition owing to the nanoparticles’ cytotoxicity to tumor cells was excluded. Furthermore, to assess their biocompatibility in vitro, DC2.4 cells were co-incubated with ZB NPs, SeB NPs, ZB NPs + SeB NPs, and ZSB NCs. The results demonstrated that ZSB NCs exhibited the highest biosafety under equivalent elemental concentrations (Fig. S14). Subsequently, histological analysis showed no obvious damage or inflammatory response in the major organs of mice (heart, liver, spleen, lung, kidney, and lymph nodes) after subcutaneous injection of ZSB NCs (Fig. S15). The above results indicated the biocompatibility and acceptable biosafety of ZSB NCs.

Conclusion

In summary, we designed ultrasmall ZSB NCs with synergistic immune activation, excellent target delivery capability, good dispersibility and biosafety. Subcutaneously injected ZSB NCs can accumulate in lymph nodes, promote dendritic cells maturation, and further stimulate T cell activation to achieve immunomodulatory effects. The multi-ETE synergistic nanoplatform realized the targeted co-delivery of Zn and Se to the lymph nodes and augmented immunomodulation with decreased dosage, ensuring the biosafety and efficacy simultaneously. Zinc ions and selenoproteins (SelK and GPX4) derived from the degradation of ZSB NCs participated in the immunomodulatory process, including the activation of the cGAS-STING pathway and the cascade immune responses. In the 4T1 orthotopic breast cancer mouse model, ZSB NCs exhibited superior tumor immunotherapy efficacy compared to ZB NPs and SeB NPs alone. The excellent immunomodulatory efficacy and biocompatibility of ZSB NCs demonstrated the significant potential of multi-ETE-based nanoplatforms for cancer immunotherapy with an expanded therapeutic window, meriting further evaluation to advance clinical translation. This synergistic nanoplatform could also offer a promising strategy for engineering next-generation vaccine adjuvants targeting malignancies, viral infections, and influenza in the future.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting the article have been included as part of the supplementary information (SI). SI contains detailed experimental methods and supplementary figures. Fig. S1: in vitro stability of ZSB NCs; Fig. S2–S3: stability and in vivo biodistribution of ZSB NCs-Cy5; Fig. S4: the schematic diagram of immune cell subtypes in a lymph node cross-section; Fig. S5–S6: cytotoxicity of ZSB NCs to dendritic cells; Fig. S7–S9: activation effect of ZSB NCs on dendritic cells in vitro; Fig. S10–S11: immune activation induced by ZSB NCs at the tumor site; Fig. S12–S13: cytotoxicity of ZSB NCs to HSF cells and 4T1 cells; Fig. 14: cytotoxicity of ZB NPs, SeB NPs, ZB NPs+ SeB NPs, and ZSB NCs to dendritic cells; Fig. S15: hematoxylin and eosin (H&E) staining images. See DOI: https://doi.org/10.1039/d5nh00372e.

Acknowledgements

This work has received support from the National Natural Science Foundation of China (32401190 and 22027810), the National Key R&D Program of China (2021YFA1200900 and 2023YFA1610200) and the New Cornerstone Science Foundation (NCI202318).

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