Ascorbic Acid-Derived Supramolecular Gels Induce Immunogenic Ferroptosis in Cancer Cells to Potentiate Tumor Immunotherapy

Abstract

High-dose ascorbic acid (AA) therapy induces cancer cell death primarily through its oxidized derivative, dehydroascorbic acid (DHA). However, maintaining therapeutic AA concentrations within tumors and overcoming intratumoral hypoxia pose critical barriers to the clinical application of AA. Herein, we develop an injectable supramolecular gel (αPD-1@Lv/HPAGel) composed of ascorbyl palmitate (an AA derivative), lovastatin-loaded hemoglobin nanoparticles (Lv/Hb-PDA), and the immune checkpoint inhibitor anti-PD-1 (αPD-1). Upon intratumoral administration, this gel system sustains high local AA concentrations and promotes efficient oxidation of AA into DHA by alleviating hypoxia via the release of oxygen from hemoglobin. Simultaneously, lovastatin inhibits glutathione peroxidase 4, synergistically amplifying AA-induced ferroptosis. The coordinated induction of ferroptosis remodels the tumor immune microenvironment and stimulates a robust antitumor immune response. In combination with αPD-1, the gel system suppresses tumor growth and metastasis, establishes durable immune memory, and effectively prevents local and distant tumor recurrence postsurgery. Collectively, these findings present an approach for translating high-dose AA therapy into practice and provide evidence for the integration of ferroptosis induction with immunotherapy for enhanced cancer treatment.

Introduction

Ascorbic acid (AA), commonly known as vitamin C, is an essential micronutrient that possesses diverse biological functions in the human body. Widely recognized for its antioxidant properties, AA is known to effectively mitigate oxidative stress caused by free radicals and is able to protect cells from oxidative damage. Beyond its conventional antioxidant role, AA has attracted considerable attention owing to its promising anticancer therapeutic potential. , Numerous preclinical studies have demonstrated that pharmacologic concentrations of AA, particularly when administered in high doses, can selectively induce cancer cell death. For instance, intraperitoneal administration of 4 g/kg of AA has been shown to substantially inhibit tumor growth in murine models of breast cancer, colorectal cancer, pancreatic cancer, and melanoma. However, despite these encouraging preclinical outcomes, translating AA into effective clinical cancer therapies remains challenging. Several early phase clinical trials, predominantly combining AA with chemotherapy, radiotherapy, or immunotherapy, have been attempted but typically have not progressed beyond Phase I or II, frequently being terminated prematurely due to treatment-related toxicity associated with sustained high-dose administration and inconsistent therapeutic responses. Furthermore, adverse events such as renal dysfunction, oxalate nephropathy, and hemolysis in patients with glucose-6-phosphate dehydrogenase deficiency have significantly hindered its clinical advancement. This paradox, wherein a high dose of AA reaches the pro-oxidant threshold necessary to exert antitumor effects but fails to deliver durable clinical benefit, underscores a critical dilemma. These inconsistencies have been largely attributed to individual patient variability, pharmacokinetic challenges, and the complex, often immunosuppressive nature of the tumor microenvironment, all of which collectively impede the reliable development of AA as a viable anticancer agent.

AA is postulated to execute its anticancer functions through multiple mechanisms, with the induction of ferroptosis prominent among those mechanisms. , Ferroptosis is a regulated, iron-dependent form of nonapoptotic cell death, characterized by overwhelming lipid peroxidation (LPO) and disruption of membrane integrity. Within tumor tissues, AA is oxidized to dehydroascorbic acid (DHA), a structural analogue of glucose. This enables its preferential uptake into cancer cells via glucose transporter 1 (GLUT1), which is often overexpressed in malignant cells due to their heightened metabolic demand. − Inside cancer cells, DHA is reduced back to AA by glutathione (GSH), substantially depleting intracellular GSH levels. This depletion disrupts cellular redox homeostasis, causing reactive oxygen species (ROS) accumulation and the initiation of oxidative stress responses. Simultaneously, the loss of GSH impairs glutathione peroxidase 4 (GPX4), a pivotal enzyme responsible for lipid peroxide neutralization, further exacerbating LPO. , Together, ROS accumulation, GSH depletion, and GPX4 inactivation culminate in irreversible damage to cellular membranes, thereby pushing cancer cells toward ferroptotic death. , Crucially, ferroptosis induced by AA is not merely a mechanism of cell elimination but also possesses immunomodulatory potential. Recent studies have shown that AA-induced ferroptosis is intimately linked to the process of immunogenic cell death (ICD). During ferroptosis, damage-associated molecular patterns (DAMPs) are released, facilitating the recruitment and activation of antigen-presenting cells such as dendritic cells (DCs). , As a result, this cascade of events primes adaptive immune responses, amplifying the efficacy of immunotherapeutic strategies, which underscores the anticancer potential of AA-induced ferroptosis through the elimination of tumor cells and the activation of adaptive immune pathways. ,

Systemic administration of AA, including intravenous injection, intraperitoneal injection, and oral intake, has been extensively explored, but substantial barriers to effective tumor targeting have remained. One of the primary limitations is the rapid clearance of AA from the bloodstream following systemic administration, resulting in only transient exposure to therapeutically relevant concentrations. This rapid pharmacokinetic profile, combined with AA’s metabolic instability, limits its accumulation in tumor tissues. Furthermore, AA exhibits poor permeability across the blood–tumor barrier, thereby restricting its ability to achieve effective intratumoral distribution. , On the other hand, oral administration of AA is hindered by low bioavailability due to limited intestinal absorption and extensive first-pass metabolism, in addition to the potential for gastrointestinal irritation at high doses. Conversely, intravenous or intraperitoneal administration of AA poses risks of systemic toxicity, including renal impairment and metabolic disturbances. To address these shortcomings, a number of advanced drug delivery platforms have been proposed to improve AA’s pharmacological performance. These include nanoparticle-based delivery systems, metal–organic frameworks, , and liposomes, , designed to enhance AA stability, prolong systemic circulation, and improve tumor accumulation. However, a critical and often overlooked challenge lies in the hypoxic nature of solid tumors, which fundamentally compromises the therapeutic mechanism of AA. Within hypoxic tumor microenvironments, the oxidative conversion of AA to DHA is severely attenuated due to insufficient molecular oxygen, thereby reducing DHA-mediated cellular uptake and subsequent ferroptosis induction. , Although strategies such as oxygen supplementation or tumor oxygenation have been explored to mitigate this barrier, , current delivery systems still fall short of achieving a coordinated solution that ensures effective AA oxidation, stable accumulation within tumor tissue, and precise temporal control over its bioactivity.

To overcome the aforementioned obstacles, we designed and developed a supramolecular gel system based on ascorbyl palmitate (AP), a lipophilic derivative of AA. Specifically, a hemoglobin (Hb)-based nanoparticle was first synthesized through dopamine polymerization on Hb, simultaneously encapsulating lovastatin (Lv), an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), forming Lv/Hb-PDA. The Hb component within these nanoparticles serves as an oxygen reservoir, alleviating tumor hypoxia. By combination of Lv/Hb-PDA with AP and the anti-PD-1 antibody (αPD-1), a supramolecular gel αPD-1@Lv/HPAGel was constructed. Following direct intratumoral (i.t.) injection, tumor-abundant esterases catalyze AP ester bond cleavage, gradually degrading the gel and sustaining the controlled release of AA, Lv/Hb-PDA, and αPD-1 into the tumor microenvironment (Figure a). Once released, Lv/Hb-PDA facilitates the oxidation of AA to DHA and produces H₂O₂ by supplying molecular oxygen. DHA is taken up via GLUT1 and is reduced back to AA by GSH, depleting intracellular GSH stores. The regenerated AA then reduces Fe³⁺ to Fe²⁺, catalyzing the Fenton reaction, in which H₂O₂ is converted into highly cytotoxic hydroxyl radicals that initiate LPO. Concurrently, intracellular Lv released from Lv/Hb-PDA inhibits HMGCR, disrupting the mevalonate pathway and downregulating GPX4 expression. Together, these synergistic interactions enhance ferroptosis in cancer cells and promote immunogenic cell death marked by the release of DAMPs (Figure b). These DAMPs reactivate immune response, facilitating DC maturation and antigen presentation. Mature DCs migrate to tumor-draining lymph nodes, where they prime cytotoxic T lymphocytes against tumor antigens. The activated T cells subsequently infiltrate the tumor, where their cytotoxic activity is further enhanced by αPD-1-mediated immune checkpoint blockade (ICB). This multilevel approach results in robust suppression of tumor growth, metastasis, and recurrence (Figure c).

1.

Schematic illustration of αPD-1@Lv/HPAGel inducing ferroptosis in cancer cells and enhancing immune checkpoint blockade therapy. (a) Schematic illustration depicting the preparation of Lv/Hb-PDA and its subsequent assembly with AP and αPD-1 to form αPD-1@Lv/HPAGel, enabling esterase-responsive sustained drug release of AA, Lv/Hb-PDA, and αPD-1 in tumors. (b) Schematic illustration depicting the mechanism by which Lv/Hb-PDA delivers oxygen into the hypoxic tumor microenvironment to facilitate AA oxidation into DHA, inducing redox imbalance in cancer cells, and subsequently triggering immunogenic ferroptosis via the synergistic inhibition of GPX4 in cooperation with intracellularly released Lv. (c) Schematic illustration depicting i.t. injection of αPD-1@Lv/HPAGel to induce cancer cell ferroptosis, promote DC maturation and antigen presentation, enhance T cell infiltration into tumors, and synergize with αPD-1 for enhanced immunotherapy efficacy. Hb, hemoglobin; Lv, lovastatin; AA, ascorbic acid; AP, ascorbyl palmitate; αPD-1, PD-1 antibody; GLUT1, glucose transporter 1; GSH, glutathione; GSSG, oxidized glutathione; GPX4, glutathione peroxidase 4; HMGCR, hydroxy methylglutaryl coenzyme A reductase; LPO, lipid peroxidation; DAMPs, damage-associated molecular patterns; i.t. injection, intratumoral injection; DC, dendritic cell.

Results

Fabrication and Characterization of Lv/Hb-PDA

Dopamine exhibits strong adhesive properties and forms stable interactions with amino acid residues in Hb. Leveraging this interaction, polydopamine-coated hemoglobin nanoparticles (Hb-PDA) were successfully synthesized through in situ dopamine polymerization. During this process, Lv was coencapsulated within Hb-PDA, resulting in the formation of Lv/Hb-PDA. The successful incorporation of Lv was verified by Fourier transform infrared (FTIR) spectroscopy, which revealed a characteristic vibrational absorption band of Lv in the 1000–1200 cm^–1 fingerprint region, confirming its presence within the nanoparticle (Figure S1). To enable oxygen storage, the methemoglobin (mHb) state within Hb-PDA was chemically reduced to its active ferrous form using sodium dithionite, subsequently introducing oxygen by continuous aeration. Consequently, the original red color of the Hb solution transitioned to black due to dopamine polymerization, with an increase in hydrated particle size from approximately 10 nm to approximately 100 nm for Hb-PDA or Lv/Hb-PDA. In parallel, a notable shift to a negative ζ potential of approximately −10 mV was observed for Lv/Hb-PDA, reflecting its increased colloidal stability (Figure a). The particle size distribution analysis revealed that Lv/Hb-PDA exhibited a hydrated diameter predominantly in the range of 50–200 nm. Transmission electron microscopy (TEM) revealed the uniform, spherical morphology of both Hb-PDA and Lv/Hb-PDA, with distinctive cocoon-like shell structures suggestive of successful surface modification (Figures b and S2). Additionally, storage stability studies demonstrated that the particle size and polydispersity index of Lv/Hb-PDA remained relatively constant over a 7-day period at ambient conditions, indicating favorable stability for practical application (Figure S3).

2.

Characterization of Lv/Hb-PDA and its capability to induce AA oxidation. (a) Diameter, ζ potential, and representative photos of Hb, Hb-PDA, and Lv/Hb-PDA. Data are shown as mean ± s.d. (n = 3). (b) Hydrated particle size distribution and representative TEM image of Lv/Hb-PDA. Scale bar, 50 nm. (c) CD spectra of the Hb and Hb-PDA. (d) UV absorption spectra of mHb-PDA and Hb-PDA before (−O₂) or after (+O₂) oxygen loading. (e) Dissolved oxygen concentration following the addition of Hb, mHb, Hb-PDA, or mHb-PDA into deoxygenated buffer. Data are shown as mean ± s.d. (n = 3). (f) Schematic illustration of the oxygen-loading and release capabilities of mHb-PDA and Hb-PDA. (g–i) Concentration of residual AA (g), and generated DHA (h) and H₂O₂ (i) after AA reaction with mHb, Hb, mHb-PDA, and Hb-PDA for 1 h. Data are shown as mean ± s.d. (n = 3). (j) Release profiles of Lv from Lv/Hb-PDA incubated at pH 7.4, 5.5, and 4.5. Data are shown as mean ± s.d. (n = 3).

To further evaluate the oxygen storage and release capabilities, a series of spectroscopic analyses was conducted to assess their structural integrity and functional performance. Circular dichroism (CD) spectroscopy was first employed to compare the secondary structures of Hb-PDA and native Hb. Notably, the CD spectrum of Hb-PDA closely resembled that of native Hb, suggesting that the dopamine polymerization process did not significantly alter the α-helical content or global folding of the Hb protein (Figure c). This preservation of the secondary structure is critical, as the oxygen-binding capacity of hemoglobin is highly dependent on its conformational integrity. The unreduced mHb state exhibited a dark red color and demonstrated the inability to bind oxygen, as indicated by a stable absorption peak at 405 nm following oxygen aeration (Figure S4a,b). In contrast, upon reduction, Hb turned bright red, with a characteristic shift in absorption peak from 412 to 405 nm after aeration, verifying successful oxygen loading (Figure S4a,c). Similarly, mHb-PDA and Hb-PDA displayed UV spectral changes akin to those observed from mHb and Hb, indicating that effective oxygen loading occurred exclusively upon reduction of mHb-PDA to Hb-PDA (Figure d). The functional performance of oxygenated Hb and Hb-PDA under hypoxic conditions was then evaluated by measuring their ability to release oxygen into a deoxygenated buffer. Both forms were found to release oxygen rapidly, effectively elevating the concentration of dissolved oxygen, which is essential for therapeutic activity in hypoxic tumor tissues. In contrast, mHb and mHb-PDA, which inherently lack oxygen-carrying capacity, were unable to increase the oxygen levels under the same conditions (Figure e,f). These findings collectively demonstrate that Hb-PDA not only retains structural and functional fidelity to native Hb but also possesses efficient oxygen-loading and release profiles.

Subsequently, the functional consequence of oxygen delivery was evaluated by assessing the oxidative conversion of AA under hypoxic conditions. To simulate a low-oxygen tumor microenvironment, AA was dissolved in a deoxygenated buffer, followed by the addition of either Hb or Hb-PDA. Our results demonstrated that oxygenated Hb and Hb-PDA rapidly oxidized AA, leading to its complete depletion, whereas mHb and mHb-PDA (lacking oxygen-binding capability) failed to facilitate this reaction (Figure g). This oxidation process was shown to be both dose-dependent and time-dependent, with increasing concentrations of Hb or Hb-PDA and longer incubation periods significantly accelerating the depletion of AA (Figure S5a,b). Moreover, under hypoxic conditions, both oxygenated Hb and Hb-PDA efficiently oxidized AA, converting it into DHA, which has the potential to enter cancer cells and initiate ferroptosis (Figure h). In parallel, concurrent H₂O₂ production was monitored throughout this reaction. Compared to the control groups, oxygenated Hb and Hb-PDA produced markedly higher concentrations of H₂O₂ during AA oxidation, while mHb and mHb-PDA showed negligible H₂O₂ production (Figure i). This outcome indicates that Hb-PDA not only enhances DHA generation but also supplies the necessary substrates for hydroxyl radical formation, further contributing to LPO and ferroptosis. The release behavior of Lv from Lv/Hb-PDA under physiologically relevant acidic conditions was then investigated. Our findings showed that Lv release was significantly accelerated under acidic conditions, particularly at pH 5.5 and 4.5, correlating with the degradation of the polydopamine shell and disintegration of the Lv/Hb-PDA (Figure j). This acid-responsive property would facilitate efficient Lv release in a mildly acidic environment within cancer cells.

Lv/Hb-PDA Enhances AA-Induced Immunogenic Ferroptosis

Triple-negative breast cancer (TNBC) is an aggressive malignancy characterized by poor prognosis, rapid metastasis, and limited immune infiltration, leading to resistance against conventional immunotherapy. To investigate the effects of AA-induced ferroptosis and its potential to trigger ICD in vitro, 4T1 mouse TNBC cells were cultured and tested under hypoxic conditions. In these hypoxic cultures, AA was oxidized to DHA with the assistance of oxygen released from Hb-PDA. As a result, intracellular accumulation of DHA was significantly enhanced in the Hb-PDA-treated group compared with cells treated with mHb-PDA, which lacks oxygen-releasing capacity. Notably, the increased uptake was abolished when cells were cotreated with BAY-876, a specific inhibitor of GLUT1 transporters, suggesting that the oxidative conversion of AA to DHA is a prerequisite for efficient GLUT1-mediated cellular internalization (Figure a). Once inside the cell, DHA rapidly underwent reduction by intracellular GSH, which led to a substantial depletion of the GSH pool. This marked reduction in GSH levels was observed exclusively in the Hb-PDA group under hypoxia, while cells treated with mHb-PDA exhibited no significant change in intracellular GSH concentration (Figure b). Intriguingly, when the same experiment was conducted under normoxic conditions, the addition of either Hb-PDA or mHb-PDA failed to induce notable GSH depletion. This observation is likely due to the presence of dissolved oxygen in normoxic buffer, which can independently promote partial AA oxidation to DHA, thereby masking the oxygen-supplying advantage conferred by Hb-PDA (Figure S6). These results collectively highlight that the therapeutic efficacy of AA in inducing ferroptosis is tightly dependent on both the tumor oxygenation status and DHA-mediated intracellular transport pathways.

3.

Ferroptosis and immunogenic cell death in 4T1 cells induced by AA combined with Lv/Hb-PDA. (a) Relative uptake of AA and DHA in 4T1 cells incubated with mHb-PDA or Hb-PDA, pretreated with or without BAY-876 (GLUT1 inhibitor). Data are shown as mean ± s.d. (n = 3). (b) Intracellular GSH levels in 4T1 cells after different treatments under hypoxic conditions for 12 h. Data are shown as mean ± s.d. (n = 3). (c, d) Quantification of Fe²⁺ levels (c) and representative confocal microscopic images (d) of 4T1 cells by FerroOrange staining after different treatments for 12 h. Data are shown as mean ± s.d. (n = 3). Scale bar, 20 μm. (e, f) Quantification (e) and representative flow cytometric analysis (f) of intracellular ROS level in 4T1 cells after different treatments for 12 h. Data are shown as mean ± s.d. (n = 3). (g) Uptake of Lv by 4T1 cells after different treatments. Data are shown as mean ± s.d. (n = 3). (h) Representative Western blot images showing GPX4 expression in 4T1 cells after different treatments for 24 h. (i) Relative oxidized lipid levels in 4T1 cells after different treatments for 12 h. Data are shown as mean ± s.d. (n = 3). (j) Representative confocal microscopic images of 4T1 cells by BODIPY581/591-C11 staining after different treatments for 12 h. Red color represents nonoxidized lipids, and green color represents oxidized lipids. Scale bar, 50 μm. (k) ATP concentration in culture medium of 4T1 cells after different treatments for 24 h. Data are shown as mean ± s.d. (n = 3). (l) Representative confocal microscopic images of 4T1 cells by immunofluorescence staining on CRT and HMGB1 after different treatments for 24 h. Scale bar, 50 μm. (m, n) Representative flow cytometric analysis (m) and quantification (n) on CD80⁺/CD86⁺ cell population of the BMDCs incubated with the culture medium from 4T1 cells after different treatments for 24 h. Data are shown as mean ± s.d. (n = 3).

Intracellular Fe²⁺ levels in 4T1 cells were subsequently evaluated by FerroOrange fluorescence staining, a sensitive probe for detecting labile Fe²⁺ pools. As anticipated, cotreatment with Hb-PDA and AA resulted in a significant elevation in intracellular Fe²⁺ accumulation, whereas cells treated with mHb-PDA showed no such significant change (Figure c,d). The elevated Fe²⁺ serves as a critical cofactor in the Fenton reaction, whereby H₂O₂ previously generated during AA oxidation is converted into highly reactive hydroxyl radicals (•OH). These radicals initiate a chain reaction of oxidative damage, culminating in elevated reactive oxygen species (ROS) levels and extensive oxidative stress. In our experiments, cotreatment with AA and Hb-PDA induced robust ROS production in 4T1 cells, as visualized by fluorescence-based ROS probes under flow cytometric analysis (Figure e,f). To further examine the drug delivery component, intracellular uptake efficiency of Lv/Hb-PDA versus free Lv was assessed. Cells treated with Lv/Hb-PDA displayed markedly higher intracellular Lv levels compared to those exposed to free drug, suggesting that nanoparticle-mediated delivery enabled enhanced internalization by cancer cells, likely through an endocytosis route rather than passive diffusion (Figure g). Upon internalization, the mildly acidic conditions of the endolysosomal compartments triggered the degradation of Lv/Hb-PDA, promoting the release of Lv into the cytosol. Released Lv effectively inhibited HMGCR, disrupting the mevalonate pathway (Figure S7a,b). The combined effects of GSH depletion, ROS generation, and mevalonate pathway suppression culminated in a significant downregulation of GPX4 expression in cancer cells (Figures h and S8).

To directly visualize lipid peroxidation and confirm ferroptotic cell death, BODIPY581/591-C11 staining was performed, which differentiates between oxidized and nonoxidized lipids based on a red-to-green fluorescence shift. As anticipated, cotreatment with Lv/Hb-PDA and AA resulted in a marked decrease in nonoxidized lipid fluorescence and a corresponding increase in oxidized lipid accumulation in 4T1 cells (Figure i,j). This outcome, together with elevated ROS and GPX4 suppression, further substantiates that observed cell death occurred via the canonical ferroptosis pathway. Cell viability and live/dead assays further confirmed ferroptosis induction. Cotreatment with Lv/Hb-PDA and AA significantly reduced the IC₅₀ of AA from 18.6 to 1.5 μM, indicating a dramatic increase in sensitivity driven by ferroptotic mechanisms (Figure S9). In addition, calcein-AM and propidium iodide dual staining assays revealed that more than 90% of the cells underwent cell death following cotreatment with Lv/Hb-PDA and AA, a rate markedly higher than observed in any other treatment group (Figure S10a,b). These results strongly suggest that the combined action of AA-induced oxidative stress and GPX4 downregulation synergistically drives ferroptosis in 4T1 cells.

ICD characteristics associated with ferroptosis were subsequently explored by evaluating the release of DAMPs from 4T1 cells, including adenosine triphosphate (ATP), calreticulin (CRT), and high-mobility group box 1 (HMGB1). Following cotreatment with Lv/Hb-PDA and AA, a significant elevation in extracellular ATP levels was observed compared to control or monotherapy groups, reflecting active DAMP release from ferroptotic cells (Figure k). Immunofluorescence (IF) staining revealed that Lv/Hb-PDA and AA treatment significantly promoted CRT translocation from the endoplasmic reticulum to the plasma membrane, a key ICD hallmark that facilitates DC recognition (Figures l and S11a). At the same time, intracellular HMGB1 levels were significantly reduced, indicating its release into the extracellular space (Figures l and S11b). Collectively, these observations indicate that ferroptosis induced by Lv/Hb-PDA and AA effectively triggered ICD, characterized by significant DAMP release. To investigate the consequences of DAMP release on immune activation, bone-marrow-derived dendritic cells (BMDCs) were cultured with conditioned media collected from treated 4T1 cells. Strikingly, the supernatants from cells treated with Lv/Hb-PDA and AA significantly enhanced DC maturation, as evidenced by an increase in the percentage of CD80⁺/CD86⁺ cells from 30.2 to 43.9% (Figure m,n). These findings indicate that immunogenic ferroptosis not only facilitates tumor cell death but also promotes the release of immune-stimulating signals that enhance antigen presentation and prime adaptive immune responses.

Construction and Characterization of Lv/HPAGel

To enable effective and sustained localized delivery of both AA and Lv/Hb-PDA within the tumor microenvironment, a supramolecular gel was developed by coassembling AP with Lv/Hb-PDA. This gel formulation, referred to as Lv/HPAGel, leverages the amphiphilic properties of AP and the abundant surface catechol groups of polydopamine to drive spontaneous gelation via noncovalent interactions (Figure S12a). Notably, AP alone was incapable of forming a stable gel matrix, highlighting the essential role of Lv/Hb-PDA in facilitating supramolecular self-assembly (Figure S12b). Residual organic solvents from the preparation process were removed by washing, and measurement of the equilibrium swelling ratio before and after washing revealed that Lv/Hb-PDA maintained a similarly high water absorption and swelling performance (Figure S12c). To investigate the mechanism underlying gel formation, we exposed the gel to various chemical disruptors. Among these, only urea was capable of significantly disrupting the gel structure, suggesting that hydrogen bonding plays a dominant role in maintaining the supramolecular network. Specifically, hydrogen bonds between the hydroxyl groups on AP and the catechol moieties on the polydopamine shell appear to serve as the key cross-linking interactions (Figure S13a,b). The resulting Lv/HPAGel demonstrated excellent stability during storage and handling, could be easily injected through standard medical syringes, adhered reliably upon administration, and maintained its gel consistency without disruption by shear forces in aqueous environments (Figure a). Morphological analysis by scanning electron microscopy (SEM) and TEM revealed a lamellar supramolecular architecture with distinct porous cross-linking features, indicative of a well-organized and interconnected internal structure (Figure b). To characterize the mechanical behavior of Lv/HPAGel, rheological measurements were performed. The storage modulus (G’) remained consistently higher than the loss modulus (G’’) across a wide range of angular frequencies (0.1–10 rad/s), confirming the viscoelastic and elastic nature of the gel network (Figure c). Under gradually increasing shear stress, the crossover point of G’ and G’’ occurred at approximately 40% strain, indicating that the gel maintains its structural integrity under moderate mechanical loading and can tolerate physiological forces (Figure d).

4.

Characterization, retention, and degradation properties of Lv/HPAGel. (a) Representative photos demonstrating gelation, injection, adhesion, and retention properties of Lv/HPAGel. (b) Representative SEM (main) and TEM (inset) image of Lv/HPAGel. Scale bar, 5 μm. (c, d) Representative rheological analysis on frequency (c) and strain (d) sweep of Lv/HPAGel. G’, storage modulus; G’’, loss modulus. (e, f) Weight loss (e) and representative photos (f) of Lv/HPAGel incubated with different concentrations of esterase. Data are shown as mean ± s.d. (n = 3). (g) Release profiles of AA from Lv/HPAGel incubated with different concentrations of esterase. Data are shown as mean ± s.d. (n = 3). (h) Representative IVIS imaging of ICG/Hb-PDA or ICG/HPAGel after i.t. injection into orthotopic 4T1 tumor-bearing mice. (i) Quantification of average radiant efficiency of ICG fluorescence over time in tumor-bearing mice after i.t. injection of ICG/Hb-PDA or ICG/HPAGel. Data are shown as mean ± s.d. (n = 3). (j) Representative H&E, Masson and PAS staining images of the skin slices from the mice after Lv/HPAGel-treated for 4 days. Scale bar, 200 μm.

Next, the enzymatic degradation behavior and drug release characteristics of Lv/HPAGel were examined to assess its suitability for intratumoral delivery. When incubated with esterase, a hydrolytic enzyme abundant in the tumor microenvironment, Lv/HPAGel underwent gradual degradation due to cleavage of the ester bonds in AP, resulting in a time-dependent reduction in gel mass (Figure e,f). The release profiles of AA and Lv from the degrading gel were subsequently assessed. Under esterase treatment, AA was continuously released from Lv/HPAGel into the surrounding medium, indicating sustained drug availability over time (Figure g). Furthermore, i.t. injection of Lv/HPAGel into 4T1 tumor-bearing mice demonstrated that gradual enzymatic degradation enabled the continuous release and accumulation of DHA within the tumor tissue, supporting the in vivo relevance of this release mechanism (Figure S13c). To evaluate the in vivo retention of the gel formulation, Lv was replaced with the fluorescent dye indocyanine green (ICG) to construct ICG/Hb-PDA or ICG/HPAGel, and an in vivo imaging system (IVIS) was used to track their distribution in 4T1 tumor-bearing mice. Remarkably, the fluorescence signal of ICG/HPAGel remained detectable at the tumor site for at least 5 days postinjection, whereas the signal from ICG/Hb-PDA rapidly diminished within 24 h, suggesting improved local retention of HPAGel (Figure h,i). This prolonged residence time is critical for maintaining therapeutic concentrations and avoiding premature systemic clearance. The biocompatibility of Lv/HPAGel following subcutaneous injection in healthy mice was further assessed. Histological analysis of skin tissues at the injection site showed no apparent inflammatory infiltration or tissue damage, as evaluated by hematoxylin and eosin (H&E), Masson’s trichrome, and periodic acid–Schiff (PAS) staining (Figure j). Additionally, longitudinal observation revealed that the gel fully degraded within 2 weeks after administration, leaving no visible residue or pathological alterations, underscoring its excellent in vivo biodegradability and safety (Figure S14a). Importantly, direct injection of Hb into mice did not result in significant changes in serum IgG or IgM levels within 1 week, indicating that Hb in the Lv/HPAGel system does not elicit notable immunogenicity (Figure S14b,c).

In Vivo Inhibition of an Orthotopic Breast Tumor Model by Lv/HPAGel

We next evaluated the therapeutic efficacy of Lv/HPAGel in vivo using an orthotopic breast tumor model. Tumor-bearing mice received a single i.t. injection of either Lv/HPAGel, a physical mixture of Lv/Hb-PDA and AA, bare HPAGel, or saline as a control. Tumor growth was monitored over a 16-day period. Compared to the control groups, Lv/HPAGel treatment led to a significant suppression of tumor growth throughout the observation period (Figure a). At the end point, excised tumor weights in the Lv/HPAGel group were reduced to approximately 39.9% of those in the saline-treated group, demonstrating more robust antitumor activity than other treatments (Figure b,c). Throughout the course of treatment, mice in the Lv/HPAGel group exhibited no significant changes in body weight or food intake, indicating good systemic tolerance and minimal toxicity (Figure S15). Histopathological examination of major organs (including liver, kidney, heart, lung, and spleen) via H&E staining revealed no overt lesions or inflammatory responses, further confirming the biocompatibility and safety profile of Lv/HPAGel (Figure S16). To investigate the underlying mechanism of tumor suppression, histological analysis of tumor tissues was conducted. H&E staining of tumor sections showed widespread necrotic areas and reduced tumor cell density in Lv/HPAGel-treated mice compared to control groups, indicating substantial cancer cell elimination (Figure d). Furthermore, immunohistochemical (IHC) staining revealed markedly reduced expression of GPX4, a critical regulator that protects cells from ferroptosis, in Lv/HPAGel-treated tumors (Figure d,e). These results align with in vitro findings and confirm that ferroptosis was effectively induced as the primary mechanism of tumor cell death in vivo.

5.

In vivo therapeutic efficacy of Lv/HPAGel in orthotopic tumor-bearing mouse model. (a) Changes in the tumor volume within 16 days after different treatments. (i) Saline; (ii) Lv/Hb-PDA and AA mixture; (iii) HPAGel; (iv) Lv/HPAGel. Data are shown as mean ± s.d. (n = 6). (b, c) Photo (b) and the weight (c) of the tumors harvested from the mice after different treatments. Data are shown as mean ± s.d. (n = 6). (d) Representative H&E, immunohistochemical (GPX4) and immunofluorescent (CRT and HMGB1) staining images of tumor sections after different treatments. Scale bar, 100 μm. (e, f) Relative GPX4 (e) and CRT (f) expression levels in tumors after different treatments. Data are shown as mean ± s.d. (n = 3). (g, h) Representative flow cytometric analysis (g) and quantification (h) of CD86⁺/F4/80⁺ M1 macrophage cell population in tumors after different treatments. Data are shown as mean ± s.d. (n = 3). (i, j) Representative flow cytometric analysis (i) and quantification (j) of CD80⁺/CD86⁺ mature DC population in lymph nodes after different treatments. Data are shown as mean ± s.d. (n = 3).

To confirm whether Lv/HPAGel treatment induced ICD in vivo, we evaluated the release of DAMPs in tumor tissues by using IF staining. As a hallmark of ICD, CRT exposure on the tumor cell membrane was markedly enhanced in Lv/HPAGel-treated tumors, indicating active translocation from the endoplasmic reticulum to the cell surface (Figure d,f). Concurrently, HMGB1 was substantially depleted from the nucleus, suggesting its release into the extracellular space (Figures d and S17). These findings collectively indicate that Lv/HPAGel-induced ferroptosis is accompanied by DAMP release, a defining feature of ICD. To assess the immunological consequences of ICD, we next evaluated the infiltration and activation of tumor-infiltrating immune cells by flow cytometry. Notably, the proportion of M1-phenotype macrophages (CD86⁺/F4/80⁺) was significantly increased in tumors from Lv/HPAGel-treated mice, rising from 21.1 to 37.1% (Figure g,h). In parallel, the maturation status of DCs within the tumor microenvironment was assessed. Flow cytometric analysis showed a substantial elevation in mature DCs (CD80⁺/CD86⁺), with their proportion increasing from 23.9% in the control group to 44.9% following Lv/HPAGel treatment (Figure i,j). This enhanced DC maturation likely reflects the immunostimulatory effects of ICD-induced DAMP release and underscores the potential of Lv/HPAGel to promote effective antigen presentation, thereby priming the adaptive immune system. These findings support the rationale for combining Lv/HPAGel with immune checkpoint blockade to further boost subsequent T cell-mediated antitumor responses.

Immunotherapy of Lung Metastatic Tumor Model by αPD-1@Lv/HPAGel

ICB therapy using αPD-1 has become a cornerstone of clinical cancer immunotherapy. To investigate whether Lv/HPAGel could enhance the efficacy of ICB, we incorporated αPD-1 into the gel formulation to form αPD-1@Lv/HPAGel. Enzymatic degradation of the gel under esterase-rich conditions enabled gradual and sustained release of αPD-1 into the tumor microenvironment, ensuring prolonged checkpoint inhibition following a single i.t. injection (Figure S18). To evaluate therapeutic efficacy against metastasis, an orthotopic lung metastatic model was established by implanting 4T1 cells into mammary fat pads and intravenously injecting 4T1-Luc cells. A single dose of αPD-1@Lv/HPAGel or other control groups was i.t. injected into the primary tumor. Metastatic progression was subsequently monitored in vivo by bioluminescence imaging (Figure a). Compared to Lv/HPAGel or αPD-1 alone, the combination formulation αPD-1@Lv/HPAGel further reduced both primary tumor growth and tumor weights (Figures b and S19a,b), demonstrating a synergistic effect between ferroptosis-induced ICD and checkpoint blockade. During the 8-day observation period, bioluminescent signals in the lungs were significantly diminished in the αPD-1@Lv/HPAGel group compared with other treatment groups, indicating effective suppression of αPD-1@Lv/HPAGel to metastatic tumor growth (Figure c–e). Moreover, no significant body weight loss was observed in the treated mice, confirming the good tolerability of the combined therapy (Figure S19c).

6.

In vivo immunotherapy efficacy of αPD-1@Lv/HPAGel in the lung metastatic tumor mouse model. (a) Treatment schedule for the immunotherapy study of αPD-1@Lv/HPAGel in the lung metastatic tumor model. (b) Changes in primary tumor volume within 8 days after different treatments. (i) Saline; (ii) αPD-1; (iii) Lv/HPAGel; (iv) αPD-1@Lv/HPAGel. Data are shown as mean ± s.d. (n = 5). (c) Bioluminescent images of the mice after different treatments. (n = 5). (d, e) Changes in the average (d) and individual (e) bioluminescent signals from the lung area of mice within 8 days after different treatments. Data are shown as mean ± s.d. (n = 5). (f) Representative image of harvested lungs after different treatments. (g) Number of lung metastatic nodules after different treatments. Data are shown as mean ± s.d. (n = 5). (h) Representative H&E staining images of the lung sections after different treatments. Scale bar, 100 μm. (i–l) Expression levels of TNF-α (i), IFN-β (j), IL-6 (k), and IL-12 (l) in the primary tumors after different treatments. Data are shown as mean ± s.d. (n = 3). (m–o) Representative flow cytometric analysis (m) and quantification of CD4 T cell (n) and CD8 T cell (o) population in lungs after different treatments. Data are shown as mean ± s.d. (n = 3).

Histological analysis and pathological staining of lung tissues demonstrated a pronounced reduction in the metastatic nodule numbers in mice receiving αPD-1@Lv/HPAGel (Figure f,g). Quantitative assessment confirmed not only a lower incidence but also a significant decrease in the overall area occupied by metastatic foci. Furthermore, H&E staining demonstrated extensive suppression of metastatic tumor cell proliferation and preservation of normal lung architecture in the treatment group (Figure h). These observations substantiate the therapeutic potential of αPD-1@Lv/HPAGel as an effective inhibitor of 4T1-Luc lung metastasis. To investigate the underlying immune mechanisms, the expression of cytokines in primary tumor tissues was analyzed. Treatment with αPD-1@Lv/HPAGel resulted in significant upregulation of proinflammatory cytokines, including TNF-α, IFN-β, IL-6, and IL-12 (Figure i–l), all of which play essential roles in promoting antigen presentation, T cell activation, and cytotoxic responses. In parallel, expression of immunosuppressive cytokines such as TGF-β and IL-10 was markedly reduced, suggesting that the tumor microenvironment was effectively shifted from an immunosuppressive to an immunostimulatory state (Figure S20a,b). The tumor microenvironment was reprogrammed and accompanied by enhanced infiltration of DCs and M1 macrophages, which collectively contribute to improved tumor antigen processing and presentation. In support of this, flow cytometric analysis of pulmonary T cell populations revealed a substantial increase in CD8⁺ T cells, rising from 30.2 to 55.8%, alongside a corresponding decrease in CD4⁺ T cells from 30.8 to 14.7% after treated with αPD-1@Lv/HPAGel (Figure m–o). This selective enrichment of CD8⁺ T cells in metastatic lung lesions suggests a robust cytotoxic immune response directed against disseminated tumor cells. Collectively, these findings demonstrate that local delivery of αPD-1@Lv/HPAGel not only suppresses primary tumor progression but also prevents systemic metastasis by enhancing tumor-specific immune responses.

Immunotherapy of a Postsurgical and Rechallenge Tumor Model by αPD-1@Lv/HPAGel

To further evaluate the long-term immunotherapeutic efficacy of αPD-1@Lv/HPAGel, we established a distant tumor rechallenge model that involved surgical excision of the primary tumors. Following complete excision of the primary tumor, a single dose of αPD-1@Lv/HPAGel or other control groups was i.t. injected into the surgical cavity. This local postoperative administration was intended to eliminate residual tumor cells and initiate in situ immune activation. Six days later, 4T1 cells were implanted into the opposite mammary fat pad to mimic distant recurrence, and tumor growth at both sites was monitored over 20 days (Figure a). The results demonstrated that local injection of αPD-1@Lv/HPAGel significantly suppressed recurrence at the surgical site compared to either monotherapy. Tumor regrowth was markedly delayed, and the growth rate was substantially reduced in the αPD-1@Lv/HPAGel group (Figure b,c). On day 20, the average tumor weights of this group were reduced to approximately 46.5% of those observed in saline-treated controls, indicating effective prevention of local recurrence (Figure S21a,b). In the distant rechallenge model, only 40% of mice treated with αPD-1@Lv/HPAGel developed secondary tumors, while the majority remained tumor-free throughout the observation period. Even in mice that did develop tumors, the growth rate was significantly slower compared to that of the control and monotherapy groups, suggesting partial immune-mediated suppression of tumor establishment at distant sites (Figure d,e). Together, these results demonstrate that αPD-1@Lv/HPAGel confers robust systemic antitumor immunity following primary tumor resection, effectively preventing both local recurrence and secondary tumor formation (Figures f,g and S21c). Importantly, mice treated with αPD-1@Lv/HPAGel exhibited stable body weights throughout the experimental period, and no signs of systemic toxicity were observed (Figure S21d). Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) remained within normal ranges, confirming the favorable safety and biocompatibility profile of αPD-1@Lv/HPAGel (Figure S22a,b).

7.

In vivo immunotherapy efficacy of αPD-1@Lv/HPAGel in postsurgical and rechallenging tumor mouse model. (a) Treatment schedule for the immunotherapy study of αPD-1@Lv/HPAGel in postsurgical and rechallenging tumor models. (b, c) Changes in the average (b) and individual (c) primary tumor volume within 20 days after different treatments. (i) Saline; (ii) αPD-1; (iii) Lv/HPAGel; (iv) αPD-1@Lv/HPAGel. Data are shown as mean ± s.d. (n = 5). (d, e) Changes in the average (d) and individual (e) distant tumor volume within 20 days after different treatments. Data are shown as mean ± s.d. (n = 5). (f) Distant tumor formation rate in the mice after different treatments (tumor formation was defined as a palpable tumor volume >10 mm³, n = 5). (g) Photos of the distant tumors harvested from the mice after different treatments. (h–k) Representative flow cytometric analysis (h,i) and quantification (j, k) of CD3⁺/CD4⁺ and CD3^+/CD8⁺ T cell populations in lymph nodes after different treatments. Data are shown as mean ± s.d. (n = 3). (l) Representative immunofluorescent (Granzyme B and FOXP3) and immunohistochemical (Ki67) staining images of the distant tumor sections after different treatments. Scale bar, 50 μm. (m–o) Representative flow cytometric analysis (m) and quantification of CD44⁺/CD62L⁺ central memory T cell (n) and CD44⁺/CD62L^– effector memory T cell (o) population in spleens after different treatments. Data are shown as mean ± s.d. (n = 3).

Post-treatment, spleens and lymph nodes were collected to analyze T cell populations by flow cytometry. In the spleen, αPD-1@Lv/HPAGel treatment significantly elevated the proportions of CD3⁺/CD4⁺ helper T cells (from 3.5 to 9.6%) and CD3⁺/CD8⁺ cytotoxic T cells (from 1.4 to 3.4%), indicating systemic immune activation and effector T cell expansion (Figures S23a,b and S24a,b). Even more notably, the flow cytometric analysis of lymph nodes revealed substantial T cell accumulation. CD3⁺/CD4⁺ T cells increased from 15.4 to 39.1%, and CD3⁺/CD8⁺ T cells from 5.6 to 12.8% in the αPD-1@Lv/HPAGel group, suggesting that these lymphoid tissues served as active sites of antigen-specific T cell priming and clonal expansion (Figure h–k). This observation supports the notion that local ferroptosis-induced ICD, in combination with a checkpoint blockade, initiated robust adaptive immune responses at both local and systemic levels. IF staining of primary tumor sections further demonstrated prominent infiltration of CD8⁺ T cells into the surgical bed following αPD-1@Lv/HPAGel treatment, indicating that primed effector T cells had successfully migrated to and persisted within the former tumor site, where they likely contributed to long-term immune surveillance and recurrence prevention (Figure S25a,b). Overall, these findings underscore the synergistic interplay between ferroptosis-induced ICD and the immune checkpoint blockade. The αPD-1@Lv/HPAGel system not only induces tumor cell ferroptosis but also enhances antigen presentation and T cell activation. This dual mechanism effectively remodels the tumor immune microenvironment, leading to robust and durable systemic antitumor immunity.

The proliferation and immune status of the distant rechallenge tumors were next evaluated to assess whether systemic immune protection had been established. IF staining showed markedly elevated expression of granzyme B, a cytotoxic effector molecule secreted by activated CD8⁺ T cells, and a simultaneous reduction in FOXP3 expression, a marker of regulatory T cells (Tregs) (Figures l and S26a,b). These findings suggest that αPD-1@Lv/HPAGel not only enhances effector T cell function at distant tumor sites but also reverses local immunosuppression, thereby reinforcing systemic immune control. IHC staining of Ki67 revealed that, even in mice that developed distant tumors following αPD-1@Lv/HPAGel treatment, the proliferation of cancer cells was significantly suppressed compared with control groups (Figure l). Finally, immunological memory development was assessed by analyzing memory T cell subsets in the spleen. Flow cytometry revealed a significant increase in the number of central memory T cells (CD44⁺/CD62L⁺), rising from 13.1% in controls to 26.8% in the αPD-1@Lv/HPAGel group. In contrast, effector memory T cells (CD44⁺/CD62L^–) showed a modest increase from 8.2 to 15.4% (Figure m–o). These results suggest that the immunotherapeutic effects of αPD-1@Lv/HPAGel extended beyond immediate tumor control by promoting the formation of a long-lived memory T cell pool, capable of mediating rapid and durable responses upon tumor re-exposure.

Conclusions

In this study, we developed a supramolecular gel system, αPD-1@Lv/HPAGel, which effectively enhanced the antitumor efficacy of AA by incorporating its derivative AP, an oxygen-supplying reservoir Lv/Hb-PDA, and an ICB antibody αPD-1. Overall, this gel system simultaneously addresses two multifaceted challenges associated with AA-based cancer therapy. The first is the rapid clearance and limited tumor accumulation associated with systemic administration of AA. The second is the insufficient oxygen supply characteristic of hypoxic tumor microenvironments, which hinders the oxidation of AA to its biologically active form, DHA. Through sustained local release of AA, the gel maintains elevated local concentrations of AA within the tumor tissue. Furthermore, oxygen provided by Lv/Hb-PDA effectively promotes the oxidation of AA to DHA, thereby enhancing intracellular uptake and initiating ferroptosis-related signaling pathways. Within cancer cells, DHA and Lv collaboratively induce LPO and ferroptosis via mechanisms involving GSH depletion, ROS generation, and GPX4 inhibition. Importantly, this strategy also induces ICD, characterized by extensive release of various DAMPs, leading to enhanced immune system activation, DC maturation, and remodeling of the tumor microenvironment. When combined with ICB mediated by αPD-1, this integrated approach significantly suppressed tumor progression, facilitated the establishment of durable immune memory, and markedly reduced tumor metastasis and postsurgical recurrence.

By effectively coupling ferroptosis induction with immune system activation, this gel system directly overcomes the immunosuppressive nature of the tumor microenvironment, thereby sustaining immune responses following tumor resection. This dual actioninducing tumor cell death while simultaneously enhancing immunogenicityenables prolonged antitumor immunity beyond local treatment. The gel promotes extensive infiltration of immune cells into the tumor and significantly increases the likelihood of a robust systemic antitumor immune response. These findings further indicate that this strategy may enhance the efficacy of immunotherapy, particularly in tumors refractory to conventional monotherapies. Additionally, our approach may be particularly beneficial in converting “cold” tumors into immunologically “hot” tumors, thereby rendering them susceptible to immune recognition and elimination. By improving tumor antigen presentation and relieving immune suppression, the gel system supports reprogramming of the tumor immune landscape. In this manner, the shortcomings of some cancer immunotherapy paradigms can be overcome. Nevertheless, several limitations remain. For example, the preclinical efficacy demonstrated here requires further validation in larger animal models, which would more accurately reflect the safety and efficacy within more complex biological contexts. Additionally, a comprehensive understanding of tumor-type-specific responses to combined ferroptosis induction and ICB therapy will be essential to optimize this approach across diverse malignancies.

In conclusion, the αPD-1@Lv/HPAGel described herein holds significant translational potential in the context of clinical immunotherapy, effectively addressing critical limitations inherent in conventional cancer treatments. This strategy significantly reduces systemic side effects, thereby rendering it particularly well-suited for integration into combination therapies within clinical settings. Furthermore, by enhancing tumor immunogenicity and remodeling the tumor immune microenvironment, this approach helps to overcome tumor-mediated immune evasion. Future studies would benefit from combining this approach with systemic immune-enhancing strategies, such as cancer vaccines or cytokine-based therapies, potentially yielding even greater therapeutic outcomes. With continued optimization and clinical validation, the therapeutic synergy achieved through ferroptosis induction and immune activation described in this work has the potential to reshape current cancer treatment paradigms, ultimately contributing to more effective and long-lasting antitumor responses. Future studies should also explore potential biomarkers that predict treatment responsiveness, which may help guide patient stratification and clinical translation.

Methods

Preparation and Characterization of Lv/Hb-PDA

Hb (3.0 mg) and dopamine (0.6 mg) were dissolved in Tris buffer (7.2 mL, pH 8.5), followed by the dropwise addition of Lv solution (0.4 mg dissolved in 0.8 mL of DMSO). The mixture was stirred for 3 h at room temperature, and the resulting suspension was then washed using an ultrafiltration centrifugal tube (50 kDa cutoff) and resuspended in 1 mL of PBS (pH 7.4). The mHb in Lv/Hb-PDA was reduced by sodium dithionite (0.4 mg/mL), and oxygen was loaded by continuous bubbling. The hydrodynamic diameter and ζ potential of nanoparticles were measured using dynamic light scattering (Malvern Panalytical, Zetasizer Pro). The morphology of Lv/Hb-PDA was examined by TEM (Thermo Fisher, TF-G20). For FTIR detection, Hb, Hb-PDA, and Lv/Hb-PDA were lyophilized overnight, pressed into pellets with KBr, and analyzed by using an infrared spectrometer (Agilent, Cary 630). To evaluate Lv release behavior, 1 mL of Lv/Hb-PDA was placed in a dialysis bag and dialyzed against 20 mL of buffer with different pH containing 0.5% SDS at 37 °C. At predetermined time points, aliquots of the dialysate were collected and replaced with an equal volume of fresh buffer. Lv concentration in dialysate was quantified by a high-performance liquid chromatograph (HPLC) system (Shimadzu, LC-40) with an ODS-SP column. The mobile phase consisted of acetonitrile/KH₂PO₄ (0.05M, pH 7.0) mixture (55.5/44.5, v/v). The detection wavelength was set at 238 nm, and the flow rate was maintained at 1 mL/min.

Detection of Oxygen Release and AA Oxidation

The secondary structure of Hb proteins or Hb-PDA was characterized by using CD spectrophotometry (JASCO, J-1500). Ultraviolet–visible (UV) absorption of Hb or Hb-PDA was performed by using UV spectrophotometry (Shimadzu, UV-2600). For oxygen release detection, Hb or Hb-PDA was injected into deoxygenated PBS, which was prepurged with nitrogen in an airtight flask. The oxygen concentration in the medium was monitored by using a dissolved oxygen meter (Leici, JPBJ-608). For AA oxidation detection, Hb-PDA or Hb was added to deoxygenated PBS and incubated with AA for 1 h. The supernatant was collected, and the residual AA concentration was measured by HPLC. The mobile phase consisted of methanol/KH₂PO₄ (5 mM, pH 2.65) mixture. The chromatographic program employed a linear gradient, starting at 5% methanol, increasing to 22% over 6 min and returning to the initial conditions over the following 9 min. The detection wavelength was set at 245 nm, and the flow rate was 0.6 mL/min. For the quantification of DHA, dithiothreitol (DTT) was added to the supernatant to reduce the oxidized AA. The DHA concentration was calculated by subtracting the remaining AA concentration from the total AA concentration after reduction. H₂O₂ levels in the supernatant were measured using a commercial H₂O₂ assay kit, following the manufacturer’s instructions.

Determination of In Vitro Ferroptosis

4T1 cells (4 × 10⁵ cells/well) were seeded in 6-well plates and incubated with Hb-PDA or Lv/Hb-PDA (100 μg/mL of Hb), with or without AA (200 μg/mL) for 24 h. After treatment, the expression level of ferroptosis-related protein was detected by Western blot assay. Briefly, cells were lysed using RIPA lysis buffer, and the protein concentration was quantified using a BCA protein quantification kit. Equal amounts of protein were separated by electrophoresis on 15% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked and incubated overnight at 4 °C with primary antibodies, including anti-β-actin, anti-GPX4, or anti-HMGCR. Subsequently, membranes were incubated with HRP-conjugated secondary antibody for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence detection method and imaged by a chemiluminescent imaging system (Azure Biosystems, Azure 400). Band intensities were quantitatively analyzed by ImageJ software. For lipid peroxide analysis, 4T1 cells were incubated with Hb-PDA or Lv/Hb-PDA (100 μg/mL of Hb), with or without AA (200 μg/mL) for 12 h. Then, the cells were stained with BODIPY581/591-C11 probe for 30 min and Hoechst 33342 for 15 min. Cells were washed with PBS and observed under a confocal laser scanning microscope (CLSM) (OLYMPUS, FV3000) to assess the presence of oxidized or nonoxidized lipids. For tracking the live/dead ratio, treated cells were stained with a calcein-AM/PI assay kit according to the manufacturer’s protocol and observed using CLSM. Oxidized lipid levels and live/dead cell ratio were quantitatively analyzed by ImageJ software. For determining the ferroptosis-associated cell viability, treated cells were incubated with CCK-8 reagent for 3 h. Absorbances at 450 nm were measured by a multifunctional microplate reader (Agilent, Synergy H1), and cell viabilities were then calculated.

Preparation and Characterization of αPD-1@Lv/HPAGel

Lv/Hb-PDA (800 μL, 2 mg/mL Hb) and αPD-1 (72 μL, 18.5 mg/mL) mixture was added dropwise into AP solution (40 mg dissolved in 200 μL of DMSO) under continuous vortexing. The resulting suspension was heated at 70 °C for 5 min and cooled to room temperature to induce gelation of αPD-1@Lv/HPAGel. The formed gel was washed with PBS five times to remove DMSO. The equilibrium swelling ratio (ESR) of the gel was calculated using ESR (%) = (m_t – m ₀) m ₀ × 100%, where m ₀ is the weight of the lyophilized gel and m_t is the weight of the PBS-swollen gel at the corresponding time point. For microstructural observation, the PBS-swollen gel was frozen with liquid nitrogen for SEM imaging (Thermo Fisher, Quanta 250), while a portion of the gel was cast onto a copper grid and negatively stained for TEM analysis (FEI, Tecnai G2 F20). To investigate the gelation mechanism, 5 mL of HCl solution (pH 2), EDTA·2Na (200 mM), NaCl (200 mM), urea (200 mM), or SDS (200 mM) were added into 1 mL of Lv/HPAGel, and their effects on gel stability were observed. The rheological properties of the gel were evaluated using a rheometer (Thermo Scientific, Haake Mars 40). For frequency sweep measurements, the Lv/HPAGel was tested over a range of angular frequencies at a constant shear strain. Dynamic oscillatory tests were also conducted over a strain range of 0.1 to 1000%. Throughout the gelation process, changes in G’ and G’’ were recorded to evaluate the viscoelastic behavior and cross-linking dynamics.

Evaluation of In Vivo Antitumor Efficacy in an Orthotopic Breast Cancer Model

To establish orthotopic breast cancer models, 1 × 10⁶ 4T1 cells suspended in PBS were mixed with Matrigel at a 3:1 ratio, and the mixture was injected into the mammary fat pad of female mice. When the tumor volume reached nearly 0.1 cm³, mice were randomly divided into four groups, and i.t. injected with saline, a mixture of Lv/Hb-PDA and AA, HPAGel, or Lv/HPAGel, respectively. The administered dosages of Lv and AA were 2.1 and 127.5 mg/kg. Body weight and tumor volume were recorded every 2 days. When the tumor volume in the saline group reached nearly 1.2 cm³, the mice were sacrificed. Different organs and tissues, including heart, liver, spleen, lung, kidney, lymph node and tumor, were harvested, and the weight of tumors was measured. Tissues were fixed, embedded in paraffin, sectioned, and subjected to histopathological analysis using H&E staining. For IHC detection, tumor sections were incubated at 4 °C with anti-GPX4, followed by incubation at room temperature with HRP labeled antibody, and visualized using DAB agent. For IF detection, tumor sections were incubated with primary antibodies, including anti-HMGB1 and anti-CRT, at 4 °C, followed by staining with a Cy3-labeled secondary antibody. All stained sections were imaged using a slide scanner (Olympus, VS200), and the expression levels of proteins were quantified using ImageJ software. To assess tumor-infiltrating immune cells, tumors were cut into small pieces and digested with collagenase IV and hyaluronidase. The digested tissues were filtered through 200-mesh membrane filters to obtain single-cell suspensions. The cells were stained with CD11c-FITC, CD80-APC, and CD86-PE antibodies for mature DC detection, and with CD45-APC/Cy7, F4/80-FITC, and CD86-PE antibodies for M1 macrophage detection, following the manufacturer’s protocol. All cell samples were measured using a flow cytometer and analyzed by FlowJo software.

Evaluation of In Vivo Immunotherapy Efficacy in a Metastatic Breast Cancer Model

An orthotopic breast cancer model was established by injecting 4T1 cells into the mammary fat pad of mice following the method described above. On the following day, mice were intravenously injected with 8 × 10⁵ 4T1-Luc cells to induce lung metastasis. To monitor and compare the metastatic burden in the lungs, bioluminescence imaging was performed by using IVIS (PerkinElmer, Lumina III). Mice were intraperitoneally injected with D-Luciferin potassium salt, and imaging was conducted 15 min postinjection. Mice were randomly divided into four groups, and i.t. injected with saline, αPD-1, Lv/HPAGel, or αPD-1@Lv/HPAGel, respectively. The administered dosages of αPD-1, Lv, and AA were 3.3, 0.7, and 42.5 mg/kg. Throughout the treatment period, cancer metastasis progression, primary tumor volume, and body weight were recorded regularly. After treatment, mice were euthanized to collect lung and tumor tissues. Lungs were fixed overnight in Bouin’s fixative solution, photographed, and the number of visible metastatic nodules was recorded. The lungs were then paraffin-embedded, sectioned, and subjected to H&E staining. The sections were imaged using a slide scanner. Tumors were homogenized and centrifuged at 4 °C. The resulting supernatants were collected for further analysis. Total protein concentrations were quantified using a BCA kit to ensure normalization. The expression levels of IL-6, IL-12, TNF-α, IFN-β, IL-10, and TGF-β were quantified using ELISA kits in accordance with the manufacturer’s instructions. To analyze T cell subpopulations, lung tissues were digested with collagenase IV (1 mg/mL) and filtered through 200-mesh nylon membranes. Red blood cells were lysed using red blood cell lysis buffer. The resulting single-cell suspensions were stained with CD45-APC/Cy7, CD3-FITC, CD8a-PB450, and CD4-APC antibodies, following the manufacturer’s protocol. All samples were measured using a flow cytometer and analyzed by FlowJo software.

Evaluation of In Vivo Immunotherapy Efficacy in a Postsurgical and Rechallenge Breast Cancer Model

An orthotopic breast cancer model was established by injecting 4T1 cells into the mammary fat pad of mice, following the method described above. When the tumor volume reached nearly 0.1 cm³, 90% of the tumor mass was surgically resected to establish the recurrence model. The mice were randomly divided into four groups. The next day after surgery, mice were i.t. injected with saline, αPD-1, Lv/HPAGel, and αPD-1@Lv/HPAGel, respectively. The administered dosages of αPD-1, Lv, and AA were, respectively, 10, 2.1, and 127.5 mg/kg. Five days after treatment, 8 × 10⁵ 4T1 cells were implanted into the distal mammary fat pad of each mouse. When the primary tumor volume in the saline group reached nearly 1.2 cm³, the mice were euthanized. Spleens, lymph nodes, and tumors were harvested, and blood samples were collected and examined using the AST assay kit and the ALT assay kit. The expression of Ki67 in the distant tumor was examined using the IHC assay according to the manufacturer’s protocols. The expression levels of granzyme B and FOXP3 in the distant tumor were examined using IF assay according to the manufacturer’s protocols. The tissue sections were observed using a slide scanner, and fluorescence intensities were quantified using ImageJ software. To analyze T cell subpopulations, as well as memory T cells, spleen and lymph nodes were filtered through a 200-mesh membrane filter to obtain single-cell suspensions. T cell subpopulations were stained with CD45-PE, CD3-FITC, CD4-APC, and CD8a-PE/Cy7 antibodies, while central memory T cells and effector memory T cells were identified using CD45-APC/Cy7, CD3-FITC, CD8a-PB450, CD44-APC, and CD62L-PE antibodies according to the manufacturer’s protocol. All cell samples were measured using a flow cytometer and analyzed by FlowJo software.

Statistical Analysis

Statistical data were analyzed by using GraphPad Prism 9.0 software. Quantitative data are presented as mean ± s.d., and significant differences between groups were analyzed using one-way analysis of variance (ANOVA) with a Tukey post hoc test. P value lower than 0.05 was deemed as statistical significance.

The authors declare that all data related to this research are available within the article and the Supporting Information. Any additional requests for information can be directed to the corresponding authors.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.5c08353.

• Supporting methods; characterization analysis of Lv/Hb-PDA; in vitro AA oxidation; intracellular GSH depletion, HMGCR and GPX4 expression; cell viability and live/dead detection; characterization and biocompatibility of Lv/HPAGel; H&E staining analysis of major organs; expression of inflammatory factors in tumors; plasma biochemical indicator level; assessment of T cells in spleen; assessment of T cells in tumor; assessment of cytotoxic T cells and regulatory T cells in tumor; and gating strategy in flow cytometry detection (PDF)

W.C. and S.S. conceived the study and designed the experiments. W.C., Y.S., W.X., X.K., Z.Z., X.C. and S.S. developed the methodology and participated in investigations. W.C., Y.H., J.X., Y.L. and S.S. analyzed the results. W.C. and S.S. wrote the draft. Y. H. and S.S. received funding and supervised the project. All authors read, corrected and approved the manuscript.

The authors declare no competing financial interest.