pH-regulating Lipiodol Pickering emulsions enhance transarterial embolization therapy via inducing ferroptosis and activating antitumor immunity.

Introduction
Hepatocellular carcinoma (HCC) accounts for approximately 80%–85% of all liver cancer cases and represents the sixth most common cancer globally.1 Owing to its minimal invasiveness, transcatheter arterial chemoembolization (TACE) is the most commonly applied intra-arterial locoregional treatment for HCC patients ineligible for surgical resection.2,3,4 However, the clinical therapeutic efficacy of TACE, which employs conventional Lipiodol and polymeric microparticles as vehicles for locoregional chemotherapeutics to simultaneously induce ischemic and cytotoxic effects, remains suboptimal due to incomplete blood vessel embolization and poorly controlled drug release kinetics of these embolic materials.5,6,7,8 Moreover, intra-arterial embolization also further aggravates tumor hypoxia and acidification, key drivers of the immunosuppressive tumor microenvironment (TME), by obstructing tumor blood supply.9,10,11 Over the past several years, numerous pioneering studies have shown that developing next-generation embolic materials with enhanced pharmacokinetic properties and/or TME-modulating capabilities represents a promising strategy to improve TACE efficacy in HCC patients.9,12,13 Given the strong potential of eliciting tumor-specific immune responses to systemically inhibit tumor growth across diverse malignancies, the strategic integration of embolic materials with immunostimulatory entities could significantly enhance TACE outcomes.
To meet their heightened metabolic demands, tumor cells undergo upregulated aerobic glycolysis (the Warburg effect), producing excessive lactate as a byproduct.14,15 To avoid intracellular acidification, which would induce apoptosis via acid stress, tumor cells evolve to express markedly high levels of the Na+/H+ exchanger (NHE1) and other plasma membrane-bound proton transporters and pumps to enhance proton efflux.16,17,18,19 Coupled with significantly reduced CO2 production due to inhibition of the tricarboxylic acid cycle and oxidative phosphorylation, tumor cells maintain an alkaline intracellular pH and an acidic extracellular pH.20,21 This pH gradient collectively promotes tumor invasion and metastasis by facilitating apoptosis evasion, immune escape, and other pathophysiological mechanisms.22 Clinical evidence has shown that such abnormal tumor acidity not only impairs the efficacy of various cancer treatments but also has emerged as a potential therapeutic target.17 In addition to using proton transport inhibitors to directly induce intracellular acidification,23 alternative strategies, such as neutralizing extracellular tumor acidity with calcium carbonate-based formulations, have been explored to enhance existing cancer therapies.24,25,26 Given their complementary mechanisms, concurrent regulation of intracellular and extracellular acidity represents a promising strategy for synergistic tumor suppression.
The Fenton reaction, which catalyzes the decomposition of hydroperoxides into highly oxidative hydroxyl radicals (⋅OH) using ferrous ions (Fe2+) as a catalyst, has been demonstrated as an effective strategy to induce ferroptosis via intracellular lipid peroxidation.27,28,29,30 Inspired by the pH-dependent Fenton-catalytic ability of Fe2+ and the potent ability of cariporide (a second-generation NHE1 inhibitor) to suppress extracellular acidification,31,32,33,34 we developed an iron nanoparticle (FeNP)-stabilized Lipiodol Pickering emulsion (LPE) for intra-arterial delivery of cariporide. In response to the acidic TME, the resulting cariporide-encapsulated LPE (CFe-LPE) exhibited sustained cariporide release, blocking proton extrusion from intracellular to extracellular spaces and significantly enhancing the ferroptosis-inducing capacity of released Fe2+. Upon intratumoral administration, CFe-LPE effectively neutralized extracellular protons and induced ferroptosis, collectively triggering potent antitumor immunity. Notably, when delivered via transarterial embolization, CFe-LPE demonstrated superior efficacy in inhibiting orthotopic N1S1 HCC progression in rats compared to conventional doxorubicin-based TACE. This study highlights CFe-LPE as a promising pH-regulating platform with dual self-enhancing ferroptosis induction and antitumor immunity activation, positioning it as a potential therapeutic candidate for HCC treatment.

Results

Preparation and characterization of CFe-LPE
FeNPs were synthesized via chemical reduction of ferric chloride (FeCl3) using sodium borohydride (NaBH4) as the reductant under a nitrogen atmosphere, following a previously reported method (Figure 1A).35,36 To prevent oxidation of the pure FeNPs, the synthesized FeNPs were immediately coated with sodium octanoate, leveraging the strong coordination interaction between iron atoms and carboxyl groups. Transmission electron microscopy (TEM) revealed that the resulting FeNPs exhibited a spherical morphology with diameters ranging from 20 to 40 nm (Figure 1B). The zeta potential of the synthesized FeNPs was measured to be −13.7 mV. X-ray diffraction (XRD) analysis confirmed their amorphous structure (Figure S1A). The contact angle of FeNPs increased significantly from 39.7° (unmodified) to ∼102.5° after surface modification (Figure S1B), placing it within the optimal range (90°–110°) for stabilizing water-in-oil (W/O) Pickering emulsions.37 Hemolysis assays further demonstrated the excellent biocompatibility of the FeNPs, with minimal hemolysis (<2%) observed in red blood cells incubated at a maximal concentration of 200 μg/mL (Figures S1C and S1D).
We then evaluated the Fenton-catalytic potential of FeNPs. Using inductively coupled plasma optical emission spectroscopy (ICP-OES), we first demonstrated that FeNPs exhibited pH-dependent decomposition, with significantly more efficient Fe2+ release at pH 5.4 compared to neutral pH conditions (Figure 1C). The pH-dependent Fe2+ release was further confirmed through a colorimetric assay using potassium permanganate as an Fe2+ indicator (Figure 1D). Methylene blue (MB) decolorization assays revealed that FeNPs incubated at pH 5.4 in the presence of 1 mM H2O2 showed pronounced time-dependent degradation ability, confirming ⋅OH generation (Figure S1E). In contrast, FeNPs incubated at pH 6.5 or 7.4 exhibited markedly reduced MB degradation efficiency under identical H2O2 conditions (Figure 1E). Together, these results demonstrate that FeNPs function as a pH-responsive Fe2+ reservoir capable of catalyzing H2O2 decomposition into ⋅OH radicals, with optimal activity under acidic conditions.
Next, we evaluated the ability of FeNPs to stabilize W/O LPE by acting as a particulate surfactant. Without FeNPs, emulsions prepared with Lipiodol and MB aqueous solution (2:1 v/v) underwent rapid phase separation within 5 min after extrusion using a medical three-way stopcock (Figures 1F, S2A, and S2B). In contrast, adding FeNPs significantly enhanced emulsion stability in a dose-dependent manner, as evidenced by prolonged phase separation times (Figure S2C). Cariporide was efficiently loaded into the aqueous phase of FeNP-stabilized LPE (Fe-LPE) with ∼97.6% encapsulation efficiency, forming CFe-LPE with excellent stability comparable to Fe-LPE (Figures 1F, S2D, and S2E). Due to the pH-dependent degradation of FeNPs, CFe-LPE incubated at pH 6.5 showed faster cariporide release than at pH 7.4 (Figure 1G). Fluorescent microscopy revealed rhodamine B-stained water droplets surrounded by curcumin-labeled Lipiodol (Figure 1H). Rotary rheometry demonstrated that CFe-LPE had a viscosity of 0.233 Pa s at 300 s−1 shear rate, similar to plain Lipiodol (Figure 1I). Additionally, comparative analysis using a universal testing machine demonstrated that the injection force required to deliver CFe-LPE through a 2.8F microcatheter was 12.6 N, comparable to the 9 N required for Lipiodol under identical conditions (Figure S2F). This further confirms its suitability for the transcatheter arterial embolization (TAE) procedure in terms of injectability. These results collectively establish CFe-LPE as a stable, pH-responsive embolic material for dual delivery of cariporide and Fe2+ Fenton catalysts.

CFe-LPE synergistically induces ferroptosis in cancer cells
Motivated by cariporide’s ability, as an NHE1 inhibitor, to induce intracellular acidosis and thereby enhance Fe2+-catalyzed Fenton reaction, we investigated the synergistic ferroptosis-inducing effects of cariporide and FeNPs, the two key components of CFe-LPE, in rat hepatoma N1S1 cells. ICP-OES analysis revealed that N1S1 cells incubated with FeNPs (25 μg/mL) exhibited a time-dependent increase in intracellular iron content (Figure 2A). We then evaluated the effects of cariporide on intracellular pH using the cell-permeable fluorescent probe 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM). After 6 h of incubation, cariporide induced a concentration-dependent decrease in BCECF-AM fluorescence, confirming intracellular acidification (Figure S3A). Confocal microscopy further demonstrated that FeNPs alone (50 μg/mL, 6 h) had a negligible impact on intracellular BCECF-AM fluorescence (Figure 2B), whereas concurrent incubation with cariporide and FeNPs reduced fluorescence to a similar extent as cariporide alone (Figures 2C and S3B). Notably, both cariporide alone and the cariporide-FeNP combination significantly elevated extracellular pH in the cell medium, as measured by cell-impermeable BCECF (Figures 2D and S3C). In contrast, FeNPs alone did not alter extracellular pH. These results collectively demonstrate that cariporide promotes concurrent intracellular acidosis and extracellular alkalization by inhibiting NHE1-mediated proton extrusion.
Next, we evaluated the synergistic ability of cariporide and FeNPs to promote intracellular reactive oxygen species (ROS) generation using confocal microscopy and flow cytometry. Using HKOU-1 as a specific ⋅OH probe, we observed that co-incubation of cariporide with FeNPs most effectively enhanced intracellular ⋅OH generation (Figure 2E). The superior capacity of co-incubation of cariporide and FeNPs to synergistically amplify intracellular ROS was also evidenced by using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) as the fluorescent ROS probe (Figures 2F, S3D, and S3E). Given that ⋅OH is effective in initiating lipid peroxidation of polyunsaturated fatty acids (PUFAs), a typical characteristic of ferroptotic cells, co-incubation of cariporide and FeNPs also exhibited the highest efficacy in promoting intracellular lipid peroxidation when detected with BODIPY-C11 (Figures 2G, 2H, and S3F). Moreover, increased malondialdehyde (MDA) levels, a marker of lipid peroxidation, in N1S1 cells treated with the combination of cariporide and FeNPs confirmed their synergistic role in inducing pronounced lipid peroxidation (Figure 2I). The BODIPY-C11 fluorescence in N1S1 cells co-treated with cariporide and FeNPs was significantly attenuated upon addition of ferrostatin-1 (Fer-1, 10 μM), a well-characterized ferroptosis inhibitor that functions as an antioxidant (Figures 2J, S3G, and S3H).38,39 In addition, N1S1 cells co-incubated with cariporide and FeNPs exhibited markedly reduced activity of glutathione peroxidase 4 (GPX4) (Figure 2K), a crucial antioxidant enzyme that suppresses ferroptosis by reducing lipid peroxidation.40,41 Moreover, both standard cell viability assay and live/dead dual staining assay demonstrated that cariporide at a safe concentration of 200 μM significantly enhanced the cytotoxicity of FeNPs against N1S1 cells (Figures S3I–S3K). Cell viability assay further demonstrated that Fer-1 treatment effectively rescued N1S1 cells treated with FeNPs (200 μg/mL) and cariporide (200 μM), with a significant increase in viability from 19.9% to 72.3% in co-treated cells (Figures 2L and S3L). In contrast, small-molecule inhibitors targeting apoptosis (Z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone, Z-DEVD, 50 μM), necroptosis (necrostatin-1, Ner-1, 5 μM), and pyroptosis (Z-Val-Ala-Asp(OMe)-fluoromethylketone, Z-VAD, 50 μM) showed minimal impact on the cytotoxicity of the FeNP and cariporide co-treatment. Similarly, the combined treatment of cariporide and FeNPs effectively induced cell death in human HepG2 and murine H22 HCC cells (Figures S3M–S3P). These findings reveal that cariporide significantly enhances the Fenton-catalytic capacity of FeNPs by suppressing intracellular proton extrusion, thereby synergistically inducing ferroptosis in cancer cells (Figure 2M).
It is established that ferroptosis can trigger pro-inflammatory antitumor immune responses by releasing distinct damage-associated molecular patterns (DAMPs).42,43 Confocal microscopic observation showed that N1S1 cells treated with cariporide and FeNPs exhibited significantly increased expression of calreticulin (CRT) and nuclear release of high-mobility group box 1 (HMGB1) (Figures 2N and 2O). The trend was further confirmed by the flow cytometric analysis (Figures S3Q–S3T). In addition, flow cytometry analysis revealed that murine H22 HCC cells pretreated with FeNPs and cariporide exhibited a greater ability to promote bone marrow-derived dendritic cell (BMDC) maturation compared to untreated H22 cells (Figure 2P), suggesting their potential to trigger tumor-specific immune responses. Moreover, ovalbumin (OVA)-expressing murine B16 melanoma cells were used as a model cell to assess how co-incubation with FeNPs and cariporide influences their capacity to activate antigen-specific T cells from OT-1 transgenic mice (Figure 2Q). Results demonstrated that pretreatment of OVA-B16 cells with co-incubation of FeNPs and cariporide significantly enhanced splenic T cell proliferation, as measured by carboxy fluorescein succinimidyl ester (CFSE) dilution, and increased secretion of interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) (Figures 2R–2T and S3U). In summary, these findings demonstrate that CFe-LPE potently induces ferroptosis in tumor cells to benefit the priming and activation of antitumor immune responses.

In vivo pharmacokinetic behaviors and therapeutic efficacy of CFe-LPE in subcutaneous H22 tumor models
We first evaluated the intratumoral retention performance of CFe-LPE in murine H22 tumor-bearing mice (Figure 3A). By using Cy5.5-tagged bovine serum albumin (Cy5.5-BSA) to label the aqueous droplets of CFe-LPE, whole-body fluorescence imaging revealed that CFe-LPE exhibited prolonged tumor retention, with approximately 73.9% of Cy5.5-BSA remaining in the tumor region at 48 h post-injection (Figures 3B and S4A). In marked contrast, H22 tumors in mice receiving intratumoral injection of Cy5.5-BSA-labeled conventional Lipiodol emulsion (LE) showed rapid fluorescence decay, with only minimal Cy5.5 signal detected by 48 h post-injection. The superior intratumoral retention of CFe-LPE was further confirmed by confocal microscopy analysis of tumor slices collected at 72 h post-injection, which revealed sustained Cy5.5 fluorescence (Figure S4B). Collectively, these results demonstrate that CFe-LPE enables sustained drug release within the tumor region.
Based on the dual pH-regulation ability of cariporide, we investigated the influence of CFe-LPE treatment on both intracellular and extracellular pH in the TME. To this end, four groups of H22 tumor-bearing mice (n = 3) received the following intratumoral injections: PBS, cariporide-loaded conventional LE (Ca-LE), Fe-LPE, or CFe-LPE at an equivalent cariporide dose of 9.36 mg/kg. At 24 h post-injection, BCECF-AM-based ex vivo fluorescence imaging revealed that H22 tumors treated with CFe-LPE exhibited the lowest intracellular pH compared to those treated with Ca-LE and Fe-LPE (Figures 3C and 3D). A similar trend was observed in BCECF-AM fluorescence measurements of tumor slices via confocal microscopy (Figure 3E). Meanwhile, BCECF-based ex vivo fluorescence imaging and microscopic examination confirmed that CFe-LPE treatment significantly increased BCECF fluorescence intensity compared to control treatments, indicating its superior capacity to promote the increase in extracellular pH of TME (Figures 3F–3H). Collectively, these results demonstrate that CFe-LPE treatment simultaneously promotes intracellular acidification and extracellular TME alkalization, which can be attributed to cariporide-mediated suppression of intracellular proton extrusion.
Next, we evaluated the efficacy of CFe-LPE in inducing ferroptosis within the TME. Confocal microscopy results revealed that tumor slices from CFe-LPE-treated mice at all time points displayed the most significant BODIPY-C11 fluorescence signal, indicating robust lipid peroxidation (Figure 3I). In contrast, Fe-LPE-treated tumor slices showed only minimal BODIPY-C11 fluorescence across the same time intervals, comparable to control groups with negligible lipid peroxidation. Furthermore, immunofluorescence staining demonstrated significantly lower GPX4 expression in CFe-LPE-treated tumors compared to other treatment groups (Figures 3J and S4C). Together, these results confirm CFe-LPE’s superior capacity to trigger ferroptotic cancer cell death.
We subsequently evaluated the therapeutic efficacy of CFe-LPE in a subcutaneous H22 tumor model (Figure 4A). Five groups of H22 tumor-bearing mice received intratumoral injections of PBS, Ca-LE, Fe-LPE, doxorubicin-LE (DOX-LE), or CFe-LPE. The dose of doxorubicin (DOX) was 2.88 mg/kg, while the doses of cariporide, FeNPs, and Lipiodol were identical to those described earlier. CFe-LPE treatment exhibited a stronger tumor growth suppression effect compared to DOX-LE and other control treatments (Figures 4B and 4C). The median survival time of CFe-LPE treatment mice was 28 days, significantly longer than that of mice treated with PBS (14 days), Fe-LPE (16 days), Ca-LE (18 days), or DOX-LE (22 days) (Figure 4D). No significant changes in body weight were observed in any treatment group (Figure 4E). Furthermore, histological analysis revealed that tumor slices from CFe-LPE-treated mice displayed the most severe histological damage, apoptosis, and Ki67 downregulation, as evidenced by hematoxylin and eosin (H&E), TdT-mediated dUTP nick-end labeling (TUNEL), and Ki67 staining, respectively (Figures 4F and S4D). Collectively, these results demonstrate that CFe-LPE effectively suppresses H22 tumor growth without causing significant side effects at the test dosage.
We next evaluated the contribution of CFe-LPE-induced ferroptosis to its overall antitumor efficacy in mice bearing subcutaneous H22 tumors (Figure 4G). Daily intraperitoneal injection of Fer-1 (5 mg/kg for 14 days) partially attenuated the tumor-suppressive effect of CFe-LPE, reflected by faster tumor growth and shorter median survival relative to mice treated with CFe-LPE alone (Figures 4H and 4I). In contrast, Fer-1 alone exhibited minimal impact on H22 tumor growth. Moreover, in immunodeficient nude mice bearing subcutaneous H22 tumors, the antitumor activity of CFe-LPE was comparable to that of DOX-LE, whereas in immunocompetent BALB/c mice with H22 tumors, CFe-LPE showed superior tumor-suppressive efficacy over DOX-LE (Figures 4J–4L). These results collectively validate the important contribution of CFe-LPE-induced ferroptosis and corresponding antitumor immunity to its overall antitumor effect.

Analysis of CFe-LPE-mediated antitumor immunity activation
We next investigated the modulation of the TME by CFe-LPE using bulk transcriptome analysis. Volcano plot analysis revealed 940 differentially expressed genes (DEGs) in CFe-LPE-treated mice compared to H22 tumor-bearing control mice, with 910 upregulated and 30 downregulated DEGs (Figure 5A). Gene Ontology (GO) enrichment analysis indicated that DEGs in CFe-LPE-treated tumors were enriched in biological processes and molecular functions related to the activation of innate/adaptive antitumor immunity and ROS metabolism, the latter being a hallmark of ferroptosis (Figure 5B). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis further confirmed the activation of ferroptosis and antitumor immunity-related signaling pathways in CFe-LPE-treated H22 tumors (Figure 5C). Among the DEGs, five ferroptosis-related genes (Acsl6, Map1lc3a, Slc40a1, Slc39a8, and CP) were significantly upregulated after CFe-LPE treatment (Figure S5A). The compensatory upregulation of iron-regulated transporter 1 (Slc40a1) and glutathione S-transferases (Gstt1, Gstm5, Gstm2, and Gstm1), which are involved in glutathione (GSH)-dependent lipid peroxide detoxification, provided compelling evidence that CFe-LPE induced robust lipid peroxidation. Additionally, gene set enrichment analysis (GSEA) confirmed the upregulation of the aforementioned KEGG pathways in CFe-LPE-treated tumors (Figure S5B). Furthermore, we observed significant upregulation of immune effector cell activation markers, including T cell signaling components (Cd33, Cd28, Lck, and Prkcq), natural killer (NK) cell receptors (Klrk1 and Ncr1), and cytotoxic effectors (Prf1, Gzmb, and Ifng). DEGs associated with immune-activating pathways, such as PD-L1 expression and PD-1 checkpoint, Th17 cell differentiation, TNF, and NF-κB, were also upregulated in CFe-LPE-treated tumors. Collectively, these findings suggest that CFe-LPE simultaneously acts as a ferroptotic cancer cell death inducer and a potent activator of antitumor immune responses.
We further investigated the impact of CFe-LPE treatment on remodeling the tumor immune microenvironment via flow cytometry. Five groups of H22 tumor-bearing BALB/c mice, receiving the same treatments as in Figure 4A, were sacrificed 4 days post-treatment, and their tumor tissues and tumor-draining lymph nodes were collected for immune analysis (Figure 5D). Flow cytometry revealed that CFe-LPE treatment significantly enhanced dendritic cell (DC) maturation in lymph nodes (Figures 5E and 5F). Concurrently, it increased the CD3+CD8+ T cell population while reducing immunosuppressive regulatory T cells (Tregs) within tumors (Figures 5G and 5H). This resulted in a CD3+CD8+ T cell/Treg ratio of 10.1, significantly higher than other treatment groups, indicating robust adaptive antitumor immunity induction (Figure 5I). Furthermore, CFe-LPE treatment elevated the proportion of cytotoxic NK cells and promoted the shift from anti-inflammatory M2 macrophages to pro-inflammatory M1 macrophages in tumors (Figures 5J–5M). The enzyme-linked immunosorbent assay (ELISA) confirmed a significant increase in IL-12p70 secretion and a decrease in IL-10 levels in CFe-LPE-treated tumors, further supporting its efficacy in M2-to-M1 macrophage repolarization (Figures 5N and 5O). Additionally, CFe-LPE-treated tumors exhibited higher levels of pro-inflammatory cytokines (TNF-α and IFN-γ) compared to untreated controls (Figures 5P and 5Q). Immunofluorescence analysis revealed that CFe-LPE treatment markedly increased the expression of cytotoxic perforin (Prf1) and granzyme B (Gzmb) in tumor tissues relative to other treatments (Figure S5C). Taken together, these results demonstrate that CFe-LPE treatment elicits both adaptive and innate antitumor immune responses within the TME.

In vivo CFe-LPE-mediated TAE treatment in orthotopic HCC tumor models
To assess the in vivo pharmacokinetic behavior of CFe-LPE after intra-arterial administration, we used orthotopic N1S1 tumor-bearing rats and monitored the fluorescence of hydrophobic DiR, a non-covalent label for Lipiodol. Ex vivo imaging at 48 h post-treatment revealed preferential tumor retention of CFe-LPE, as shown by stronger DiR fluorescence in N1S1 tumor tissues compared with other major organs (Figures S6A and S6B). Quantitative analysis of DiR fluorescence intensities in homogenized tumors and major organs at 48 h time point further confirmed this selective tumor accumulation (Figure S6C), consistent with previous reports that Lipiodol can persist in tumor regions for over 1 month.44
We next evaluated the antitumor efficacy of CFe-LPE-mediated TAE treatment in an orthotopic N1S1 HCC rat model (Figure 6A). Four groups of rats with N1S1 tumors implanted in the left hepatic lobes received intra-arterial injections of PBS, Fe-LPE, DOX-LE, or CFe-LPE. The administered doses per rat were as follows: 1 mg FeNPs, 0.41 mg DOX, 1.33 mg cariporide, and 100 μL Lipiodol. Tumor progression was monitored via small-animal ultrasound imaging. The results showed that CFe-LPE treatment resulted in the most significant tumor growth suppression, whereas Fe-LPE and DOX-LE only partially inhibited tumor growth (Figures 6B, 6C, S6D, and S6E). Notably, rats treated with CFe-LPE showed no significant body weight fluctuations throughout the study (Figure S6F), suggesting minimal systemic toxicity. Then, positron emission tomography (PET)/computed tomography (CT) imaging with 2-deoxy-2-[fluorine-18]-fluoro-D-glucose (18F-FDG) at 7 days post-treatment revealed markedly reduced glucose uptake in CFe-LPE-treated tumors compared to baseline, indicating significant suppression of tumor metabolism (Figures 6D and S6G). In contrast, tumors treated with conventional DOX-LE-based TACE exhibited only a moderate reduction in 18F-FDG uptake. Histopathological H&E and Ki67 staining further demonstrated that CFe-LPE-mediated TAE induced the most severe tumor cell damage and proliferation inhibition compared to other treatments (Figures 6E and S6H), further underscoring its superior therapeutic efficacy. Immunofluorescence staining further showed that CFe-LPE treatment led to significantly increased tumor infiltration of CD8+ T cells, CD4+ T cells, and NK cells, along with a reduced proportion of Tregs, compared with other treatments in rat HCC tumors (Figures 6E and S6I). Concurrently, it induced a phenotypic shift from anti-inflammatory M2 macrophages to pro-inflammatory M1 macrophages within the rat HCC tumors, as indicated by the increase in inducible nitric oxide synthase (iNOS) expression and decrease in CD206, respectively (Figure 6E). These results further confirm that CFe-LPE-mediated TAE robustly stimulates antitumor immunity and effectively reverses the immunosuppressive TME.
Thereafter, we assessed the biosafety of CFe-LPE-mediated TAE in orthotopic N1S1 tumor-bearing rats. Blood analysis revealed that only white blood cell (WBC) count and aspartate aminotransferase (AST) levels showed a transient increase on day 7 post-treatment, likely due to postoperative inflammation and embolization-induced liver injury. However, both parameters normalized by day 14, and no significant abnormalities were detected in other blood chemistry or routine blood tests throughout the study (Figure S7A). Furthermore, histopathological examination of major organs (heart, liver, spleen, lung, and kidney) from CFe-LPE-treated rats showed no evident tissue damage (Figure S7B). Collectively, these findings demonstrate that CFe-LPE-mediated TAE exhibits a favorable biosafety profile.
We next evaluated the therapeutic effect of CFe-LPE in a VX2 rabbit tumor model, established by direct implantation of VX2 tumor tissue into the left hepatic lobe (Figure 6F). Under digital subtraction angiography (DSA) guidance, three groups of VX2 tumor-bearing rabbits received intra-arterial embolization with PBS, DOX-LE, or CFe-LPE via selective tumor vasculature delivery. The doses of FeNPs, cariporide, and DOX were 1.5, 3.45, and 1.06 mg/kg, respectively. DSA imaging confirmed stable arterial embolization with both CFe-LPE and conventional DOX-LE, as no signs of recanalization were observed 24 h after treatment (Figure 6G). Contrast-enhanced CT imaging revealed no marked tumor progression in the CFe-LPE group, whereas DOX-LE only partially suppressed tumor growth during the monitoring period (Figures 6H–6J). Histopathological analysis using H&E and Ki67 staining further validated the superior efficacy of CFe-LPE-mediated TAE in inducing tumor cell damage and proliferation inhibition compared with other treatments (Figure 6K). Moreover, histopathological examination of major organs (heart, liver, spleen, lungs, and kidneys) from CFe-LPE-treated rabbits showed no obvious tissue damage (Figure S7C). Collectively, these results demonstrate that CFe-LPE, as a TAE embolic agent, exhibits favorable therapeutic efficacy and safety.

Discussion
TACE has emerged as a minimally invasive treatment for patients with intermediate to advanced HCC. However, its efficacy is often limited by suboptimal pharmaceutical properties and exacerbation of tumor immunosuppression.5,7,10,11 Therefore, developing new embolic materials capable of controllable drug release and potent activation of antitumor immunity represents a promising strategy to enhance TACE outcomes. Capitalizing on the tunable stability of nanoparticle-stabilized Pickering emulsions, we developed an FeNP-stabilized LPE that enabled efficient encapsulation and pH-responsive release of cariporide for TAE therapy of HCC (Figure 7). Beyond their role as a particular surfactant, FeNPs also acted as effective Fenton reaction inducers by gradually releasing Fe2+ in response to tumor acidity. Combined with the intracellular acidification strategy mediated by cariporide via NHE1 inhibition, the obtained CFe-LPE induced robust ferroptosis through a mechanism distinct from our previously reported self-fueling LPE driven by lipoxygenase and hemin.24
Moreover, consistent with reports that ferroptosis constitutes an immunogenic cell death modality that can initiate tumor-specific immune responses,42,43 CFe-LPE-induced ferroptotic cancer cells effectively promoted DC maturation via DAMP release and activated tumor-specific T cells. Furthermore, leveraging the ability of cariporide to neutralize extracellular tumor acidity, CFe-LPE treatment also effectively reversed the acidic TME, unlike our previously reported calcium carbonate-based embolic materials that consume extracellular protons through chemical reaction.24 As a result, CFe-LPE treatment exerted potent antitumor effects by synergistically inducing ferroptosis in cancer cells and activating antitumor immunity across multiple HCC models.
In summary, we report the rational design of a multifunctional LPE capable of simultaneously modulating extracellular/intracellular acidity and inducing self-amplified ferroptosis, offering an immunogenic strategy for HCC embolization therapy. Owing to its favorable biocompatibility, CFe-LPE-based TAE therapy represents a promising and translationally viable approach for the clinical treatment of HCC.

Limitations of the study
The therapeutic efficacy of CFe-LPE-mediated TAE in this study was assessed using only small-animal HCC embolization models. Owing to the restricted vascular diameter in such models, the clinical conditions of superselective embolization could not be fully recapitulated, potentially influencing the evaluation of both efficacy and safety. Consequently, studies employing large-animal (e.g., porcine) or non-human primate models would be more appropriate to examine the therapeutic performance and translational potential of CFe-LPE. Additionally, while tumor extracellular and intracellular pH modulation here relied solely on NHE inhibition, further investigation is needed to elucidate the efficacy and safety of co-targeting multiple pH-regulatory pathways.

Resource availability

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Liangzhu Feng (lzfeng@suda.edu.cn).

Materials availability
This study did not generate new unique reagents.

Data and code availability

•All data needed to evaluate the conclusion of this work are presented in the paper and the supplemental information.

•This paper does not report original code.

•Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments
This article was partially supported by the 10.13039/501100001809National Natural Science Foundation of China (grant nos. 82272094, 82472078, 82302330, and 32322046); the Foundation of Suzhou Municipal Health Commission (grant no. MSXM2024002); China Zhongguancun Precision Medicine Science and Technology Foundation (grant no. GYLZH06); Prof. Changgeng Ruan's Research and Innovation Fund for Graduate Students, the First Affiliated Hospital of 10.13039/501100007824Soochow University (grant no. 2024006902001); the Suzhou Key Laboratory of Nanotechnology and Biomedicine; the Collaborative Innovation Center of Suzhou Nano Science and Technology; 2024 Jiangsu Province Health Innovation Team Project; and the Higher Education Discipline Innovation Introduction Project (“111” project) from the Ministry of Education. Y.N. is currently a Yangtze River Chair Professor assigned by Chinese Ministry of Education. We also thank the website of app.biorender.com for assistance in creating the illustration figures.

Author contributions
L.Z., L.F., and C.N. designed the research. W.Y., C.W., C.Y., N.J., Y.N., and B.Y. performed the research. All authors analyzed and interpreted the data. W.Y., L.Z., L.F., Y.N., and C.N. wrote the paper.

Declaration of interests
The authors declare no competing interests.

STAR★Methods

Key resources table

Experimental model and study participant details

Cell lines and culture
Murine H22 HCC cells were obtained from Shanghai Zhongqiao Xinzhou Biological Technology Co., Ltd., and maintained in RPMI 1640 medium supplemented with 20% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin. Rat N1S1 HCC cells were obtained from Lishui Hospital of Zhejiang University as a gift, and maintained in DMEM high-glucose medium supplemented with 10% FBS and 1% penicillin/streptomycin. OVA-B16 and human HepG2 HCC cells, obtained as a gift from Zhongda Hospital of Southeast University, were cultured in RPMI-1640 and high-glucose DMEM medium, respectively. Each culture medium was supplemented with 10% FBS and 1% penicillin/streptomycin. All cell lines were cultured in a 5% CO2 environment at 37°C.

Animals
Female Balb/c mice (Wild-type, 6-8 weeks old), female Balb/c nude mice (Foxn1nu/Foxn1nu, 6-8 weeks old), and male Sprague-Dawley (SD) rats (outbred, 8-10 weeks old, 300-350 g) were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. All mice were housed under specific pathogen-free (SPF) conditions in a controlled environment (temperature: 22 ± 2°C; humidity: 50 ± 10%; 12-hour light/dark cycle) with free access to standard rodent chow and autoclaved water. All mice were used under the protocol approved by the Laboratory Animal Center of Soochow University (Protocol Number: 20241015019).
All rats were housed under standard laboratory conditions (temperature: 22 ± 2°C; 12-hour light/dark cycle) with ad libitum access to food and water. All procedures involving rats were approved by the Laboratory Animal Center of Soochow University (Protocol Number: 20241105003).
Female New Zealand White rabbits (outbred, 10-12 weeks old, 2-2.5 kg) were purchased from Shanghai Jiagan Biotechnology Co., Ltd. and housed individually in standard rabbit cages under controlled environmental conditions. Rabbits were provided with a standard diet and water ad libitum. All rabbits were used under the protocol approved by the Laboratory Animal Center of Soochow University (Protocol Number: 202511A0482).

Method details

Synthesis and characterization of FeNP
FeNPs were synthesized according to previously reported methods.35,36 The obtained FeNPs were then modified with sodium octanoate by dispersing them (20 mg/mL, 20 mL) in a sodium octanoate solution (80 mg/mL, 10 mL) under stirring in a nitrogen atmosphere for 30 minutes. The modified FeNPs were isolated via magnetic separation and stored in absolute ethanol for subsequent experiments.
The morphology and size of the modified FeNPs were characterized under transmission electron microscopy (CM300, Philips). The XRD spectrum of the modified FeNPs was acquired with an X-ray diffractometer (XRD-6000, Shimadzu). The contact angles of the FeNPs, with and without surface modification, were measured using an optical contact angle analyzer with contour analysis (OCA 20, DataPhysics, Filderstadt, Germany).

pH-dependent ferrous ion release of FeNPs
The pH-dependent ability of FeNPs to release ferrous ions (Fe2+) was confirmed using a potassium permanganate degradation assay. Briefly, supernatants from FeNPs incubated under different pH conditions (7.4, 6.5, 5.4) for 5 minutes were collected via high-speed centrifugation and mixed with potassium permanganate (100 μM) for 5 minutes. The degradation of potassium permanganate was monitored using a UV-Vis spectrophotometer, with a mass extinction coefficient of ∼200 L·mol-1·cm-1 at 525 nm, to quantify Fe2+ release based on the reaction: MnO4− + 5Fe2+ + 8H+ → Mn2+ + 5Fe3+ + 4H2O.
To further evaluate pH-dependent Fe2+ release, FeNPs solution (333 μg/mL, 300 μL) sealed in dialysis bags (molecular weight cutoff: 3000 Da) was immersed in 10 mL of buffer media at varying pH (7.4, 6.5 and 5.4) and shaken at 100 rpm (37°C). At predetermined time intervals, Fe2+ concentrations in the buffer were analyzed via ICP-OES (Aurora M90, Bruker, Germany).

pH-dependent Fenton catalytic ability of FeNPs
To evaluate FeNPs-catalyzed generation of hydroxyl radicals (⋅OH), FeNPs (50 μg/mL) were incubated with MB solutions (30 μg/mL) containing H2O2 (1 mM) at different pH values (7.4, 6.5, 5.4) for predetermined time intervals. After removing the FeNPs via high-speed centrifugation, the supernatants were collected for recording the UV-vis spectra of MB.

Preparation and characterization of CFe-LPE
The conventional Lipiodol emulsion was prepared by mixing ultrapure water with Lipiodol at a volume ratio of 1:2 using a medical three-way stopcock. To prepare CFe-LPE, FeNPs were first dispersed with Lipiodol, and then mixed with ultrapure water containing cariporide, as mentioned above.
Phase separation profiles of different Lipiodol emulsions were monitored using an optical microscope (DM4000M, Leica), with MB added to label aqueous droplets. The water-in-oil morphology of LPE was confirmed via confocal microscopy by tracking fluorophores dissolved in the aqueous droplets and Lipiodol phase. The viscosity of as-prepared CFe-LPE was measured with a rotary rheometer (HAAKE MARS 40). The injection force required for CFe-LPE to pass through a 2.8 F microcatheter was measured using a universal testing machine (34TM-50, Instron).

The release of cariporide from CFe-LPE
To evaluate the cariporide release profile, CFe-LPE (1.2 mL) was immersed in 5 mL of PBS (pH 6.5 and 7.4) and incubated at 37°C. At predetermined time intervals, the supernatant was collected, and the released cariporide was quantified by measuring its characteristic absorbance at 254 nm using a UV-Vis spectrophotometer (GENESYM™ 10S, Thermo Fisher Scientific).

Cell experiments
The cytotoxicity of FeNPs, with and without cariporide, was assessed in N1S1 cells using a commercial CCK-8 assay. To evaluate cellular uptake efficacy, N1S1 cells were incubated with FeNPs (25 μg/mL) for various time intervals (1, 2, 4, and 6 h). Following incubation, cells were collected and digested with aqua regia, and the iron content was quantified using ICP-OES.
The BCECF fluorescent probe was utilized to evaluate the effect of cariporide incubation on extracellular pH (cell culture medium) in treated N1S1 cells. To this end, N1S1 cells were seeded in 12-well plates (105 cells per well) and treated for 6 hours with either FeNPs (50 μg/mL), cariporide (200 μM), or their combination. Following incubation, culture supernatants were collected by centrifugation and incubated with the BCECF probe for 30 minutes at 37°C. Fluorescence intensity was subsequently measured using a microplate reader (BioTek Synergy H1, USA).
Intracellular pH changes in treated N1S1 cells were assessed using the BCECF-AM fluorescent probe. After identical treatments, N1S1 cells were loaded with BCECF-AM (5 μM in fresh medium) for 30 minutes, washed twice with PBS, and analyzed using confocal laser scanning microscopy (CLSM; Zeiss, LSM 800) and flow cytometry (BD Accuri™ C6 Plus).
N1S1 cells subjected to the same treatments were also stained with 2′,7′-Dichlorofluorescindiacetate (DCFH-DA), BODIPY-C11 or HKOH-1 for 30 minutes to measure intracellular ROS generation and lipid peroxidation, respectively, using confocal microscopy and flow cytometry according to the manufacturers’ procedure. To test intracellular GPX4 activity, treated N1S1 cells were analyzed using a commercial Glutathione Peroxidase Assay Kit (Beyotime, Jiangsu, China) following the manufacturer’s instructions. Intracellular malondialdehyde (MDA) generation of N1S1 cells post different treatments were analyzed using a commercial Lipid Peroxidation MDA Assay Kit (Beyotime, Jiangsu, China) following the manufacturer’s instructions.
To evaluate the ability of combined FeNPs and cariporide treatment in inducing immunogenic cell death, N1S1 cells with the same treatments as mentioned above sequentially stained with primary anti-HMGB1 antibody (dilution: 1:1000) or anti-CRT antibody for 1 h, followed by being stained with respective fluorophore-conjugated secondary antibody (dilution: 1:500) for additional 30 min and then analyzed via confocal microscopy and flow cytometry.
BMDCs were isolated from the hind limbs of female C57BL/6 mice by using our previously used method.45 Then, BMDCs (2 × 105 cells per well) were incubated with the supernatant of H22 cells pre-treated by FeNPs and cariporide for 24 h, followed by analyzing the maturation levels of BMDCs via flow cytometric analysis their surface expression of CD80 and CD86 in CD11c+ cells.
To evaluate the capacity of dead cancer cells to specifically activate T cells in vitro, single-cell splenocytes were first isolated from OT-I transgenic mice and labeled with CFSE before being subjected to B16-OVA tumor cells pretreated by FeNPs and cariporide for 24 h at a ratio of 5 : 1 ratio. 48 h later, splenic cells were collected for elevating intracellular CFSE quantification via flow cytometry. The concentrations of TNF-α and IFN-γ in the supernatants were also quantified using commercially available ELISA kits according to the manufacturers’ protocols.

Animal experiments
A subcutaneous H22 tumor model was established by subcutaneously injecting H22 cells (2 × 106) suspended in 50 μL of PBS into the right flank of each Balb/c mouse. An orthotopic N1S1 tumor model was established by injecting N1S1 cells (6 × 106) suspended in 75 μL of PBS containing 30% Matrigel (Corning) into the left lower liver lobe of each SD rat under anesthesia. Orthotopic VX2 rabbit HCC model was established via direct injection of VX2 tumor tissue homogenates (100 μL) into the left hepatic lobe of New Zealand White rabbits.
To evaluate the in vivo locoregional cargo release profile of CFe-LPE, two groups of H22 tumor-bearing mice were intratumorally injected with equal doses of either conventional lipodol emulsion or Fe-LPE loaded with Cy5.5-labeled BSA. Cy5.5 fluorescence intensity was monitored at predetermined time intervals using the IVIS® in vivo imaging system (Lumina III, PerkinElmer). Additionally, at 24, 48, and 72 h post-injection, one mouse from each group was euthanized, and their tumors were collected for cryosectioning and subsequent microscopic examination of intratumoral Cy5.5 fluorescence distribution.
To evaluate the pH-modulation capacity of CFe-LPE, H22 tumor-bearing mice received intratumoral injections of PBS, Ca-LE, Fe-LPE, or CFe-LPE. The administered doses of cariporide, FeNPs, and lipodol were 9.36 mg/kg, 2.5 mg/kg, and 2.5 mL/kg, respectively. Twenty-four hours post-injection, mice were intratumorally administered either BCECF-AM (20 μM, 50 μL) or BCECF (20 μM, 50 μL), followed by surgical tumor resection for ex vivo fluorescence imaging under the IVIS® imaging. Imaging was performed using excitation wavelengths of 440 and 480 nm with an emission wavelength of 535 nm. Subsequently, tumors were cryosectioned for confocal microscopic analysis.
To evaluate intratumoral lipid peroxidation and GPX4 expression, subcutaneous H22 tumor-bearing mice receiving the aforementioned treatments were euthanized, and their tumors were harvested, cryosectioned, and processed for BODIPY-C11 staining and GPX4 immunofluorescence staining according to the manufacturers’ protocols.
To assess the therapeutic efficacy of CFe-LPE, five groups of H22 tumor-bearing mice were administered intratumoral injections of PBS, Ca-LE, Fe-LPE, DOX-LE, or CFe-LPE. The DOX dose was 2.88 mg/kg, while the doses of cariporide, FeNPs, and Lipiodol remained consistent with previous experiments. Tumor dimensions (length and width) were measured using digital calipers, with volumes calculated using the formula: (width∗width∗length)/2. Body weights were monitored using a precision digital balance. Mice were presumed dead when their tumor volumes reached 1500 mm3. On day 4 post-injection, one randomly selected mouse from each group was euthanized for tumor collection and subsequent histological analysis, including H&E staining, TUNEL assay, and Ki67 immunohistochemistry, performed according to standard protocols.
The same experimental protocol was employed to evaluate the effect of Fer-1 on the antitumor efficacy of CFe-LPE in the subcutaneous H22 tumor model. Fer-1 (5 mg/kg) was intraperitoneally administered daily for a total of 14 times since the intratumoral injection of CFe-LPE. The tumor suppression effects of CFe-LPE and DOX-LE were evaluated in H22 tumor-bearing nude-Balb/c mice under the identical experimental conditioned mentioned above.
For transcriptomic analysis, H22 tumor-bearing mice (n = 3 per group) treated with either PBS or CFe-LPE were euthanized four days post-treatment, and tumor specimens were immediately snap-frozen in liquid nitrogen for preservation. Following RNA extraction, high-throughput sequencing of the mRNAs was performed at GENEWIZ Co., Ltd., Suzhou, China. Differential gene expression analysis employed stringent thresholds: |log2FC| ≥ 2 with q-value (FDR, p adj) ≤ 0.05. We considered GO terms with p ≤ 0.05 and KEGG pathways as statistically significant. Subsequent bioinformatic analysis was performed using the online platform at www.bioinformatics.com.
For antitumor immune mechanism study, five groups of H22 tumor-bearing mice (n = 5 per group) received the aforementioned treatments and were euthanized four days post-treatment. Tumors and tumor-draining lymph nodes were harvested and processed into single-cell suspensions following established protocols.46 Cell suspensions were stained with fluorophore-conjugated antibody panels for flow cytometric analysis. Cytokine levels (TNF-α, IFN-γ, IL-10, and IL-12) in tumor lysates were quantified using commercially available ELISA kits according to the manufacturers’ protocols. Additionally, H22 HCC tumors were harvested for immunofluorescence staining of Prf1 and Gzmb four days post-treatment following standard protocol.
To trace the in vivo distribution of CFe-LPE, DiR labeled CFe-LPE was intraarterially administrated into the orthotopic N1S1 tumor bearing rats, followed by ex vivo imaging of dissected major organs and tumor tissues 5min, 24 h and 48 h post embolization. Meanwhile, these major organs and tumors were also homogenized for quantifying the DiR fluorescence intensity using a microplate reader.
To evaluate the TAE treatment efficacy of CFe-LPE in orthotopic N1S1 rat HCC model, four groups of tumor-bearing rats received intra-arterial injections of PBS, Fe-LPE, DOX-LE, or CFe-LPE by using a modified method of transabdominal catherization of hepatic artery.47 The administered doses were 1 mg FeNPs, 0.41 mg DOX, 1.33 mg cariporide, and 100 μL Lipiodol per rat. Tumor progression was monitored using small-animal ultrasound imaging (ULTIMUS-9LAB, VINNO) before treatment and at 3, 7, and 14 days post-treatment. At day 4 post-treatment, tumors were collected for histological evaluation through H&E and Ki67 staining following standard protocols. Tumor metabolic activity was evaluated via 18F-FDG PET/CT imaging (Discovery LS, GE Healthcare) at baseline and 7 days after treatments.
To evaluate intratumoral immune cell infiltration, orthotopic N1S1 HCC tumors were harvested from rats on day 4 post-treatment for immunofluorescence staining of CD8 (cytotoxic T cells), CD4, NKp44 (NK), Foxp3 (Tregs), iNOS (M1 macrophages), and CD206 (M2 macrophages) following standard protocols.
To evaluate the in vivo biosafety profile of CFe-LPE-mediated TAE treatment, N1S1 tumor-bearing rats were euthanized 4 days post-treatment, and major organs (heart, liver, spleen, kidneys, and lungs) were harvested for histopathological evaluation via H&E staining. In addition, serial blood samples were collected from N1S1 tumor-bearing rats at three time points: pre-treatment baseline, and at 7 and 14 days following CFe-LPE administration, for comprehensive hematological analysis and blood chemistry profiling.
To evaluate the vascular embolization efficacy of CFe-LPE and DOX-LE was injected into the hepatic artery through the femoral artery using a 2.0-F microcatheter under DSA guidance. The tumor vasculature perfusion behaviors were also visualized via DSA imaging 24 h post embolization treatment. The administered doses of cariporide, FeNPs, and DOX were 3.45 mg/kg, 1.5 mg/kg, and 1.06 mg/kg, respectively. Tumor progression was monitored by contrast-enhanced CT at designated time intervals. Finally, rabbits were euthanized with their tumor tissues along with major organs harvested 14 days post-treatment for histopathological evaluation.

Quantification and statistical analysis
All results are expressed as a mean ± standard deviation unless specified otherwise. Statistical analyses were conducted with GraphPad Prism 9.5.0. The significant differences between groups were determined by one-way analysis of variance (ANOVA) and Student’s t test. When the p-value is less than 0.05, indicating a significant difference. Further statistical details, including n values, are provided in the figure legends.