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American Association for the Advancement of Science

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10.1126/sciadv.adn3002
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Applied Sciences and Engineering
Hybrid nanoparticle–mediated simultaneous ROS scavenging and STING activation improve the antitumor immunity of in situ vaccines
Hybrid in situ nanovaccine boosts antitumor immunity
https://orcid.org/0009-0007-8608-3319
Li Jianing Conceptualization Data curation Formal analysis Investigation Methodology Project administration Resources Software Supervision Validation Visualization Writing - original draft Writing - review & editing 1
Wu Tianze Data curation Formal analysis Investigation Methodology Resources Software Validation Visualization 1
https://orcid.org/0009-0003-1777-3704
Wang Weidong Conceptualization Data curation Formal analysis Investigation Methodology Project administration Resources Software Supervision Validation Visualization Writing - original draft Writing - review & editing 1
https://orcid.org/0000-0002-4749-1420
Gong Yimin Conceptualization Formal analysis Resources Software Visualization 1
Lu Mingzhu Conceptualization Data curation Formal analysis Investigation Methodology Project administration Resources Software Supervision Validation Visualization Writing - original draft Writing - review & editing 1
Zhang Mengmeng Formal analysis Investigation Project administration Resources Visualization Writing - original draft Writing - review & editing 1
Lu Wanyue Formal analysis Methodology Project administration Resources Validation Visualization 1
https://orcid.org/0000-0003-2720-9536
Zhou Yaming Conceptualization Data curation Formal analysis Funding acquisition Methodology Project administration Resources Supervision Validation Writing - review & editing 1 *
https://orcid.org/0000-0001-6696-3879
Yang Yannan Conceptualization Data curation Funding acquisition Methodology Project administration Resources Supervision Validation Visualization Writing - original draft Writing - review & editing 2 3 *
1 Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials, Department of Chemistry, Fudan University, Shanghai 200433, China.
2 Institute of Optoelectronics, Fudan University, Shanghai 200433, China.
3 South Australian immunoGENomics Cancer Institute, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, South Australia 5005, Australia.
* Corresponding author. Email: yannan.yang@adelaide.edu.au (Y.Y.); ymzhou@fudan.edu.cn (Y.Z.)
20 9 2024
18 9 2024
10 38 eadn300203 12 2023
12 8 2024
Copyright © 2024 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).
2024
The Authors
https://creativecommons.org/licenses/by-nc/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

In situ vaccine (ISV) is a versatile and personalized local immunotherapeutic strategy. However, the compromised viability and function of dendritic cells (DCs) in a tumor microenvironment (TME) largely limit the therapeutic efficacy. We designed a hybrid nanoparticle–based ISV, which accomplished superior cancer immunotherapy via simultaneously scavenging reactive oxygen species (ROS) and activating the stimulator of interferon genes (STING) pathway in DCs. This ISV was constructed by encapsulating a chemodrug, SN38, into diselenide bond–bridged organosilica nanoparticles, followed by coating with a Mn2+-based metal phenolic network. We show that this ISV can activate the STING pathway through Mn2+ and SN38 comediated signaling and simultaneously scavenge preexisting H2O2 in the TME and Mn2+-catalyzed •OH by leveraging the antioxidant property of diselenide and polyphenol. This ISV effectively activated DCs and protected them from oxidative damage, leading to remarkable downstream T cell activation and systemic antitumor immunity. This work highlights a nanoparticle design that manipulates DCs in the TME for improving the ISV.

An in situ nanovaccine simultaneously manipulates STING signaling and oxidative stress of intratumoral dendritic cells.

http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 22275037 Australia National Health and Medical Research Council 2010100
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pmcINTRODUCTION

Cancer vaccines are one of the most important immunotherapeutic strategies and has reshaped the landscape of cancer treatment over the past decade (1, 2). As a unique type of vaccination strategy, in situ cancer vaccines are pursued to harness endogenous antigens from tumors to elicit systemic cytotoxic T lymphocyte (CTL) response to eradicate both primary and distant/metastatic tumors (3–5). Natural or engineered oncolytic viruses have been the most widely used approach for constructing in situ vaccines (ISVs) against cancer (6, 7). Despite their excellent efficacy in enhancing tumor immunogenicity, the activation of systemic antitumor immune responses and the accompanied adverse effects pose notable concerns for their clinical use (8). Recently, chemotherapy, radiotherapy, photodynamic/photothermal therapy, and other therapeutics are applied in ISVs to induce the release of tumor antigens in situ to provoke antitumor immune responses but usually with insufficient immune efficacy (9–13).

It has been well investigated that a tumor microenvironment (TME) in solid tumors play vital roles in determining the efficacy of immunotherapy, which demands accurate activation of a complex immunological system at multiple spacetime (14, 15). Dendritic cells (DCs) are considered to be the most potent antigen-presenting cells (APCs) that play crucial roles in coordinating innate and adaptive immunity (16, 17). However, the overproduction of reactive oxygen species (ROS) in the TME largely compromises the viability and function of DCs as well as subsequent T cell response in tumors, contributing to the immunosuppression in cold tumors (18, 19). In addition, regulating the intracellular environment of DCs to initiate cascade immunosignaling is a crucial approach to boost immune responses (20–22). Stimulator of interferon genes (STING) is a cytosolic pattern recognition receptor that stimulates innate immune responses (23, 24). It has been known that certain chemotherapeutic approaches (e.g., camptothecin, cisplatin, and topotecan) can induce DNA damage, which can potentially activate cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS) and STING (cGAS-STING) pathway (25, 26). Besides, manganese ions (Mn2+) are also reported to directly activate the cGAS-STING pathway by improving the sensitivity of cGAS for double-stranded DNA and enhancing the affinity of STING for various cyclic dinucleotides (27, 28), but the Fenton-like catalytic activity of Mn2+ is likely to increase the ROS level and induce DC immunosuppression (29). Given the aforementioned research progress in tumor immunology, we hypothesize that simultaneously activating DCs and protecting them from oxidative damage would cooperatively improve the therapeutic outcome of the ISV.

To test our hypothesis, in the present work, we designed a hybrid nanoparticle–based ISV (nano-ISV). This hybrid nano-ISV was constructed by encapsulating SN38 (7-ethyl-10-hydroxycamptothecin), a DNA-targeting chemodrug, into diselenide bond–bridged mesoporous organosilica nanoparticles (Se-MONs) and then coated with metal phenolic networks (MPNs) consisted of Mn2+ and epigallocatechin gallate (EGCG) (denoted Se-MON@SN38@MPN) (Fig. 1A). Therefore, this rationally designed ISV encompasses two major modules: STING activation module and ROS scavenging module. In response to the acidic TME, the STING module was initiated, releasing SN38 and Mn2+ ions from MPNs. The former killed tumor cells and caused release of damaged DNA, which, together with the latter, promoted the maturation of DCs by activating the cGAS-STING signaling pathway. Concomitantly, the ROS scavenging module, i.e., diselenide groups and EGCG molecules, effectively deplete H2O2 in the TME as well as the Mn2+-mediated production of free radicals, consequently protect DCs from oxidative damage, and facilitate reversal of the immunosuppression in the TME (Fig. 1B). Owing to the cooperative working mechanism of these two modules, in situ vaccination with Se-MON@SN38@MPN not only resulted in a localized antitumor effect but also, when combined with anti-programmed death-ligand 1 (aPD-L1) immune checkpoint blockade (ICB), elicited potent systemic antitumor immunity for suppressing the progression of distant tumors.

Fig. 1. Schematic illustration of the synthesis and biological mechanism of Se-MON@SN38@MPN in situ nanovaccine for cancer immunotherapy.

(A) Preparation process of the hybrid in situ nanovaccine. (B) Schematic illustration of nanovaccine-mediated ROS scavenging and activation of the cGAS-STING pathway for antitumor immunotherapy. dsDNA, double-stranded DNA; ER, endoplasmic reticulum; cGAMP, cyclic guanosine monophosphate–adenosine monophosphate; NF-κB, nuclear factor κB.

RESULTS

Preparation and characterization

Se-MONs with a high Se density (9.7%) were fabricated by a previously reported sol-gel method (Fig. 2A) (30). Transmission electron microscopy (TEM) (Fig. 2B) image and scanning electron microscopy (SEM) (fig. S1) of Se-MONs revealed the spherical morphology and mesoporous structure with mesopores. A uniform particle size of ~60 nm was also observed. Furthermore, the energy-dispersive x-ray (EDX) elemental mapping images showed that the elements of Si, O, and Se signals were uniformly distributed in Se-MONs (Fig. 2C). The solid-state nuclear magnetic resonance spectra of Se-MONs revealed five peaks of bis[3-(triethoxysilyl)propyl]diselenide (BTESePD), indicating the successful doping of Se-Se (fig. S2). N2 adsorption-desorption isotherms revealed the pore size of 12 nm, Brunauer-Emmett-Teller surface areas of 505 m2 g−1, and the pore volume of 1.3 cm3 g−1 (Fig. 2, D and E).

Fig. 2. Preparation and characterization of Se-MON@SN38@MPN.

(A) Synthetic route for Se-MONs. (B) Representative TEM image of Se-MONs. (C) TEM mapping image of Se-MONs indicated the distribution behaviors of Si, O, and Se. (D) N2 adsorption-desorption isotherms of Se-MONs. (E) Pore size distribution of Se-MONs. (F) Representative TEM image of Se-MON@SN38@MPN. (G) Representative SEM image of Se-MON@SN38@MPN. (H) Zeta potentials of Se-MONs, Se-MON@SN38, Se-MON@SN38@MPN. Means ± SEM, n = 3. (I) Time-dependent release curves of Mn2+ from Se-MON@SN38@MPN incubated in various pH buffer solutions. (J) Time-dependent release curves of SN38 from Se-MON@SN38@MPN incubated in various pH buffer solutions.

We selected SN38 as the chemodrug for constructing the ISV because this drug was identified as the most potent drug for stimulating interferon-β (IFN-β) secretion via activating the STING pathway in comparison to other DNA-targeting chemodrugs such as doxorubicin, cisplatin, and topotecan (25). SN38 loading was performed in a dimethyl sulfoxide (DMSO) solution (Se-MON@SN38), and the loading capacity of SN38 in Se-MONs was 69 μg mg−1 (fig. S3). Next, Se-MON@SN38@MPN was prepared by mixing the Mn2+ and EGCG in a basic solution. TEM images of MPN coating under different concentrations of Mn2+ and EGCG are shown in fig. S4, which confirmed that a thin layer was formed on the surface of Se-MONs. We chose an optimal content of MPNs, i.e., 2.5% for Mn2+ and 2.3% for EGCG (fig. S5), in which the release of drugs was not considerably blocked while the amounts of Mn2+ and EGCG were sufficient to exert biological functions. Characterizations of TEM, SEM, and zeta potentials of Se-MON@SN38@MPN were performed (Fig. 2, F to H). EDX mapping images showed that Mn signals were uniformly distributed in Se-MON@SN38@MPN (fig. S6). Moreover, the product appears pink in color, which is due to the presence of Mn2+ (fig. S7). It has been reported that the coordination between Mn2+ and polyphenols is pH-sensitive (31, 32). Under pH = 6.5 (mimicking the acidic TME), the MPN coating layer was rapidly degraded, which led to the release of Mn2+ and SN38 (Fig. 2, I and J). In addition, dynamic light scattering (DLS) measurements showed that the nanoparticles exhibited an average of ~200 nm in hydrodynamic particle size (fig. S8), indicating their excellent physiological stability after being incubated under different pH conditions. Considering the diselenide bond structure in the ISV, we also proved that the release of SN38 would be more under the acidic glutathione (GSH) conditions due to the degradation of Se-Se (figs. S9 and S10).

In vitro cytotoxicity and STING activation

SN38 is a fluorescent drug with the maximum excitation wavelength at 408 nm and the maximum emission wavelength at 426 nm (fig. S11). To investigate the cellular uptake of the nano-ISV, SN38 fluorescence was used to detect the cellular uptake of the nano-ISV without any additional marker, and confocal laser scanning microscopy (CLSM) was used to track the internalization of drugs into 4T1 cells (fig. S12). The results revealed that stronger fluorescent signals could be observed intracellularly for Se-MON@SN38 and Se-MON@SN38@MPN compared to free SN38, suggesting that these hybrid nanoparticles could effectively promote the uptake of drugs. We next assessed the cytotoxicity of Se-MON@SN38@MPN to tumor cells. As shown in Fig. 3A, while Se-MON@MPN was well tolerated by tumor cells, Se-MON@SN38 and Se-MON@SN38@MPN showed potent inhibitory activity to 4T1 cells at the concentrations above 125 μg/ml, which was due to the cytotoxicity of SN38 (fig. S13). The slightly lower cytotoxicity of Se-MON@SN38 than Se-MON@SN38@MPN is due presumably to a premature drug leakage. Furthermore, calcein AM/propidium iodide (PI) staining (Fig. 3B and fig. S14) in 4T1 cells showed a similar trend. Se-MON@SN38@MPN exhibited the most effective anticancer activity as evidenced by the high portion of PI-stained cells. Annexin V–fluorescein isothiocyanate (FITC)/PI double staining assay further confirmed that Se-MON@SN38@MPN induced a considerably enhanced apoptosis rate in 4T1 cells (Fig. 3, C and D, and fig. S15). In addition, the cell viability study of the nano-ISV in a mouse fibroblast cell line (NIH/3T3) and a human umbilical vein endothelial cell (HUVEC) showed limited cytotoxicity at a dosage below 125 μg/ml (fig. S16).

Fig. 3. In vitro cytotoxicity and STING activation of Se-MON@SN38@MPN.

(A) Cell viability of 4T1 cells after 48-hour treatment of various formulations. Means ± SEM, n = 3. (B) CLSM images of calcein-AM/PI double-stained 4T1 cells treated with control (Ctrl), Se-MON@MPN, Se-MON@SN38, and Se-MON@SN38@MPN for 48 hours. The calcein green fluorescence and PI red fluorescence indicated living and dead cells, respectively. (C and D) Apoptosis levels determined by flow cytometry of 4T1 cells treated with various formulations for 48 hours. Means ± SEM, n = 2. (E) Schematic illustration of the coincubation experiment of 4T1 and DC2.4 cells. NPs, nanoparticles. (F) Western blot images indicated the expression levels of IRF-3 and P-IRF3 extracted from DC2.4 cells after incubation with the 4T1 supernatant medium for 24 hours. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (G) Fold increase in IFN-β concentration in supernatants of DC2.4 incubated with the 4T1 supernatant medium for 24 hours. Means ± SEM, n = 3. (H and I) Flow cytometry analysis and the corresponding quantification of maturation markers CD80. Means ± SEM, n = 3. (J and K) Flow cytometry analysis and the corresponding quantification of maturation markers CD86. Means ± SEM, n = 3. (P values were determined by unpaired Student’s t tests, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.)

It has been known that Mn2+ could directly activate the cGAS-STING pathway to promote the secretion of type I IFNs (IFN-I) and proinflammatory cytokines, such as IFN-β, tumor necrosis factor–α (TNF-α), and interleukin-6 (IL-6) (28, 33). Moreover, SN38 could induce more DNA damage and DNA cytosol leakage, leading to STING activation (25). To test if Mn2+ and SN38 cotreated tumor cells can stimulate the activation of the cGAS-STING pathway in immune cells, we collected the conditioned culture medium of 4T1 cells treated with different formulations and used this conditioned culture medium to culture DC2.4 cells for another 24 hours of incubation (Fig. 3E). Then, we investigated the STING pathway–related proteins expression in DC2.4 cells after various treatments via Western blotting. As shown in Fig. 3F and fig. S17, the downstream phosphorylated interferon regulatory factor 3 (IRF-3) (P-IRF3) protein expression was increased in the Se-MON@SN38@MPN group. Enzyme-linked immunosorbent assay (ELISA) tests of DC2.4 supernatants showed that the secretion of IFN-β in the Se-MON@SN38@MPN group increased (Fig. 3G), indicating that the conditioned culture medium of 4T1 treated with Mn2+- and SN38-containing nanoparticles could synergistically elicit cGAS-STING pathway activation in DCs. Furthermore, the conditioned culture medium of Se-MON@SN38@MPN–treated 4T1 cells resulted in elevated expression of CD80 and CD86 on DC2.4 cells (Fig. 3, H to K), suggesting an increased maturation of DCs.

ROS scavenging capability

We next studied the ROS scavenging capability of the nano-ISV. H2O2 is the major type of ROS in the TME and is overexpressed therein. It is known that Se-Se can reduce H2O2 and be converted to seleninic acid (34), thus is able to eliminate H2O2 in the TME. The ability of Se-MONs and Se-MON@MPN to consume H2O2 was investigated (Fig. 4, A and B). Both of them showed a dose-dependent H2O2 consumption behavior. Notably, at the concentration above 500 μg/ml, Se-MON@MPN could consume more than 80% of H2O2, suggesting their strong ROS scavenging ability. In addition, the mesoporous structure of Se-MONs was gradually collapsed upon reaction with H2O2 due to the cleavage of the Se-Se bond (fig. S18). It is worth mentioning that EGCG also showed a moderate reactivity with H2O2, leading to an enhanced H2O2 consumption capability of Se-MON@MPN compared to Se-MONs. To investigate the radical ROS scavenging capability, terephthalic acid (TA) was used, which can be oxidized into fluorescent 2-hydroxyterephthalic acid (TAOH) in the presence of reactive hydroxyl radicals (•OH), and the level of •OH can be quantified based on the fluorescence intensity (35). As shown in Fig. 4C, Mn2+ could act as a Fenton-like catalyst to convert H2O2 into •OH. However, the addition of Se-MONs or EGCG largely reduced the production of •OH, and Se-MON@MPN exhibited the highest capacity in •OH removal, confirming its ROS scavenging capability.

Fig. 4. In vitro and in vivo ROS scavenging capability.

(A and B) H2O2 depletion induced by Se-MONs, EGCG, and Se-MON@MPN. Means ± SEM, n = 2. (C) Fluorescence intensity of various formulations after reacting with TA for 12 hours. Means ± SEM, n = 3. (D) Cell viability of DC2.4 cells after 24-hour treatment of different formulations. Means ± SEM, n = 3. (E) Quantitative measurement by using a microplate reader of the intracellular ROS level in DC2.4 cells after 2-hour treatment of various formulations and H2O2. Means ± SEM, n = 3. (F) Cell viability of DC2.4 cells after 6-hour treatment of various formulations and H2O2. Means ± SEM, n = 3. (G) CLSM images of the intracellular ROS level in DC2.4 cells after 2-hour treatment of various formulations and H2O2. The green fluorescence is generated by the oxidized ROS probe. (H) Mean fluorescence intensity (MFI) of the ROS probe in CLSM images. Means ± SEM, n = 2. (I) ROS (red, ROS; blue, 4′,6-diamidino-2-phenylindole) levels in the tumor tissue of the 4T1 xenograft tumor model. (J) Flow cytometry quantification of the MFI of ROS in the tumor tissue of the 4T1 xenograft tumor model. Means ± SEM, n = 3. (P values were determined by unpaired Student’s t tests, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.)

Next, we investigated the cytotoxicity of various formulations to DC2.4 cells. As shown in Fig. 4D, compared to Se-MONs, the addition of Mn2+ (Se-MON+Mn2+) caused higher cytotoxicity, whereas the inclusion of EGCG in the formulation (i.e., Se-MON@MPN and Se-MON@SN38@MPN) increased the cell viability. To evaluate the ROS scavenging capacity in the intracellular environment of immune cells, various formulations were incubated in DC2.4 cells in the presence of H2O2, and 2′,7′-dichlorofluorescein (DCF) was used as the intracellular ROS indicator. After incubation for 2 hours, Mn2+ ions significantly increased the fluorescent signal of DCF owing to their Fenton-like catalytic activity (Fig. 4E). However, the fluorescent signal of DCF was significantly decreased by Se-MON@MPN treatment, indicative of reduced intracellular oxidative stress as a result of ROS scavenging. This, in turn, effectively rescued DC2.4 cells (Fig. 4F), and the ROS scavenging of Se-MON@MPN is comparable to that of a molecular antioxidant, GSH (fig. S19). The ROS scavenging effect of Se-MON@MPN as reflected by the decreased DCF fluorescence in DC2.4 cells was further confirmed by CLSM (Fig. 4, G and H).

The ROS scavenging capability was further tested in a tumor model, and DCF was used as the indicator. As shown in Fig. 4I, compared with the control group and Se-MON+Mn2+ group, an obvious decrease in the ROS level was observed after treatment with Se-MONs and Se-MON@MPN. Flow cytometry provided quantitative analysis of the intratumoral ROS, showing that the oxidative stress in the tumor was significantly decreased upon Se-MON@MPN treatment (Fig. 4J). These results collectively evidence the Se-MON@MPN-mediated effective ROS elimination in the TME.

Antitumor activity of Se-MON@SN38@MPN

To investigate the antitumor effect of the nanoformulations, a 4T1 xenograft tumor model in BALB/c mice was established by subcutaneously injecting 2.0 × 106 4T1 cells (Fig. 5A). The intratumoral injection allows much higher concentrations of drugs or adjuvants in the TME to improve the effect of immunotherapy while limiting the risk of systemic exposure and associated toxicity. Notably, neutrophils and macrophages are prominent phagocytes that act as contributors to the in vivo clearance of injected nanoparticles. We investigated the uptake of FITC-modified Se-MON@SN38@MPN by neutrophils and macrophages in the tumors. As represented in figs. S20 and S21, only less than 6% of these cells showed uptake of nanoparticles, indicating that these phagocytes had a minimum impact on the interaction between the nano-ISV and their target cells, i.e., DCs and tumor cells.

Fig. 5. Antitumor activity of Se-MON@SN38@MPN.

(A) Schematic illustration of the timeline for 4T1 subcutaneous tumor model establishment and cancer immunotherapy. Mice were assigned to four groups as follows: (i) PBS, (ii) SN38, (iii) Se-MON@SN38, (iv) Se-MON@MPN, and (v) Se-MON@SN38@MPN. s.c., subcutaneous; i.t., intratumoral. (B) Sizes changes of tumors since first administration. Means ± SD, n = 6. (C) Photograph of dissected 4T1 tumors at the end point. (D) Body weight changes of mice since first administration. Means ± SD, n = 6. (E) Individual mouse tumor growth profile. (F) Representative H&E images and tumor immunofluorescence images of each group after being stained with CD4 (red), CD8 (red), and p-STING (red). (P values were determined by one-way ANOVA with a Tukey post hoc test, **P < 0.01 and ****P < 0.0001.)

When the tumor volume reached around 50 mm3, the mice were randomly divided into five groups: phosphate-buffered saline (PBS), SN38, Se-MON@SN38, Se-MON@MPN, and Se-MON@SN38@MPN. The mice were intratumorally injected four times with different formulations every 3 days (days 0, 3, 6, and 9). As shown in Fig. 5 (B, C, and E), the free SN38 treatment only resulted in a moderate tumor suppression. The Se-MON@SN38 showed an enhanced inhibition of the tumor growth, which is attributable to a controlled release of SN38 and ROS scavenging property of Se-MONs. Moreover, the Se-MON@SN38@MPN shows the most potent tumor growth inhibition due to the cooperative effect of Mn2+/SN38 comediated STING activation and EGCG/Se-Se comediated ROS scavenging. The survival time in each group correlated well with the tumor inhibition result (fig. S22). Meanwhile, the body weight of mice in all five groups showed no significant changes during the treatment process, indicating good biosafety of the nano-ISV (Fig. 5D). The biodistribution and intratumoral retention profile of the nano-ISV was then evaluated by detecting the Si content in the major organs and tumors (fig. S23). It was found that the nano-ISV remained inside tumors at 3 days after intratumoral injection and there is almost no distribution in the organs. Hematoxylin and eosin (H&E) staining suggested that Se-MON@SN38@MPN induced extensive necrosis of tumor cells (Fig. 5F). The immunofluorescence staining suggests that the expression of p-STING and the infiltration of CD4 and CD8 T cells in tumors were considerably elevated in the Se-MON@SN38@MPN group compared with other groups, suggesting the activation of STING signaling and T cell immunity (Fig. 5F and fig. S24).

In addition, tumor metastasis or recurrence account for major cancer deaths. To demonstrate if our nano-ISV can inhibit lung metastasis, the lungs were collected after various treatments and the number of pulmonary metastatic nodules in the lungs was quantified. Our results show that the mice treated with Se-MON@SN38@MPN showed a significantly less number of metastatic lung nodules than other groups, indicating that the ISV effectively inhibited lung metastasis (fig. S25).

Antitumor activity of the ISV in a bilateral tumor model

Encouraged by the excellent antitumor performance of Se-MON@SN38@MPN, we next aimed to understand if the nano-ISV could elicit robust systemic immune response to suppress distant/metastatic tumors. We intended to seek for a suitable ICB to improve the therapeutic efficacy, and we found that the Se-MON@SN38@MPN induced significant up-regulation of programmed death-ligand 1 (PD-L1) in 4T1 cells (fig. S26). A study has shown that enhanced expression of PD-L1 in tumor cells can promote the sensitivity of tumor cells toward aPD-L1 immunotherapy (36); thus, we explored if combining the nano-SIV with aPD-L1 could lead to an optimal treatment outcome.

A 4T1 bilateral tumor model was established by subcutaneous injection of 4T1 cells into the left and right flank regions of the BALB/c mice (Fig. 6A). The tumors at the left flank were defined as the primary tumor and received ISV treatment, while the tumors at the right flank were defined as the distant tumor and did not receive such a treatment. When the primary tumor sizes increased to ~50 mm3, the tumor-bearing mice were divided into five groups (PBS, aPD-L1, SN38 + aPD-L1, Se-MON@SN38@MPN, and Se-MON@SN38@MPN + aPD-L1). As shown in Fig. 6 (B to D), the Se-MON@SN38@MPN + aPD-L1 group exhibited an excellent antitumor effect for both primary tumors and distant tumors. Notably, the inhibition rate of distant tumors in the Se-MON@SN38@MPN + aPD-L1 group reached 80.7%, which was 3.4-fold higher than that in the aPD-L1 group (24.0%) (Fig. 6, E to G), indicating that the combination therapy of the nano-ISV and aPD-L1 induced a potent systemic antitumor immunity.

Fig. 6. Antitumor activity of the ISV in a 4T1 bilateral tumor model.

(A) Schematic illustration of the antitumor strategy of Se-MON@SN38@MPN combined with aPD-L1 in the 4T1 tumor model. i.p., intraperitoneal. (B to D) Primary tumor growth curve, photograph, and tumor weight of mice from each group. Means ± SD, n = 6. (E to G) Distant tumor growth curve, photograph, and tumor weight of mice from each group. (H) Body weight changes of mice since first administration. Means ± SD, n = 6. (I) Spleen weight of mice from each group. Means ± SD, n = 6. (J) Representative distant tumor immunofluorescence images of CD4 (red)/CD8 (green), IFN-γ (green), and TUNEL (green; apoptotic cell) staining. (P values were determined by one-way ANOVA with a Tukey post hoc test, *P < 0.05, **P < 0.01, and ****P < 0.0001.)

The terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay further demonstrated that the Se-MON@SN38@MPN + aPD-L1 treatment induced extensive cell apoptosis in distant tumor tissues (Fig. 6J). Immunofluorescence staining demonstrated that Se-MON@SN38@MPN + aPD-L1 induced the highest infiltration of CD4 and CD8 T cells and the secretion of IFN-γ in tumor tissues (Fig. 6J), indicating the successful induction of immune response for suppressing the growth of distant tumors. In addition, the body weights of mice did not change significantly in each group, indicating the biosafety of the nano-ISV (Fig. 6H). Notably, the nanovaccine slightly reduced the weight of the spleens in mice, which is due to the migration of T cells from the spleens into tumor tissues upon immune activation (Fig. 6I). On day 14, the heart, liver, spleen, lung, and kidney were dissected for H&E staining, which further confirmed the biosafety of the nanovaccine (fig. S27). In addition, we collected blood samples after treatment to separate the serum and analyze alanine aminotransferase (ALT), aspartate aminotransferase (AST), UREA, creatinine (CREA), and uric acid (UA) levels. The whole blood was also taken for blood cell counts including white blood cell, red blood cell, hemoglobin, and platelet. The results showed that the biochemical indicators were not affected (figs. S28 and S29).

Characterization of the TME

Last, we sought to gain comprehensive insights into the nano-ISV–regulated immune activation and TME. The spleen is the most crucial immune tissue and organ, containing numerous lymphocytes that are the centers of the body’s immunity. BALB/c mice bearing 4T1 bilateral tumors were intratumorally injected with the nano-ISV, and then the spleens and primary and distant tumors were resected for flow cytometry analysis to evaluate systemic immune response (Fig. 7A). Mature DCs are well recognized as professional APCs that can stimulate systemic antitumor immune responses. As shown in Fig. 7B and fig. S30, the expression of costimulatory markers CD80 and CD86 on DCs in spleens were significantly up-regulated, indicating that the combination of the nano-ISV and aPD-L1 promoted the DC maturation markedly. Upon activation of the STING pathway in DCs, the secreted IFN-I and proinflammatory cytokines could further prime the CTLs. The frequencies of CD4+ and CD8+ T cells were significantly increased in both primary tumors (Fig. 7, C and D, and figs. S31 and S32) (CD45+CD3+CD4+ T: I: 2.42, II: 2.61, III: 3.18, IV: 4.00, and V: 5.68; CD45+CD3+CD8+ T: I: 1.65, II: 1.77, III: 2.17, IV: 2.85, and V: 4.26) and distant tumors (Fig. 7, E and F, and figs. S33 and S34) (CD45+CD3+CD4+ T: I: 0.95, II: 1.52, III: 1.50, IV: 4.35, and V: 5.29; CD45+CD3+CD8+ T: I: 3.16, II: 5.28, III: 5.03, IV: 8.48, and V: 12.16). Furthermore, the highest CD8+ IFN-γ+ T cells in the distant tumors were detected in Se-MON@SN38@MPN + aPD-L1 group (Fig. 7G and fig. S35), indicating the successful activation and infiltration of CTLs (I: 3.94, II: 6.13, III: 6.76, IV: 8.91, V: 13.46). In addition, the frequencies of CD4+, CD8+, and CD8+ IFN-γ+ T cells also increased in the peripheral blood (fig. S36). Moreover, the activation of STING induced nuclear factor κB translocation into the nucleus and interacted with IFN regulatory factors to coinduce the expression of IFN-I and inflammatory cytokines such TNF-α and IL-6. As shown in Fig. 7 (H to J), the contents of TNF-α, IL-6, and IFN-β in the primary tumors increased after the indicated treatments and the highest concentration was obtained in the Se-MON@SN38@MPN + aPD-L1 group. IFN-γ produced from CTLs has the capacity to kill tumor cells. Similarly, cytokines TNF-α, IL-6, and IFN-γ in the distant tumors also showed the highest level for this group (Fig. 7, K to M). These results suggest that the nano-ISV in combination with aPD-L1 could reshape a proinflammatory TME in breast cancer tumors normally classified as cold tumors, which evidences the superior performance of our nano-ISV that integrates STING activation and ROS scavenging for provoking robust systemic immune response.

Fig. 7. In vivo characterization of the TME.

(A) Schematic illustration of the immune activation analysis in vivo. (B) Flow cytometry analysis of CD80 and CD86 expression in spleens of 4T1 tumor-bearing mice with various treatments. (C and D) Flow cytometry analysis of CD3+ CD4+ T cells and CD3+ CD8+ T cells in primary tumors. (E to G) Flow cytometry analysis of CD3+ CD4+ T cells, CD3+ CD8+ T cells, and CD8+ IFN-γ+ T cells in distant tumors. (H to J) Cytokine secretion levels of TNF-α, IL-6, and IFN-β in primary tumors. (K to M) Cytokine secretion levels of TNF-α, IL-6, and IFN-γ in primary tumors. (Means ± SEM, n = 4. P values were determined by unpaired Student’s t tests, *P < 0.05.)

DISCUSSION

Here, we developed a hybrid nano-ISV—Se-MON@SN38@MPN, which is capable of simultaneously activating the cGAS-STING pathway and protecting DCs from oxidative damage by scavenging both radical and nonradical ROS, leading to potent antitumor immune response. Upon intratumoral administration, this nano-ISV in combination with aPD-L1 efficiently inhibited the growth of both primary and distant tumors by enhancing tumor-specific T cell response and improving T cell infiltration.

At present, oncolytic virotherapy has been used as a major ISV for treating various types of tumors, but the pathogenic effect of live oncolytic viruses causes the concern about their safety (3). A chemodrug-based ISV represents a promising technology with great prospects for clinical translation. However, the limited immune response due to insufficient immune cell activation and the immunosuppressive TME hinder their therapeutic efficacy compared with oncolytic viruses. In this work, an idea of the nano-ISV is proposed by constructing two functional modules: (i) STING activation and (ii) ROS scavenging, to significantly improve the efficacy of immunotherapy with chemodrug-based ISVs. We have shown that our ISV formulation in combination with an immune checkpoint inhibitor (Se-MON@SN38@MPN + aPD-L1) achieve an excellent inhibition rate of 80.7% for untreated distant tumors. However, it is still challenging to elicit a sufficiently strong immune response that can completely eradicate the tumors. Ideally, a good cancer vaccine should completely eradicate cancer and induce long-term immune memory to prevent the metastasis or recurrence of tumor. This is the ultimate goal of all the cancer vaccine researchers. However, due to the existence of multiple mechanisms, such as tumor heterogeneity and immunosuppressive microenvironment of tumors, the ideal scenario of the radical cure of cancer is extremely difficult in both preclinical and clinical settings. In our future design, additional mechanisms within a tumor immune microenvironment, such as immunosuppressive cytokines (e.g., transforming growth factor–β), chemokines (e.g., CCL2 and CCL5), and myeloid-derived suppressor cells, should also be taken into consideration to further improve the therapeutic outcome and accelerate the clinical translation.

Although systemic drug delivery is the current clinically preferred route for cancer therapy, systemic administration–related safety risks limit the clinical effectiveness of this route. Intratumoral treatment is an emerging viable alternative to be widely used in the clinical treatment of cancer due to its high potency in maximizing treatment outcomes. In situ antitumor vaccination is designed to target the tumor immune microenvironment, which strives to elicit a robust systemic antitumor immune response through an intratumoral injection route. Such a local administration–induced systemic effect potentially offers a solution for addressing the dilemmatic issues of therapeutic efficacy and biosafety. The current ISV primarily focuses on treating superficial tumors and still has limitations for applying in tumors located in visceral organs and deep tissues. However, with the advancement of imaging and biopsy techniques, the applicability of the ISV is broadening. We envision that the ISV will be suitable for more types of tumor in the near future and can be used a supplementary or complementary approach for current first-line treatments for the sake of improving the therapeutic outcome and the patient’s quality of life.

MATERIALS AND METHODS

Experimental design

The objective of this study was to develop a hybrid nanoparticle–based in situ nanovaccine for simultaneously scavenging ROS and activating the STING pathway in DCs to boost systemic antitumor immunity. For in vitro study, H2O2 depletion, DC maturation, IFN-β detection, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining, and other experiments were performed strictly following the standard or manufacturer’s protocol. For in vivo experiments, mice were randomized to each group before treatment to verify the therapeutic effect (n = 6 mice per group). The tumor tissue and other organs are used to study the immune cell populations by flow cytometry and immunofluorescence staining (n = 4 mice per group) or other experiments.

Preparation and characterization

Materials

Tetraethyl orthosilicate (TEOS), SN38, and doxorubicin hydrochloride (DOX·HCl) were purchased from Aladdin. Cetyltrimethylammonium tosylate (CTAT) was purchased from Macklin. BTESePD was purchased from Xaribio. Manganese(II) nitrate tetrahydrate [Mn(NO3)2·4H2O] was purchased from Adamas. EGCG was purchased from Bide Pharmatech. Triethanolamine (TEAH3), hydrogen peroxide 30% aqueous solution (30% H2O2), and benzoic acid (TA) were purchased from Sinopharm Chemical Reagent.

Synthesis of nanoparticles

For the preparation of Se-MONs, 0.6 g of CTAT and 0.15 g of TEAH3 were dissolved in 40 ml of deionized water and stirred at 80°C. After 30 min, 4.0 g of TEOS and 1.0 g of BTESePD were mixed with 3 ml of ethanol and added dropwise. The mixture was continued to stir for another 4 hours. The products were collected and washed three times with ethanol. To remove the surfactants, 50 mg of products was added in 20 ml of a NaCl/MeOH solution (10 mg/ml) and stirred three times at 45°C for 8 hours. The products were collected by high-speed centrifugation (10,000 rpm, 5 min), washed three times with ethanol and water, and dried in vacuum overnight.

For drug loading, SN38 was dissolved in DMSO to obtain a stock solution (2 mg/ml). The mixture of Se-MONs (2 mg/ml in DMSO) and SN38 solution was stirred in a 1:1 ratio at room temperature for 24 hours to obtain Se-MON@SN38. Se-MON@SN38 was collected, washed with PBS, and dried in vacuum overnight.

For the preparation of Se-MON@MPN and Se-MON@SN38@MPN, 1 ml of Mn(NO3)2·4H2O (50 mM) and 1 ml of EGCG (50 mM) were added to 1 ml of Se-MONs or Se-MON@SN38 (10 mg/ml), respectively. The mixture was vigorously mixed under ultrasonication for 30 s immediately. Last, 5 ml of tris-HCl (pH = 8.5) was added to adjust the pH of the solution, then collected by centrifugation and washed three times with deionized water, and dried in vacuum overnight.

For the preparation of FITC-modified Se-MON@SN38@MPN, the FITC conjugation with 3-aminopropyltriethoxysilane (APTES) was performed firstly. Two hundred microliters of FITC–N-hydroxysuccinimide (10 mg/ml in DMSO) and 10 μl of APTES were dispersed in 300 μl of DMSO, and the mixture was stirred for 24 hours in a dark environment. Then, 50 μl of the FITC-APTES solution was added to 2 ml of ethanol solution with Se-MON@SN38@MPN, followed by stirring for 12 hours in a dark environment. FITC was successfully conjugated onto the nanoparticles.

Characterizations

The morphologies of the samples were characterized with a Zeiss Ultra 55 field-emission SEM and a HT7800 TEM. High-resolution TEM was carried on an FEI Tecnai F20 microscope. Inductively coupled plasma atomic emission spectrometry (ICP-AES) results were acquired from a PE Optima 8000 inductively coupled plasma mass spectrometer. The DLS and zeta potential of nanoparticles was measured on a Zetasizer Nano-ZS size analyzer. Ultraviolet-visible spectroscopy (UV-Vis) absorption spectra were recorded on a PerkinElmer.

Mn2+ and drug release

The Mn2+ releasing behavior from Se-MON@SN38@MPN under different pH values (5.5, 6.5, and 7.5) was monitored. Briefly, Se-MON@SN38@MPN nanoparticles were soaked in tris-HCl and incubated at 37°C. The concentrations of Mn2+ of the collected supernatant solution at 0.5, 1, 3, 8, and 24 hours were determined via ICP-AES.

The SN38 releasing behavior from Se-MON@SN38@MPN under different pH values (5.5, 6.5, and 7.5) was measured. Briefly, Se-MON@SN38@MPN nanoparticles were soaked in tris-HCl and incubated at 37°C. The concentrations of SN38 of the collected supernatant solution at 3, 12, 24, 48, and 72 hours were determined via UV-Vis.

The SN38 releasing behavior from Se-MON@SN38@MPN under a range of GSH concentrations (0, 0.5, 1, 2, 5, and 10 mM) was measured. Briefly, Se-MON@SN38@MPN nanoparticles were soaked in PBS (pH 6.5) solution containing GSH and incubated at 37°C. The concentrations of SN38 of the collected supernatant solution at 24 hours were determined via UV-Vis.

In vitro cytotoxicity and STING activation

Cell culture

4T1, DC2.4, NIH/3T3, and HUVECs were obtained from Hunan Fenghui Biotechnology Co. Ltd. 4T1 cells were cultured in RPMI 1640 medium (Genomcell Bio). DC2.4, NIH/3T3, and HUVECs were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Genomcell Bio). All media were supplemented with 10% fetal bovine serum (Excell) and 1% penicillin-streptomycin solution (Sigma-Aldrich). All cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C.

Cell uptake

A total of 2 × 105 4T1 cells were seeded per dish in confocal glass bottom dishes and incubated overnight. The culture media were replaced with fresh media containing SN38, Se-MON@SN38, and Se-MON@SN38@MPN (100 μg/ml) for another 4 hours of incubation. Next, the 4T1 cells were washed three times with PBS. Images were taken by CLSM (Nikon C2+).

Cytotoxicity assay

The 4T1 cell toxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Beyotime) colorimetric assay. Briefly, 4T1 cells were seeded on 96-well plates at a density of 5 × 103 per well and cultured overnight. Nanoparticles (Se-MONs, Se-MON@MPN, Se-MON@SN38, and Se-MON@SN38@MPN) in a dose-dependent manner were added and further cultured for 48 hours. Then, 4T1 cells were incubated with the MTT solution (20 μl per well from the 5 mg/ml solution in PBS) for 4 hours. Then, 100 μl of DMSO was added to each well to dissolve the formazan crystals. The optical density was recorded at 570 nm in a microplate reader (BioTek Synergy H1), and the percentage of cell viability was determined.

Cell apoptosis and necrosis

Cell apoptosis of 4T1 cells was examined using the Annexin V-FITC/PI Apoptosis Detection Kit (Elabscience). 4T1 cells were seeded at 2 × 105 cells per well in 6-well plates and incubated overnight. Then, Se-MON@MPN, Se-MON@SN38, and Se-MON@SN38@MPN at a concentration of 400 μg/ml were added to incubate for another 48 hours. Afterward, the cells were collected and stained with annexin V–FITC and PI according to the manufacturer’s protocol. Last, the cells were detected by flow cytometry.

Besides, 4T1 cells were seeded at 1.5 × 105 cells per dish in confocal glass bottom dishes overnight and coincubated with various nanomaterials (200 μg/ml) for 48 hours. Last, the cell necrosis was observed by CLSM, after staining with calcein-AM and PI according to the manufacturer’s instructions (Solarbio).

Western blot, ELISA test, and DC maturation experiment

4T1 cells were seeded in 6-well plates (2 × 105 cells per well) and cultured overnight. Then, Se-MON@MPN, Se-MON@SN38, and Se-MON@SN38@MPN at a concentration of 200 μg/ml were added and further cultured for 48 hours. Afterward, nanoparticle-treated tumor cell supernatant media were obtained after centrifugation at a speed of 12,000 rpm for 5 min. DC2.4 (2 × 105 cells per well in 6-well plates) were cultured with 50% obtained media plus 50% fresh media containing different formulations for 24 hours. DC2.4 cell lysates were boiled after loading on SDS–polyacrylamide gel electrophoresis gels and then transferred onto a polyvinylidene fluoride membrane (MEGSEN). The membranes were blocked with a blocking buffer for 1.5 hours and incubated with the primary antibodies overnight at 4°C: IRF-3, P-IRF3, and glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology) was set as controls. The membranes were washed three times for 10 min in a mixture of 1×Tris Buffered Saline with Tween 20 (TBST) (Sangon Biotech), and an horseradish peroxidase–linked goat anti-rabbit immunoglobulin G (Cell Signaling Technology) was used as secondary antibodies for 1 hour and then washed again three times for 10 min in TBST. The blot signals were visualized using Vilber Bio Imaging after being incubated with a SuperSignal West Pico Plus Chemiluminescent Substrate (Thermo Fisher Scientific).

Besides, to test the IFN-β cytokines levels in the supernatant of DC2.4 cells, the supernatants were centrifuged at a speed of 12,000 rpm for 5 min for ELISA tests (Multi Sciences). The test procedures strictly followed the manufacturer’s protocol.

The 4T1 tumor cells supernatant was obtained by the same treatment as described above and used to treat DC2.4 cells. After 24 hours, the cells were trypsinized, rinsed, and harvested. Before the surface antibody staining, the cells were incubated with Fc block for 15 min at room temperature. Last, CD80 and CD86 antibodies (Thermo Fisher Scientific) were used to stain the cells in a flow cytometry staining buffer for 30 min according to the manufacturer’s instruction, and flow cytometry (Beckman) was used to analyze the maturation of DC2.4 cells.

ROS scavenging study

H2O2 depletion

For in vitro reactivity of Se-MONs and EGCG toward H2O2, various concentrations (10, 20, 50, 100, 200, and 500 μg/ml) of Se-MONs, EGCG, and Se-MON@MPN were reacted with a H2O2 solution (200 μM) at 37°C for 24 hours. Afterward, the concentration of H2O2 in different wells was determined by a H2O2 assay kit (Beyotime Biotechnology).

Generation of •OH

Generation of •OH from various formulations was detected by fluorescence spectra due to the capturing of •OH by TA to produce the fluorescent product TAOH. Typically, a TA solution (1.0 mM) was added into PBS (pH = 5.5), and then various formulations were added into the above mixture. After reaction for 12 hours, the fluorescence intensity was measured via a microplate reader with excitation at 315 nm and emission at 435 nm.

Cytotoxicity assay

The cell viability of DC2.4 cells was measured using the MTT assay. Briefly, DC2.4 cells were seeded in 96-well plates (8 × 103 cells per well) and cultured overnight and then treated with Se-MONs, Se-MON+Mn2+, Se-MON@MPN, Se-MON@SN38, and Se-MON@SN38@MPN at various concentrations. The cell viability was determined after 48 hours.

To explore the protection mechanism of Se-MON@MPN, DC2.4 cells were stimulated with fresh media containing Mn2+, Mn2++Se-MON, Mn2++EGCG, Se-MON@MPN (25 μg/ml), and GSH after adding 400 μM H2O2. The cell viability was determined after 6 hours following a similar protocol.

Intracellular ROS detection

The ROS level in DC2.4 cells was detected by using DCFH-DA (Sigma-Aldrich) as a fluorescent probe. Briefly, DC2.4 cells were seeded in black 96-well plates (5 × 103 cells per well) and cultured overnight. Then, the cells were stimulated with Mn2+, Se-MON+Mn2+, EGCG+Mn2+, and Se-MON@MPN (25 μg/ml) for 2 hours after adding 400 μM H2O2. Last, the culture medium was removed and washed twice with DMEM followed by incubation with 10 μM DCFH-DA in DMEM for 20 min. The intensity of fluorescence was quantified by a microplate reader with the excitation and emission peak of 488 and 525 nm, respectively.

Besides, DC2.4 cells were seeded at 1 × 105 cells per well in confocal dishes and cultured overnight. Then, cells were stimulated with fresh media containing nanoparticles (50 μg/ml) or other formulations after adding 400 μM H2O2. To observe the fluorescence, cells were examined by CLSM following a similar method.

In vivo ROS detection

The unilateral 4T1 tumor model was established by subcutaneously injecting 2 × 106 4T1 cells into the left flank of BALB/c mice in 100 μl of PBS. When tumor sizes reached ~50 mm3, the mice were randomly divided into four groups: PBS (control), Se-MONs, Se-MON+Mn2+, and Se-MON@MPN. The prepared nanoparticles (2 mg) and Mn2+ were intratumorally injected once. To evaluate the ROS level in vivo, mice were euthanized 48 hours after the treatment, and the tumors were harvested for flow cytometry analysis and DCFH-DA staining analysis.

Animal studies

Animals

Female BALB/c nude mice (6 to 8 weeks old) were purchased from Shanghai BK/KY Biotechnology Co. Ltd. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Fudan University and approved by the Animal Ethics Committee of Fudan University (approval no. 202106046S).

Animal models and antitumor study

To construct the unilateral tumor models, the subcutaneous 4T1 tumor model was established by injecting 2 × 106 4T1 cells into the left flank of BALB/c mice in 100 μl of PBS. The tumors were allowed to grow to ~50 mm3 for further use. In the in vivo tumor inhibition experiment, 4T1 tumor-bearing BALB/c nude mice were randomly divided into groups (each group contained six mice): PBS (control), SN38, Se-MON@SN38, Se-MON@MPN, and Se-MON@SN38@MPN. The mice were intratumorally injected with 30 μl of PBS or prepared nanoparticles (1.2 mg) per mouse on days 0, 3, 6, and 9. The SN38 solution was prepared by firstly dissolving in DMSO, then added 80% PEG-300 (polyethylene glycol, molecular weight 300) as an adjuvant, and lastly mixed with 20% PBS to reduce the viscosity. The mouse weight and tumor size were measured every day. The tumor volume was calculated as length × (width)2 × 1/2. Thirteen days later, the tumors were collected for H&E staining analysis and immunofluorescence analysis (CD4, CD8, and p-STING).

To construct the bilateral tumor model, cells (1.5 × 106) were injected into the left flanks (primary tumor), and 0.5 × 106 4T1 cells were injected into the right flanks (distant tumor). When primary tumors sizes reached ~50 mm3, the primary tumors of mice were randomly divided into experimental groups and treated four times as follows: PBS (control), anti–PD-L1 (aPD-L1), SN38 + aPD-L1, Se-MON@SN38@MPN, and Se-MON@SN38@MPN + aPD-L1. The prepared nanoparticles (2 mg) and SN38 were intratumorally injected per mouse on days 0, 2, 4, and 6. The mice groups of aPD-L1, SN38 + aPD-L1, and Se-MON@SN38@MPN + aPD-L1 were treated with intraperitoneal administration of the aPD-L1 antibody (10 mg/kg per mouse) on days 1, 3, and 5, respectively. The mouse weight and tumor size were measured every 2 days. Fourteen days later, the mice were euthanized, and the main organs (heart, liver, spleen, lung, and kidney) were collected for H&E staining analysis. Tumors were collected for immunofluorescence analysis of TUNEL, IFN-γ, CD4, and CD8.

Analysis of immune cells

In the bilateral tumor model, the mice were intratumorally treated twice with PBS, aPD-L1, SN38 + aPD-L1, Se-MON@SN38@MPN, and Se-MON@SN38@MPN + aPD-L1 on days 0 and 2, and the aPD-L1 antibody was intraperitoneally injected on day 1. To evaluate the DC maturation in vivo, mice were euthanized 48 hours after the last treatment, and the spleens were harvested for a flow cytometry analysis. Briefly, the single-cell suspensions were prepared via passing through cell strainers and washed twice with PBS on ice. Then, the single cells were treated with 2 ml of an RBC lysis buffer for 10 min to lyse RBCs and stopped the process with 2 ml of PBS. Before the surface antibody staining, the cells were incubated with Fc block for 15 min at room temperature. Afterward, cells were stained with fluorescence-labeled antibodies APC-CD45, PE-CD11C, PerCP/Cyanine5.5-CD80, FITC-CD86 (Multi Sciences), and Zombie Aqua Fixable Viability Kit (BioLegend) for 30 min in a flow cytometry staining buffer to analyze the level of DCs in the spleen.

Besides, the primary and distant tumors were harvested 72 hours after the last treatment following a similar protocol. Single cells were stained with fluorescence-labeled antibodies APC-CD45, FITC-CD3, PE-CD4, PerCP/Cyanine5.5-CD8 (Multi Sciences), Brilliant Violet 421 IFN-γ (BioLegend), and Zombie Aqua Fixable Viability Kit in a flow cytometry staining buffer for 30 min to analyze the level of effector T cells in tumors.

In addition, the peripheral blood of mice was acquired 72 hours after the last treatment. The single cells were treated with the RBC lysis buffer for 10 min to lyse RBCs. Then, single cells were stained with fluorescence-labeled antibodies APC-CD45, FITC-CD3, PE-CD4, PerCP/Cyanine5.5-CD8 (Multi Sciences), Brilliant Violet 421 IFN-γ (BioLegend), and Zombie Aqua Fixable Viability Kit (BioLegend) in a flow cytometry staining buffer for 30 min to analyze the level of effector T cells in the peripheral blood.

To evaluate the changes of cytokine level in tumors, tumors were excised from the mice with different treatments, and 50 mg of tumor tissues was grinded in 250 ml of the PBS solution containing 1% phenylmethanesulfonyl fluoride solution by a tissue homogenizer on ice. The supernatants of primary tumor tissues were centrifuged for ELISA tests of TNF-α, IL-6, and IFN-β (Multi Sciences) by centrifuging at a speed of 12,000 rpm/min for 10 min. Similarly, the supernatants of distant tumor tissues were centrifuged for ELISA tests of TNF-α, IL-6, and IFN-γ. The test procedures strictly followed the manufacturer’s instructions.

Survival study and lung metastasis study

In the 4T1 unilateral tumor model, the mice were intratumorally treated with PBS, SN38, Se-MON@SN38, Se-MON@MPN, and Se-MON@SN38@MPN following a similar protocol. Mice were euthanized before reaching the maximal tumor size (1000 mm3), and the lungs were euthanized to analyze the metastasis.

In vivo uptake study

The subcutaneous 4T1 tumor model was established by injecting 2 × 106 4T1 cells into the left flank of BALB/c mice in 100 μl of PBS. When tumor sizes reached ~50 mm3, the tumors were injected with FITC-modified Se-MON@SN38@MPN. Tumors were harvested 24 hours after the treatment, and the single-cell suspensions were then stained using APC-CD45, PE-CD11b, PE-Cy7-F4/80 (Multi Sciences), and PerCP-Fluor710-Ly6G (Thermo Fisher Scientific) to quantify the uptake of the nanoparticles by neutrophils and macrophages via flow cytometry.

Biodistribution

4T1 tumor-bearing mice were intratumorally injected with Se-MON@SN38@MPN (2 mg). Heart, liver, spleen, lung, kidney, and tumor samples were collected at 12, 24, and 48 hours after injection. The organs were weighed and digested using aqua regia (3 ml) for 8 hours at 80°C. The silica in the digested solution was dissolved by further adding hydrofluoric acid (0.1 ml). Subsequently, the solution was neutralized using deionized water to 10 ml, and Si concentrations were quantified by inductively coupled plasma optical emission spectrometry.

Biosafety study

In the bilateral tumor model, the mice were intratumorally treated twice with PBS, aPD-L1, SN38 + aPD-L1, Se-MON@SN38@MPN, and Se-MON@SN38@MPN + aPD-L1 on days 0 and 2, and the aPD-L1 antibody was intraperitoneally injected on day 1. In addition, the blood of mice was acquired 72 hours after the last treatment. The whole blood was used to detect cell counts (white blood cell, red blood cell, hemoglobin, and platelet), and the serum was used to detect blood enzymes (ALT, AST, CREA, UA, and UREA).

Statistical analysis

The data were expressed as means ± SD or SEM. All statistical analyses were performed using GraphPad Prism 8.0. Statistical analysis was performed by using Student’s t test or one-way analysis of variance (ANOVA) with Tukey’s post hoc test. P values <0.05 were considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

Acknowledgments

Funding: We gratefully acknowledge financial support from the National Natural Science Foundation of China [grant 22275037 (Y.Z.)] and the Australia National Health and Medical Research Council [2010100 (Y.Y.)].

Author contributions: Writing—original draft: Y.Y., J.L., M.Z., W.W., and M.L. Conceptualization: Y.Y., J.L., Y.Z., W.W., M.L., and Y.G. Investigation: J.L., M.Z., W.W., T.W., and M.L. Writing—review and editing: Y.Y., J.L., Y.Z., M.Z., W.W., and M.L. Methodology: Y.Y., J.L., Y.Z., W.W., T.W., M.L., and W.L. Resources: Y.Y., J.L., Y.Z., M.Z., W.W., T.W., M.L., Y.G., and W.L. Funding acquisition: Y.Y. and Y.Z. Data curation: Y.Y., J.L., Y.Z., W.W., T.W., and M.L. Validation: Y.Y., J.L., Y.Z., W.W., T.W., M.L., and W.L. Supervision: Y.Y., J.L., Y.Z., W.W., and M.L. Formal analysis: J.L., Y.Z., M.Z., W.W., T.W., M.L., Y.G., and W.L. Software: J.L., W.W., T.W., M.L., and Y.G. Project administration: Y.Y., J.L., Y.Z., M.Z., W.W., M.L., and W.L. Visualization: Y.Y., J.L., M.Z., W.W., T.W., M.L., Y.G., and W.L.

Competing interests: The authors declare that they have no competing interests.

Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.

Supplementary Materials

This PDF file includes:

Figs. S1 to S36
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