The plasmid GSDMD was obtained from TSINGKE Biological Technology (Guangzhou, China). Lipofectamine 3000 Transfection Reagent was purchased from Thermo Fisher (Waltham, MA, USA). Methoxy polyethylene glycol-g-polyethyleneimine (mPEG_5k-g-PEI_25k) was bought from Tanshtech Co., Ltd. (Guangzhou, China). Cy5-NHS was purchased from Meilunbio (MB12193, Dalian, China). Iron (II) chloride (FeCl₂), Na₂S, and WSP-1 were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI), collagenase I, and IV were obtained from Sigma–Aldrich (Saint Louis, and MO, USA). LDC7559 (BD01225006) and Necrostatin-1 (BD14657) were bought from Bidepharm (Shanghai, China). Z-VAD-FMK (JX517845) was obtained from Energy Chemical (Shanghai, China). Ferrostatin-1 (HY-100579) was purchased from MedChem Express LLC. (Monmouth Junction, and NJ, USA). Apoptosis and Necrosis Detection Kit with YO-PRO-1 and PI (YP1/PI) was bought from Beyotime Biotechnology (Shanghai, China). NF-κB p65 Antibody (T55034), Phospho-NF-κB p65 (Ser536) Antibody (TA2006), STAT3 Antibody (T55292), Phospho-STAT3 (Y705) Antibody (T56566), Calreticulin Antibody (T55353), GPX4 Antibody (T56959), HMGB1 Antibody (T55060), Phospho-MLKL Antibody (p-MLKL, TA7420), total and cleaved N-terminal GSDMD Antibody (P30823) and caspase-3 Antibody (TA6311) were purchased from Abmart (Shanghai, China). Indoleamine 2,3-dioxygenase Antibody (IDO1, ab277522), Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150073), and Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 568) (ab175470) were bought from Abcam (Shanghai, China). ELISA kits of TNF-α, IL-6, and IFN-γ were purchased from DAKEWE (Shenzhen, China). ATP assay kit (S0027) was purchased from Beyotime Biotechnology (Shanghai, China). The flow cytometry antibodies (anti-CD16/32, BV510-conjugated anti-CD45, BV421 anti-mouse CD3, PE-Cy7 anti-mouse CD4, BV650 anti-mouse CD8, PE anti-mouse Foxp3, FITC anti-mouse CD44, PerCP-Cy5.5 anti-mouse CD11c, PE anti-mouse CD62L, Alexa Fluor® 488 anti-mouse CD80, and APC/Cy7 anti-mouse CD86) were purchased from Biolegend (San Diego, CA, USA).

At first, 1.0 mL of FeCl₂ aqueous solution (1.0 mmol/L), 1.0 mL of PPE aqueous solution (10 mg/mL), and 1.0 mL of mPEG_5k-PEI_25k (0, 0.1, 0.2, 0.5 and 1.0 mg/mL) were mixed with stirring in a flask at 25 ℃ for 0.5 h. Then, 0.2 mL of an aqueous solution containing Na₂S (1.0 mmol/L) and GSDMD plasmid (0.1 mg/mL) was added dropwise for another 0.5 h. After the reaction, the mixed solution was dialyzed in pure water overnight, and the samples collected after dialysis were concentrated in an ultrafiltration tube and centrifuged at 3000 rpm (Sorvall Legend Micro 17 Centrifuge, ThermoScientific, Germany) for 5 min to collect the supernatant. The NP_FeS was prepared using the method mentioned above without the GSDMD plasmid. The NP_GD was constructed without the use of FeCl₂ and Na₂S. The NP_FeS/GD-Cy5 was prepared using the abovementioned method, with Cy5-NHS (0.2 mg) added for conjugation with mPEG-PEI. The zeta potential and size distribution of the nanocomplex were detected by dynamic light scattering (DLS, Zetasizer, and Nano ZS90, Worcestershire, UK). The morphology and mapping analysis of the nanocomplex were obtained using a transmission electron microscope (TEM, JEM-2011, JEOL, Japan).

NP_GD, NP_FeS, and NP_FeS/GD (20 μg/mL with 1.0 μg/mL of plasmid) were co-incubated with 4T1 cells in 12-well plates for 12 h. The cells were collected and stained with YP1/PI staining kits to analyze the types of cell death. Meanwhile, the expression of key proteins p-MLKL, GSDMD, and caspase-3 related to cell death patterns was analyzed by immunoblotting and CLSM. Apoptotic cells demonstrated green fluorescence; necroptotic cells showed red or green fluorescence; pyroptotic cells displayed red fluorescence; and PANopototic cells exhibited yellow fluorescence.

To verify the ability of different formulations to induce immunogenic cell death (ICD) in vitro, the release of high mobility group box 1 (HMGB1), secretion of adenosine triphosphate (ATP), and exposure of calreticulin (CRT) were examined. NP_GD, NP_FeS, and NP_FeS/GD (20 μg/mL with 1.0 μg/mL plasmid) were co-incubated with 4T1 cells in 12-well plates for 12 h. The extracellular ATP secreted from the cells in the medium was measured using an ATP bioluminescent assay kit. The cells were collected and stained with 4% paraformaldehyde (PFA, Macklin). After being washed three times with PBS, the cells were incubated with CRT and HMGB1 antibodies (ICD markers). The exposure of CRT and release of HMGB1 were measured using CLSM.

All experimental procedures were executed according to the protocols approved by the Jiangnan University Animal Care and Use Committee (JN. No20231115b0980531[544]). 4T1 tumor-bearing mice were intravenously injected with PBS, NP_GD, NP_FeS, and NP_FeS/GD (5 mg/kg with 1.0 mg/kg plasmid) on Days 10,13, and 16. The tumor volume and mouse body weight were recorded every 3 days. On Day 22, the spleens, tumor-draining lymph nodes, and tumors of the tumor-bearing mice were collected to obtain the isolated immune cells following a previous study. The cells were stained with antibodies to measure the proportion of CD8 T cells, Treg cells, and memory T cells using FACS. More detailed experimental details are included in the Supporting Information

To prepare the nanocomplex of NP_FeS/GD for inducing immunogenic PANoptosis and ferroptosis, biocompatible polyphosphate ester (PPE, ¹H NMR was shown in Supporting Information Fig. S1) and mPEG_5k-PEI_25k were synthesized to stabilize the nanomedicine structures during the construction of FeS nanoparticles (NP_FeS) and to facilitate the binding of gasdermin D (GSDMD) plasmid. The hydrophilic polymer PPE could provide a hydration layer to the nanocomplex to protect the FeS core from oxidation. As shown in Supporting Information Fig. S2, when the polymer PPE was not present during the NP_FeS/GD preparation process, the obtained nanocomplex was highly unstable, leading to oxidative aggregation and precipitation of the nanocomplex within a short period. Firstly, the expression of GSDMD proteins in the engineered vehicles was successfully verified by immunoblot analysis (Supporting Information Fig. S3). The binding efficiency of the GSDMD plasmid and NP_FeS was obtained through electrophoresis. The nanocomplex of NP_FeS could effectively bind the GSDMD plasmid through electrostatic interactions when the weight ratio between the cationic mPEG_5k-PEI_25k and the plasmid was 5 or higher (Fig. 2A). As the weight ratios increased, the zeta potentials of the nanocomplexes also increased (Fig. 2B). Therefore, the mPEG_5k-PEI_25k and GSDMD plasmid were chosen with a weight ratio of 5 to form a nanocomplex in the following experiments, known as NP_FeS/GD. The size distribution and zeta potential of the NP_FeS were approximately 190 nm and 18 mV. Meanwhile, after binding the GSDMD plasmid to NP_FeS, the size distribution and zeta potential of NP_FeS/GD were approximately 220 nm and −0.45 mV. As a control, when the mPEG_5k-PEI_25k and GSDMD plasmid were combined without FeS to form NP_GD, the size distribution and zeta potential of NP_GD were approximately 230 nm and 1.60 mV (Fig. 2C and Supporting Information Fig. S4). Following a 24 h incubation with PBS buffer (pH 7.4), the sizes of NP_FeS and NP_FeS/GD showed minimal changes, indicating their excellent stability (Supporting Information Fig. S5). The results indicated that the neutral-charged PPE played a crucial role in stabilizing the NP_FeS structures and preventing aggregation, forming stable nanocomplexes capable of efficiently delivering the GSDMD plasmid. The morphology and composition of NP_FeS and NP_FeS/GD were measured by transmission electron microscopy (TEM). As shown in Fig. 2D, the NP_FeS and NP_FeS/GD structures displayed consistent morphologies. Analysis of the mapping data revealed a uniform distribution of Fe and S elements within the nanocomplex. In PBS buffer at pH 7.4 and 6.8, the measurement of Fe²⁺ released from NP_FeS/GD was conducted. It was observed that 98.2% and 84.8% of NP_FeS/GD degraded in the acidic PBS buffer at pH 5.0 and 6.8, while only 68.3% of Fe²⁺ was released at pH 7.4 (Fig. 2E and Supporting Information Fig. S6). Additionally, the released H₂S content from NP_FeS/GD in the acidic solution reached up to 86.9% after 5 h of incubation, which was significantly higher compared to the release in PBS at pH 7.4 (Fig. 2F). The NP_FeS/GD nanocomplex was able to effectively respond to the acidic tumor microenvironment, leading to the release of Fe²⁺ and H₂S. This pH-responsive behavior was crucial for targeted drug delivery and therapeutic efficacy. Overall, the results confirmed the successful synthesis and characterization of the NP_FeS/GD nanocomplex, which possessed desirable stability and pH-responsiveness.

To investigate the intracellular uptake of the prepared nanocomplex, confocal laser scanning microscopy (CLSM) was performed. The fluorescent signal was observed after incubation with NP_FeS/GD-Cy5 for different times (2, 4, and 8 h). As shown in Fig. 2G and H, the mean fluorescence intensity (MFI) of Cy5 in cells increased gradually with longer incubation times. However, there was no notable difference in the Cy5 signal between 4 and 8 h of incubation. This indicated that the NP_FeS/GD nanocomplex was efficiently internalized by the cells within the first 4 h, and the uptake reached a plateau after 8 h. Then the Fe content in the cells was measured after incubation with NP_FeS/GD at different times (2, 4, 8, and 12 h). The results showed a time-dependent increase in Fe content, with the highest amount detected at 12 h of incubation (Fig. 2I). Furthermore, the generated H₂S from intracellular NP_FeS/GD was measured using the WSP-1 method. As shown in Fig. 2J and Supporting Information Fig. S7, the green signal was observed in the NP_FeS and NP_FeS/GD groups after incubation with cells for 4 h, while the other groups showed a low signal. This demonstrated that NP_FeS and NP_FeS/GD could effectively release H₂S in tumor cells. These results suggested that the bioactive NP_FeS/GD exhibited efficient cellular uptake and effectively released Fe²⁺ and H₂S intracellularly, making it a promising nanoplatform for targeted cancer therapy.

Based on the design, the nanocomplex NP_FeS/GD could trigger necrosis and apoptosis by generating H₂S at the tumor site, causing a redox imbalance and increasing ROS. It could also induce pyroptosis by the nanocomplex NP_FeS/GD-mediated GSDMD expression, leading to PANoptotic cell death of tumors. Firstly, intracellular ROS generation was also detected. The cells treated with NP_FeS and NP_FeS/GD showed a stronger green signal after 12 h incubation than the groups treated with PBS and NP_GD, indicating that H₂S release could effectively induce a redox imbalance and increase ROS (Supporting Information Fig. S8). To verify the activation of PANoptosis, the cells were stained with the Apoptosis and Necrosis Detection Kit (YO-PRO-1 and PI, YP1/PI) after co-incubation for 12 h. Apoptotic cells exhibited green fluorescence (YP1), while necroptotic cells displayed either red (PI) or green fluorescence (YP1). Pyroptotic cells were characterized by red fluorescence (PI), whereas PANoptotic cells emitted yellow fluorescence (YP1/PI). In Fig. 3A, cells exposed to NP_FeS/GD displayed significantly higher levels of green and red fluorescence signals compared to the other groups, indicating an increase in the fractions of dead cell encompassing YO-PRO-1-positive cells (indicative of necroptosis or apoptosis) and PI-positive cells (indicative of pyroptosis or necroptosis). In addition, the cell viability of the normal human hepatocyte line (LO2) and the 4T1 tumor cells was measured after various treatments. Due to the lack of tumor-targeting ability, NP_FeS/GD could also cause minimal damage to the normal cells of LO2. In contrast, tumor cells demonstrated increased malignancy, faster reproduction rates, and stronger phagocytosis ability, contributing to a more significant killing effect of nanomedicines on 4T1 tumor cells (Supporting Information Fig. S9). The expression levels of key proteins involved in the pyroptotic pathway (GSDMD), apoptotic pathway (caspase-3), and necroptotic pathway (phosphor mixed-lineage kinase domain-like, p-MLKL) were assessed following various treatments using confocal laser scanning microscopy (CLSM) and immunoblot analysis. The expression levels of GSDMD, caspase-3, and p-MLKL were significantly increased in 4T1 cells treated with NP_FeS/GD compared to the control groups in CLSM imaging (Fig. 3B). The MFI of GSDMD indicated a significant increase in expression levels in the NP_FeS/GD-treated group, showing a 1.55- and 1.66-fold higher expression compared to the NP_GD-treated group and the NP_FeS-treated group, respectively (Supporting Information Fig. S10A). An increase in expression levels of caspase-3 and p-MLKL was significantly observed in the NP_FeS/GD treated group, as indicated by the MFI (Fig. S10B and S10C). Immunoblot analysis revealed consistent findings regarding the increased expression of caspase-3 and GSDMD in cells treated with NP_FeS/GD compared to other groups. Additionally, a notable increase in the expression of the necroptotic protein p-MLKL was observed in the NP_FeS/GD group in comparison to the PBS group (Fig. 3C and Supporting Information Fig. S11). Furthermore, the expression levels of other key molecules implicated in PANoptosis, such as AIM2, ZBP1, and RIPK1, were measured in vitro. As illustrated in Supporting Information Fig. S12, a decline in the expression levels of AIM2, ZBP1, and RIPK1 was detected via immunoblot analysis, suggesting that NP_FeS/GD treatment elicited AIM2, ZBP1, and RIPK1-dependent PANoptosis in tumor cells. The findings indicated that cells exposed to NP_FeS/GD might induce PANoptotic cell death through the coordinated activation of necroptotic, apoptotic, and pyroptotic pathways. Due to the incorporation of FeS in the nanocomplex NP_FeS/GD and NP_FeS, it could accumulate Fe²⁺ intracellularly, potentially triggering an alternative pathway to immunogenic cell death known as ferroptosis. The level of ferroptosis-related protein GPX4 was measured using Western blotting after treatment in 4T1 cells. The Western blot analysis revealed a reduced expression of GPX4 in cells treated with NP_FeS/GD or NP_FeS, suggesting the partial induction of ferroptosis (Fig. 3D and Supporting Information Fig. S13). To further confirm ferroptosis, lipid peroxidation was measured using flow cytometry after treatment in 4T1 cells. The flow cytometry results and MFI measurements demonstrated a significant increase in lipid peroxidation in cells treated with NP_FeS/GD compared to the control groups, indicating the initiation of ferroptosis (Fig. 3E and F). The transcriptome sequencing results of the PBS group and NP_FeS/GD group revealed that differentially expressed genes were abnormally abundant in signaling pathways related to PANoptosis (pyroptosis, necroptosis, apoptosis), ferroptosis, and immune response regulation, further demonstrating the potential of PANoptosis in tumor immune sensitization therapy (Supporting Information Figs. S14‒S16). To verify the contribution of each role in pyroptosis, necroptosis, apoptosis, and ferroptosis, the GSDMD inhibitor LDC7559, the apoptosis inhibitor Z-VAD-FMK, the necroptosis inhibitor Necrostatin-1, and the ferroptosis inhibitor Ferrostatin-1 were added to cells with different formulations to detect cell viability using Cell-Counting-Kit-8. As shown in Supporting Information Fig. S17, there was a 46.3% reduction in cell viability after cells were treated with Ferrostatin-1 and NP_FeS/GD, demonstrating that PANoptosis could damage cells more effectively. The cell viability was reduced by 65.6% after treatment with LDC7559, Z-VAD-FMK, Necrostatin-1, and NP_FeS/GD. Additionally, the cells treated with NP_FeS/_GD showed a significantly greater inhibition with an 87.6% reduction. The coefficient of drug interaction⁵⁶ for PANoptosis and ferroptosis was 0.677, indicating a significant synergistic effect. The findings indicated that NP_FeS/GD could effectively activate multiple immunogenic PANoptosis and ferroptosis cell death pathways simultaneously, offering a multi-faceted strategy for inducing cell death. This approach may enhance the efficacy of cancer cell treatment and mitigate the risk of resistance development.

To verify the potential of NP_FeS/GD to enhance the induction of immunogenic cell death (ICD) across multiple immunogenic PANoptosis and ferroptosis cell death pathways in vitro, the presence of danger-associated molecular patterns (DAMPs) such as high mobility group box 1(HMGB1), adenosine triphosphate (ATP) and the exposure of calreticulin (CRT) were investigated after treatment. Following treatment with different formulations, immunofluorescence staining was employed to measure the release of HMGB1 and the exposure of CRT. As shown in Fig. 4A, NP_FeS/GD resulted in a substantial extracellular release of HMGB1 post-treatment. Moreover, NP_FeS/GD effectively decreased HMGB1 levels by 64.6% and reduced positive staining of HMGB1 in the cell nucleus (Fig. 4B). A notable presence of CRT was identified on the surface of 4T1 cells following treatment with NP_FeS/GD in Fig. 4C, with consistent findings corroborated through MFI analysis in Fig. 4D. The results indicated efficient extracellular release of HMGB1 and exposure of CRT in 4T1 cells. The ATP assay kit was used to assess ATP secretion, measuring extracellular ATP levels in 4T1 cells as an additional indicator of ICD. The results showed that ATP levels in the NP_FeS/GD group were significantly higher than those in the PBS, NP_GD, and NP_FeS groups (Fig. 4E). A noticeable decrease in HMGB1 expression, an increase in CRT expression, and enhanced ATP secretion were observed after treatment with NP_FeS/GD, providing evidence of a strong ICD response in tumor cells for triggering a subsequent immune response.

However, previous studies had shown that H₂S could downregulate IDO1 expression by blocking the NF-κB and STAT3 pathways, inhibiting IDO1 activity through H₂S/NO crosstalk, inducing the differentiation of cytotoxic T-effector cells and inhibiting MDSCs, effective inhibiting tumor development⁵³. To further confirm whether the H₂S generated by NP_FeS/GD could inhibit IDO expression and reverse the immunosuppressive microenvironment, we measured the accumulation of kynurenine (Kyn), a byproduct of IDO1 catalyzed tryptophan (Trp) consumption, as previously reported⁵⁷. As shown in Supporting Information Fig. S18, the Kyn generation of NP_FeS/GD and NP_FeS was lower than that of NP_GD, indicating that the production of H₂S inhibited the function of IDO1. The expression of IDO1 in cells was detected by immunofluorescence staining after different treatments. As shown in Fig. 4F and Supporting Information Fig. S19, it was demonstrated that IDO1 expression was regulated by the STAT3 and NF-κB pathways and that treatment with NP_FeS/GD or NP_FeS could downregulate NF-κB phosphorylation and STAT3 phosphorylation to reduce the protein and mRNA expression levels of IDO1. As depicted in Fig. 4G and H, the expression of IDO1 in cells treated with NP_FeS/GD or NP_FeS was comparable to that in the control group treated with PBS but lower than in the NP_GD group. This suggests that the released H₂S effectively reduces immune suppression by downregulating the expression of IDO1 via the STAT3 and NF-κB pathways (Fig. 4I). Hence, the experimental findings presented above offer compelling evidence that the nanocomplex NP_FeS/GD was capable of inducing strong ICD and alleviating immune suppression.

Additionally, we investigated whether ICD triggered by multiple immunogenic PANoptosis and ferroptosis cell deaths could activate an innate immune response by releasing DAMPs that signal "eat me” to antigen-presenting cells (APCs), leading to the maturation and activation of dendritic cells (DCs). After the mouse bone marrow-derived dendritic cells (BMDCs) were incubated with 4T1 cells treated with PBS, NP_GD, NP_FeS, and NP_FeS/GD, the percentage of mature DCs was measured by flow cytometry. The results of FACS analysis revealed a significant increase in the expression of costimulatory molecules (CD80 and CD86) in BMDCs following treatment with NP_FeS/GD during co-incubation (Fig. 4J). The NP_FeS/GD-treated group exhibited a 65.9% induction of mature DCs, representing a 2.11- and 1.83-fold increase compared to the NP_GD-treated group and NP_FeS-treated group, respectively. These results indicated that treatment with NP_FeS/GD could enhance the activation and maturation of DCs (Fig. 4K). Consequently, the nanocomplex NP_FeS/GD could trigger CRT exposure on the surface of tumor cells, enhance the extracellular release of HMGB1 and ATP, reduce the expression of IDO1, and thereby initiate ICD responses, leading to DC maturation and the initiation of anti-tumor immune responses.

Before evaluating the immune activation and anti-tumor capability of NP_FeS/GD to enhance the immune response in vivo, its biodistribution and tumor accumulation were investigated. Following intravenous injection of NP_FeS/GD-Cy5, the 4T1 tumor-bearing mice were analyzed using an IVIS (In Vivo Imaging System) instrument to track their fluorescence signal via the method in the previous study⁵⁸. As shown in Fig. 5A and B, the Cy5 signal was still detectable in the serum after 48 h of NP_FeS/GD-Cy5 injection, indicating that NP_FeS/GD exhibited a long circulation time and providing evidence for high enrichment in tumors. After systemic administration of NP_FeS/GD-Cy5, the tumoral Cy5 signal intensity gradually increased within 12 h and then gradually decayed after 12 h, demonstrating effective retention and accumulation of NP_FeS/GD in tumors. The fluorescent signal of Cy5 in tumors was much higher than in other organs, confirming the outstanding capability of NP_FeS/GD to be retained and accumulated in tumors (Fig. 5C and D). We also investigated the pharmacokinetics and biodistribution of free Cy5 in tumor-bearing mice with intravenous injection through the tail vein. As shown in Supporting Information Fig. S20, the free Cy5 decreased rapidly in the blood after 2 h of injection and disappeared at 12 h. In contrast, the fluorescence of NP_FeS/GD remained after 48 h injection. The accumulation of free Cy5 in the tumor was further measured with in vivo imaging. After intravenous injection, a lower fluorescence signal of Cy5 was observed at 4 h post-injection of free Cy5. The biodistribution and tumor accumulation study further supported the potential of NP_FeS/GD as a promising candidate for immune activation in cancer therapy.

After confirming the tumor accumulation and retention of NP_FeS/GD, we assessed its capacity to induce immunogenic PANoptosis and ferroptosis cell death in vivo. 4T1 tumor-bearing mice were intravenously administered with PBS, NP_GD, NP_FeS, and NP_FeS/GD at a plasmid dosage of 2 mg/kg. After 48 h, the tumors were harvested to prepare frozen tissue sections for further analysis. Subsequently, to confirm the PANoptosis, the expression of GSDMD, caspase-3, and p-MLKL in tumor tissues from the different groups was assessed through immunofluorescent staining. The NP_FeS/GD treatment group showed a significant increase in PANoptosis proteins GSDMD, caspase-3, and p-MLKL, consistent with cellular-level observations (Fig. 5E). The results also reinforced the capacity of NP_FeS/GD to trigger PANoptosis in tumors. Additionally, the levels of CRT and HMGB1, two key markers of ICD, were measured in the tumor tissues. The images showed a notable increase in CRT exposure and HMGB1 release in the tumor microenvironment of the NP_FeS/GD group. The expression of IDO-1, as an immunosuppressive factor in the tumor microenvironment, was also measured using immunofluorescent staining. The observed downregulation of IDO-1 expression suggested that NP_FeS/GD could effectively induce ICD in tumor cells and reverse immunosuppression in vivo (Fig. 5F). As shown in Supporting Information Fig. S21, the content of Trp in tumors treated with NP_FeS/_GD and NP_FeS was higher than that in the NP_GD group, indicating their potential to enhance T-cell responses. Meanwhile, the pro-inflammatory cytokines IL-6, TNF-α, and IFN-γ were detected in the serum at 48 h post-virous treatments. Following NP_FeS/GD treatment, higher levels of TNF-α, IL-6, and IFN-γ cytokines were observed at the 48 h mark compared to the other groups (Fig. 5G and Supporting Information Fig. S22). To validate the immune activation of NP_FeS/GD, tumor-bearing mice were intravenously injected with PBS, NP_GD, NP_FeS, and NP_FeS/GD at a plasmid dosage of 2 mg/kg. Subsequently, the activation of DCs in tumors and tumor-draining lymph nodes was assessed using flow cytometry post-treatment. The NP_FeS/GD-treatment group demonstrated a significantly higher proportion of mature DCs (58.1%) compared to the PBS group (21.2%), indicating a 2.74-fold increase (Fig. 5H and I). The proportion of M1-type macrophages (CD86⁺CD11b⁺F4/80⁺) within tumor tissues was measured. As depicted in Supporting Information Fig. S23, the percentage of CD86-positive CD11b⁺F4/80⁺ macrophages (M1-type macrophages) was 36.2% after PBS treatment, which increased to 64.5% after NP_FeS/GD treatment. The results indicated that NP_FeS/GD treatment effectively triggers an immune response by promoting DC maturation and M1-type macrophage polarization. Moreover, the percentage of mature DCs (44.5%) observed in tumor-draining lymph nodes (TDLN) after NP_FeS/GD treatment surpassed that of all other groups (Fig. 5J and K). The NP_FeS/GD compound could trigger robust immunogenic cell death, leading to the release of DAMPs and depletion of IDO-1. The process induces an immune response by stimulating DC maturation and increasing cytokine expression, leading to the activation of anti-tumor immunity and the reversal of tumor immunosuppression.

After demonstrating the efficient immune activation effect in vivo mediated by NP_FeS/GD, the subsequent evaluation focuses on the antitumor therapy effect. Female Balb/C mice were subcutaneously implanted with 4T1 cells on the left side and then randomly allocated tumor-bearing mice into treatment groups. The in vivo antitumor investigation comprised the systemic administration of treatment formulations on Days 3, 6, and 9, with monitoring every 3 days for 15 days post-injection. The mice treated with NP_GD (Group II) and NP_FeS (Group III) showed a 35.5% and 48.2% inhibition of tumor growth, respectively, compared to the PBS group (Group I). NP_FeS/GD (Group IV) showed significantly greater inhibition of tumors compared to the NP_FeS and NP_GD groups, as evidenced by an 82.2% reduction in tumor burden compared to the PBS group (Fig. 6A). Moreover, images of dissected tumor tissues from various treatment groups (Fig. 6B) and the tumor tissue mass (Fig. 6C) displayed a consistent pattern in tumor volume. The TUNEL apoptosis assay conducted on excised tumors further confirmed that NP_FeS/GD exhibited the highest efficacy in suppressing tumor growth (Fig. 6D). Despite the various treatments administered during the therapeutic experiment, there was no significant decrease in the body weight of the mice as monitored. The observation indicated a remarkable biosafety profile of the NP_FeS/GD treatment (Supporting Information Fig. S24). Furthermore, the heart, liver, spleen, lung, and kidney were collected to assess their pathological condition by hematoxylin and eosin (HE) staining. As shown in Supporting Information Fig. S25, the tissue structures and cell morphology were visible. It was evident that there was minimal tissue damage in the major organs following treatment with various formulations. The data collectively confirmed the capability of the nanocomplex NP_FeS/GD to inhibit tumor growth, induce tumor cell death, and show satisfactory safety.

For a more comprehensive insight into immune response mechanisms after different treatments in vivo, the percentage of immune cells in tumors and tumor-draining lymph nodes was assessed through flow cytometry (Supporting Information Fig. S26). The NP_FeS/GD-treated group displayed the most notable infiltration of cytotoxic CD8⁺ T lymphocytes within tumor tissues (23.0%), aligning with its role in tumor suppression (Fig. 6E and F). A significant increase in cytotoxic CD8⁺ T cells was observed in the tumor-draining lymph nodes following NP_FeS/GD treatments compared to the other groups (Fig. 6G and H). As illustrated in Supporting Information Fig. S27, the proportion of CD8⁺ T lymphocytes within the spleen was around 11.5% following NP_FeS/GD treatment, exhibiting a notable increase compared to other groups. The results were aligned with those observed in tumors and tumor-draining lymph nodes, which collectively demonstrated that NP_FeS/GD treatment could elicit systemic CD8⁺T cell antitumor immunity. Furthermore, there was a notable reduction in the proportion of regulatory T cells (Tregs), the primary immunosuppressive cells, in tumor-draining lymph nodes treated with NP_FeS/GD (Fig. 6I and Supporting Information Fig. S28). The infiltration of central memory T cells within tumor-draining lymph nodes after NP_FeS/GD treatment demonstrated a marked increase, indicating a durable immune memory response (Fig. 6J and Supporting Information Fig. S29). The results collectively demonstrated that the NP_FeS/GD group effectively induced antitumor T cell immunity and reversed tumor immunosuppression.

Metastasis of breast cancer to the lung was a primary factor contributing to the ineffectiveness of traditional cancer therapies. Hence, it was imperative to assess the efficacy of NP_FeS/GD in inhibiting 4T1 tumor metastasis. The aggressive whole-body 4T1 tumor metastasis model was established by intravenous injection of 4T1-luciferase tumor cells into BALB/c mice via the tail vein. Subsequently, the mice received intravenous injections of PBS (Group I), NP_GD (Group II), NP_FeS (Group III), and NP_FeS/GD (Group IV) on Days 1, 4, and 7. Monitoring included assessment of whole-body luciferase expression and survival of the mice (Fig. 7A). The results showed that the NP_FeS/GD-treated group exhibited slightly lower whole-body luciferase expression compared to the other groups on Day 10 (Fig. 7B). On Days 20 and 30, there were a particularly obvious difference in decreased bioluminescence signals in mice treated with NP_FeS/GD compared to NP_GD and NP_FeS, as evidenced by the effective suppression of metastasis (Fig. 7C and D). Additionally, the survival rate of the NP_FeS/GD-treated mice was significantly higher than that of the other groups. The treatments were continued for up to 47 days. The survival studies revealed a considerably extended animal survival, with 70% of mice in Group IV remaining alive-whereas all the mice died within 38 days in the other groups (Fig. 7E). Moreover, the bioluminescence signals and HE staining of entire lungs validated the anti-metastatic efficacy of NP_FeS/GD. Mice administered with NP_FeS/GD displayed reduced luciferase activity in the lungs, and histological analysis of lung tissues revealed a significant reduction in the number and size of metastatic nodules in the NP_FeS/GD-treated group (Fig. 7F and G). These findings suggested that NP_FeS/GD effectively suppresses breast cancer metastasis in vivo. Collectively, the results verified that the antitumor immune response induced by NP_FeS/GD offers a promising approach for inhibiting tumor growth and suppressing metastasis.