MnUA–DOX–Artesunate Hydrogel Remodels Immunosuppressive Tumor Microenvironment and Prevents Postoperative Recurrence in Triple-negative Breast Cancer

MnUA–DOX–Artesunate Hydrogel Remodels Immunosuppressive Tumor Microenvironment and Prevents Postoperative Recurrence in Triple-negative Breast Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article MnUA–DOX–Artesunate Hydrogel Remodels Immunosuppressive Tumor Microenvironment and Prevents Postoperative Recurrence in Triple-negative Breast Cancer Keneng Cai, Run Xia, Mengyao Xu, Wanying Chen, Xinyu Liu, Tiantian Guo, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7975044/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Apr, 2026 Read the published version in Journal of Nanobiotechnology → Version 1 posted 16 You are reading this latest preprint version Abstract Triple-negative breast cancer (TNBC) remains a formidable clinical challenge because of its high aggressiveness, metastatic potential, and lack of effective therapeutic options. In this study, we developed an injectable, multifunctional hydrogel, MND-ART-GEL, by encapsulating immunostimulatory manganese urate (MnUA), the chemotherapeutic agent doxorubicin (DOX), and an artemisinin derivative artesunate (ART) within a thermosensitive Pluronic F127 matrix. Uric acid crystals (MSU) and manganese ions (Mn²⁺) were coordinated and assembled into manganese urate (MnUA). The MSU–Mn²⁺ system exhibits a synergistic immunoactivating effect, combining the DAMPs-like immune stimulation of MSU with the STING pathway activation ability of Mn²⁺. Owing to its simple chemical composition, low cost, and easy accessibility, this combination offers a more practical and scalable alternative to conventional synthetic immune agonists. ART synergizes with DOX to exert potent antitumor effects. MND-ART-GEL synergistically elevated intracellular ROS levels, inducing oxidative stress, mitochondrial damage, and subsequent tumor cell apoptosis, immunogenic cell death (ICD), and exposure of immune markers such as MHCI and Fas. Moreover, MND-ART-GEL downregulated STAT3 expression, thereby suppressing tumor cell invasion and migration. In orthotopic and postsurgical TNBC mouse models, MND-ART-GEL significantly inhibited tumor growth and recurrence, remodeled the immunosuppressive tumor microenvironment, enhanced CD8 + T-cell infiltration, dendritic cell maturation, and M1 macrophage polarization, while reducing PD-1/PD-L1 expression on CD8 + T cells and promoting the secretion of immune-related cytokines. This study presents a localized drug delivery strategy integrating chemotherapy, immune modulation, and multitarget synergy, offering a promising approach to overcome therapeutic resistance and reduce recurrence in TNBC. Triple-negative breast cancer Hydrogel Manganese urate Artesunate Immunosuppressive tumor microenvironment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Breast cancer is one of the most common cancers and the second leading cause of cancer-related deaths among women worldwide 1 . In 2022, approximately 2.3 million new cases of breast cancer were reported and 670,000 breast cancer-related deaths occurred globally 2 . Triple-negative breast cancer (TNBC), a breast cancer subtype characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), lacks specific therapeutic targets 3 . Consequently, endocrine therapy and HER2-targeted drugs are ineffective for TNBC. Moreover, TNBC is highly aggressive, prone to metastasis, and presents a narrow therapeutic window 4 . Chemotherapy remains the primary treatment for TNBC; however, it often leads to drug resistance and significant adverse effects upon prolonged administration at high doses 5 , 6 . Although a subset of TNBC patients responds to immunotherapy, such as programmed cell death ligand 1/programmed cell death protein 1 (PD-L1/PD-1) blockade, the high heterogeneity of TNBC results in many patients exhibiting immunosuppressive “cold tumor” phenotypes, rendering the immunotherapy less effective 7 , 8 . Surgical resection is the cornerstone for potential cure of early-stage TNBC; however, postoperative recurrence is frequent and poses a serious threat. Clinical data indicate that 30–40% of patients with TNBC relapse within five years of surgery, with poor prognosis and high metastatic potential 9 . Recurrent TNBC tumors are often resistant to chemotherapy, with predominantly suppressive immune microenvironment, which limits the efficacy of immunotherapy 10 . These challenges highlight the inadequacy of monotherapies and underscore the urgent need for devising novel therapeutic strategies. Manganese ions (Mn² + ), as natural cGAS agonists, induce robust immune responses by activating the cGAS-STING pathway 11 . This is indicative of their potential as immune adjuvants capable of modulating cellular immunity, which can be leveraged to overcome the limitation of traditional aluminum-based adjuvants that predominantly elicit humoral immunity 12 . Unlike immune checkpoint inhibitors such as PD-1/PD-L1 blockers that mainly enhance the functioning of T cells, Mn² + can directly reprogram the tumor microenvironment (TME)—it promotes the maturation and differentiation of tumor-associated dendritic cells (DCs) and stimulates substantial production of type I interferon, thereby enhancing the infiltration of CD8 + T cells into tumors 13 – 15 . This effectively reverses the immunosuppressive TME and converts poorly immunogenic “cold tumors” into immune-responsive “hot tumors.” Recent evidence indicates that NF-κB activation can prevent the intracellular degradation of activated STING, thereby amplifying STING-associated signaling cascades 16 . As a key regulator of immune homeostasis, NF-κB plays a crucial role in coordinating innate and adaptive immune responses. NF-κB signaling can be triggered by Toll-like receptors (TLRs), while monosodium urate (MSU) crystals, acting as a typical damage-associated molecular pattern (DAMP), are capable of activating TLRs 17 . These findings suggest that MSU may enhance Mn²⁺-induced immune activation, highlighting its potential as an adjuvant candidate for antitumor immunotherapy. Importantly, both MSU and Mn²⁺ are common endogenous or inorganic components with wide availability, facile preparation, and low cost, offering excellent accessibility and scalability. Compared with other expensive or synthetically demanding immune activators, this combination exhibits superior economic and translational advantages, making it highly promising for large-scale preparation and future clinical applications. Artesunate (ART), a derivative of artemisinin, has been extensively investigated for its antitumor potential in recent years. The STAT3 pathway, which is highly activated in TNBC, plays a pivotal role in promoting tumor proliferation, metastasis, and immune evasion 18 , 19 . ART can inhibit phosphorylation and nuclear translocation of STAT3 20 , thereby disrupting the positive feedback loop between STAT3 and IL-6/JAK2 in various tumor models, including colon and liver cancers 21 , 22 . ART suppresses tumor stemness and reverses immunosuppressive TME. Moreover, it promotes mitochondrial damage via reactive oxygen species (ROS) accumulation 23 , and synergizes with doxorubicin (DOX) to exert potent antitumor effects 24 . These properties make ART a promising candidate for overcoming chemoresistance. In this study, we synthesized manganese urate (MnUA) as an immune adjuvant by combining Mn² + and MSU. We coencapsulated MnUA with the chemotherapeutic agents DOX and ART into a thermosensitive Pluronic F127 hydrogel (Scheme 1 ). This multimodal strategy, integrating chemotherapy, immune modulation, and STAT3 suppression, was aimed at enhancing the therapeutic efficacy against TNBC by inhibiting tumor aggressiveness and metastasis, reversing the immunosuppressive TME, and improving treatment outcomes for both primary and postoperative recurrent breast cancers. Methods Preparation of MnUA. MnUA particles were prepared using a reverse microemulsion technique. Briefly, 10 mL of cyclohexane, 2 mL of Triton X-100, and 1 mL of n -hexanol were mixed in two separate glass vials and vigorously stirred for 10 min. Thereafter, 1 mL of 2 mg/mL MSU suspension in phosphate-buffered saline (PBS) was added to one vial (MSU phase), and 1 mL of 4 mg/mL MnCl 2 aqueous solution was added to the other (Mn phase). The contents of each vial were stirred vigorously for 30 min. The two phases were then combined and stirred vigorously for 1 h to allow the reaction to proceed. The organic solvent was then removed by rotary evaporation. An equal volume of absolute ethanol was added to break the emulsion, followed by centrifugation at 10,000 rpm for 10 min to collect the precipitate. The precipitate was washed once with absolute ethanol and twice with ultrapure water, resuspended in an appropriate amount of ultrapure water, and stored at 4°C. Preparation of MnUA-DOX (MND). MnUA particles were resuspended in a 2 mg/mL aqueous solution of doxorubicin hydrochloride and incubated at 37°C for 6–12 h with shaking at 100 rpm. The incubation was terminated when the particles turned dark red, indicating successful drug loading. The resulting MND particles were collected by centrifugation at 10,000 rpm for 10 min. The supernatant was collected for determining the drug loading efficiency. Preparation of MND-ART-GEL. ART was weighed at eight-times the mass of DOX in MND. ART and Pluronic F127 were dissolved together in absolute ethanol at a mass ratio of 1:5. The ethanol was evaporated at 37°C to allow the formation of a thin film, which was then hydrated using an appropriate volume of PBS with ultrasonication. After centrifuging 1 mL of MND suspension at 10,000 rpm to remove the supernatant, 1 mL of the hydrated ART/F127 suspension was added. The mixture was probe-sonicated at an ice bath at 200 W for 5 min to ensure uniform dispersion of ART and MND. The final concentration was to adjusted to 22% with Pluronic F127. The mixture was stirred overnight at 4°C to ensure complete dissolution, yielding the final thermosensitive hydrogel formulation, which was designated “MND-ART-GEL.” Drug Release. DOX solution, MND, and MND-ART-GEL were prepared separately and adjusted to the same DOX concentration. For each group, 1 mL of the formulation was loaded into a dialysis bag (molecular weight cut-off: 8000–14000 Da), with both ends sealed tightly. The dialysis bags were immersed in 100 mL of PBS and incubated at 37°C on a shaker at 100 rpm. At predetermined time points (0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 24, 48, 72, 96, 120, and 168 h), 1 mL of release medium was withdrawn and immediately replenished with 1 mL of fresh PBS. The collected samples were filtered through a 0.22 µm membrane filter and analyzed via high-performance liquid chromatography (HPLC). The cumulative release percentage of DOX was calculated and plotted as a release profile over time. Cell Culture. 4T1 (mouse triple-negative breast cancer cells) and RAW264.7 (mouse monocyte-macrophage leukemia cells) cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). 4T1 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in a humidified 5% CO 2 incubator. RAW264.7 cells were cultured in Dulbecco’s modified Eagle medium with the same supplements and under identical conditions. Cytotoxicity Assay. The cytotoxicity was assessed using the Cell Counting Kit (CCK)-8 assay. 4T1 cells were seeded in 96-well plates at a density of 5 × 10³ cells per well and incubated overnight. The medium was then replaced with drug-containing medium at MND:ART mass ratios of 1:2, 1:4, 1:6, 1:8, and 1:10. The MND dosage was calculated based on the DOX concentration, and final DOX concentrations were set at 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.813, and 0 ng/mL. After 48 h of incubation, CCK-8 solution was added to each well, followed by incubation for an additional 1 h. The absorbance at 450 nm was then measured using a microplate reader (Varioskan LUX; Thermo Fisher, USA). Apoptosis Assay. 4T1 cells were seeded in 6-well plates at a density of 2 × 10 5 cells per well and incubated overnight. The cells were then treated with culture medium containing DOX, MND, ART, MND-ART, or MND-ART-GEL. The final concentration of DOX and ART in the medium was 4 and 32 µg/mL, respectively. After 48 h of incubation, the cells were collected and washed with PBS. They were then stained with Annexin V-AF647 and 4′,6-diamidino-2-phenylindole (DAPI) (1:200 Annexin V, 2 µg/mL DAPI, diluted in 1× binding buffer) at 4°C for 30 min. The cells were subsequently washed with PBS and analyzed via flow cytometry (LSRFortessa; BD, USA). Mitochondrial Membrane Potential Assay. 4T1 cells were seeded in 6-well plates at a density of 2 × 10 5 cells per well and incubated overnight. The cells were then treated with culture medium containing DOX, MND, ART, MND-ART, or MND-ART-GEL. The final concentration of DOX and ART was 4 and 32 µg/mL, respectively. After 48 h of incubation, the cells were collected and washed with PBS. They were then stained with JC-10 dye (15 µM, diluted in Cell Staining Buffer) at 37°C for 30 min, washed with PBS, and analyzed via flow cytometry. Caspase-3 Activity Assay. 4T1 cells were seeded in 6-well plates at a density of 2 × 10 5 cells per well and incubated overnight. Thereafter, they were treated with culture medium containing DOX, MND, ART, MND-ART, or MND-ART-GEL. The final concentration of DOX and ART was 2 and 16 µg/mL, respectively. After 24 h of incubation, the cells were collected, washed with PBS, and stained with Z-DEVD-AFC (2.5 µg/mL, diluted in Cell Staining Buffer) at 37°C for 30 min. The cells were subsequently washed with PBS and analyzed via flow cytometry. Detection of Intracellular ROS. 4T1 cells were seeded in 12-well plates at a density of 2 × 10 5 cells per well and incubated overnight. They were then treated with culture medium containing DOX, MND, ART, MND-ART, or MND-ART-GEL. The final concentration of DOX and ART was 2 and 16 µg/mL, respectively. After 16 h of incubation, the cells were collected, washed with PBS, and stained with 2′,7′-dichlorodihydrofluorescein diacetate (10 µM, diluted in serum-free medium) at 37°C for 30 min. Thereafter, the cells were washed with PBS and analyzed via flow cytometry. Western Blot Analysis. 4T1 cells were seeded in 6-well plates at a density of 3 × 10 5 cells per well. After overnight incubation, the cells were treated with culture medium containing DOX, MND, ART, MND-ART, or MND-ART-GEL. The concentration of DOX and ART was 240 ng/mL and 1.92 µg/mL, respectively. After 48 h of incubation, the cells were washed with PBS and lysed using RIPA lysis buffer supplemented with protease and phosphatase inhibitors. For tumor tissue samples, approximately 100 mg of tumor tissue was weighed and homogenized in 1 mL of RIPA buffer containing protease and phosphatase inhibitors using a precooled tissue homogenizer. The protein concentrations of both cell and tissue lysates were determined using a BCA protein assay kit. Equal amounts of protein were subjected to SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with protein-free blocking buffer and incubated overnight at 4°C with primary antibodies. After washing, the membranes were incubated with appropriate secondary antibodies for 1 h at room temperature. The signals were detected using the Amersham ImageQuant 800 imaging system (Cytiva, Sweden). Scratch Assay. 4T1 cells were seeded in 6-well plates at a density of 1 × 10 6 cells per well and cultured overnight until reaching full confluence. A scratch was made in the well surface using a 200 µL pipette tip, and detached cells were removed by washing with PBS. The cells were then treated with culture medium containing DOX, MND, ART, MND-ART, or MND-ART-GEL. The final concentration of DOX and ART was 2 and 16 µg/mL, respectively. Wound closure was monitored and photographed under a microscope at 0, 12, and 24 h. Matrigel Invasion Assay. 4T1 cells were seeded in the upper chamber of a Transwell insert (8 µm pore size) at a density of 5 × 10 4 cells per well in serum-free medium and starved overnight. After removing the medium, the cells were treated with serum-free medium containing DOX, MND, ART, MND-ART, or MND-ART-GEL. The final concentration of DOX and ART was 1 and 8 µg/mL, respectively. After 48 h of incubation, cells on the lower surface of the membrane were fixed with 4% paraformaldehyde and stained with 1% crystal violet. After washing with distilled water, the migrated cells were imaged under a microscope. Colony Formation Assay. 4T1 cells were seeded in 6-well plates at a density of 2 × 10³ cells per well and cultured overnight. They were then treated with culture medium containing DOX, MND, ART, MND-ART, or MND-ART-GEL. The final concentration of DOX and ART was 240 ng/mL and 1.92 µg/mL, respectively. The cells were cultured until visible colonies (> 50 cells per colony) formed. The colonies were fixed with 4% paraformaldehyde and stained with 1% crystal violet, followed by washing with distilled water and air-drying. Images of each well were captured using a digital camera, and the number of colonies was counted. Analysis of Immunogenic Cell Death (ICD)-Related Markers. 4T1 cells were seeded in 6-well plates at a density of 2 × 10 5 cells per well and cultured overnight. The cells were then treated with culture medium containing DOX, MND, ART, MND-ART, or MND-ART-GEL, with the final concentration of DOX and ART being 240 ng/mL and 1.92 µg/mL, respectively. After 48 h of incubation, the cells were harvested and washed with PBS. The expression levels of calreticulin (CRT), major histocompatibility complex class I (MHCI), and Fas cell surface death receptor (Fas) were analyzed via flow cytometry. In parallel, cell lysates were collected for western blot analysis of HMGB1 expression. After the different treatments, cells were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100, followed by blocking with 5% bovine serum albumin (BSA) for 60 min. The cells adhered to coverslips were then incubated overnight at 4°C in a humidified chamber with primary antibodies against CRT or HMGB1 (1:200 dilution). Next day, the cells were incubated with Alexa Fluor 488-conjugated secondary antibodies (1:400 dilution) for 1 h at room temperature, followed by counterstaining of nuclei with Hoechst 33342. After washing with PBS, coverslips were mounted on glass slides with glycerol gelatin and imaged using a BX53 fluorescence microscope (Olympus, Japan). Bone marrow-derived dendritic cell (BMDC) Maturation and Coculture with Treated Tumor Cells. Bone marrow cells were harvested from the femur and tibia of BALB/c mice and resuspended in RPMI-1640 complete medium after lysing the red blood cells. The cells were cultured in the presence of 20 ng/mL granulocyte-macrophage colony-stimulating factor, with half of the medium replaced every other day. On day 6, loosely adherent immature BMDCs were collected and seeded in 12-well plates at a density of 5 × 10 5 cells per well. The cells were then treated with MSU (4 µg/mL), Mn² + (1.5 µg/mL), or the MnUA combination. Lipopolysaccharide (LPS; 1 µg/mL) was used as a positive control. After 48 h of incubation, the cells were harvested, washed with PBS, and stained with anti-CD11c-APC, anti-CD86-PE, and anti-MHC II-PerCP/Cy5.5 antibodies (1:100 dilution) at 4°C for 30 min. After washing, the cells were analyzed via flow cytometry to determine the proportion of mature BMDCs (MHC II + CD86 + ). For the coculture assay, 4T1 cells were pretreated with DOX, MND, ART, MND-ART, or MND-ART-GEL for 48 h, with the DOX and ART concentration being 240 ng/mL and 1.92 µg/mL, respectively. Treated 4T1 cells were then cocultured with BMDCs; a group treated with LPS (1 µg/mL) served as a positive control. After 24 h of coculture, the cells were collected, washed with PBS, and stained with anti-CD11c-APC, anti-CD86-PE, and anti-MHC II-PerCP/Cy5.5 antibodies (1:100 dilution) at 4°C for 30 min. After washing, the samples were analyzed via flow cytometry to evaluate the maturation of BMDCs (MHC II + CD86 + ). Macrophage Polarization Assay. RAW264.7 cells were seeded in 12-well plates at a density of 1 × 10 6 cells per well and incubated overnight. The cells were then treated with serum-free medium containing MSU (4 µg/mL), Mn² + (1.5 µg/mL), or the MnUA combination. A group treated with LPS (1 µg/mL) was used as a positive control. After 24 h of incubation, the cells were harvested, washed with PBS, and stained with an anti-CD86-APC antibody (1:100 dilution) at 4°C for 30 min. After washing with PBS, the cells were analyzed via flow cytometry to determine the proportion of M1-polarized macrophages (CD86 + ). In Vivo Antitumor Study in BALB/c Mice. An orthotopic 4T1 breast cancer model was established using female BALB/c mice (6–8-weeks-old; weight, 18–22 g), purchased from the Guangdong Medical Laboratory Animal Center. The mice were housed under specific pathogen-free conditions at 25 ± 2°C with a 12 h light/dark cycle and had ad libitum access to food and water. All animal procedures were approved by the Institutional Animal Care and Use Committee of Guangzhou University of Chinese Medicine (Approval No. PZ24115). 4T1 cells (1 × 10 6 cells in 100 µL PBS) were injected into the mammary fat pad of female BALB/c mice after washing with PBS to remove serum. Once tumors reached a volume of approximately 80 mm³, the mice were randomly divided into six groups ( n = 5 per group): PBS, DOX, MND, ART, MND-ART, and MND-ART-GEL. Treatments were administered via intratumoral injection every other day for a total of eight doses. The administered doses were equivalent to 4 mg/kg DOX or 32 mg/kg ART. Tumor dimensions (length and width) and body weight were measured using a caliper every two days, and tumor volumes were calculated according to the following formula: Tumor volume (mm³) = length × width² / 2. At the end of the experiment, the mice were euthanized, and whole blood, heart, liver, spleen, lungs, kidneys, tumor-draining lymph nodes (TDLNs), and tumors were harvested for imaging and weighing. Major organs (heart, liver, spleen, lungs, and kidneys) were fixed in 4% paraformaldehyde for histological analysis. Tumor tissues were divided into three portions for flow cytometry, western blotting, and paraffin embedding (for hematoxylin and eosin (H&E) and immunohistochemical staining). TDLNs were processed for flow cytometry analysis. Post-Surgical Tumor Recurrence Model and In Situ Treatment. To simulate post-surgical recurrence, an incomplete tumor resection was performed when tumor volume reached approximately 200 mm³. Mice were anesthetized with isoflurane, and the surgical area was sterilized. An incision was made on one side of the tumor, and the skin was separated from the tumor using surgical scissors. The majority of the tumor was excised, leaving a residual tumor mass of approximately 20 mm³. The wound was then sutured and the mice were placed in a warming chamber for recovery. The mice were randomly divided into three groups ( n = 5): PBS, DOX, and MND-ART-GEL (DOX: 4 mg/kg; ART: 32 mg/kg). Treatments were administered via in situ injection every other day for a total of five doses. Tumor size was measured using a digital caliper, and tumor volume was calculated using the formula provided under subheading “In Vivo Antitumor Study in BALB/c Mice”. Flow Cytometry of Tumor and Lymph Node Immune Cells. At the end of treatment, tumors were excised and digested with tumor tissue digestion buffer at 37°C for 1 h. The resulting tumor cell suspensions were passed through a 200-mesh nylon cell strainer. After lysing red blood cells, the single-cell suspension was blocked with 1% BSA at room temperature for 15 min. The cells were then divided into four parts for the analysis of tumor cells, DCs, macrophages, and T cells. Lymph nodes were harvested and gently ground on a 200-mesh nylon cell strainer in PBS to obtain single-cell suspensions. After passing through the strainer, the cells were blocked with 1% BSA at room temperature for 15 min and divided into three parts for the detection of DCs, macrophages, and T cells. All samples were incubated with specific antibodies at 4°C for 30 min in the dark, followed by flow cytometry analysis. Enzyme-linked Immunosorbent Assay for Cytokines. Tumor tissue (100 mg) was weighed and homogenized in 0.9 mL of cold PBS containing protease inhibitors using a precooled tissue homogenizer. The tissue lysate was centrifuged to remove debris, and the supernatant was collected for analysis. The levels of IL-6, TNF-α, and IFN-γ in the tumor homogenates were measured using commercially available ELISA kits (Biolegend, USA) according to the manufacturer’s instructions. Statistical Analysis . Statistical analysis was performed using IBM SPSS Statistics 26, and graphs were generated with GraphPad Prism 10. For comparison between two groups, an independent-sample t -test was used, with Levene’s test applied to assess variance homogeneity. For comparisons among three or more groups, normality was tested using the Shapiro–Wilk test. Data with P > 0.05 were considered normally distributed and expressed as mean ± standard deviation (SD), shown in bar graphs. Data with P < 0.05 were considered non-normally distributed and presented as median (P25–P75), shown in box plots. Nonparametric tests were used for non-normal data: Mann–Whitney U test for two groups and Kruskal–Wallis H test for multiple groups. For normally distributed data, one-way ANOVA was used. If variances were homogeneous (P > 0.05), Tukey’s test was applied for more than three groups, and Bonferroni correction for three or fewer groups with equal sample sizes. When sample sizes were unequal, Scheffé’s test was used. If variances were unequal (P < 0.05), Welch’s ANOVA followed by Dunnett T3 post hoc test was performed. Statistical significance was indicated as follows: ns (not significant), *P < 0.05 (significant), and **P < 0.01 (highly significant). Asterisks placed above the bars indicate comparisons versus the PBS group. Results and Discussion Preparation and Characterization of MND-ART-GEL. Pluronic F127 is a triblock copolymer consisting of a central hydrophobic polypropylene oxide block and two hydrophilic polyethylene oxide blocks, exhibiting temperature-dependent self-assembly 25 . The blank hydrogel remained in the liquid form at temperatures between 15 and 25°C but gelled at temperatures > 30°C (Fig. 1 A). MND-ART-GEL retained this behavior, indicating its suitability for in situ gelation at near physiological temperatures—ideal for local and postoperative tumor therapy. The MND and ART were uniformly dispersed into a 22% Pluronic F127 thermosensitive hydrogel by ultrasonic mixing with 6% Pluronic film hydration, forming an injectable MND-ART-GEL (Fig. 1 B). Scanning electron microscopy (SEM) images revealed a uniform porous gel network (~ 20 µm, Fig. 1 C), and energy dispersive spectroscopy (EDS) mapping showed homogeneous distribution of Mn, N, and Cl (Fig. 1 D), indicating even dispersion of MND within the gel. Drug release profiles were evaluated via HPLC. As shown in Fig. 1 E, ~ 90% of free DOX was released within 6 h, and complete release was eventually achieved. In contrast, MND achieved a sustained release plateau at ~ 80% over 48 h. MND-ART-GEL exhibited the slowest initial release and a sustained profile, with ~ 76% release achieved by day 7. As shown in Figure S1 , free ART was almost completely released within 2 h, while ART in the MND-ART-GEL formulation reached a plateau at 48 h, with approximately 70% of the drug released. These results indicated that both MnUA particle encapsulation and hydrogel formulation conferred sustained-release properties, enabling prolonged drug exposure and potentially reducing systemic toxicity associated with conventional high-dose chemotherapy. Meanwhile, the relatively safe drug ART was released at a faster rate from the hydrogel, allowing for an early therapeutic effect. MnUA microparticles were first synthesized via a reverse microemulsion method. MSU and MnCl 2 formed an MnUA microemulsion upon vigorous stirring, which was washed to remove organic solvents and surfactants to obtain MnUA particles. FTIR spectra of MSU, MnCl 2 , and MnUA (Fig. 1 F) revealed that the characteristic peaks of MSU at 1770–440 cm − ¹—corresponding to C = O stretching of the ketone group and purine ring vibrations—were replaced by two new peaks at 1160–880 cm − ¹ (C–O–Mn) and 560 cm − ¹ (Mn–O) in MnUA. The enhanced 560 cm − ¹ peak in MnUA compared with that in MnCl 2 confirmed the formation of Mn–O coordination bonds. Additionally, the disappearance of the broad peak at 3200–2600 cm − ¹ in MSU indicated that the amino group (–NH) on the purine ring may also coordinate with Mn² + , forming Mn–N bonds that stabilize the MnUA structure. MnUA was then incubated with DOX solution to form drug-loaded MnUA-DOX (MND). HPLC analysis of the supernatant after centrifugation revealed a DOX loading content of 35.45%, indicating that MnUA achieved high drug-loading efficiency in aqueous solution without requiring activation with a base (e.g., triethylamine) 26 – 29 . The FTIR spectrum (Fig. 1 G) indicated that the drug loading occurred primarily via physical adsorption: the characteristic peaks of DOX (e.g., –NH/–OH at 3530 and 3310 cm − ¹, and anthraquinone ring vibrations between 1740 and 400 cm − ¹) disappeared, whereas the MnUA skeleton peaks at 1010 and 560 cm − ¹ remained unchanged, which was suggestive of physical adsorption of DOX rather than its chemical binding. Evaluation of the Synergistic Cytotoxicity of MND and ART Against 4T1 Cells. The CCK-8 assay was performed to determine the optimal drug ratio for combining MND and ART. As shown in Fig. 2 A, DOX, a first-line chemotherapeutic agent, effectively inhibited the viability of 4T1 cells, whereas ART exerted a weaker cytotoxic effect, acting primarily as an adjuvant. Compared with DOX alone, MND exhibited a lower half-maximal inhibitory concentration (IC 50 ), indicative of enhanced efficacy of DOX-loaded MnUA microparticles at reduced doses. When MND was combined with ART, the IC 50 values at 1:8 and 1:10 ratios were further reduced, with the lowest value observed at the 1:8 ratio. The combination index (CI), a quantitative metric for drug interaction, was used to evaluate synergism (CI 1). As shown in Fig. 2 B, the CI values at the 1:8 combination ratio were consistently less than 1 across most fractional affected (FA) levels, with the lowest CI at IC 50 , indicating a favorable synergistic effect. Therefore, the 1:8 ratio was selected for subsequent experiments. MND-ART-GEL Induces Apoptosis and ROS Accumulation in 4T1 Cells In Vitro. Apoptosis and caspase-3 activation in 4T1 cells treated with various formulations were assessed via flow cytometry. All the treatments induced apoptosis and activation of caspase-3 to varying degrees. Compared with free DOX, the MND formulation and its combination with ART in the hydrogel form significantly increased the proportion of apoptotic cells, with treatment with MND-ART-GEL resulting in the highest apoptosis rate and caspase-3 activation (Fig. 2 C-F). These results indicated that, at equivalent drug dosages, the hydrogel formulation was more effective in inducing apoptosis of tumor cells. DOX induces ROS accumulation, which damages tumor cell DNA, thereby contributing to cytotoxicity 30 . Moreover, ART contains an endoperoxide bridge that reacts with intracellular iron ions, leading to additional ROS production 31 . These ROS species exacerbate DNA damage and mitochondrial dysfunction, indicating that ROS generation is one of the primary mechanisms underlying MND-ART-GEL–induced apoptosis. To further confirm this hypothesis, intracellular ROS levels and mitochondrial membrane potential (MMP) were evaluated via flow cytometry. Compared with DOX and MND groups, MND-ART-GEL significantly elevated the ROS levels (Fig. 2 G-H) and increased the proportion of cells with depolarized mitochondrial membrane (low MMP) (Fig. 2 I-J). These findings confirmed that MND-ART-GEL induces tumor cell death primarily through ROS-mediated mechanisms. MND-ART-GEL Induces ICD and Surface Immunomarker Exposure in 4T1 Cells. DOX is a well-established inducer of ICD, known to trigger endoplasmic reticulum (ER) stress in tumor cells, thereby promoting ICD 32 . As MND-ART-GEL elevates intracellular ROS levels, it may enhance ER stress-mediated ICD 33 , 34 . The expression levels of CRT and high mobility group box 1 (HMGB1), two hallmark ICD-associated DAMPs, were evaluated to assess the ICD-inducing ability of MND-ART-GEL. Among all treatment groups, MND-ART-GEL induced the highest expression levels of both CRT and HMGB1, significantly exceeding those observed in the DOX, MND, and ART groups (Fig. 3 A-D). This was further validated by immunofluorescence imaging, wherein 4T1 cells treated with MND-ART-GEL displayed the brightest green fluorescence signal (Fig. 3 E, F). Flow cytometry analysis of surface immune markers revealed that 4T1 cells treated with MND-ART-GEL exhibited increased expression of apoptosis-associated factor Fas and MHCI molecules (Fig. 3 G–J). This enhancement in the expression of immunogenic markers indicated that MND-ART-GEL treatment rendered tumor cells more recognizable and susceptible to immune cell-mediated cytotoxicity. MND-ART-GEL Activates Immune Cells In Vitro. DCs are the most potent antigen-presenting cells (APCs). Mature DCs express high levels of MHC I/II molecules and costimulatory molecules, such as CD80 and CD86, enabling them to present tumor antigens to T cells and initiate immune responses. Tumor-associated macrophages (TAMs) are a major component of the TME, which exhibit two phenotypes: M1 and M2. M1-like TAMs exert antitumor activity by sustaining inflammation, recruiting immune cells, and phagocytosing tumor cells, whereas M2-like TAMs generally promote tumor progression. Mn² + is a widely recognized immune adjuvant capable of activating the cGAS-STING pathway and inducing robust adaptive immunity. MSU serves as a DAMP that can regulate immune responses via TLR/NF-κB signaling and enhance STING-associated signaling cascades. To evaluate the immunostimulatory effects of MnUA, primary BMDCs and RAW264.7 macrophages from Balb/c mice were incubated with MnUA. Flow cytometry analysis revealed that MnUA treatment significantly increased the proportion of CD86 + MHCII + BMDCs (Fig. 3 K-L), surpassing the effects observed in MSU or Mn² + single-agent groups and even exceeding those in the LPS-treated positive control group. Similarly, the percentage of CD86 + RAW264.7 macrophages was markedly increased following MnUA exposure (Fig. 3 M-N). These findings indicated that MnUA acts as a potent immune adjuvant capable of promoting DC maturation and polarizing TAMs toward the M1 phenotype. Given the fact that MND-ART-GEL could induce robust ICD, we cocultured 4T1 cells and BMDCs to evaluate whether ICD-induced signals could activate DCs. As shown in Figure S2, compared with the blank control, BMDCs cocultured with PBS-treated 4T1 cells exhibited marked immunosuppression, with reduced maturation likely due to suppressive cytokines or metabolites secreted by tumor cells 35 , 36 . In contrast, the MND-ART-GEL–treated group exhibited the highest DC maturation, significantly exceeding that in other groups, indicating that ICD triggered by MND-ART-GEL could reverse the immunosuppressive TME. MND-ART-GEL Suppresses STAT3 Activation and Inhibits the Invasion and Metastasis of 4T1 Cells. In TNBC, the STAT3 signaling pathway is frequently hyperactivated, promoting tumor proliferation, invasion, metastasis, and immune evasion, while contributing to chemoresistance 18 , 19 . ART acts as an inhibitor of STAT3 by preventing its phosphorylation and nuclear translocation, thereby reducing its DNA-binding ability 20 . This mechanism may be one of the key contributors to the antitumor activity of the MND-ART-GEL system. Western blot analysis was used to detect total STAT3 and phosphorylated STAT3 (Tyr705 and Ser727) levels in the treated 4T1 cells. As shown in Fig. 4 A–D, the drugs containing ART (free ART, MND-ART, and MND-ART-GEL) significantly downregulated the levels of both p-STAT3 and total STAT3. Scratch assays, colony formation, and Transwell invasion assays further revealed that MND-ART-GEL effectively inhibited the migration, invasion, proliferation, and clonogenic potential of 4T1 cells (Fig. 4 E–J), indicating its strong potential to suppress TNBC metastasis. Transcriptomic Analysis of 4T1 Cells Treated with MND-ART-GEL. To elucidate the molecular mechanisms underlying the antitumor effects of MND-ART-GEL, transcriptome sequencing and differential gene expression analysis were performed on 4T1 cells treated with various formulations. As evident from the principal component analysis (PCA) and correlation matrix results (Figure S3A, B), samples within the same group exhibited high consistency, with clear intergroup differences, justifying downstream analysis. Volcano plots revealed that, compared with PBS treatment, DOX treatment resulted in the upregulation of 2,025 and downregulation of 2,057 genes (Figure S3C). In contrast, the gene expression profiles for the DOX and MND groups were relatively similar, with only 74 upregulated and 95 downregulated genes, indicating that MnUA alone had minimal impact on gene expression. However, a comparison between the MND and MND-ART-GEL groups led to the identification of 2,292 upregulated and 2,029 downregulated genes, indicating substantial transcriptomic alterations driven by ART addition and hydrogel delivery. The Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis of differentially expressed genes in the PBS vs. DOX, DOX vs. MND, and MND vs. MND-ART-GEL comparison (Figure S4A) revealed that DOX primarily influenced TNF, NOD-like, Notch, MAPK, and PI3K-Akt signaling pathways, which was suggestive of its role in direct tumor cytotoxicity, activation of inflammation, and inhibition of proliferation. MND showed a trend toward regulating antigen processing and presentation; however, the q-values were relatively high, which limited confidence in these changes. ART treatment was associated with modulation of TNF, MAPK, IL-17, glutathione metabolism, glyoxylate and dicarboxylate metabolism, cysteine and methionine metabolism, HIF-1, arginine and proline metabolism, and PI3K-Akt pathways—reflecting its multifaceted effects on cytotoxicity, inhibition of proliferation, redox metabolism, and ferroptosis. The KEGG pathway mapping and classification (Fig. 5 A) revealed significant enrichment of ferroptosis, glutathione metabolism, HIF-1, MAPK, and PI3K-Akt signaling pathways in the MND-ART-GEL group compared with that in the controls. These findings indicated that MND-ART-GEL induced oxidative stress and depleted glutathione to trigger ferroptosis 37 , while also activating adaptive responses to hypoxia and stress 38 . To investigate the global biological impact of MND-ART-GEL, gene set enrichment analysis (GSEA) was performed using selected genes related to oxidative stress, immunity, and tumor metastasis (Fig. 5 B). Wnt and TGF-β signaling pathways, which are often implicated in epithelial-mesenchymal transition (EMT) and metastatic potential, were also modulated, indicating a possible role of MND-ART-GEL in suppressing invasion and migration 39 , 40 . Immune-related pathways, including antigen processing and presentation, IL-17 signaling, NOD-like receptor signaling, and cytosolic DNA sensing, were significantly upregulated, suggesting that the ICD triggered by MND-ART-GEL effectively activated both innate and adaptive immune responses 41 . Further gene ontology (GO)-GSEA analysis (Figure S4B) confirmed that gene sets, such as “antigen processing and presentation of endogenous peptide antigen via MHC class I,” “activation of innate immune response,” “positive regulation of interferon-beta production,” and “cellular response to interferon-beta,” were highly enriched. These findings imply activation of the cGAS-STING–mediated type I interferon pathway, thereby enhancing innate immune signaling 42 . Moreover, biological processes related to cell migration—such as “positive regulation of cell migration,” “positive regulation of endothelial cell migration,” and “DNA integration”—were significantly downregulated, further supporting the notion on the inhibitory effect of MND-ART-GEL on tumor invasiveness. The transcriptomic analysis of the effects of MND-ART-GEL was consistent with in vitro results. In Vivo Antitumor Efficacy and Immune Activation of MND-ART-GEL in an Orthotopic 4T1 Breast Cancer Model. Given the potent in vitro antitumor and immunostimulatory effects of MND-ART-GEL, its in vivo efficacy was further evaluated using an orthotopic 4T1 breast tumor model in Balb/c mice. As shown in Fig. 6 A, mice received intratumoral injections of different formulations (DOX 4 mg/kg or ART 32 mg/kg) every other day for a total of seven treatments. The ART group showed minimal tumor inhibition, whereas DOX, MND, MND-ART, and MND-ART-GEL groups significantly suppressed tumor growth, with MND-ART-GEL exhibiting the greatest tumor inhibition as evidenced by the lowest tumor weights and volumes (Fig. 6 B–E). Histological analyses, including H&E, TUNEL, and Ki67 staining, were performed to assess tumor tissue morphology, apoptosis, and proliferation. H&E staining revealed only minor nuclear condensation in the ART group, whereas the DOX and MND groups showed increased areas of nuclear condensation, with MND also displaying nuclear dissolution, indicative of apoptosis and necrosis. Notably, MND-ART and MND-ART-GEL treatments led to extensive nuclear condensation, fragmentation, and dissolution, with the MND-ART-GEL group exhibiting near-complete nuclear degradation (Fig. 6 F). TUNEL and Ki67 staining results corroborated these findings, demonstrating the highest levels of apoptosis and inhibition of proliferation in the MND-ART-GEL group. To validate the in vitro findings of ICD induction and STAT3 suppression, HMGB1 and CRT expression were examined via immunohistochemistry, whereas STAT3 and phosphorylated STAT3 levels were analyzed via western blotting. As shown in Fig. 6 F, HMGB1 release and CRT membrane translocation were significantly increased in the MND-ART and MND-ART-GEL groups compared with that in PBS, DOX, MND, and ART control groups. Furthermore, STAT3 and its phosphorylated forms (Tyr705 and Ser727) were significantly downregulated in ART-containing groups, consistent with in vitro data (Fig. 6 G–J). These results confirmed that MND-ART-GEL induced strong ICD and inhibited STAT3 signaling in vivo. To further evaluate the immunomodulatory effects of MND-ART-GEL, flow cytometry was conducted to analyze immune cell populations in tumors and TDLNs (Figure S5). Surface expression of MHCI and Fas on tumor cells was first examined. Compared with the PBS group, the MND-ART-GEL group showed a two-fold increase in the percentage of Fas + cells and a 1.8-fold increase in that of MHCI + cells, both significantly higher than the respective percentages in the DOX and ART groups (Fig. 7 A-B). These results indicated that MND-ART-GEL effectively enhances tumor cell immunogenicity in vivo, facilitating immune recognition and cytotoxicity. Based on our findings that MnUA acted as an immune adjuvant and that MND-ART-GEL induced robust ICD, we next evaluated DC activation in the TDLNs and tumor tissue. The MND-ART-GEL group exhibited the highest proportion of mature DCs (CD86 + MHCII + ) in TDLNs, approximately 6.87-fold higher than that in the PBS group and significantly greater than that in the MND and ART groups (Fig. 7 C-D). Although DC infiltration in tumors was relatively low, MND-ART-GEL treatment increased the abundance of CD11c + DCs from 1.4% to 27.6% (Fig. 7 E), providing a favorable immune environment. A similar trend was observed in MHCII + DCs, the abundance of which increased from 0.4% in the PBS group to approximately 9.5% in the MND-ART-GEL group (Fig. 7 F). TAM profiling revealed that MND-ART-GEL significantly promoted M1 polarization and suppressed M2 polarization, compared with that in other treatments (Fig. 7 G-H). The M1/M2 ratio in tumors treated with MND-ART-GEL was approximately 4.5, which was 12.5-times higher than that in the PBS group (Fig. 7 I). We next assessed the percentage of CD8 + T cells, which are crucial for tumor cell killing and correlate with favorable prognosis. MND-ART-GEL induced the highest CD8 + T-cell infiltration, which reached 43.3%—about 5.5-times that in the PBS group (Fig. 7 J-K). Notably, MND-ART-GEL also reduced the expression of PD-1 and PD-L1 on CD8 + T and tumor cells, respectively (Fig. 7 L, M). This indicated that MND-ART-GEL not only promoted T-cell infiltration but also alleviated T-cell exhaustion and immune suppression, thereby restoring cytotoxic activity. Notably, the ART group alone also exhibited low PD-L1 and PD-1 levels, indicative of ART-mediated STAT3 inhibition contributing to this effect. Finally, proinflammatory cytokines including TNF-α, IFN-γ, and IL-6 were quantified in tumor tissues via ELISA. These cytokines play essential roles in antitumor immunity by activating cytotoxic T cells, enhancing antibody production, and promoting immune cell recruitment to the TME 43 – 45 . The MND-ART-GEL group exhibited the highest levels of TNF-α, IFN-γ, and IL-6 among all groups (Fig. 7 N–P), indicating a favorable shift in the immune microenvironment. Throughout the treatment period, body weight of mice was monitored every two days. At the study endpoint, major organs were harvested, weighed, and analyzed after H&E staining to assess biosafety. No significant differences in body weight or organ coefficients were observed between groups (Figure S6A, B), suggesting good biocompatibility. Although splenomegaly was observed—likely due to tumor-induced immune activation—this was excluded from toxicity evaluation. Apart from mild inflammatory infiltration in the lungs (likely due to TNBC-associated pre-metastatic niches 46 ), no significant histopathological changes were detected in major organs (Figure S6C). Overall, MND-ART-GEL exhibited excellent biosafety in vivo. In summary, MND-ART-GEL effectively triggered ICD and downregulated STAT3, thereby remodeling the immunosuppressive microenvironment of TNBC, eliciting a robust antitumor immune response, and demonstrating favorable biosafety in vivo. Evaluation of the Anti-Recurrence Efficacy of MND-ART-GEL After Surgery. Surgical resection remains the primary curative treatment for TNBC; however, the risk of postoperative recurrence is high and often detrimental. To assess whether MND-ART-GEL can prevent tumor relapse after surgery, a postsurgical recurrence model was established. As illustrated in Fig. 8 A, mice were randomly assigned to three groups (PBS, DOX, and MND-ART-GEL; DOX: 4 mg/kg; ART: 32 mg/kg; n = 5) and received five intratumoral injections every other day following surgery. One mouse in the PBS group died from relapse before the study endpoint, and the remaining PBS group mice exhibited severe tumor regrowth. In contrast, MND-ART-GEL treatment effectively suppressed the growth of residual tumor cells, resulting in significantly reduced tumor weights and volumes compared with that in the PBS and DOX groups (Fig. 8 B-E), indicating its robust anti-recurrence capability. Postoperative tumors typically present with an immunosuppressive microenvironment. Therefore, flow cytometry was used to assess immune cell profiles in relapsed tumors. MND-ART-GEL markedly enhanced DC maturation (Fig. 8 F, G), CD8 + T-cell infiltration (Fig. 8 H, I), and M1 macrophage polarization while reducing the abundance of M2-type TAMs (Fig. 8 J–M). The M1/M2 ratio in the MND-ART-GEL group was 16.25-times of that in the PBS group (Fig. 8 K). These results indicated that MND-ART-GEL successfully reversed the immunosuppressive state of postoperative residual tumors and reactivated antitumor immunity. Conclusion In this study, MnUA microparticles were successfully synthesized via reverse microemulsion, wherein urate molecules coordinate with Mn² + through metal–ligand interactions. MnUA exhibited intrinsic immunostimulatory capability and served as a drug carrier by physically adsorbing the chemotherapeutic drug, DOX. Codelivery of DOX and ART within a Pluronic F127-based thermosensitive hydrogel resulted in the generation of a multifunctional platform—MND-ART-GEL. Both in vitro and in vivo studies revealed that MND-ART-GEL exerted potent therapeutic efficacy in primary and postsurgical 4T1 TNBC mouse models. DOX and ART synergistically increased intracellular ROS levels, causing oxidative stress, mitochondrial damage, and subsequent tumor cell apoptosis and ICD. These changes enhanced the exposure of immunogenic markers (MHCI and Fas) on tumor cells. Simultaneously, MnUA acted as an immune adjuvant to promote infiltration and maturation of DCs, while ICD further activated antigen presentation and T-cell responses. MND-ART-GEL also reduced PD-L1/PD-1 expression, alleviated T-cell exhaustion, and enhanced CD8 + T-cell–mediated cytotoxicity. Moreover, STAT3 inhibition by ART suppressed tumor invasion and metastasis. This study introduces a novel application of MSU as an immune adjuvant, integrating chemotherapy, immunomodulation, and multitarget therapy to reshape the tumor immune microenvironment. The platform was found to effectively inhibit tumor growth and recurrence and offers a promising therapeutic strategy for TNBC, which is expected to overcome treatment resistance while improving patient outcomes. Declarations Ethics approval and consent to participate All animal experiments were approved by the Ethics Committee of Science and Technology Industrial Park, Guangzhou University of Chinese Medicine (PZ24115). All applicable institutional guidelines for the care and use of animals were followed. Consent for publication All authors agree with the submission and publication of this paper. Competing interests All authors declare no conflicts of interest. Funding This work was supported by Natural Science Foundation of China (82204628), the Special Projects in Key Areas of Colleges and Universities in Guangdong Province (2022ZDZX2015), Natural Science Foundation of Guangdong Province (2022A1515011312), the Science and Technology Program of Guangzhou (2024A04J4899), and Young Talent Project of Guangzhou University of Chinese Medicine (No. A1-2601-24-414-110Z76). Author Contribution 1. L. designed the project and supervised its implementation. K. C., R. X., M. X., W. C., X. L., T. G. W. J. C. Y. J. F. and S. Z. completed all experiments. Q. X., Y. C. and C. L. contributed to data analysis. K. C. wrote the manuscript and J. L. revised the manuscript. All authors read and approved the final manuscript. Acknowledgements The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. We also acknowledge the Lingnan Medical Research Center of Guangzhou University of Chinese Medicine for the support on facilities. Data Availability All data analyzed during this study are included in this article. References Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, Jemal A. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74(3):229–63. 10.3322/caac.21834 . From NLM. Kim J, Harper A, McCormack V, Sung H, Houssami N, Morgan E, Mutebi M, Garvey G, Soerjomataram I, Fidler-Benaoudia MM. Global patterns and trends in breast cancer incidence and mortality across 185 countries. Nat Med. 2025;31(4):1154–62. 10.1038/s41591-025-03502-3 . From NLM. 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Supplementary Files Supplementarymaterial.docx Onlinefloatimage1.png Scheme 1 Schematic illustration of the anti-tumor mechanism of MND-ART-GEL. Created with BioGDP.com 47 Cite Share Download PDF Status: Published Journal Publication published 18 Apr, 2026 Read the published version in Journal of Nanobiotechnology → Version 1 posted Editorial decision: Revision requested 23 Nov, 2025 Reviews received at journal 21 Nov, 2025 Reviews received at journal 21 Nov, 2025 Reviewers agreed at journal 18 Nov, 2025 Reviews received at journal 17 Nov, 2025 Reviews received at journal 16 Nov, 2025 Reviews received at journal 16 Nov, 2025 Reviewers agreed at journal 13 Nov, 2025 Reviewers agreed at journal 11 Nov, 2025 Reviewers agreed at journal 10 Nov, 2025 Reviewers agreed at journal 10 Nov, 2025 Reviewers agreed at journal 10 Nov, 2025 Reviewers invited by journal 10 Nov, 2025 Editor assigned by journal 29 Oct, 2025 Submission checks completed at journal 29 Oct, 2025 First submitted to journal 28 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":1550822,"visible":true,"origin":"","legend":"\u003cp\u003ePreparation and Characterization of MND-ART-GEL. (A) Inversion test of blank GEL and MND-ART-GEL at different temperatures. (B) The injectability of blank GEL and MND-ART-GEL. (C) SEM images of blank GEL and MND-ART-GEL. (D) Elemental mapping of MND-ART-GEL. (E) Cumulative in vitro release profiles of DOX (n = 3). FTIR spectra of MnCl\u003csub\u003e2\u003c/sub\u003e, MSU, MnUA (F) and MnUA, DOX, MND (G).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7975044/v1/2835d3addfd15076e041314b.png"},{"id":96454927,"identity":"93f3e740-afc5-4945-bc24-535489b016ec","added_by":"auto","created_at":"2025-11-21 10:03:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1441327,"visible":true,"origin":"","legend":"\u003cp\u003eIn Vitro Antitumor Efficacy Evaluation of MND-ART-GEL. (A) Cell viability curves and IC₅₀ values of 4T1 cells treated with different drug combinations (n = 3). (B) CI-FA plot and CI values at IC₅₀ for different drug combinations. Representative dot plots (C) and quantitative analysis (D) of Annexin V-DAPI staining to assess apoptosis in 4T1 cells (n = 3). Quantitative analysis (E) and representative histograms (F) of Caspase-3 activity in 4T1 cells (n = 3). Representative histograms (G) and quantitative analysis (H) of intracellular ROS levels in 4T1 cells (n = 3). Representative dot plots (I) and quantitative analysis (J) of JC-10 staining to evaluate mitochondrial membrane potential (MMP) in 4T1 cells (n = 3). Statistical annotations: *P \u0026lt; 0.05, **P \u0026lt; 0.01 vs. PBS group; ns, not significant.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7975044/v1/1bc177d404608898edb49cf0.png"},{"id":96412626,"identity":"592f3950-655a-4995-ba62-e2e2cc63834d","added_by":"auto","created_at":"2025-11-20 19:14:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2295626,"visible":true,"origin":"","legend":"\u003cp\u003eMND-ART-GEL Induces ICD and Promotes Immune Responses In Vitro. Quantitative analysis (A) and representative histogram (B) of CRT expression in 4T1 cells (n=3). Semi-quantitative analysis (C) and western blot images (D) of HMGB1 expression in 4T1 cells (n = 3). Immunofluorescence images of CRT (E) and HMGB1 (F) expression in 4T1 cells. Quantitative analysis of Fas (G) and MHCI (H) expression in 4T1 cells. Representative flow cytometry histogram of Fas (I) and MHCI (J) expression in 4T1 cells. Representative flow cytometry scatter plots (K) and quantitative analysis (L) of BMDC maturation (CD86⁺MHCII⁺ cells%, n = 3). Representative flow cytometry scatter plots (M) and quantitative analysis (N) of M1 polarization in RAW264.7 cells (CD86⁺ cells%, n = 3). Statistical annotations: *P \u0026lt; 0.05, **P \u0026lt; 0.01 vs. PBS group; ns, not significant.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7975044/v1/a38f6f687f5bc67c6439b1ed.png"},{"id":96412629,"identity":"d2b2eb54-4beb-479c-ab40-23fcfdc2c30a","added_by":"auto","created_at":"2025-11-20 19:14:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1594383,"visible":true,"origin":"","legend":"\u003cp\u003eMND-ART-GEL Inhibits 4T1 Cell total STAT3 and p-STAT3 levels, Invasion and Migration by Downregulating STAT3. Western blot analysis of p-STAT3 (Tyr705(A) and Ser727 (B)) and total STAT3 (C) protein expression in 4T1 cells (n = 3). (D) Western blot images of 4T1 cells with drugs treated. (E) Bright-field images of wound healing assay at 12 h and 24 h after treatment. (F) Quantification of migration rate at 24 h (n = 3). (G) Quantification of invaded cells (n = 3). (H) Quantification of colony numbers (n = 3). (I) Images of colony formation assay. (J) Bright-field images of transwell invasion assay. Statistical annotations: *P \u0026lt; 0.05, **P \u0026lt; 0.01 vs. PBS group; ns, not significant.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7975044/v1/a72734b40448bf5c75884d66.png"},{"id":96454554,"identity":"9d653f0e-cb3d-40e4-9569-2cb7640a648e","added_by":"auto","created_at":"2025-11-21 10:02:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1804376,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptomic Analysis of 4T1 Cells Treated with MND-ART-GEL (n=3). (A) KEGG pathway classification of differentially expressed genes between PBS and MND-ART-GEL groups. (B) GSEA enrichment analysis based on KEGG pathways between PBS and MND-ART-GEL groups.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7975044/v1/c33034aad7f2d652f878becc.png"},{"id":96412636,"identity":"2840a39d-4a3b-4451-8920-b342c6aa1fc8","added_by":"auto","created_at":"2025-11-20 19:14:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3450241,"visible":true,"origin":"","legend":"\u003cp\u003eIn Vivo Antitumor Efficacy of MND-ART-GEL in an Orthotopic 4T1 Breast Cancer Model. (A) Schematic diagram of tumor model establishment and treatment regimen. (B) Tumor growth curves in each treatment group (n = 5). Tumor volume (C) and weight (D) post-treatment (n = 5). (E) Representative tumor images after treatment. (F) TUNEL, Ki67, H\u0026amp;E, CRT, and HMGB1 staining of tumor sections. Western blot analysis of p-STAT3 (Tyr705(G) and Ser727 (H)) and total STAT3 (I) expression in tumor tissues from different groups (n = 3). (J) Western blot images of tumor tissues in difference drugs treated groups. Statistical annotations: *P \u0026lt; 0.05, **P \u0026lt; 0.01 vs. PBS group; ns, not significant.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7975044/v1/506910591cf614ab90b67bef.png"},{"id":96412632,"identity":"040e80af-4a0a-42d3-a49c-867c19d7de50","added_by":"auto","created_at":"2025-11-20 19:14:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1689035,"visible":true,"origin":"","legend":"\u003cp\u003eIn Vivo Immune Profiling in the Orthotopic 4T1 Breast Cancer Model. Percentages of Fas⁺ (A) and MHCI⁺ (B) in tumor cells post-treatment (n = 5). Maturation of DCs in TDLNs (CD86⁺MHCII⁺ cells%, n = 5): representative plots (C) and quantification (D). Tumor-infiltrating DCs (CD11c⁺ cells%, n = 5) (E) and mature DCs (CD11c⁺MHCII⁺ cells%, n = 5) (F). Percentages of M1 macrophages (CD86⁺CD206⁻) (G), M2 TAMs (CD86⁺CD206⁺ and CD86⁻CD206⁺) (H), and M1/M2 ratio in tumors (n = 5) (I). Representative flow cytometry plots (J) and quantification (K) of CD8⁺ T cells (CD3⁺CD8⁺%, n=5) in tumors post-treatment. CD8⁺PD-1⁺ (L), and CD8⁺PD-L1⁺ T cells% (M) in tumors (n = 5). Quantification of TNF-α (N), IFN-γ (O), and IL-6 (P) in tumor tissues (n = 3). Statistical annotations: *P \u0026lt; 0.05, **P \u0026lt; 0.01 vs. PBS group; ns, not significant.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7975044/v1/8ad3cdfa76e4c35592614b33.png"},{"id":96412634,"identity":"95112446-1258-4583-a1f0-57438878103f","added_by":"auto","created_at":"2025-11-20 19:14:15","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1417683,"visible":true,"origin":"","legend":"\u003cp\u003ePostoperative Anti-Recurrence Efficacy and Immunological Evaluation of MND-ART-GEL. (A) Schematic diagram of the 4T1 postsurgical tumor recurrence model and treatment plan. Tumor weight (B), growth curves (C), and volume (D) in each group (n = 5, PBS group n = 4). (E) Representative tumor images post-treatment. DC maturation in tumor (CD86⁺MHCII⁺ cells%, n = 3): representative plots (F) and quantification (G). CD8⁺ T cell infiltration in tumors (CD3⁺CD8⁺ cells%, n = 3): representative plots (H) and quantification (I). Representative flow plots of macrophage subpopulations in tumor (J), percentages of M1/M2 ratio (K), M2 TAMs (CD86⁺CD206⁺ and CD86⁻CD206⁺) (L), and M1 macrophages (CD86⁺CD206⁻) (M) in tumors (n = 3). Statistical annotations: *P \u0026lt; 0.05, **P \u0026lt; 0.01 vs. PBS group; ns, not significant.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7975044/v1/888f3592c626115cca897d4b.png"},{"id":107350761,"identity":"5553b315-e2e6-4a21-8413-2037f293a8d7","added_by":"auto","created_at":"2026-04-20 16:03:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16019593,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7975044/v1/d0801902-1ed5-499c-8f4e-47974ca85a1a.pdf"},{"id":96412638,"identity":"c2e97c94-f14d-4e9d-9fe6-233ce0def613","added_by":"auto","created_at":"2025-11-20 19:14:15","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5931898,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7975044/v1/1b6de7a1ddca0dffede2483d.docx"},{"id":96412635,"identity":"c3ce80fc-c936-4681-baea-a9d2da2fb952","added_by":"auto","created_at":"2025-11-20 19:14:15","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":376309,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003e Schematic illustration of the anti-tumor mechanism of MND-ART-GEL. Created with BioGDP.com\u003csup\u003e47\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7975044/v1/f8649b8a461c15d1d9c3131b.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"MnUA–DOX–Artesunate Hydrogel Remodels Immunosuppressive Tumor Microenvironment and Prevents Postoperative Recurrence in Triple-negative Breast Cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer is one of the most common cancers and the second leading cause of cancer-related deaths among women worldwide\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In 2022, approximately 2.3\u0026nbsp;million new cases of breast cancer were reported and 670,000 breast cancer-related deaths occurred globally\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Triple-negative breast cancer (TNBC), a breast cancer subtype characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), lacks specific therapeutic targets\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Consequently, endocrine therapy and HER2-targeted drugs are ineffective for TNBC. Moreover, TNBC is highly aggressive, prone to metastasis, and presents a narrow therapeutic window\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Chemotherapy remains the primary treatment for TNBC; however, it often leads to drug resistance and significant adverse effects upon prolonged administration at high doses\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Although a subset of TNBC patients responds to immunotherapy, such as programmed cell death ligand 1/programmed cell death protein 1 (PD-L1/PD-1) blockade, the high heterogeneity of TNBC results in many patients exhibiting immunosuppressive \u0026ldquo;cold tumor\u0026rdquo; phenotypes, rendering the immunotherapy less effective\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Surgical resection is the cornerstone for potential cure of early-stage TNBC; however, postoperative recurrence is frequent and poses a serious threat. Clinical data indicate that 30\u0026ndash;40% of patients with TNBC relapse within five years of surgery, with poor prognosis and high metastatic potential\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Recurrent TNBC tumors are often resistant to chemotherapy, with predominantly suppressive immune microenvironment, which limits the efficacy of immunotherapy\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. These challenges highlight the inadequacy of monotherapies and underscore the urgent need for devising novel therapeutic strategies.\u003c/p\u003e\u003cp\u003eManganese ions (Mn\u0026sup2;\u003csup\u003e+\u003c/sup\u003e), as natural cGAS agonists, induce robust immune responses by activating the cGAS-STING pathway\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. This is indicative of their potential as immune adjuvants capable of modulating cellular immunity, which can be leveraged to overcome the limitation of traditional aluminum-based adjuvants that predominantly elicit humoral immunity\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Unlike immune checkpoint inhibitors such as PD-1/PD-L1 blockers that mainly enhance the functioning of T cells, Mn\u0026sup2;\u003csup\u003e+\u003c/sup\u003e can directly reprogram the tumor microenvironment (TME)\u0026mdash;it promotes the maturation and differentiation of tumor-associated dendritic cells (DCs) and stimulates substantial production of type I interferon, thereby enhancing the infiltration of CD8\u003csup\u003e+\u003c/sup\u003e T cells into tumors\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. This effectively reverses the immunosuppressive TME and converts poorly immunogenic \u0026ldquo;cold tumors\u0026rdquo; into immune-responsive \u0026ldquo;hot tumors.\u0026rdquo;\u003c/p\u003e\u003cp\u003eRecent evidence indicates that NF-κB activation can prevent the intracellular degradation of activated STING, thereby amplifying STING-associated signaling cascades\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. As a key regulator of immune homeostasis, NF-κB plays a crucial role in coordinating innate and adaptive immune responses. NF-κB signaling can be triggered by Toll-like receptors (TLRs), while monosodium urate (MSU) crystals, acting as a typical damage-associated molecular pattern (DAMP), are capable of activating TLRs\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. These findings suggest that MSU may enhance Mn\u0026sup2;⁺-induced immune activation, highlighting its potential as an adjuvant candidate for antitumor immunotherapy. Importantly, both MSU and Mn\u0026sup2;⁺ are common endogenous or inorganic components with wide availability, facile preparation, and low cost, offering excellent accessibility and scalability. Compared with other expensive or synthetically demanding immune activators, this combination exhibits superior economic and translational advantages, making it highly promising for large-scale preparation and future clinical applications.\u003c/p\u003e\u003cp\u003eArtesunate (ART), a derivative of artemisinin, has been extensively investigated for its antitumor potential in recent years. The STAT3 pathway, which is highly activated in TNBC, plays a pivotal role in promoting tumor proliferation, metastasis, and immune evasion\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. ART can inhibit phosphorylation and nuclear translocation of STAT3\u003csup\u003e20\u003c/sup\u003e, thereby disrupting the positive feedback loop between STAT3 and IL-6/JAK2 in various tumor models, including colon and liver cancers\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. ART suppresses tumor stemness and reverses immunosuppressive TME. Moreover, it promotes mitochondrial damage via reactive oxygen species (ROS) accumulation\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, and synergizes with doxorubicin (DOX) to exert potent antitumor effects\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. These properties make ART a promising candidate for overcoming chemoresistance.\u003c/p\u003e\u003cp\u003eIn this study, we synthesized manganese urate (MnUA) as an immune adjuvant by combining Mn\u0026sup2;\u003csup\u003e+\u003c/sup\u003e and MSU. We coencapsulated MnUA with the chemotherapeutic agents DOX and ART into a thermosensitive Pluronic F127 hydrogel (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This multimodal strategy, integrating chemotherapy, immune modulation, and STAT3 suppression, was aimed at enhancing the therapeutic efficacy against TNBC by inhibiting tumor aggressiveness and metastasis, reversing the immunosuppressive TME, and improving treatment outcomes for both primary and postoperative recurrent breast cancers.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003ePreparation of MnUA.\u003c/b\u003e MnUA particles were prepared using a reverse microemulsion technique. Briefly, 10 mL of cyclohexane, 2 mL of Triton X-100, and 1 mL of \u003cem\u003en\u003c/em\u003e-hexanol were mixed in two separate glass vials and vigorously stirred for 10 min. Thereafter, 1 mL of 2 mg/mL MSU suspension in phosphate-buffered saline (PBS) was added to one vial (MSU phase), and 1 mL of 4 mg/mL MnCl\u003csub\u003e2\u003c/sub\u003e aqueous solution was added to the other (Mn phase). The contents of each vial were stirred vigorously for 30 min. The two phases were then combined and stirred vigorously for 1 h to allow the reaction to proceed. The organic solvent was then removed by rotary evaporation. An equal volume of absolute ethanol was added to break the emulsion, followed by centrifugation at 10,000 rpm for 10 min to collect the precipitate. The precipitate was washed once with absolute ethanol and twice with ultrapure water, resuspended in an appropriate amount of ultrapure water, and stored at 4\u0026deg;C.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation of MnUA-DOX (MND).\u003c/b\u003e MnUA particles were resuspended in a 2 mg/mL aqueous solution of doxorubicin hydrochloride and incubated at 37\u0026deg;C for 6\u0026ndash;12 h with shaking at 100 rpm. The incubation was terminated when the particles turned dark red, indicating successful drug loading. The resulting MND particles were collected by centrifugation at 10,000 rpm for 10 min. The supernatant was collected for determining the drug loading efficiency.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation of MND-ART-GEL.\u003c/b\u003e ART was weighed at eight-times the mass of DOX in MND. ART and Pluronic F127 were dissolved together in absolute ethanol at a mass ratio of 1:5. The ethanol was evaporated at 37\u0026deg;C to allow the formation of a thin film, which was then hydrated using an appropriate volume of PBS with ultrasonication. After centrifuging 1 mL of MND suspension at 10,000 rpm to remove the supernatant, 1 mL of the hydrated ART/F127 suspension was added. The mixture was probe-sonicated at an ice bath at 200 W for 5 min to ensure uniform dispersion of ART and MND. The final concentration was to adjusted to 22% with Pluronic F127. The mixture was stirred overnight at 4\u0026deg;C to ensure complete dissolution, yielding the final thermosensitive hydrogel formulation, which was designated \u0026ldquo;MND-ART-GEL.\u0026rdquo;\u003c/p\u003e\u003cp\u003e\u003cb\u003eDrug Release.\u003c/b\u003e DOX solution, MND, and MND-ART-GEL were prepared separately and adjusted to the same DOX concentration. For each group, 1 mL of the formulation was loaded into a dialysis bag (molecular weight cut-off: 8000\u0026ndash;14000 Da), with both ends sealed tightly. The dialysis bags were immersed in 100 mL of PBS and incubated at 37\u0026deg;C on a shaker at 100 rpm. At predetermined time points (0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 24, 48, 72, 96, 120, and 168 h), 1 mL of release medium was withdrawn and immediately replenished with 1 mL of fresh PBS. The collected samples were filtered through a 0.22 \u0026micro;m membrane filter and analyzed via high-performance liquid chromatography (HPLC). The cumulative release percentage of DOX was calculated and plotted as a release profile over time.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell Culture.\u003c/b\u003e 4T1 (mouse triple-negative breast cancer cells) and RAW264.7 (mouse monocyte-macrophage leukemia cells) cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). 4T1 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 \u0026micro;g/mL streptomycin at 37\u0026deg;C in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. RAW264.7 cells were cultured in Dulbecco\u0026rsquo;s modified Eagle medium with the same supplements and under identical conditions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCytotoxicity Assay.\u003c/b\u003e The cytotoxicity was assessed using the Cell Counting Kit (CCK)-8 assay. 4T1 cells were seeded in 96-well plates at a density of 5 \u0026times; 10\u0026sup3; cells per well and incubated overnight. The medium was then replaced with drug-containing medium at MND:ART mass ratios of 1:2, 1:4, 1:6, 1:8, and 1:10. The MND dosage was calculated based on the DOX concentration, and final DOX concentrations were set at 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.813, and 0 ng/mL. After 48 h of incubation, CCK-8 solution was added to each well, followed by incubation for an additional 1 h. The absorbance at 450 nm was then measured using a microplate reader (Varioskan LUX; Thermo Fisher, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eApoptosis Assay.\u003c/b\u003e 4T1 cells were seeded in 6-well plates at a density of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well and incubated overnight. The cells were then treated with culture medium containing DOX, MND, ART, MND-ART, or MND-ART-GEL. The final concentration of DOX and ART in the medium was 4 and 32 \u0026micro;g/mL, respectively. After 48 h of incubation, the cells were collected and washed with PBS. They were then stained with Annexin V-AF647 and 4\u0026prime;,6-diamidino-2-phenylindole (DAPI) (1:200 Annexin V, 2 \u0026micro;g/mL DAPI, diluted in 1\u0026times; binding buffer) at 4\u0026deg;C for 30 min. The cells were subsequently washed with PBS and analyzed via flow cytometry (LSRFortessa; BD, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMitochondrial Membrane Potential Assay.\u003c/b\u003e 4T1 cells were seeded in 6-well plates at a density of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well and incubated overnight. The cells were then treated with culture medium containing DOX, MND, ART, MND-ART, or MND-ART-GEL. The final concentration of DOX and ART was 4 and 32 \u0026micro;g/mL, respectively. After 48 h of incubation, the cells were collected and washed with PBS. They were then stained with JC-10 dye (15 \u0026micro;M, diluted in Cell Staining Buffer) at 37\u0026deg;C for 30 min, washed with PBS, and analyzed via flow cytometry.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCaspase-3 Activity Assay.\u003c/b\u003e 4T1 cells were seeded in 6-well plates at a density of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well and incubated overnight. Thereafter, they were treated with culture medium containing DOX, MND, ART, MND-ART, or MND-ART-GEL. The final concentration of DOX and ART was 2 and 16 \u0026micro;g/mL, respectively. After 24 h of incubation, the cells were collected, washed with PBS, and stained with Z-DEVD-AFC (2.5 \u0026micro;g/mL, diluted in Cell Staining Buffer) at 37\u0026deg;C for 30 min. The cells were subsequently washed with PBS and analyzed via flow cytometry.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetection of Intracellular ROS.\u003c/b\u003e 4T1 cells were seeded in 12-well plates at a density of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well and incubated overnight. They were then treated with culture medium containing DOX, MND, ART, MND-ART, or MND-ART-GEL. The final concentration of DOX and ART was 2 and 16 \u0026micro;g/mL, respectively. After 16 h of incubation, the cells were collected, washed with PBS, and stained with 2\u0026prime;,7\u0026prime;-dichlorodihydrofluorescein diacetate (10 \u0026micro;M, diluted in serum-free medium) at 37\u0026deg;C for 30 min. Thereafter, the cells were washed with PBS and analyzed via flow cytometry.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern Blot Analysis.\u003c/b\u003e 4T1 cells were seeded in 6-well plates at a density of 3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well. After overnight incubation, the cells were treated with culture medium containing DOX, MND, ART, MND-ART, or MND-ART-GEL. The concentration of DOX and ART was 240 ng/mL and 1.92 \u0026micro;g/mL, respectively. After 48 h of incubation, the cells were washed with PBS and lysed using RIPA lysis buffer supplemented with protease and phosphatase inhibitors. For tumor tissue samples, approximately 100 mg of tumor tissue was weighed and homogenized in 1 mL of RIPA buffer containing protease and phosphatase inhibitors using a precooled tissue homogenizer. The protein concentrations of both cell and tissue lysates were determined using a BCA protein assay kit. Equal amounts of protein were subjected to SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with protein-free blocking buffer and incubated overnight at 4\u0026deg;C with primary antibodies. After washing, the membranes were incubated with appropriate secondary antibodies for 1 h at room temperature. The signals were detected using the Amersham ImageQuant 800 imaging system (Cytiva, Sweden).\u003c/p\u003e\u003cp\u003e\u003cb\u003eScratch Assay.\u003c/b\u003e 4T1 cells were seeded in 6-well plates at a density of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per well and cultured overnight until reaching full confluence. A scratch was made in the well surface using a 200 \u0026micro;L pipette tip, and detached cells were removed by washing with PBS. The cells were then treated with culture medium containing DOX, MND, ART, MND-ART, or MND-ART-GEL. The final concentration of DOX and ART was 2 and 16 \u0026micro;g/mL, respectively. Wound closure was monitored and photographed under a microscope at 0, 12, and 24 h.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMatrigel Invasion Assay.\u003c/b\u003e 4T1 cells were seeded in the upper chamber of a Transwell insert (8 \u0026micro;m pore size) at a density of 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well in serum-free medium and starved overnight. After removing the medium, the cells were treated with serum-free medium containing DOX, MND, ART, MND-ART, or MND-ART-GEL. The final concentration of DOX and ART was 1 and 8 \u0026micro;g/mL, respectively. After 48 h of incubation, cells on the lower surface of the membrane were fixed with 4% paraformaldehyde and stained with 1% crystal violet. After washing with distilled water, the migrated cells were imaged under a microscope.\u003c/p\u003e\u003cp\u003e\u003cb\u003eColony Formation Assay.\u003c/b\u003e 4T1 cells were seeded in 6-well plates at a density of 2 \u0026times; 10\u0026sup3; cells per well and cultured overnight. They were then treated with culture medium containing DOX, MND, ART, MND-ART, or MND-ART-GEL. The final concentration of DOX and ART was 240 ng/mL and 1.92 \u0026micro;g/mL, respectively. The cells were cultured until visible colonies (\u0026gt;\u0026thinsp;50 cells per colony) formed. The colonies were fixed with 4% paraformaldehyde and stained with 1% crystal violet, followed by washing with distilled water and air-drying. Images of each well were captured using a digital camera, and the number of colonies was counted.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of Immunogenic Cell Death (ICD)-Related Markers.\u003c/b\u003e 4T1 cells were seeded in 6-well plates at a density of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well and cultured overnight. The cells were then treated with culture medium containing DOX, MND, ART, MND-ART, or MND-ART-GEL, with the final concentration of DOX and ART being 240 ng/mL and 1.92 \u0026micro;g/mL, respectively. After 48 h of incubation, the cells were harvested and washed with PBS. The expression levels of calreticulin (CRT), major histocompatibility complex class I (MHCI), and Fas cell surface death receptor (Fas) were analyzed via flow cytometry. In parallel, cell lysates were collected for western blot analysis of HMGB1 expression. After the different treatments, cells were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100, followed by blocking with 5% bovine serum albumin (BSA) for 60 min. The cells adhered to coverslips were then incubated overnight at 4\u0026deg;C in a humidified chamber with primary antibodies against CRT or HMGB1 (1:200 dilution). Next day, the cells were incubated with Alexa Fluor 488-conjugated secondary antibodies (1:400 dilution) for 1 h at room temperature, followed by counterstaining of nuclei with Hoechst 33342. After washing with PBS, coverslips were mounted on glass slides with glycerol gelatin and imaged using a BX53 fluorescence microscope (Olympus, Japan).\u003c/p\u003e\u003cp\u003e\u003cb\u003eBone marrow-derived dendritic cell (BMDC) Maturation and Coculture with Treated Tumor Cells.\u003c/b\u003e Bone marrow cells were harvested from the femur and tibia of BALB/c mice and resuspended in RPMI-1640 complete medium after lysing the red blood cells. The cells were cultured in the presence of 20 ng/mL granulocyte-macrophage colony-stimulating factor, with half of the medium replaced every other day. On day 6, loosely adherent immature BMDCs were collected and seeded in 12-well plates at a density of 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well. The cells were then treated with MSU (4 \u0026micro;g/mL), Mn\u0026sup2;\u003csup\u003e+\u003c/sup\u003e (1.5 \u0026micro;g/mL), or the MnUA combination. Lipopolysaccharide (LPS; 1 \u0026micro;g/mL) was used as a positive control. After 48 h of incubation, the cells were harvested, washed with PBS, and stained with anti-CD11c-APC, anti-CD86-PE, and anti-MHC II-PerCP/Cy5.5 antibodies (1:100 dilution) at 4\u0026deg;C for 30 min. After washing, the cells were analyzed via flow cytometry to determine the proportion of mature BMDCs (MHC II\u003csup\u003e+\u003c/sup\u003eCD86\u003csup\u003e+\u003c/sup\u003e).\u003c/p\u003e\u003cp\u003eFor the coculture assay, 4T1 cells were pretreated with DOX, MND, ART, MND-ART, or MND-ART-GEL for 48 h, with the DOX and ART concentration being 240 ng/mL and 1.92 \u0026micro;g/mL, respectively. Treated 4T1 cells were then cocultured with BMDCs; a group treated with LPS (1 \u0026micro;g/mL) served as a positive control. After 24 h of coculture, the cells were collected, washed with PBS, and stained with anti-CD11c-APC, anti-CD86-PE, and anti-MHC II-PerCP/Cy5.5 antibodies (1:100 dilution) at 4\u0026deg;C for 30 min. After washing, the samples were analyzed via flow cytometry to evaluate the maturation of BMDCs (MHC II\u003csup\u003e+\u003c/sup\u003eCD86\u003csup\u003e+\u003c/sup\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMacrophage Polarization Assay.\u003c/b\u003e RAW264.7 cells were seeded in 12-well plates at a density of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per well and incubated overnight. The cells were then treated with serum-free medium containing MSU (4 \u0026micro;g/mL), Mn\u0026sup2;\u003csup\u003e+\u003c/sup\u003e (1.5 \u0026micro;g/mL), or the MnUA combination. A group treated with LPS (1 \u0026micro;g/mL) was used as a positive control. After 24 h of incubation, the cells were harvested, washed with PBS, and stained with an anti-CD86-APC antibody (1:100 dilution) at 4\u0026deg;C for 30 min. After washing with PBS, the cells were analyzed via flow cytometry to determine the proportion of M1-polarized macrophages (CD86\u003csup\u003e+\u003c/sup\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn Vivo Antitumor Study in BALB/c Mice.\u003c/b\u003e An orthotopic 4T1 breast cancer model was established using female BALB/c mice (6\u0026ndash;8-weeks-old; weight, 18\u0026ndash;22 g), purchased from the Guangdong Medical Laboratory Animal Center. The mice were housed under specific pathogen-free conditions at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C with a 12 h light/dark cycle and had ad libitum access to food and water. All animal procedures were approved by the Institutional Animal Care and Use Committee of Guangzhou University of Chinese Medicine (Approval No. PZ24115). 4T1 cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells in 100 \u0026micro;L PBS) were injected into the mammary fat pad of female BALB/c mice after washing with PBS to remove serum. Once tumors reached a volume of approximately 80 mm\u0026sup3;, the mice were randomly divided into six groups (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5 per group): PBS, DOX, MND, ART, MND-ART, and MND-ART-GEL. Treatments were administered via intratumoral injection every other day for a total of eight doses. The administered doses were equivalent to 4 mg/kg DOX or 32 mg/kg ART. Tumor dimensions (length and width) and body weight were measured using a caliper every two days, and tumor volumes were calculated according to the following formula:\u003c/p\u003e\u003cp\u003eTumor volume (mm\u0026sup3;)\u0026thinsp;=\u0026thinsp;length \u0026times; width\u0026sup2; / 2.\u003c/p\u003e\u003cp\u003eAt the end of the experiment, the mice were euthanized, and whole blood, heart, liver, spleen, lungs, kidneys, tumor-draining lymph nodes (TDLNs), and tumors were harvested for imaging and weighing. Major organs (heart, liver, spleen, lungs, and kidneys) were fixed in 4% paraformaldehyde for histological analysis. Tumor tissues were divided into three portions for flow cytometry, western blotting, and paraffin embedding (for hematoxylin and eosin (H\u0026amp;E) and immunohistochemical staining). TDLNs were processed for flow cytometry analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePost-Surgical Tumor Recurrence Model and In Situ Treatment.\u003c/b\u003e To simulate post-surgical recurrence, an incomplete tumor resection was performed when tumor volume reached approximately 200 mm\u0026sup3;. Mice were anesthetized with isoflurane, and the surgical area was sterilized. An incision was made on one side of the tumor, and the skin was separated from the tumor using surgical scissors. The majority of the tumor was excised, leaving a residual tumor mass of approximately 20 mm\u0026sup3;. The wound was then sutured and the mice were placed in a warming chamber for recovery. The mice were randomly divided into three groups (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5): PBS, DOX, and MND-ART-GEL (DOX: 4 mg/kg; ART: 32 mg/kg). Treatments were administered via in situ injection every other day for a total of five doses. Tumor size was measured using a digital caliper, and tumor volume was calculated using the formula provided under subheading \u0026ldquo;In Vivo Antitumor Study in BALB/c Mice\u0026rdquo;.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFlow Cytometry of Tumor and Lymph Node Immune Cells.\u003c/b\u003e At the end of treatment, tumors were excised and digested with tumor tissue digestion buffer at 37\u0026deg;C for 1 h. The resulting tumor cell suspensions were passed through a 200-mesh nylon cell strainer. After lysing red blood cells, the single-cell suspension was blocked with 1% BSA at room temperature for 15 min. The cells were then divided into four parts for the analysis of tumor cells, DCs, macrophages, and T cells. Lymph nodes were harvested and gently ground on a 200-mesh nylon cell strainer in PBS to obtain single-cell suspensions. After passing through the strainer, the cells were blocked with 1% BSA at room temperature for 15 min and divided into three parts for the detection of DCs, macrophages, and T cells. All samples were incubated with specific antibodies at 4\u0026deg;C for 30 min in the dark, followed by flow cytometry analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEnzyme-linked Immunosorbent Assay for Cytokines.\u003c/b\u003e Tumor tissue (100 mg) was weighed and homogenized in 0.9 mL of cold PBS containing protease inhibitors using a precooled tissue homogenizer. The tissue lysate was centrifuged to remove debris, and the supernatant was collected for analysis. The levels of IL-6, TNF-α, and IFN-γ in the tumor homogenates were measured using commercially available ELISA kits (Biolegend, USA) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical Analysis\u003c/b\u003e. Statistical analysis was performed using IBM SPSS Statistics 26, and graphs were generated with GraphPad Prism 10. For comparison between two groups, an independent-sample \u003cem\u003et\u003c/em\u003e-test was used, with Levene\u0026rsquo;s test applied to assess variance homogeneity. For comparisons among three or more groups, normality was tested using the Shapiro\u0026ndash;Wilk test. Data with P\u0026thinsp;\u0026gt;\u0026thinsp;0.05 were considered normally distributed and expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD), shown in bar graphs. Data with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered non-normally distributed and presented as median (P25\u0026ndash;P75), shown in box plots. Nonparametric tests were used for non-normal data: Mann\u0026ndash;Whitney U test for two groups and Kruskal\u0026ndash;Wallis H test for multiple groups.\u003c/p\u003e\u003cp\u003eFor normally distributed data, one-way ANOVA was used. If variances were homogeneous (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), Tukey\u0026rsquo;s test was applied for more than three groups, and Bonferroni correction for three or fewer groups with equal sample sizes. When sample sizes were unequal, Scheff\u0026eacute;\u0026rsquo;s test was used. If variances were unequal (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), Welch\u0026rsquo;s ANOVA followed by Dunnett T3 post hoc test was performed. Statistical significance was indicated as follows: ns (not significant), *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (significant), and **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (highly significant). Asterisks placed above the bars indicate comparisons versus the PBS group.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cb\u003ePreparation and Characterization of MND-ART-GEL.\u003c/b\u003e Pluronic F127 is a triblock copolymer consisting of a central hydrophobic polypropylene oxide block and two hydrophilic polyethylene oxide blocks, exhibiting temperature-dependent self-assembly\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The blank hydrogel remained in the liquid form at temperatures between 15 and 25\u0026deg;C but gelled at temperatures\u0026thinsp;\u0026gt;\u0026thinsp;30\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). MND-ART-GEL retained this behavior, indicating its suitability for in situ gelation at near physiological temperatures\u0026mdash;ideal for local and postoperative tumor therapy. The MND and ART were uniformly dispersed into a 22% Pluronic F127 thermosensitive hydrogel by ultrasonic mixing with 6% Pluronic film hydration, forming an injectable MND-ART-GEL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Scanning electron microscopy (SEM) images revealed a uniform porous gel network (~\u0026thinsp;20 \u0026micro;m, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), and energy dispersive spectroscopy (EDS) mapping showed homogeneous distribution of Mn, N, and Cl (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), indicating even dispersion of MND within the gel.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDrug release profiles were evaluated via HPLC. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, ~\u0026thinsp;90% of free DOX was released within 6 h, and complete release was eventually achieved. In contrast, MND achieved a sustained release plateau at ~\u0026thinsp;80% over 48 h. MND-ART-GEL exhibited the slowest initial release and a sustained profile, with ~\u0026thinsp;76% release achieved by day 7. As shown in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, free ART was almost completely released within 2 h, while ART in the MND-ART-GEL formulation reached a plateau at 48 h, with approximately 70% of the drug released. These results indicated that both MnUA particle encapsulation and hydrogel formulation conferred sustained-release properties, enabling prolonged drug exposure and potentially reducing systemic toxicity associated with conventional high-dose chemotherapy. Meanwhile, the relatively safe drug ART was released at a faster rate from the hydrogel, allowing for an early therapeutic effect.\u003c/p\u003e\u003cp\u003eMnUA microparticles were first synthesized via a reverse microemulsion method. MSU and MnCl\u003csub\u003e2\u003c/sub\u003e formed an MnUA microemulsion upon vigorous stirring, which was washed to remove organic solvents and surfactants to obtain MnUA particles. FTIR spectra of MSU, MnCl\u003csub\u003e2\u003c/sub\u003e, and MnUA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) revealed that the characteristic peaks of MSU at 1770\u0026ndash;440 cm\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026sup1;\u0026mdash;corresponding to C\u0026thinsp;=\u0026thinsp;O stretching of the ketone group and purine ring vibrations\u0026mdash;were replaced by two new peaks at 1160\u0026ndash;880 cm\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026sup1; (C\u0026ndash;O\u0026ndash;Mn) and 560 cm\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026sup1; (Mn\u0026ndash;O) in MnUA. The enhanced 560 cm\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026sup1; peak in MnUA compared with that in MnCl\u003csub\u003e2\u003c/sub\u003e confirmed the formation of Mn\u0026ndash;O coordination bonds. Additionally, the disappearance of the broad peak at 3200\u0026ndash;2600 cm\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026sup1; in MSU indicated that the amino group (\u0026ndash;NH) on the purine ring may also coordinate with Mn\u0026sup2;\u003csup\u003e+\u003c/sup\u003e, forming Mn\u0026ndash;N bonds that stabilize the MnUA structure.\u003c/p\u003e\u003cp\u003eMnUA was then incubated with DOX solution to form drug-loaded MnUA-DOX (MND). HPLC analysis of the supernatant after centrifugation revealed a DOX loading content of 35.45%, indicating that MnUA achieved high drug-loading efficiency in aqueous solution without requiring activation with a base (e.g., triethylamine)\u003csup\u003e\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The FTIR spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG) indicated that the drug loading occurred primarily via physical adsorption: the characteristic peaks of DOX (e.g., \u0026ndash;NH/\u0026ndash;OH at 3530 and 3310 cm\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026sup1;, and anthraquinone ring vibrations between 1740 and 400 cm\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026sup1;) disappeared, whereas the MnUA skeleton peaks at 1010 and 560 cm\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026sup1; remained unchanged, which was suggestive of physical adsorption of DOX rather than its chemical binding.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEvaluation of the Synergistic Cytotoxicity of MND and ART Against 4T1 Cells.\u003c/b\u003e The CCK-8 assay was performed to determine the optimal drug ratio for combining MND and ART. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, DOX, a first-line chemotherapeutic agent, effectively inhibited the viability of 4T1 cells, whereas ART exerted a weaker cytotoxic effect, acting primarily as an adjuvant. Compared with DOX alone, MND exhibited a lower half-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e), indicative of enhanced efficacy of DOX-loaded MnUA microparticles at reduced doses. When MND was combined with ART, the IC\u003csub\u003e50\u003c/sub\u003e values at 1:8 and 1:10 ratios were further reduced, with the lowest value observed at the 1:8 ratio.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe combination index (CI), a quantitative metric for drug interaction, was used to evaluate synergism (CI\u0026thinsp;\u0026lt;\u0026thinsp;1) or antagonism (CI\u0026thinsp;\u0026gt;\u0026thinsp;1). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, the CI values at the 1:8 combination ratio were consistently less than 1 across most fractional affected (FA) levels, with the lowest CI at IC\u003csub\u003e50\u003c/sub\u003e, indicating a favorable synergistic effect. Therefore, the 1:8 ratio was selected for subsequent experiments.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMND-ART-GEL Induces Apoptosis and ROS Accumulation in 4T1 Cells In Vitro.\u003c/b\u003e Apoptosis and caspase-3 activation in 4T1 cells treated with various formulations were assessed via flow cytometry. All the treatments induced apoptosis and activation of caspase-3 to varying degrees. Compared with free DOX, the MND formulation and its combination with ART in the hydrogel form significantly increased the proportion of apoptotic cells, with treatment with MND-ART-GEL resulting in the highest apoptosis rate and caspase-3 activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-F). These results indicated that, at equivalent drug dosages, the hydrogel formulation was more effective in inducing apoptosis of tumor cells.\u003c/p\u003e\u003cp\u003eDOX induces ROS accumulation, which damages tumor cell DNA, thereby contributing to cytotoxicity\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Moreover, ART contains an endoperoxide bridge that reacts with intracellular iron ions, leading to additional ROS production\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. These ROS species exacerbate DNA damage and mitochondrial dysfunction, indicating that ROS generation is one of the primary mechanisms underlying MND-ART-GEL\u0026ndash;induced apoptosis.\u003c/p\u003e\u003cp\u003eTo further confirm this hypothesis, intracellular ROS levels and mitochondrial membrane potential (MMP) were evaluated via flow cytometry. Compared with DOX and MND groups, MND-ART-GEL significantly elevated the ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-H) and increased the proportion of cells with depolarized mitochondrial membrane (low MMP) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI-J). These findings confirmed that MND-ART-GEL induces tumor cell death primarily through ROS-mediated mechanisms.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMND-ART-GEL Induces ICD and Surface Immunomarker Exposure in 4T1 Cells.\u003c/b\u003e DOX is a well-established inducer of ICD, known to trigger endoplasmic reticulum (ER) stress in tumor cells, thereby promoting ICD\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. As MND-ART-GEL elevates intracellular ROS levels, it may enhance ER stress-mediated ICD\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The expression levels of CRT and high mobility group box 1 (HMGB1), two hallmark ICD-associated DAMPs, were evaluated to assess the ICD-inducing ability of MND-ART-GEL. Among all treatment groups, MND-ART-GEL induced the highest expression levels of both CRT and HMGB1, significantly exceeding those observed in the DOX, MND, and ART groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D). This was further validated by immunofluorescence imaging, wherein 4T1 cells treated with MND-ART-GEL displayed the brightest green fluorescence signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFlow cytometry analysis of surface immune markers revealed that 4T1 cells treated with MND-ART-GEL exhibited increased expression of apoptosis-associated factor Fas and MHCI molecules (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG\u0026ndash;J). This enhancement in the expression of immunogenic markers indicated that MND-ART-GEL treatment rendered tumor cells more recognizable and susceptible to immune cell-mediated cytotoxicity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMND-ART-GEL Activates Immune Cells In Vitro.\u003c/b\u003e DCs are the most potent antigen-presenting cells (APCs). Mature DCs express high levels of MHC I/II molecules and costimulatory molecules, such as CD80 and CD86, enabling them to present tumor antigens to T cells and initiate immune responses. Tumor-associated macrophages (TAMs) are a major component of the TME, which exhibit two phenotypes: M1 and M2. M1-like TAMs exert antitumor activity by sustaining inflammation, recruiting immune cells, and phagocytosing tumor cells, whereas M2-like TAMs generally promote tumor progression. Mn\u0026sup2;\u003csup\u003e+\u003c/sup\u003e is a widely recognized immune adjuvant capable of activating the cGAS-STING pathway and inducing robust adaptive immunity. MSU serves as a DAMP that can regulate immune responses via TLR/NF-κB signaling and enhance STING-associated signaling cascades.\u003c/p\u003e\u003cp\u003eTo evaluate the immunostimulatory effects of MnUA, primary BMDCs and RAW264.7 macrophages from Balb/c mice were incubated with MnUA. Flow cytometry analysis revealed that MnUA treatment significantly increased the proportion of CD86\u003csup\u003e+\u003c/sup\u003eMHCII\u003csup\u003e+\u003c/sup\u003e BMDCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK-L), surpassing the effects observed in MSU or Mn\u0026sup2;\u003csup\u003e+\u003c/sup\u003e single-agent groups and even exceeding those in the LPS-treated positive control group. Similarly, the percentage of CD86\u003csup\u003e+\u003c/sup\u003e RAW264.7 macrophages was markedly increased following MnUA exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM-N). These findings indicated that MnUA acts as a potent immune adjuvant capable of promoting DC maturation and polarizing TAMs toward the M1 phenotype.\u003c/p\u003e\u003cp\u003eGiven the fact that MND-ART-GEL could induce robust ICD, we cocultured 4T1 cells and BMDCs to evaluate whether ICD-induced signals could activate DCs. As shown in Figure S2, compared with the blank control, BMDCs cocultured with PBS-treated 4T1 cells exhibited marked immunosuppression, with reduced maturation likely due to suppressive cytokines or metabolites secreted by tumor cells\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In contrast, the MND-ART-GEL\u0026ndash;treated group exhibited the highest DC maturation, significantly exceeding that in other groups, indicating that ICD triggered by MND-ART-GEL could reverse the immunosuppressive TME.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMND-ART-GEL Suppresses STAT3 Activation and Inhibits the Invasion and Metastasis of 4T1 Cells.\u003c/b\u003e In TNBC, the STAT3 signaling pathway is frequently hyperactivated, promoting tumor proliferation, invasion, metastasis, and immune evasion, while contributing to chemoresistance\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. ART acts as an inhibitor of STAT3 by preventing its phosphorylation and nuclear translocation, thereby reducing its DNA-binding ability\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. This mechanism may be one of the key contributors to the antitumor activity of the MND-ART-GEL system.\u003c/p\u003e\u003cp\u003eWestern blot analysis was used to detect total STAT3 and phosphorylated STAT3 (Tyr705 and Ser727) levels in the treated 4T1 cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;D, the drugs containing ART (free ART, MND-ART, and MND-ART-GEL) significantly downregulated the levels of both p-STAT3 and total STAT3. Scratch assays, colony formation, and Transwell invasion assays further revealed that MND-ART-GEL effectively inhibited the migration, invasion, proliferation, and clonogenic potential of 4T1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u0026ndash;J), indicating its strong potential to suppress TNBC metastasis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTranscriptomic Analysis of 4T1 Cells Treated with MND-ART-GEL.\u003c/b\u003e To elucidate the molecular mechanisms underlying the antitumor effects of MND-ART-GEL, transcriptome sequencing and differential gene expression analysis were performed on 4T1 cells treated with various formulations. As evident from the principal component analysis (PCA) and correlation matrix results (Figure S3A, B), samples within the same group exhibited high consistency, with clear intergroup differences, justifying downstream analysis.\u003c/p\u003e\u003cp\u003eVolcano plots revealed that, compared with PBS treatment, DOX treatment resulted in the upregulation of 2,025 and downregulation of 2,057 genes (Figure S3C). In contrast, the gene expression profiles for the DOX and MND groups were relatively similar, with only 74 upregulated and 95 downregulated genes, indicating that MnUA alone had minimal impact on gene expression. However, a comparison between the MND and MND-ART-GEL groups led to the identification of 2,292 upregulated and 2,029 downregulated genes, indicating substantial transcriptomic alterations driven by ART addition and hydrogel delivery.\u003c/p\u003e\u003cp\u003eThe Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis of differentially expressed genes in the PBS vs. DOX, DOX vs. MND, and MND vs. MND-ART-GEL comparison (Figure S4A) revealed that DOX primarily influenced TNF, NOD-like, Notch, MAPK, and PI3K-Akt signaling pathways, which was suggestive of its role in direct tumor cytotoxicity, activation of inflammation, and inhibition of proliferation. MND showed a trend toward regulating antigen processing and presentation; however, the q-values were relatively high, which limited confidence in these changes. ART treatment was associated with modulation of TNF, MAPK, IL-17, glutathione metabolism, glyoxylate and dicarboxylate metabolism, cysteine and methionine metabolism, HIF-1, arginine and proline metabolism, and PI3K-Akt pathways\u0026mdash;reflecting its multifaceted effects on cytotoxicity, inhibition of proliferation, redox metabolism, and ferroptosis.\u003c/p\u003e\u003cp\u003eThe KEGG pathway mapping and classification (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) revealed significant enrichment of ferroptosis, glutathione metabolism, HIF-1, MAPK, and PI3K-Akt signaling pathways in the MND-ART-GEL group compared with that in the controls. These findings indicated that MND-ART-GEL induced oxidative stress and depleted glutathione to trigger ferroptosis\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, while also activating adaptive responses to hypoxia and stress\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. To investigate the global biological impact of MND-ART-GEL, gene set enrichment analysis (GSEA) was performed using selected genes related to oxidative stress, immunity, and tumor metastasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWnt and TGF-β signaling pathways, which are often implicated in epithelial-mesenchymal transition (EMT) and metastatic potential, were also modulated, indicating a possible role of MND-ART-GEL in suppressing invasion and migration\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Immune-related pathways, including antigen processing and presentation, IL-17 signaling, NOD-like receptor signaling, and cytosolic DNA sensing, were significantly upregulated, suggesting that the ICD triggered by MND-ART-GEL effectively activated both innate and adaptive immune responses\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFurther gene ontology (GO)-GSEA analysis (Figure S4B) confirmed that gene sets, such as \u0026ldquo;antigen processing and presentation of endogenous peptide antigen via MHC class I,\u0026rdquo; \u0026ldquo;activation of innate immune response,\u0026rdquo; \u0026ldquo;positive regulation of interferon-beta production,\u0026rdquo; and \u0026ldquo;cellular response to interferon-beta,\u0026rdquo; were highly enriched. These findings imply activation of the cGAS-STING\u0026ndash;mediated type I interferon pathway, thereby enhancing innate immune signaling\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Moreover, biological processes related to cell migration\u0026mdash;such as \u0026ldquo;positive regulation of cell migration,\u0026rdquo; \u0026ldquo;positive regulation of endothelial cell migration,\u0026rdquo; and \u0026ldquo;DNA integration\u0026rdquo;\u0026mdash;were significantly downregulated, further supporting the notion on the inhibitory effect of MND-ART-GEL on tumor invasiveness. The transcriptomic analysis of the effects of MND-ART-GEL was consistent with in vitro results.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn Vivo Antitumor Efficacy and Immune Activation of MND-ART-GEL in an Orthotopic 4T1 Breast Cancer Model.\u003c/b\u003e Given the potent in vitro antitumor and immunostimulatory effects of MND-ART-GEL, its in vivo efficacy was further evaluated using an orthotopic 4T1 breast tumor model in Balb/c mice. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, mice received intratumoral injections of different formulations (DOX 4 mg/kg or ART 32 mg/kg) every other day for a total of seven treatments. The ART group showed minimal tumor inhibition, whereas DOX, MND, MND-ART, and MND-ART-GEL groups significantly suppressed tumor growth, with MND-ART-GEL exhibiting the greatest tumor inhibition as evidenced by the lowest tumor weights and volumes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u0026ndash;E).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHistological analyses, including H\u0026amp;E, TUNEL, and Ki67 staining, were performed to assess tumor tissue morphology, apoptosis, and proliferation. H\u0026amp;E staining revealed only minor nuclear condensation in the ART group, whereas the DOX and MND groups showed increased areas of nuclear condensation, with MND also displaying nuclear dissolution, indicative of apoptosis and necrosis. Notably, MND-ART and MND-ART-GEL treatments led to extensive nuclear condensation, fragmentation, and dissolution, with the MND-ART-GEL group exhibiting near-complete nuclear degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). TUNEL and Ki67 staining results corroborated these findings, demonstrating the highest levels of apoptosis and inhibition of proliferation in the MND-ART-GEL group.\u003c/p\u003e\u003cp\u003eTo validate the in vitro findings of ICD induction and STAT3 suppression, HMGB1 and CRT expression were examined via immunohistochemistry, whereas STAT3 and phosphorylated STAT3 levels were analyzed via western blotting. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, HMGB1 release and CRT membrane translocation were significantly increased in the MND-ART and MND-ART-GEL groups compared with that in PBS, DOX, MND, and ART control groups. Furthermore, STAT3 and its phosphorylated forms (Tyr705 and Ser727) were significantly downregulated in ART-containing groups, consistent with in vitro data (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG\u0026ndash;J). These results confirmed that MND-ART-GEL induced strong ICD and inhibited STAT3 signaling in vivo.\u003c/p\u003e\u003cp\u003eTo further evaluate the immunomodulatory effects of MND-ART-GEL, flow cytometry was conducted to analyze immune cell populations in tumors and TDLNs (Figure S5). Surface expression of MHCI and Fas on tumor cells was first examined. Compared with the PBS group, the MND-ART-GEL group showed a two-fold increase in the percentage of Fas\u003csup\u003e+\u003c/sup\u003e cells and a 1.8-fold increase in that of MHCI\u003csup\u003e+\u003c/sup\u003e cells, both significantly higher than the respective percentages in the DOX and ART groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-B). These results indicated that MND-ART-GEL effectively enhances tumor cell immunogenicity in vivo, facilitating immune recognition and cytotoxicity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBased on our findings that MnUA acted as an immune adjuvant and that MND-ART-GEL induced robust ICD, we next evaluated DC activation in the TDLNs and tumor tissue. The MND-ART-GEL group exhibited the highest proportion of mature DCs (CD86\u003csup\u003e+\u003c/sup\u003eMHCII\u003csup\u003e+\u003c/sup\u003e) in TDLNs, approximately 6.87-fold higher than that in the PBS group and significantly greater than that in the MND and ART groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-D). Although DC infiltration in tumors was relatively low, MND-ART-GEL treatment increased the abundance of CD11c\u003csup\u003e+\u003c/sup\u003e DCs from 1.4% to 27.6% (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE), providing a favorable immune environment. A similar trend was observed in MHCII\u003csup\u003e+\u003c/sup\u003e DCs, the abundance of which increased from 0.4% in the PBS group to approximately 9.5% in the MND-ART-GEL group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eTAM profiling revealed that MND-ART-GEL significantly promoted M1 polarization and suppressed M2 polarization, compared with that in other treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG-H). The M1/M2 ratio in tumors treated with MND-ART-GEL was approximately 4.5, which was 12.5-times higher than that in the PBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI).\u003c/p\u003e\u003cp\u003eWe next assessed the percentage of CD8\u003csup\u003e+\u003c/sup\u003e T cells, which are crucial for tumor cell killing and correlate with favorable prognosis. MND-ART-GEL induced the highest CD8\u003csup\u003e+\u003c/sup\u003e T-cell infiltration, which reached 43.3%\u0026mdash;about 5.5-times that in the PBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ-K). Notably, MND-ART-GEL also reduced the expression of PD-1 and PD-L1 on CD8\u003csup\u003e+\u003c/sup\u003e T and tumor cells, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eL, M). This indicated that MND-ART-GEL not only promoted T-cell infiltration but also alleviated T-cell exhaustion and immune suppression, thereby restoring cytotoxic activity. Notably, the ART group alone also exhibited low PD-L1 and PD-1 levels, indicative of ART-mediated STAT3 inhibition contributing to this effect.\u003c/p\u003e\u003cp\u003eFinally, proinflammatory cytokines including TNF-α, IFN-γ, and IL-6 were quantified in tumor tissues via ELISA. These cytokines play essential roles in antitumor immunity by activating cytotoxic T cells, enhancing antibody production, and promoting immune cell recruitment to the TME\u003csup\u003e\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. The MND-ART-GEL group exhibited the highest levels of TNF-α, IFN-γ, and IL-6 among all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eN\u0026ndash;P), indicating a favorable shift in the immune microenvironment.\u003c/p\u003e\u003cp\u003eThroughout the treatment period, body weight of mice was monitored every two days. At the study endpoint, major organs were harvested, weighed, and analyzed after H\u0026amp;E staining to assess biosafety. No significant differences in body weight or organ coefficients were observed between groups (Figure S6A, B), suggesting good biocompatibility. Although splenomegaly was observed\u0026mdash;likely due to tumor-induced immune activation\u0026mdash;this was excluded from toxicity evaluation. Apart from mild inflammatory infiltration in the lungs (likely due to TNBC-associated pre-metastatic niches\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e), no significant histopathological changes were detected in major organs (Figure S6C). Overall, MND-ART-GEL exhibited excellent biosafety in vivo.\u003c/p\u003e\u003cp\u003eIn summary, MND-ART-GEL effectively triggered ICD and downregulated STAT3, thereby remodeling the immunosuppressive microenvironment of TNBC, eliciting a robust antitumor immune response, and demonstrating favorable biosafety in vivo.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEvaluation of the Anti-Recurrence Efficacy of MND-ART-GEL After Surgery.\u003c/b\u003e Surgical resection remains the primary curative treatment for TNBC; however, the risk of postoperative recurrence is high and often detrimental. To assess whether MND-ART-GEL can prevent tumor relapse after surgery, a postsurgical recurrence model was established. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, mice were randomly assigned to three groups (PBS, DOX, and MND-ART-GEL; DOX: 4 mg/kg; ART: 32 mg/kg; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5) and received five intratumoral injections every other day following surgery. One mouse in the PBS group died from relapse before the study endpoint, and the remaining PBS group mice exhibited severe tumor regrowth. In contrast, MND-ART-GEL treatment effectively suppressed the growth of residual tumor cells, resulting in significantly reduced tumor weights and volumes compared with that in the PBS and DOX groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB-E), indicating its robust anti-recurrence capability.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePostoperative tumors typically present with an immunosuppressive microenvironment. Therefore, flow cytometry was used to assess immune cell profiles in relapsed tumors. MND-ART-GEL markedly enhanced DC maturation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF, G), CD8\u003csup\u003e+\u003c/sup\u003e T-cell infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH, I), and M1 macrophage polarization while reducing the abundance of M2-type TAMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eJ\u0026ndash;M). The M1/M2 ratio in the MND-ART-GEL group was 16.25-times of that in the PBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eK). These results indicated that MND-ART-GEL successfully reversed the immunosuppressive state of postoperative residual tumors and reactivated antitumor immunity.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, MnUA microparticles were successfully synthesized via reverse microemulsion, wherein urate molecules coordinate with Mn\u0026sup2;\u003csup\u003e+\u003c/sup\u003e through metal\u0026ndash;ligand interactions. MnUA exhibited intrinsic immunostimulatory capability and served as a drug carrier by physically adsorbing the chemotherapeutic drug, DOX. Codelivery of DOX and ART within a Pluronic F127-based thermosensitive hydrogel resulted in the generation of a multifunctional platform\u0026mdash;MND-ART-GEL.\u003c/p\u003e\u003cp\u003eBoth in vitro and in vivo studies revealed that MND-ART-GEL exerted potent therapeutic efficacy in primary and postsurgical 4T1 TNBC mouse models. DOX and ART synergistically increased intracellular ROS levels, causing oxidative stress, mitochondrial damage, and subsequent tumor cell apoptosis and ICD. These changes enhanced the exposure of immunogenic markers (MHCI and Fas) on tumor cells. Simultaneously, MnUA acted as an immune adjuvant to promote infiltration and maturation of DCs, while ICD further activated antigen presentation and T-cell responses. MND-ART-GEL also reduced PD-L1/PD-1 expression, alleviated T-cell exhaustion, and enhanced CD8\u003csup\u003e+\u003c/sup\u003e T-cell\u0026ndash;mediated cytotoxicity. Moreover, STAT3 inhibition by ART suppressed tumor invasion and metastasis.\u003c/p\u003e\u003cp\u003eThis study introduces a novel application of MSU as an immune adjuvant, integrating chemotherapy, immunomodulation, and multitarget therapy to reshape the tumor immune microenvironment. The platform was found to effectively inhibit tumor growth and recurrence and offers a promising therapeutic strategy for TNBC, which is expected to overcome treatment resistance while improving patient outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003e All animal experiments were approved by the Ethics Committee of Science and Technology Industrial Park, Guangzhou University of Chinese Medicine (PZ24115). All applicable institutional guidelines for the care and use of animals were followed.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eAll authors agree with the submission and publication of this paper.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eAll authors declare no conflicts of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by Natural Science Foundation of China (82204628), the Special Projects in Key Areas of Colleges and Universities in Guangdong Province (2022ZDZX2015), Natural Science Foundation of Guangdong Province (2022A1515011312), the Science and Technology Program of Guangzhou (2024A04J4899), and Young Talent Project of Guangzhou University of Chinese Medicine (No. A1-2601-24-414-110Z76).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e1. L. designed the project and supervised its implementation. K. C., R. X., M. X., W. C., X. L., T. G. W. J. C. Y. J. F. and S. Z. completed all experiments. Q. X., Y. C. and C. L. contributed to data analysis. K. C. wrote the manuscript and J. L. revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eThe manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. We also acknowledge the Lingnan Medical Research Center of Guangzhou University of Chinese Medicine for the support on facilities.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data analyzed during this study are included in this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, Jemal A. 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Oncologist. 2005;10(6):427\u0026ndash;37. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1634/theoncologist.10-6-427\u003c/span\u003e\u003cspan address=\"10.1634/theoncologist.10-6-427\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Y, Chen W, Kang Y, Zhen X, Zhou Z, Liu C, Chen S, Huang X, Liu HJ, Koo S, et al. Nanosensitizer-mediated augmentation of sonodynamic therapy efficacy and antitumor immunity. Nat Commun. 2023;14(1):6973. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41467-023-42509-7\u003c/span\u003e\u003cspan address=\"10.1038/s41467-023-42509-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSpranger S. Mechanisms of tumor escape in the context of the T-cell-inflamed and the non-T-cell-inflamed tumor microenvironment. Int Immunol. 2016;28(8):383\u0026ndash;91. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/intimm/dxw014\u003c/span\u003e\u003cspan address=\"10.1093/intimm/dxw014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiedtke C, Mazouni C, Hess KR, Andr\u0026eacute; F, Tordai A, Mejia JA, Symmans WF, Gonzalez-Angulo AM, Hennessy B, Green M, et al. Response to neoadjuvant therapy and long-term survival in patients with triple-negative breast cancer. J Clin Oncol. 2008;26(8):1275\u0026ndash;81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1200/jco.2007.14.4147\u003c/span\u003e\u003cspan address=\"10.1200/jco.2007.14.4147\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKrall JA, Reinhardt F, Mercury OA, Pattabiraman DR, Brooks MW, Dougan M, Lambert AW, Bierie B, Ploegh HL, Dougan SK, et al. The systemic response to surgery triggers the outgrowth of distant immune-controlled tumors in mouse models of dormancy. Sci Transl Med. 2018;10(436). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/scitranslmed.aan3464\u003c/span\u003e\u003cspan address=\"10.1126/scitranslmed.aan3464\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang C, Guan Y, Lv M, Zhang R, Guo Z, Wei X, Du X, Yang J, Li T, Wan Y, et al. Manganese Increases the Sensitivity of the cGAS-STING Pathway for Double-Stranded DNA and Is Required for the Host Defense against DNA Viruses. Immunity. 2018;48(4):675\u0026ndash;e687677. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.immuni.2018.03.017\u003c/span\u003e\u003cspan address=\"10.1016/j.immuni.2018.03.017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao Z, Ma Z, Wang B, Guan Y, Su XD, Jiang Z. Mn(2+) Directly Activates cGAS and Structural Analysis Suggests Mn(2+) Induces a Noncanonical Catalytic Synthesis of 2'3'-cGAMP. Cell Rep. 2020;32(7):108053. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.celrep.2020.108053\u003c/span\u003e\u003cspan address=\"10.1016/j.celrep.2020.108053\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDemaria O, De Gassart A, Coso S, Gestermann N, Di Domizio J, Flatz L, Gaide O, Michielin O, Hwu P, Petrova TV, et al. STING activation of tumor endothelial cells initiates spontaneous and therapeutic antitumor immunity. Proc Natl Acad Sci U S A. 2015;112(50):15408\u0026ndash;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.1512832112\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1512832112\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDeng L, Liang H, Xu M, Yang X, Burnette B, Arina A, Li XD, Mauceri H, Beckett M, Darga T, et al. STING-Dependent Cytosolic DNA Sensing Promotes Radiation-Induced Type I Interferon-Dependent Antitumor Immunity in Immunogenic Tumors. Immunity. 2014;41(5):843\u0026ndash;52. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.immuni.2014.10.019\u003c/span\u003e\u003cspan address=\"10.1016/j.immuni.2014.10.019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSivick KE, Desbien AL, Glickman LH, Reiner GL, Corrales L, Surh NH, Hudson TE, Vu UT, Francica BJ, Banda T, et al. Magnitude of Therapeutic STING Activation Determines CD8(+) T Cell-Mediated Anti-tumor Immunity. Cell Rep. 2018;25(11):3074\u0026ndash;e30853075. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.celrep.2018.11.047\u003c/span\u003e\u003cspan address=\"10.1016/j.celrep.2018.11.047\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang L, Wei X, Wang Z, Liu P, Hou Y, Xu Y, Su H, Koci MD, Yin H, Zhang C. NF-κB activation enhances STING signaling by altering microtubule-mediated STING trafficking. Cell Rep. 2023;42(3):112185. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.celrep.2023.112185\u003c/span\u003e\u003cspan address=\"10.1016/j.celrep.2023.112185\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSo AK, Martinon F. Inflammation in gout: mechanisms and therapeutic targets. Nat Rev Rheumatol. 2017;13(11):639\u0026ndash;47. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nrrheum.2017.155\u003c/span\u003e\u003cspan address=\"10.1038/nrrheum.2017.155\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuynh J, Chand A, Gough D, Ernst M. Therapeutically exploiting STAT3 activity in cancer - using tissue repair as a road map. Nat Rev Cancer. 2019;19(2):82\u0026ndash;96. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41568-018-0090-8\u003c/span\u003e\u003cspan address=\"10.1038/s41568-018-0090-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJohnson DE, O'Keefe RA, Grandis JR. Targeting the IL-6/JAK/STAT3 signalling axis in cancer. Nat Rev Clin Oncol. 2018;15(4):234\u0026ndash;48. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nrclinonc.2018.8\u003c/span\u003e\u003cspan address=\"10.1038/nrclinonc.2018.8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIlamathi M, Santhosh S, Sivaramakrishnan V. Artesunate as an Anti-Cancer Agent Targets Stat-3 and Favorably Suppresses Hepatocellular Carcinoma. Curr Top Med Chem. 2016;16(22):2453\u0026ndash;63. 10.2174/1568026616666160212122820 From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDeng D, Xu N, Wang M, Zhang G, Su Y, Fang H, Su Z. An artesunate-modified half-sandwich iridium(iii) complex inhibits colon cancer cell proliferation and metastasis through the STAT3 pathway. RSC Chem Biol. 2025;6(2):218\u0026ndash;26. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/d4cb00114a\u003c/span\u003e\u003cspan address=\"10.1039/d4cb00114a\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu X, Cai Y, Zhang Y, Zhang H, Tian S, Gong Y, Song Q, Chen X, Ma X, Wen Y, et al. Artesunate: A potential drug for the prevention and treatment from hepatitis to hepatocellular carcinoma. Pharmacol Res. 2024;210:107526. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.phrs.2024.107526\u003c/span\u003e\u003cspan address=\"10.1016/j.phrs.2024.107526\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJiang Z, Wang Z, Chen L, Zhang C, Liao F, Wang Y, Wang Y, Luo P, Luo M, Shi C. Artesunate induces ER-derived-ROS-mediated cell death by disrupting labile iron pool and iron redistribution in hepatocellular carcinoma cells. Am J Cancer Res. 2021;11(3):691\u0026ndash;711. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHe Z, chen F, Liu H, Liu T, He D, Wang Y, Li J, Chen D, Tian Z, Li M, et al. Dual-targeted self-delivery micelle co-loading doxorubicin and artesunate for enhanced AML treatment via mitochondrial damage induction. Nano Res. 2025. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.26599/NR.2025.94907663\u003c/span\u003e\u003cspan address=\"10.26599/NR.2025.94907663\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShriky B, Kelly A, Isreb M, Babenko M, Mahmoudi N, Rogers S, Shebanova O, Snow T, Gough T. Pluronic F127 thermosensitive injectable smart hydrogels for controlled drug delivery system development. J Colloid Interface Sci. 2020;565:119\u0026ndash;30. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jcis.2019.12.096\u003c/span\u003e\u003cspan address=\"10.1016/j.jcis.2019.12.096\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLei L, Song Y, Yang L, Wang Y, Xia X, Zhang Y, Zhang X, Zhang X, Duggal I, He B, et al. Triethylamine-mediated protonation-deprotonation unlocks dual-drug self assembly to suppress breast cancer progression and metastasis. Proc Natl Acad Sci U S A. 2025;122(5):e2416796122. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.2416796122\u003c/span\u003e\u003cspan address=\"10.1073/pnas.2416796122\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTiburcius S, Krishnan K, Jose L, Patel V, Ghosh A, Sathish CI, Weidenhofer J, Yang JH, Verrills NM, Karakoti A, et al. Egg-yolk core-shell mesoporous silica nanoparticles for high doxorubicin loading and delivery to prostate cancer cells. Nanoscale. 2022;14(18):6830\u0026ndash;45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/d2nr00783e\u003c/span\u003e\u003cspan address=\"10.1039/d2nr00783e\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWei H, Chen F, Chen J, Lin H, Wang S, Wang Y, Wu C, Lin J, Zhong G. Mesenchymal Stem Cell Derived Exosomes as Nanodrug Carrier of Doxorubicin for Targeted Osteosarcoma Therapy via SDF1-CXCR4 Axis. Int J Nanomed. 2022;17:3483\u0026ndash;95. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2147/ijn.S372851\u003c/span\u003e\u003cspan address=\"10.2147/ijn.S372851\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYan Y, Wang R, Hu Y, Sun R, Song T, Shi X, Yin S. Stacking of doxorubicin on folic acid-targeted multiwalled carbon nanotubes for in vivo chemotherapy of tumors. Drug Deliv. 2018;25(1):1607\u0026ndash;16. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/10717544.2018.1501120\u003c/span\u003e\u003cspan address=\"10.1080/10717544.2018.1501120\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKciuk M, Gielecińska A, Mujwar S, Kołat D, Kałuzińska-Kołat Ż, Celik I, Kontek R. Doxorubicin-An Agent with Multiple Mechanisms of Anticancer Activity. \u003cem\u003eCells\u003c/em\u003e 2023, \u003cem\u003e12\u003c/em\u003e (4). DOI: 10.3390/cells12040659 From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang Z, Gan S, Zhuang X, Chen Y, Lu L, Wang Y, Qi X, Feng Q, Huang Q, Du B et al. Artesunate Inhibits the Cell Growth in Colorectal Cancer by Promoting ROS-Dependent Cell Senescence and Autophagy. \u003cem\u003eCells\u003c/em\u003e 2022, \u003cem\u003e11\u003c/em\u003e (16). DOI: 10.3390/cells11162472 From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKuai R, Yuan W, Son S, Nam J, Xu Y, Fan Y, Schwendeman A, Moon JJ. Elimination of established tumors with nanodisc-based combination chemoimmunotherapy. Sci Adv. 2018;4(4):eaao1736. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/sciadv.aao1736\u003c/span\u003e\u003cspan address=\"10.1126/sciadv.aao1736\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGarg AD, Agostinis P. ER stress, autophagy and immunogenic cell death in photodynamic therapy-induced anti-cancer immune responses. \u003cem\u003ePhotochem Photobiol Sci\u003c/em\u003e 2014, \u003cem\u003e13\u003c/em\u003e (3), 474\u0026ndash;487. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/c3pp50333j\u003c/span\u003e\u003cspan address=\"10.1039/c3pp50333j\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKepp O, Menger L, Vacchelli E, Locher C, Adjemian S, Yamazaki T, Martins I, Sukkurwala AQ, Michaud M, Senovilla L, et al. Crosstalk between ER stress and immunogenic cell death. Cytokine Growth Factor Rev. 2013;24(4):311\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cytogfr.2013.05.001\u003c/span\u003e\u003cspan address=\"10.1016/j.cytogfr.2013.05.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBegley J, Ribas A. Targeted therapies to improve tumor immunotherapy. Clin Cancer Res. 2008;14(14):4385\u0026ndash;91. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1158/1078-0432.Ccr-07-4804\u003c/span\u003e\u003cspan address=\"10.1158/1078-0432.Ccr-07-4804\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eH\u0026ouml;lzel M, Bovier A, T\u0026uuml;ting T. Plasticity of tumour and immune cells: a source of heterogeneity and a cause for therapy resistance? Nat Rev Cancer. 2013;13(5):365\u0026ndash;76. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nrc3498\u003c/span\u003e\u003cspan address=\"10.1038/nrc3498\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSong Q, Peng S, Che F, Zhu X. Artesunate induces ferroptosis via modulation of p38 and ERK signaling pathway in glioblastoma cells. J Pharmacol Sci. 2022;148(3):300\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jphs.2022.01.007\u003c/span\u003e\u003cspan address=\"10.1016/j.jphs.2022.01.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBae T, Hallis SP, Kwak MK. Hypoxia, oxidative stress, and the interplay of HIFs and NRF2 signaling in cancer. Exp Mol Med. 2024;56(3):501\u0026ndash;14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s12276-024-01180-8\u003c/span\u003e\u003cspan address=\"10.1038/s12276-024-01180-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDrabsch Y, ten Dijke P. TGF-β signaling in breast cancer cell invasion and bone metastasis. J Mammary Gland Biol Neoplasia. 2011;16(2):97\u0026ndash;108. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10911-011-9217-1\u003c/span\u003e\u003cspan address=\"10.1007/s10911-011-9217-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu X, Zhang M, Xu F, Jiang S. Wnt signaling in breast cancer: biological mechanisms, challenges and opportunities. 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From NLM.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJiang S, Li H, Zhang L, Mu W, Zhang Y, Chen T, Wu J, Tang H, Zheng S, Liu Y, et al. Generic Diagramming Platform (GDP): a comprehensive database of high-quality biomedical graphics. Nucleic Acids Res. 2025;53(D1):D1670\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/nar/gkae973\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkae973\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. From NLM.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme ","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Triple-negative breast cancer, Hydrogel, Manganese urate, Artesunate, Immunosuppressive tumor microenvironment","lastPublishedDoi":"10.21203/rs.3.rs-7975044/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7975044/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTriple-negative breast cancer (TNBC) remains a formidable clinical challenge because of its high aggressiveness, metastatic potential, and lack of effective therapeutic options. In this study, we developed an injectable, multifunctional hydrogel, MND-ART-GEL, by encapsulating immunostimulatory manganese urate (MnUA), the chemotherapeutic agent doxorubicin (DOX), and an artemisinin derivative artesunate (ART) within a thermosensitive Pluronic F127 matrix. Uric acid crystals (MSU) and manganese ions (Mn\u0026sup2;⁺) were coordinated and assembled into manganese urate (MnUA). The MSU\u0026ndash;Mn\u0026sup2;⁺ system exhibits a synergistic immunoactivating effect, combining the DAMPs-like immune stimulation of MSU with the STING pathway activation ability of Mn\u0026sup2;⁺. Owing to its simple chemical composition, low cost, and easy accessibility, this combination offers a more practical and scalable alternative to conventional synthetic immune agonists. ART synergizes with DOX to exert potent antitumor effects. MND-ART-GEL synergistically elevated intracellular ROS levels, inducing oxidative stress, mitochondrial damage, and subsequent tumor cell apoptosis, immunogenic cell death (ICD), and exposure of immune markers such as MHCI and Fas. Moreover, MND-ART-GEL downregulated STAT3 expression, thereby suppressing tumor cell invasion and migration. In orthotopic and postsurgical TNBC mouse models, MND-ART-GEL significantly inhibited tumor growth and recurrence, remodeled the immunosuppressive tumor microenvironment, enhanced CD8\u003csup\u003e+\u003c/sup\u003e T-cell infiltration, dendritic cell maturation, and M1 macrophage polarization, while reducing PD-1/PD-L1 expression on CD8\u003csup\u003e+\u003c/sup\u003e T cells and promoting the secretion of immune-related cytokines. This study presents a localized drug delivery strategy integrating chemotherapy, immune modulation, and multitarget synergy, offering a promising approach to overcome therapeutic resistance and reduce recurrence in TNBC.\u003c/p\u003e","manuscriptTitle":"MnUA–DOX–Artesunate Hydrogel Remodels Immunosuppressive Tumor Microenvironment and Prevents Postoperative Recurrence in Triple-negative Breast Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-20 19:14:10","doi":"10.21203/rs.3.rs-7975044/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-23T20:08:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-21T13:04:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-21T10:12:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9314976012983013730461409270405947305","date":"2025-11-18T14:30:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-17T14:20:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-16T23:53:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-16T20:56:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326010950044418602503911351987926096205","date":"2025-11-13T09:59:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"57951874003603603086526338441105788753","date":"2025-11-11T09:49:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"218844465415050538743270828474372104959","date":"2025-11-11T04:13:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"88922884069909523623395147853851214681","date":"2025-11-11T01:52:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"278227072301620739711147884687204341092","date":"2025-11-10T23:23:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-10T18:21:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-29T11:30:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-29T11:30:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2025-10-29T03:15:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bcb311a2-e03f-4ed3-982d-8212c18bc6f5","owner":[],"postedDate":"November 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-20T16:01:26+00:00","versionOfRecord":{"articleIdentity":"rs-7975044","link":"https://doi.org/10.1186/s12951-026-04381-7","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2026-04-18 15:57:13","publishedOnDateReadable":"April 18th, 2026"},"versionCreatedAt":"2025-11-20 19:14:10","video":"","vorDoi":"10.1186/s12951-026-04381-7","vorDoiUrl":"https://doi.org/10.1186/s12951-026-04381-7","workflowStages":[]},"version":"v1","identity":"rs-7975044","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7975044","identity":"rs-7975044","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}