Mesoporous polydopamine nanoplatform enhances IL-2 immunotherapy for hepatocellular carcinoma via mild photothermal therapy and lactate regulation

Abstract

Hepatocellular carcinoma (HCC) is commonly diagnosed at intermediate or advanced stages. While transcatheter arterial embolization (TAE) is the accepted locoregional therapy, embolization inevitably induces severe intratumoral hypoxia, leading to excessive lactate accumulation, metabolic reprogramming and the formation of an immunosuppressive tumor microenvironment (TME), factors that collectively limit durable therapeutic efficacy and promote tumor recurrence. Although immunotherapy has shown promise in HCC, the efficacy of combining TAE with immune-based strategies remains suboptimal, largely due to metabolic constraints and T cell dysfunction within the post-embolization TME. Interleukin-2 (IL-2) therapy can stimulate effector T cells but suffers from the drawbacks of systemic toxicity, a short half-life and TME-induced dysfunction. Recent evidence indicates that lactate-rich, acidic TMEs impair IL-2 stability and signaling, suppress CD8⁺ T cell function, and promote Treg cell activity, thereby further compromising IL-2-based immunotherapy following TAE. Here, we developed a nano-immunotherapy platform (Syr/IL-2@MPDA) using mesoporous polydopamine (MPDA) nanoparticles co-loaded with His-tagged IL-2 and the monocarboxylate transporter inhibitor Syrosingopine (Syr). Syr-mediated lactate regulation alleviates TME acidity and restores IL-2 activity, while MPDA enables mild photothermal therapy (mPTT) to transform immunologically “cold” tumors into “hot” phenotypes, thereby enhancing the immune response. This integrated strategy significantly enhanced antitumor immunity, achieving a tumor growth inhibition rate of 70.87% in a murine subcutaneous tumor model. In an orthotopic N1S1 HCC rat model, the treatment demonstrated strong therapeutic efficacy in combination with αPD-L1 and TAE, overcoming the hazards of TAE-induced lactate accumulation and immune suppression. Overall, this study demonstrates a safe and effective strategy that enhances IL-2 immunotherapy through mPTT and metabolic modulation, offering a promising combination treatment for advanced HCC.

Graphical abstract

MPDA contains IL-2 and Syr, which served as T cell growth factor and MCT inhibitor, respectively. Upon laser exposure, MPDA generated mPTT to stimulate DC maturation and activate further T cell mediated anticancer immunity. Syr inhibited lactate excretion thereby promoting IL-2 mediated T cell activation and proliferation.

1. Introduction

Hepatocellular carcinoma (HCC) is one of the most malignant tumors, at present ranking as the second leading cause of cancer-related deaths globally [[1], [2], [3]]. Owing to its insidious onset and rapid progression, the majority of HCC patients are diagnosed at intermediate or advanced stages, when curative treatments such as surgical resection or ablation are no longer effective [4]. In this clinical context, transcatheter arterial embolization (TAE), including conventional TAE and related embolization-based strategies, has been widely adopted for unresectable or advanced HCC.

Despite its clinical utility, the therapeutic efficacy of TAE is frequently compromised by embolization-induced hypoxia that promotes the accumulation of lactic acid, metabolic reprogramming, and the creation of an immunosuppressive tumor microenvironment (TME), ultimately leading to tumor recurrence and treatment resistance [5,6]. To address these limitations, cancer immunotherapy has emerged as a promising therapeutic paradigm, capable of inducing lasting antitumor responses by reactivating the host immune system [7]. Recent clinical studies have demonstrated that combining arterial embolization with immune checkpoint blockade (ICB) or other immunotherapeutic approaches can achieve synergistic antitumor effects in HCC patients [[7], [8], [9], [10]]. However, the overall clinical benefit of embolization-immunotherapy combinations remains suboptimal. Accumulating evidence indicates that embolization-induced hypoxia, excessive lactate production, and impaired immune cell function within the TME limit T cell infiltration, activation and persistence, thereby diminishing the efficacy of immunotherapy [[11], [12], [13]]. These challenges highlight an urgent need for therapeutic strategies that not only integrate locoregional embolization with immunotherapy but also actively remodel the hostile metabolic and immunological microenvironment following TAE.

Interleukin-2 (IL-2) is a classical pleiotropic cytokine that promotes T cell activation and proliferation and has been used in cancer immunotherapy for decades [14,15]. Since IL-2 activates both cytotoxic CD8⁺ T cells and regulatory T (Treg) cells, and the latter have higher affinity, high doses of IL-2 are required to achieve anti-cancer effects; however, this may cause severe cytotoxic effects in patients [16,17]. Moreover, the short half-life of IL-2 limits its immunotherapeutic effects [18]. To address these limitations, Zhang et al. developed a surface-anchored immunoliposome to achieve accumulation of IL-2 and anti-CD137 in tumors with reduced systemic toxicity [19]. Fu et al. synthesized an IL-2 prodrug that localized and cleaved within tumors, thereby reducing the toxicity of IL-2 and enhancing its antitumor effect [20]. Pu et al. developed a sonodynamic IL-2 nanocomplex that specifically activates cytotoxic CD8⁺ T cells, thereby enhancing antitumor immunotherapy [21]. These studies demonstrate that localized or intratumoral IL-2 delivery represents a more clinically translatable strategy than systemic administration, as it enables high cytokine concentrations while minimizing off-target immune activation and systemic toxicity. Nevertheless, effector T cell dysfunction and exhaustion within the TME remain major barriers to effective IL-2-based immunotherapy. Recent studies have indicated that excessive lactate in the TME is a key factor in IL-2 therapy, for several reasons: (1) excessive lactate accumulation leads to an acidic microenvironment that inactivates IL-2 and disrupts IL-2 signaling [22]; (2) lactate in TME impairs CD8⁺ T cell function but supports Treg cells [[23], [24], [25]]. Thus, we hypothesized that regulating lactate in the TME could confer advantages by enhancing IL-2 and CD8⁺ T cell function.

Photothermal therapy (PTT), which converts light energy into heat to achieve precise anti-cancer effects, has emerged as a novel approach in cancer treatment with the advantages of non-invasiveness and controllable irradiation [26]. However, direct tumor killing by high temperatures (>50 °C) during PTT can destroy healthy tissues and cause intolerance in patients, thus limiting its practical application [27]. Therefore, increasing attention is being focused on mild PTT (mPTT) using fever-like temperatures of 40∼43 °C as a potential means of modulating the immunosuppressive environment, and thus can be utilized as an adjunct to immunotherapy [[28], [29], [30]]. mesoporous polydopamine (MPDA) is an excellent PTT agent and drug delivery vehicle with high biocompatibility and photothermal conversion efficiency [31]. The functional groups indole and amine of PDA can chelate with various metal ions, including Cu²⁺, Ni²⁺, Mn²⁺, Gd³⁺ and Fe³⁺, and the chelation with Ni²⁺ enables MPDA to reversibly adsorb a His-tagged label [32,33].

In this study, we devised and fabricated an mPTT-enhanced immune agent based on Ni-MPDA nanoparticles (NPs) loaded with His-tagged IL-2 and Syrosingopine (Syr), with the aim of using MPDA NPs to amplify the anti-tumor immune response and achieve optimal treatment for HCC. MPDA NPs not only served as a drug delivery vehicle but also as a photothermal agent, generating mPTT to turn “cold” tumors “hot”, thereby facilitating immunotherapy. Furthermore, Syr, a dual MCT1 and MCT4 inhibitor [34], can reduce lactate levels in the TME, thereby releasing IL-2 from an acidic environment, reversing T cell exhaustion, and restoring an immunologically active environment. In summary, this nano-immune agent exhibits a synergistic effect of mPTT and lactate regulation that boosts effector T cell activation by IL-2, and combines ICB therapy and TAE for powerful HCC treatment (Scheme 1).

Scheme 1.

MPDA contains IL-2 and Syr that serve as a T cell growth factor and an MCT inhibitor, respectively. Upon laser exposure, MPDA generates mPTT to stimulate DC maturation and activate further T cell-mediated anticancer immunity. Syr inhibits lactate excretion, thereby promoting IL-2-mediated T cell activation and proliferation.

2. Materials and methods

2.1. Materials

Syr and IL-2 (mouse, His, HY-P70646AF) were purchased from MedChemExpress. NH₃·H₂O (25%–28%), ethanol, and 1,3,5-trimethy1 benzene (TMB) were purchased from Shanghai Sinopharm Group. Pluronic F-127 (F127) and Pluronic P-123 (P123) were purchased from Sigma-Aldrich. Dopamine hydrochloride (DA·HCl) and NiCl₂·6H₂O were purchased from Aladdin. MPEG-NH₂ (5000 Da) was purchased from ToYongBio. Mouse IL-2 (KE10126, 12.5–800 pg/ml) and mouse IFN-γ (KE10094, 7.8–500 pg/ml) ELISA Kits were purchased from Proteintech. CFSE Cell Proliferation Tracker Kits were purchased from Solarbio. All reagents and solvents employed in this study were of analytical or chromatographic purity.

2.2. Cell lines and animals

Hepa 1–6 cells were obtained from the Cell Bank of the Shanghai Institutes for Biological Sciences (Chinese Academy of Sciences) and cultured in DMEM (Gibco, USA) with 10% (v/v) FBS and 100 U/ml Pen-Strep (Gibco, USA). The cells were cultured in a humidified incubator at 37 °C with 5% CO₂. N1S1 cells were purchased from ATCC (USA) and grown in IMDM (Gibco, USA) containing 10% (v/v) FBS and 100 U/mL Pen-Strep under the same incubation conditions (37 °C, 5% CO₂).

Female C57BL/6 mice (18–22 g) and male Sprague-Dawley (SD) rats (250–300 g) were supplied by Slack Laboratory Animal Co., Ltd. (China). All animal procedures involving animals were approved by Lishui University (Approval No. 2024YD0054).

2.3. Synthesis and Ni²⁺ loading of Ni²⁺-MPDA

MPDA was synthesized following a previously described procedure [26]. Briefly, a mixture containing 75 mg F127, 30 mg P123, and 150 mg DA·HCl was prepared by dissolving the components in 10 ml of 40% (v/v) ethanol. Then, 0.4 ml TMB was incorporated, and the mixture was sonicated to produce a well-dispersed emulsion. Finally, 0.4 ml aqueous ammonia (25%–28%) was added, and the reaction was maintained under vigorous agitation for 4 h. The resulting MPDA products were collected and washed with deionized water and ethanol. To prepare Ni²⁺-MPDA, 50 mg mPEG-NH₂ and 40 mg NiCl₂·6H₂O were added to 10 ml MPDA solution (1 mg/ml), and the mixture was stirred for 24 h. Ni²⁺-MPDA was collected by centrifugation and subsequently washed with deionized water and ethanol. The Ni²⁺ content in MPDA was measured by ICP-MS (iCAP RQ, Thermo Fisher). Briefly, Ni²⁺-MPDA was digested in a microwave system (M6, PreeKem) and treated with concentrated HNO₃ and H₂O₂ to ensure complete oxidation of the Ni²⁺-MPDA. After digestion, the excess acid was evaporated by heating at ∼100 °C. Then, the solutions were diluted with ultrapure water. The concentrations of metal elements were quantified by ICP-MS using external standard calibration, with procedural blanks included for background correction.

2.4. Preparation of Syr/il-2@MPDA

The as-prepared Ni²⁺-MPDA (1 mg/ml) was mixed with Syr dichloromethane solution (0.2 mg/ml) and stirred at room temperature overnight. The Syr@MPDA was obtained after centrifugation and subsequent washing with deionized water and ethanol for subsequent use. Subsequently, 100 µL His-tagged IL-2 (10 µg/ml) was added to a tube with 1 mg Ni²⁺-MPDA or Syr@MPDA. IL-2@MPDA and Syr/IL-2@MPDA were obtained after 1 h co-incubation at 4 °C.

2.5. Characterization of Syr/il-2@MPDA

The particle size, distribution and zeta potential of Syr/IL-2@MPDA were determined using dynamic light scattering (DLS) analysis (Zetasizer 3000HS, Malvern, UK). A transmission electron microscope (TEM, JEM-1200EX, JEOL, Japan) was employed to observe cell morphology. The UV–vis spectra of Syr/IL-2@MPDA were recorded using a UV1900i spectrophotometer (UV1900i, Shimadzu, Japan).

2.6. Preparation and release of Syr/il-2@MPDA in vitro

The encapsulation efficiency (EE) and drug loading (DL) of Syr/IL-2@MPDA were measured by HPLC and ELISA kits. The EE and DL were calculated based on Eqs. (1) and (2).

$$\text{EE}(\%)=\frac{\text{Mass}\text{of}\text{drug}\text{encapulated}\text{in}\text{MPDA}}{\text{Mass}\text{of}\text{drug}\text{added}}\times 100\%$$ (1)

$$\text{DL}(\%)=\frac{\text{Mass}\text{of}\text{drug}\text{encapulated}\text{in}\text{MPDA}}{\text{Mass}\text{of}\text{drug}\text{loaded}\text{in}\text{MPDA}}\times 100\%$$ (2)

The release profiles of Syr from Syr/IL-2@MPDA were obtained as follows. First, 1 mg Syr/IL-2@MPDA was dispersed in 10 ml PBS, then incubated on an orbital shaker at 200 rpm and 37 °C. At a determined time point, 5 ml of the medium was collected, and an equal volume of fresh PBS solution was added. In addition, to verify the effect of the laser on drug release, Syr/IL-2@MPDA was treated with 808 nm laser (0.5 W/cm²) for 10 min after 24 h of culture. The concentration of Syr was quantified by HPLC using a C₁₈ column (4.6 µm, 25 cm) with a mobile phase of acetonitrile/water containing 0.1% KH₂PO₄ (70:30, v/v), a flow rate of 1.0 ml/min, a column temperature of 30 °C, an injection volume of 10 µL, and UV detection at 259 nm. His-tagged IL-2 was quantified by using an ELISA kit. The cumulative release of Syr and His-tagged IL-2 was calculated using the Eq. (3):

$$\text{Drug}\text{release}(\%)=\frac{\text{Mass}\text{of}\text{the}\text{released}\text{drugs}}{\text{Mass}\text{of}\text{the}\text{encapulated}\text{drugs}}\times 100\%$$ (3)

2.7. Photothermal performance in vitro

To evaluate the photothermal ability of Syr/IL-2@MPDA, 1 ml of the sample with different Syr/IL-2@MPDA concentrations was placed in a 10 mm cuvette and exposed to laser light (2.0 W/cm²) for 10 min. The laser system (B0T808-3K-TEC-D200S-S4) was purchased from Xi’an Lei Ze Electronic Technology Co., Ltd. The temperature of the solution was recorded at 30-s intervals using a digital thermometer and a thermal imaging camera (FLIR E96). In addition, 1 ml Syr/IL-2@MPDA (0.25 mg/ml) was placed in a 10 mm cuvette and treated with different powers of an 808 nm laser for 10 min (0.5, 1.0 and 2.0 W/cm²). The solution temperature was monitored at 30-s intervals using a digital thermometer. The photothermal stability was evaluated by recording the temperature variation of Syr/IL-2@MPDA (0.25 mg/ml) after four cycles of laser irradiation (2.0 W/cm²).

2.8. Cytotoxicity and lactate regulation in vitro

Hepa 1–6 cancer cells were seeded in 96-well plates at a density of 5 × 10³ and cultured for 24 h. Then, the cells were incubated with PBS, MPDA, Syr@MPDA, IL-2@MPDA or Syr/IL-2@MPDA (100 µg/ml) for 24 h. Subsequently, for the mPTT treatment group, the cells were irradiated with an 808 nm laser (0.5 W/cm²) for 10 min and incubated for 24 h. Then, 20 µl of 5% MTT solution was added to each well and incubated for 4 h. The medium was then removed, and 200 µl DMSO was added to dissolve the purple formazan crystals. The absorbance of each well was measured at 570 nm using a microplate reader (BioTek, USA). For calcein/PI staining, the medium of cells subjected to different treatments was replaced with a 100 µl solution containing calcein AM and PI. Live (green fluorescence) and dead (red fluorescence) cells were observed using a fluorescence microscope (Leica DMi8, Wetzlar, Germany). For the lactate regulation experiments, Hepa 1–6 cancer cells were seeded into plates and cultured for 24 h. The cells were treated with PBS, MPDA, Syr@MPDA, IL-2@MPDA or Syr/IL-2@MPDA (100 µg/ml) for 24 h. Subsequently, for the mPTT treatment group, the cells were irradiated with an 808 nm laser (0.5 W/cm²) for 10 min and incubated for 24 h. The supernatants were collected for pH and lactate measurements.

2.9. Bone marrow-derived dendritic cells maturation in vitro

Bone marrow-derived dendritic cells (BMDCs) were obtained following a previously described procedure [35]. Bone marrow cells were flushed from dissected femurs using RPMI-1640, then filtered through a 70 µm strainer. After red blood cell lysis, the cells were cultured in RPMI-1640 supplemented with 20 ng/ml GM-CSF and IL-4 at 37 °C in 5% CO₂. On Day 3, equal volumes of fresh cytokine-supplemented medium were added, and the cultures were maintained for another 3 d Non-adherent and loosely adherent cells were collected as BMDCs.

To study DC maturation, Hepa 1–6 cancer cells were first seeded into six-well plates at a density of 5 × 10⁴ cells/well and cultured for 24 h. Then, the cells were incubated with PBS, MPDA, Syr@MPDA, IL-2@MPDA or Syr/IL-2@MPDA (100 µg/ml) for 24 h. For the mPTT treatment group, the cells were irradiated with an 808 nm laser (0.5 W/cm²) for 10 min and incubated for 24 h. The supernatants obtained from various treatment groups were added to the BMDC culture plates and incubated for 24 h. Subsequently, the BMDCs were harvested, stained with antibodies against CD11c, CD80 and CD86, and analyzed by flow cytometry (BD FACSCanto II).

2.10. T cell activation and proliferation in vitro

Naïve CD8⁺ T cells were obtained using a BeaverBeads Mouse CD8⁺ T Cell Isolation Kit. Isolated T cells were cultured, activated with anti-CD3/anti-CD28 antibodies, and incubated for 48 h; they were then labeled with CFSE for subsequent use. The cell compatibility of MPDA was assessed using the CCK-8 assay. The activated CD8⁺ T cells were seeded into 96-well plates at a density of 5 × 10³ cells/well and cultured for 24 h. Subsequently, the cells were incubated with PBS or MPDA for 24 h, and the culture medium in each well was replaced with fresh medium containing 10% CCK-8. After 3 h, the absorbance at 450 nm was measured using a microplate reader.

To evaluate the bioactivity of IL-2 loaded on MPDA, STAT5 phosphorylation (p-STAT5) was analyzed by Western blotting. Activated CD8⁺ T cells were treated with IL-2@MPDA or Syr/IL-2@MPDA for 24 h, followed by washing with cold PBS and lysis in RIPA buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were determined using a BCA assay, and equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes. After blocking, the membranes were incubated with primary antibodies against pSTAT5, followed by HRP-conjugated secondary antibodies. Protein bands were visualized using an enhanced chemiluminescence detection system (iBright FL1500, Thermo Fisher).

To study T cell activation and proliferation, Transwell systems were established to coculture the T cells with Hepa 1–6 cells subjected to different treatments. First, Hepa 1–6 cancer cells were seeded in the lower chamber, and incubated with PBS, MPDA, Syr@MPDA, IL-2@MPDA or Syr/IL-2@MPDA (100 µg/ml) for 24 h. CD8⁺T cells were seeded in the upper chamber. Subsequently, the cells were treated with laser light (0.5 W/cm²) for 10 min and incubated for 48 h. The supernatants were extracted to measure the concentration of IFN-γ via ELISA, and the CFSE-labeled CD8⁺ T cells were extracted to measure the proliferation via CFSE. In a separate experiment, CD8⁺ T cells were stimulated with a cell stimulation cocktail (00-4975-93, eBioscience) for intracellular cytokine analysis. After fixation and permeabilization, the CD8⁺ T cells were stained with PE-IFN-γ and APC-TNF-α, and subjected to flow cytometric analysis.

To study the activated CD8⁺ T cell-mediated cytotoxicity, Hepa 1–6 cancer cells were incubated with PBS, MPDA, Syr@MPDA, IL-2@MPDA or Syr/IL-2@MPDA (100 µg/ml) for 24 h. Then, the CD8⁺ T cells were co-cultured with tumor cells at the indicated 2:1 effector-to-target ratio. The cells were subsequently treated with laser light (0.5 W/cm²) for 10 min and incubated for 24 h. Tumor cell killing was assessed by flow cytometry. Apoptosis of tumor cells as a measure of CD8⁺ T cell-mediated cytotoxicity was assessed by flow cytometry.

2.11. In vivo fluorescence imaging and photothermal performance of Hepa 1–6 in tumor-bearing mice

To explore the release behavior of IL-2 in MPDA in vivo, His-tagged IL-2 was labeled with Cy5.5 and incubated with MPDA to obtain Cy5.5-His-tagged IL-2@MPDA. Real-time in vivo fluorescence imaging was conducted in Hepa 1–6 tumor-bearing mice at 0, 1, 3,5, 12, 24, 48 and 96 h after intratumoral injection of Cy5.5-His-tagged IL-2 or Cy5.5-His-tagged IL-2@MPDA. Fluorescence imaging of the mice was performed using an IVIS Spectrum CT system. At 96 h after injection, the mice were sacrificed, and the collected organs were imaged ex vivo to evaluate the biodistribution of the fluorescence. The fluorescence intensities of the tumors and excised organs were quantified by region-of-interest analysis using Living Image software. MPDA labeled with Cy5.5-NH₂ was used to explore its biodistribution. Real-time in vivo fluorescence imaging was performed in Hepa 1–6 tumor-bearing mice at 1, 2, 4, 12, 24, 48, 72, 96 and 120 h. The mice were sacrificed, and the collected organs were imaged ex vivo for evaluation.

To examine the photothermal capability of Syr/IL-2@MPDA in vivo, Hepa 1–6 tumor-bearing mice were intratumorally injected with PBS or Syr/IL-2@MPDA, and the surface temperature of the tumor site during laser irradiation was recorded using a thermal imaging camera.

2.12. Anti-tumor efficacy in vivo

To develop an animal tumor model, tumors were generated in female C57/BL6 mice by subcutaneous injection of Hepa 1–6 cells (1 × 10⁵ cells in 100 µl PBS) into the right posterior leg. Once the tumor volume reached approximeately100 mm³, the mice were randomly assigned to seven groups (n = 5 per group): (1) PBS; (2) MPDA; (3) MPDA+L; (4) Syr@MPDA; (5) IL-2@MPDA; (6) Syr/IL-2@MPDA; (7) Syr/IL-2@MPDA+L, (intratumoral injection of MPDA 20 mg/kg, IL-2 20 µg/kg, and Syr 3.8 mg/kg). NPs were injected on Day 0, 3 and 6, and mPTT treatment was performed on Day 1, 4 and 7. An 808 nm near-infrared laser was used to irradiate the tumor region with a spot diameter of 10 mm. The distance between the laser source and the tumor surface was maintained at 30 cm throughout the irradiation. The length and width of tumors were measured by a digital caliper every 2 d, and the volume was calculated as Eq. (4):

$$\text{Tumor}\text{volume}=\text{Tumor}\text{Length}\times {\text{Width}}^2/2$$ (4)

2.13. Evaluation of immune microenvironment in vivo

The tumor-bearing mice were randomly assigned to seven groups (n = 5 per group): (1) PBS; (2) MPDA; (3) MPDA+L; (4) Syr@MPDA; (5) IL-2@MPDA; (6) Syr/IL-2@MPDA; (7) Syr/IL-2@MPDA+L, intratumoral injection of MPDA 20 mg/kg, IL-2 20 µg/kg and Syr 3.8 mg/kg. NPs were injected on Day 0, 3 and 6, and mPTT treatment was performed on Day 1, 4 and 7. On Day 8, the mice from each group were euthanized, and the tumors and spleens were harvested. For T-cell analysis, the cells were stained with antibodies against CD3, CD4 and CD8. To evaluate Treg cells, the cells were first stained with anti-CD4 and anti-CD25 antibodies, then fixed in 0.5 ml fixation buffer, and subsequently incubated with anti-FOXP3 antibodies. For dendritic cell (DC) maturation analysis, cells were stained with antibodies against CD11c, CD80 and CD86.

2.14. Evaluation of anti-tumor efficacy in combination with αPD-L1 in vivo

To develop the animal tumor model, right-side tumors were generated in female C57/BL6 mice by subcutaneously injecting Hepa 1–6 cells (1 × 10⁵ cells, 100 µl PBS) into the right hind legs. Four days post-injection, left side tumors were generated in female C57/BL6 mice by subcutaneously injecting Hepa 1–6 cells (2 × 10⁵ cells, 100 µl PBS) into the left rear legs of the mice. Once the right side tumor volume reached approximately 100 mm³, the mice bearing Hepa1–6 tumors were randomly assigned to four groups (n = 5 per group): (1) PBS; (2) αPD-L1; (3) Syr/IL-2@MPDA+L; (4) Syr/IL-2@MPDA+L+αPD-L1 (intratumoral injection, MPDA 20 mg/kg, IL-2 20 µg/kg, Syr 3.8 mg/kg; αPD-L1 by intravenous injection, 1 mg/kg). NPs were injected on Day 0, 3 and 6; mPTT treatment was performed on Day 1, 4 and 7, and αPD-L1 was injected on Day 1, 4 and 7 after mPTT treatment. The length and width of tumors were measured by a digital caliper every 2 days.

2.15. Evaluation of immune microenvironment in combination with αPD-L1 in vivo

The mice bearing Hepa1–6 tumors were randomly assigned to four groups (n = 5 per group): (1) PBS; (2) αPD-L1; (3) Syr/IL-2@MPDA + L; (4) Syr/IL-2@MPDA + L + αPD-L1 (intratumoral injection, MPDA 20 mg/kg, IL-2 20 µg/kg, Syr 3.8 mg/kg; αPD-L1 intravenous injection, 1 mg/kg). NPs were injected on Day 0, 3 and 6; mPTT treatment was performed on Day 1, 4 and 7, and αPD-L1 was injected on Day 1, 4 and 7 after mPTT treatment. The mice in each group were euthanized on Day 8, and the tumors and spleens were collected. Flow cytometry was performed to analyze T cells (CD3, CD4, CD8), Treg (CD4, CD25, FOXP3), and DC maturation (CD11c, CD80, CD86), as described in Section 2.13.

2.16. Establishment of a rat model for orthotopic HCC tumors

Orthotopic HCC models were generated in SD rats as described previously. Under isoflurane anesthesia (4% induction, 1.8% maintenance), N1S1 cells (5 × 10⁶) were implanted into the left liver lobe, followed by gentle compression with a gelatin sponge for 3 min to prevent cell leakage. The wound was then closed in layers. Tumor growth was verified 1 week later using MRI (Ingenia, Philips Healthcare, Netherlands).

2.17. In vivo fluorescence imaging and photothermal performance of the rat HCC model

MPDA was labeled with Cy5.5 and incubated with MPDA to obtain Cy5.5-His-tagged IL-2@MPDA, which was then used to investigate the distribution of IL-2 in MPDA rats. Real-time in vivo fluorescence imaging was performed on the rats at 1, 2, 12, 24 and 48 h after TAE surgery. Fluorescence imaging was carried out using an IVIS Spectrum CT system.

To examine the photothermal capability of Syr/IL-2@MPDA in the orthotopic HCC rat model, after 24 h of treatment with iodized oil or iodized oil + Syr/IL-2@MPDA, the rats underwent laser irradiation, and the changes in their surface temperature were recorded by a thermal imaging camera. The irradiated area was determined based on the above fluorescence imaging results.

2.18. Evaluation of anti-tumor efficacy and biocompatibility in combination with TAE in a rat HCC model

TAE was performed under DSA guidance in accordance with established protocols. The rats were anesthetized with isoflurane (4% induction, 1.8% maintenance) and placed in a supine position. Following a midline laparotomy, the liver and surrounding vascular structures were carefully exposed. The common hepatic artery (CHA), the proper hepatic artery (PHA), and the gastroduodenal artery (GDA) were sequentially identified and isolated. A PE-10 catheter (Scientific Commodities Inc., USA) was inserted into the proximal GDA under DSA monitoring (AlluraXper, Philips, Netherlands). Before embolization, 0.1 mL heparin was administered to prevent coagulation. Then, 5 mg Syr/IL-2@MPDA in 0.2 mL iodized oil was injected, followed by flushing with saline. After embolization, the catheter was removed, the GDA was ligated with 3–0 suture, and the incision was closed in layers.

Magnetic resonance imaging (MRI) was obtained on Day 2, 6, 10 and 14 post-treatment for therapeutic evaluation. T2-weighted images were recorded by MRI to determine tumor dimensions, and the volume was calculated as Eq. (4). For biocompatibility assessment, the main organs of the rats were collected and sliced into sections. The sections were stained with H&E and examined under a light microscope for assessment of histological damage.

2.19. Transcriptomic analysis

One week after the inoculation of N1S1 cells, the rats were randomly assigned to two treatment groups: (1) PBS; (2) Syr/IL-2@MPDA + L + TAE (n = 3 per group). On Day 5 after receiving treatment, the rats were euthanized, and the tumors were collected for transcriptome sequencing conducted by BGI Genomics Co., Ltd.

2.20. Image analysis and quantification

Image quantification was performed using ImageJ. At least three fields per sample were analyzed using identical thresholds. Fluorescence intensity or positive area was quantified and statistically analyzed.

2.21. Statistical analysis

All data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism (10.0.0). An unpaired two-tailed Student’s t-test was used for comparisons between two groups. For experiments involving more than two groups, one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc multiple-comparison test was applied. Significance levels were defined as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

3. Results and discussion

3.1. Preparation and characterization of Syr/il-2@MPDA

The synthetic route of Syr/IL-2@MPDA is illustrated in Fig. 1A. First, MPDA NPs were synthesized by self-polymerization of dopamine using F127 and P123 as templates under alkaline conditions, with oxygen as the oxidant, in a mixture of deionized water and ethanol (v/v, 3:2). Then, mPEG-NH₂, which reacts with carbonyl groups (C O), was used for improving the stability of MPDA by providing a hydrophilic outer layer. Successful PEGylation was demonstrated by an increased hydrodynamic diameter in the DLS measurements, together with the appearance of characteristic PEG-related bands in the FTIR spectra (Figs. S1 and S2). Further reaction with NiCl₂ endows MPDA with the ability to adsorb His-tagged labels. The obtained NPs exhibited a number-weighted particle size of approximately 169.9 nm and a zeta potential of −9.623 ± 0.262 mV. After reaction with NiCl₂, the particle size exhibited no significant variation, whereas the zeta potential was altered to 12.307 ± 1.579 mV, indicating the successful incorporation of Ni²⁺ (Fig. S3). Quantitative analysis by ICP-MS further revealed a Ni loading amount of 19.8 µg/mg MPDA. Finally, Syr/IL-2@MPDA was obtained by loading Syr and His-tagged IL-2 by mesoporous structures and coordinated bonding with Ni²⁺, respectively (Fig. S4).

Fig. 1.

Characterization of Syr/IL-2@MPDA NPs. (A) Synthetic route of Syr/IL-2@MPDA; (B) TEM image of Syr/IL-2@MPDA; (C) Elemental analysis of Syr/IL-2@MPDA by EDS-HAADF; (D) Number-weighted size distribution of Syr/IL-2@MPDA; (E) UV–vis spectra of Ni-MPDA, Syr/IL-2@MPDA and pure Syr; (F) EE and DL of Syr by MPDA (1 mg) at different Syr inputs; (G) Percentage of loaded His-tagged IL-2 (1 µg) after 1 h of co-incubation with MPDA or Ni-MPDA (1 mg) at 4 °C; Relative release curves of (H) Syr and (I) IL-2 from Syr/IL-2@MPDA in PBS; (J) Intensity-weighted hydrodynamic size and PDI of Syr/IL-2@MPDA for 7 d in PBS containing 10% FBS; (K) Infrared thermography of a 10 mm cuvette containing different concentrations of Syr/IL-2@MPDA solution under laser irradiation (2.0 W/cm²); (L) Photothermal heating curves of the Syr/IL-2@MPDA solution under laser irradiation (2.0 W/cm²) at various concentrations; (M) Photothermal heating curves of the Syr/IL-2@MPDA solution (250 µg/ml) under laser irradiation; (N) Photothermal stability under four cycles of laser irradiation and natural cooling. Error bars represent means ± SD (n = 3).

TEM images showed that Syr/IL-2@MPDA possessed a spherical particle shape with mesoporous properties and a size of 170.0 nm (Fig. 1B). The number-weighted average diameter of Syr/IL-2@MPDA determined by DLS was approximately 170.0 nm, in good agreement with the TEM observations (Fig. 1D). Furthermore, the images from the elemental analysis by EDS showed that nickel was uniformly distributed among the particles, confirming the successful binding of Ni²⁺ to MPDA (Figs. 1C and S5). The ICP-MS analysis revealed a Ni²⁺ content of 19.8 µg/mg in Syr/IL-2@MPDA.

3.2. Drug loading and release properties in vitro

The UV–vis spectra of pure Syr, Ni²⁺-MPDA and Syr/IL-2@MPDA were obtained to examine the loading of the Syr. The absorption peaks at 212 and 257 nm of Syr/IL-2@MPDA matched those of pure Syr, indicating successful drug loading (Fig. 1E). Meanwhile, to explore the encapsulation efficiency of Syr and the loading ability of MPDA, different mass ratios of Syr: MPDA ranging from 1:1 to 1:4 were examined. At Syr:MPDA mass ratios of 1:2 or 1:4, the EE of Syr achieved over 88% (Fig. 1F). Next, the loading efficiency of Ni²⁺-MPDA toward His-tagged IL-2 was verified. Quantification of IL-2 in the supernatant was detected after co-incubation of Ni²⁺-MPDA and His-tagged IL-2 at 4 °C using an ELISA kit. First, to validate the chelation between Ni²⁺ and His tag label, 100 ng His-tagged IL-2 was incubated with 1 mg MPDA or Ni²⁺-MPDA. More than 52% of His-tagged IL-2 remained in the supernatant of the MPDA group (Fig. 1G). In comparison, Ni²⁺-MPDA exhibited an extremely high loading efficiency of up to 99.9% for His-tagged IL-2. Various amounts of His-tagged IL-2 were subsequently incubated with 1 mg Ni²⁺-MPDA, and the results showed that 99.9% of the IL-2 was absorbed in each group, resulting from the Ni²⁺-rich MPDA (Fig. S6). In addition, different incubation times were investigated, and Ni²⁺-MPDA rapidly adsorbed IL-2 within 1 h (Fig. S7).

In vitro release of Syr and His-tagged IL-2 from Syr/IL-2@MPDA was investigated in PBS at 37 °C using HPLC and ELISA (Fig. 1H). The Syr/IL-2@MPDA exhibited a rapid release of 50% of the loaded Syr within 24 h, and more than 70% within 120 h. Syr/IL-2@MPDA showed a sustained release of His-tagged IL-2, ∼30% within 120 h, potentially mitigating the side effects caused by high doses of IL-2. Notably, when the release study was conducted under mildly acidic conditions (pH 6.5), there was a markedly accelerated release of His-tagged IL-2, whereas the release behavior of Syr was only moderately affected. This phenomenon is likely attributable to the partial weakening of Ni²⁺-histidine coordination under acidic conditions, facilitating IL-2 dissociation from the MPDA (Fig. 1I). Furthermore, the effect of mPTT treatment on the release properties was explored; the results showed that it slightly enhanced drug release. The long-term colloidal stability of Syr/IL-2@MPDA was assessed in PBS containing 10% FBS. The hydrodynamic diameter and polydispersity index (PDI) of Syr/IL-2@MPDA showed no discernible changes over 7 d of incubation (Fig. 1J), indicating excellent serum stability of the nanoplatform. These results suggest that Ni²⁺-MPDA is not only an efficient carrier for Syr and His-tagged IL-2 but is also advantageous for drug release.

3.3. Photothermal performance and photothermal stability in vitro

MPDA-based materials exhibit excellent photothermal conversion efficiency and stability under NIR irradiation; as such, they are widely used as PTT agents in cancer therapy. To investigate the photothermal performance of Syr/IL-2@MPDA, the temperatures of Syr/IL-2@MPDA nanosuspensions were recorded under continuous NIR light by a digital thermometer and infrared thermography (Fig. 1K). The temperature was increased by 25.8 °C at a Syr/IL-2@MPDA concentration of 250 µg/mL (Fig. 1L). Moreover, the temperature changes were directly related to the changes in Syr/IL-2@MPDA concentrations, suggesting that Syr/IL-2@MPDA has a concentration-dependent photothermal effect, which was proportional to the irradiation power intensity (Fig. 1M). In addition, Syr/IL-2@MPDA nanosuspensions could be heated to similar temperatures in four cycles of irradiation/cooling, indicating that Syr/IL-2@MPDA is photostable (Fig. 1N).

3.4. Cytotoxicity and lactate regulation in vitro

The cytotoxicity of MPDA NPs was assessed using the MTT assay. The results showed that high doses of MPDA and IL-2@MPDA (100 µg/ml MPDA containing IL-2) exhibited low cytotoxicity regardless of 808 nm laser irradiation, indicating that mPTT had no significant tumor-killing effect (Fig. S8). Additionally, cell viability of the Syr@MPDA and Syr/IL-2@MPDA groups was reduced to approximately 85%, largely due to Syr blocking lactate transport in tumor cells. Calcein-AM/PI staining experiments were conducted to confirm the cytotoxicity of MPDA NPs, and the results were generally consistent with the MTT results (Fig. S9). These results demonstrated that the MPDA NPs were non-cytotoxic and safe when applied in vitro. In addition, mPTT treatment had no significant cytotoxic effect but did kill a subset of the tumor cells and promoted tumor-associated antigen release.

To explore the lactate regulation ability of Syr/IL-2@MPDA, Hepa 1–6 cells were incubated with different MPDA NPs, and the lactate concentration and pH value of the cell culture media were measured. The results showed that the extracellular lactate level was significantly reduced in the Syr@MPDA and Syr/IL-2@MPDA groups due to strong inhibition of MCT1/4 by the released Syr (Fig. 2A). In addition, the pH value declined to 6.2–6.4 due to the lactate secreted by the tumor cells in the PBS, MPDA and IL-2@MPAD groups, while the Syr@MPDA and Syr/IL-2@MPDA groups maintained a pH of 7.2–7.4 as a result of Syr blocking lactate efflux (Fig. 2B). Syr/IL-2@MPDA regulated extracellular pH in a clear dose-dependent manner (Fig. 2C). At lower concentrations, Syr/IL-2@MPDA effectively maintained a near-neutral pH during the initial 24 h. Notably, increasing the concentration of Syr/IL-2@MPDA prolonged the pH-stabilizing effect, with concentrations ≥ 25 µg/ml sustaining near-physiological pH levels throughout the 96 h observation period.

Fig. 2.

Syr/IL-2@MPDA mediated therapy in vitro. (A) Lactic acid content of Hepa 1–6 cells of each group; (B) pH value of Hepa 1–6 cells of each group; (C) Concentration-dependent pH value of Hepa 1–6 cells treated with Syr/IL-2@MPDA; (D) Schematic illustration of experiment for CD8⁺ T cells activation and proliferation; (E-F) Quantification analysis of CFSE-labeled (E) CD8⁺ T cells and (F) IFN-γ⁺TNF-α⁺ cells in CD8⁺ T cells after different treatments; (G) Flow cytometry histogram of CFSE-labeled CD8⁺ T cells; (H) Concentration of IFN-γ in the medium of each group; (I) Quantification analysis of tumor cell viability in a co-culture system; (J) Flow cytometry histogram of IFN-γ⁺TNF-α⁺ cells in CD8⁺ T cells; (K) Schematic illustration of the experiment for BMDC maturation; (L) quantification analysis of mature DCs (CD80⁺CD86⁺CD11c⁺ cells) of each group and (M) flow cytometry histogram. Data were expressed as mean ± SD.

3.5. T cell activation and proliferation in vitro

The cytocompatibility of MPDA was assessed using the CCK-8 assay. The results showed that MPDA did not cause a significant reduction in CD8⁺ T cell viability, as this remained above 95%, even at an MPDA concentration of 200 µg/ml (Fig. S10). To further verify whether IL-2 retains its biological activity after adsorption onto MPDA, isolated CD8⁺ T cells were treated with IL-2@MPDA or Syr/IL-2@MPDA. The biological activity of IL-2 in MPDA was assessed by analyzing p-STAT5, a canonical downstream signaling event of IL-2 receptor activation. Both IL-2@MPDA and Syr/IL-2@MPDA induced robust p-STAT5 in CD8⁺ T cells. Notably, the extent of STAT5 phosphorylation increased progressively with the concentration of IL-2@MPDA or Syr/IL-2@MPDA, indicating a clear dose-dependent activation of the IL-2R-STAT5 signaling pathway and further demonstrating that IL-2 retained its signaling activity after chelation-based loading onto MPDA.

Having established the biosafety of MPDA and the bioactivity of IL-2 on MPDA, a Transwell system was used to co-culture Hepa 1–6 cells and CFSE-labeled isolated T cells subjected to various treatments (Fig. 2D). The proliferation of CD8⁺ T cells was evaluated by flow cytometry using CFSE labeling. The IL-2@MPDA exhibited optimal capacity for CD8⁺ T cell proliferation, indicating that IL-2 remains biologically active through metal-chelation loading in MPDA (Fig. 2E and 2G). Notably, the Syr/IL-2@MPDA treated CD8⁺T cells exhibited 1.23-fold greater cell proliferation compared to the IL-2@MPDA-treated group due to the regulation of lactate by Syr. Additionally, the proliferation rate of the MPDA NPs groups treated with 808 nm laser light was slightly higher than that of groups not treated with light, potentially due to the mPTT enhancement of drug release. Flow cytometric analysis showed that the Syr/IL-2@MPDA treatment markedly increased the proportion of IFN-γ⁺TNF-α⁺CD8⁺ T cells in the tumor co-culture system. In comparison, this proportion was significantly higher than that observed in the IL-2@MPDA-treated group (Fig. 2F and 2J), indicating that modulation of the acidic TME in the co-culture system effectively enhances CD8⁺ T cell effector function. IFN-γ levels in the supernatant were further detected by ELISA (Fig. 2H). The results indicated that IFN-γ levels increased in the IL-2@MPDA group due to the proliferation of CD8⁺ T cells. In contrast, the Syr/IL-2@MPDA group showed a 2.91-fold higher IFN-γ level compared to the IL-2@MPDA group; this was attributed to a decrease in the extracellular lactate level that alleviated CD8⁺ T cell exhaustion. Under laser irradiation, the IFN-γ levels in the IL-2@MPDA and Syr/IL-2@MPDA groups were further increased. To determine whether the enhanced CD8⁺ T cell responses translated into improved tumor cell killing, the viability of Hepa1–6 cells was evaluated by Annexin V/PI staining in a direct co-culture system, in which tumor cells were cultured in direct contact with CD8⁺ T cells. Flow cytometric analysis revealed a reduced proportion of viable Hepa1–6 cells in the Syr/IL-2@MPDA-treated group (Figs. 2I and S12), indicating that Syr/IL-2@MPDA-mediated activation of CD8⁺ T cells effectively enhanced tumor cell apoptosis. The above results indicate that Syr/IL-2@MPDA with mPTT effectively promoted CD8⁺ T cell proliferation and function, thereby effectively enhancing anti-tumor immune effects.

3.6. DC maturation in vitro

DCs are essential for initiating and regulating T cell activation and proliferation, and mPTT and lactate have been reported to be associated with DC function [36,37]. Therefore, for in vitro DC maturation analysis, Hepa1–6 cells were pre-incubated with different treatments, and the supernatants were collected for co-incubation with BMDCs (Fig. 2K). DC maturation was evaluated by flow cytometry of CD11c⁺CD86⁺CD80⁺ DCs. The proportions of CD86⁺CD80⁺ DCs in the NIR light-treated groups were higher than those in the dark-treated groups (Fig. 2L and 2M), attributed to the MPDA photothermal effect. Meanwhile, treatment with Syr@MPDA and Syr/IL-2@MPDA effectively matured DCs by inhibiting lactate efflux via Syr. Therefore, we conclude that mPTP and lactate modulation affect DC maturation, thereby further facilitating IL-2-mediated T cell activation and proliferation.

3.7. Anti-tumor immunotherapy by Syr/il-2@MPDA in vivo

The pharmacokinetics of Syr/IL-2@MPDA were examined in vivo. His-tagged IL-2 was labeled with Cy5.5 and adsorbed onto MPDA to obtain Cy5.5-labeled His-tagged IL-2@MPDA. Cy5.5-labeled His-tagged IL-2 and Cy5.5-labeled His-tagged IL-2@MPDA were intratumorally injected into C57BL/6 mice, followed by monitoring using an in vivo optical imaging system (IVIS). The results showed that the fluorescence signals of Cy5.5-labeled His-tagged IL-2@MPDA were more localized to the tumors, while those of Cy5.5-labeled His-tagged IL-2 diffused out of the tumors (Fig. S13). In addition, the tumors of mice with the injected Cy5.5-labeled His-tagged IL-2@MPDA exhibited a stronger fluorescence signal than that of Cy5.5-labeled His-tagged IL-2 at the same post-injection time (Fig. S14). In parallel, Cy5.5-labeled MPDA (Figs. S15 and S16) exhibited prolonged retention and gradual degradation within the tumor after intratumoral injection. Ex vivo fluorescence imaging further revealed that the majority of the fluorescence signal was localized at the tumor site, while a small fraction was detected in the liver, implying limited systemic distribution and hepatic clearance. These results indicate that MPDA NPs exhibit prolonged retention in tumors and sustained release of IL-2, thereby preventing the leakage of IL-2.

To achieve mPTT treatment in vivo, tumor-bearing mice were intratumorally injected Syr/IL-2@MPDA (20 mg/kg) and received laser irradiation (0.5 W/cm², 10 min). The tumor temperature in mice treated with Syr/IL-2@MPDA after 10 min of 808 nm laser irradiation rose by approximately 4 to 6 °C, whereas the control group only rose by less than 1 °C, as recorded by infrared thermography (Fig. S17). Next, to evaluate anticancer efficiency of Syr/IL-2@MPDA in vivo, tumor-bearing mice were assigned to seven groups (n = 5): (1) control; (2) MPDA; (3) MPDA + L; (4) Syr@MPDA; (5) IL-2@MPDA; (6) Syr/IL-2@MPDA; (7) Syr/IL-2@MPDA + Light, and subjected to treatments according to the timeline shown in Fig. 3A. In addition, at 24 h after injecting MPDA NPs, the temperature of tumors treated with NIR light (808 nm, 0.5 W/cm², 10 min) were increased to about 40–41 °C, as monitored by an infrared camera. Body weights in all groups showed no apparent changes during treatment, suggesting that MPDA NPs are not toxic (Fig. S18). The MPDA and MPDA + L groups showed no clear differences in tumor volume, demonstrating that mPTT treatment alone had no significant antitumor effect (Figs. 3C and S19). The Syr/IL-2@MPDA treatment significantly suppressed tumor growth compared to the IL-2@MPDA and Syr@MPDA groups, indicating that lactate regulation enhanced the antitumor effect of IL-2. Under NIR irradiation, the tumor growth in the Syr/IL-2@MPDA + L group was further reduced, and the rate of tumor volume growth was the lowest among all groups. H&E and Ki-67 staining were used to further assess the therapeutic effect (Fig. 3D). No significant tumor tissue damage was observed in the PBS or MPDA groups. The mPTT treatment of the MPDA + L group and LA regulation of Syr@MPDA group induced a small amount of cell death, indicating their limited anti-tumor effect. However, the IL-2@MPDA, Syr/IL-2@MPDA, and Syr/IL-2@MPDA + L groups showed extensive apoptotic and necrosis characteristics, among which the Syr/IL-2@MPDA + L group was the most significant. In addition, the Syr/IL-2@MPDA + L group exhibited the lowest Ki-67 levels, further indicating that the combination of mPTT treatment and LA regulation enhanced the anti-tumor effect of IL-2. Meanwhile, H&E images of major organs revealed no apparent pathological abnormalities in any treatment group, and blood biochemical and hematological parameters showed no significant differences compared to the control group, indicating the systemic biosafety of Syr/IL-2@MPDA (Figs. S21 and S22).

Fig. 3.

Syr/IL-2@MPDA mediated cancer immunotherapy in vivo. (A) Schematic illustration and timeline of Syr/IL-2@MPDA in a Hepa1–6 tumor model; (B) Illustration of mPTT treatment under infrared thermography monitoring; (C) Tumor volume growth curves of each group (PBS, MPDA, MPDA + Light, Syr@MPDA, IL-2@MPDA, Syr/IL-2@MPDA, and Syr/IL-2@MPDA + Light, n = 5); (D) Tumor histological analysis of each group. Scale bar: 200 µm: (E-H) Corresponding quantification of (E) matured DCs, (F) CD3⁺CD8⁺ T cells, (G) GZMB⁺CD8⁺ T cells, and (H) Treg cells in tumors. Data are expressed as mean ± SD.

To further investigate in vivo immune activation, tumors from mice receiving different treatments were collected and analyzed by flow cytometry to assess mature DCs, the effector T cells and the Treg cells (Fig. 3E–3H). Syr/IL-2@MPDA resulted in a percentage of high DC maturation (49.1%), and mPTT treatment further promoted DC maturation in the Syr/IL-2@MPDA + L group (67.3%) (Fig. S23). At the same time, the percentage of CD8⁺ T cells in tumors treated with Syr/IL-2@MPDA + L was 2.31-fold higher than in the PBS group (Fig. S24), accompanied by a significant reduction in immunosuppressive Treg cells compared to the control group. (Fig. S25). To evaluate the functional status of tumor-infiltrating CD8⁺ T cells, intracellular granzyme B (GZMB) expression was analyzed by flow cytometry. The proportion of GZMB⁺CD8⁺ T cells in tumors treated with Syr/IL-2@MPDA + L was markedly higher than in the PBS and other control groups (Fig. S26), indicating enhanced cytotoxic effector activity of CD8⁺ T cells. These results suggest that Syr/IL-2@MPDA is a potential antitumor agent that acts through enhancing the antitumor immune effects of IL-2 via lactate regulation and mPTT treatment.

3.8. Anti-tumor immunotherapy with αPD-L1 in vivo

The therapeutic efficacy of ICB therapies is limited by low T cell infiltration and PD-L1 expression in tumor tissue [38]. Encouraged by the above anti-tumor efficacy of Syr/IL-2@MPDA, we hypothesized that Syr/IL-2@MPDA could promote T cell activation and PD-L1 expression via IL-2 and mPTT treatment, thereby producing a synergistic effect with ICB therapy. Therefore, a bilateral mouse tumor model was established and subjected for different treatments: (1) control; (2) αPD-L1; (3) Syr/IL-2@MPDA + L, and (4) Syr/IL-2@MPDA + L + αPD-L1, according to the timeline shown in Fig. 4A. Compared to the control group, single use αPD-L1 or Syr/IL-2@MPDA + L moderately suppressed the growth of both primary and distal tumors (Fig. 4C and 4D). The combination of Syr/IL-2@MPDA and αPD-L1 exhibited the most potent anti-tumor effect, which may have been due to their synergistic effects on T cell function. The relative inhibition rates of each group calculated according to tumor weight were 63.6%, 47.9% and 84.3% for primary tumors and 43.1%, 61.2% and 93.8% for distal tumors (Fig. 4E), indicating that the combined application of Syr/IL-2@MPDA and αPD-L1 under light promotion demonstrated a tumor inhibition effect.

Fig. 4.

Syr/IL-2@MPDA mediated enhanced ICB therapy in vivo. (A) Schematic illustration and timeline of Syr/IL-2@MPDA combined with αPD-L1 in the bilateral Hepa1–6 tumor model; (B) Body weight of each group after different treatments (PBS, αPD-L1, Syr/IL-2@MPDA + L and Syr/IL-2@MPDA + L + αPD-L1, n = 5); (C) Tumor volume, (D) digital image and (E) tumor weight of primary tumor and distant tumor in each group. (F-H) Corresponding quantification of CD3⁺CD8⁺ T cells and Treg cells in (F) tumors and (G) spleen. (H) PD-L1 expression in the tumors of each group was assessed by immunohistochemistry. Scale bar: 100 µm.

To explore systemic immune activation in vivo, tumors and spleens from mice were harvested for flow cytometry analysis of effector T cells and Treg cells (Figs. S27–S30). The ratio of effector T cells increased, and Treg cells decreased in both tumors and the spleen (Fig. 4F and 4G). We conducted an additional immunohistochemistry experiment to assesst the expression of PD-L1 in the tumor tissue. The PD-L1 expression in the Syr/IL-2@MPDA + L and Syr/IL-2@MPDA + L + αPD-L1 groups increased due to the mPTT effect of Syr/IL-2@MPDA (Figs. 4H and S31). The above results indicated that Syr/IL-2@MPDA could turn “cold tumors” into immune-sensitive “hot tumors” and amplify the effect of ICB therapy.

3.9. Enhanced TAE by Syr/il-2@MPDA

TAE has been widely adopted as the first-line treatment for advanced HCC. However, lactate accumulation caused by hypoxia after TAE is an important cause of tumor recurrence [[39], [40], [41], [42]]. We hypothesized that Syr/IL-2@MPDA could enhance the antitumor efficacy of TAE by improving the acidic microenvironment and promoting immune-cell infiltration after TAE. In this study, iodized oil was used to perform TAE surgery and deliver MPDA NPs. The compatibility and stability of MPDA nanoparticles with iodized oil were initially evaluated. MPDA could be homogeneously dispersed in iodized oil without visible phase separation, producing a uniform and stable embolic formulation suitable for intra-arterial administration. DLS and visual inspection showed that the particle size distribution and dispersion state of MPDA remained unchanged during prolonged storage (Fig. S32). Then, we used Cy5.5-labeled MPDA mixed with iodized oil, to perform TAE surgery on N1S1 rat models, aiming to determine the distribution of MPDA NPs after TAE. Cy5.5 fluorescence signals localized around the liver and were strengthened within 48 h after TAE, indicating that MPDA NPs could be released into the liver by injecting via the PHA (Fig. S33). We also assessed the feasibility of mPTT treatment using rats. The body surface temperature near the liver in the Syr/IL-2@MPDA group increased after laser irradiation, indicating that the 808 nm laser light could penetrate rat skin to trigger mPTT (Fig. S34).

The therapeutic potency was examined in an N1S1 rat model. N1S1 tumor-bearing rats were assigned to five groups as follows: (1) control; (2) TAE; (3) αPD-L1; (4) TAE + Syr/IL-2@MPDA + L, and (5) TAE + Syr/IL-2@MPDA + L + αPD-L1. By monitoring the tumor volume via MRI, we found that tumors in the αPD-L1 and TAE groups progressed within 2 weeks, even though tumor progression was slower in the TAE group (Fig. 5B). In contrast, the treatment of TAE + Syr/IL-2@MPDA + L gwas effective in suppressing the tumor growth and showed a better anti-tumor effect when combined with αPD-L1 (Fig 5D). The H&E staining results showed severe destruction of the tumor tissue structure in the TAE + Syr/IL-2@MPDA + L + αPD-L1 group, and there were no apparent pathological changes in the major organs of all treatment groups (Figs. 5E and S35). In addition, blood biochemical and routine hematological analyses indicated no significant differences among the treatment groups compared to the control group, further indicating systemic biosafety (Fig. S36). Moreover, the infiltration of CD8⁺ T cells into tumor tissues was assessed to evaluate immune activation. The Syr/IL-2@MPDA + L treatment group displayed CD8⁺ T cell infiltration, and the recruitment of CD8⁺ T cells in the TAE+Syr/IL-2@MPDA + L + αPD-L1 group was the highest, confirming the activation of an anti-tumor immune response (Figs. 5F and S37).

Fig. 5.

Syr/IL-2@MPDA-mediated enhanced TAE therapy in vivo. (A) Schematic illustration and timeline of Syr/IL-2@MPDA combined with TAE in the orthotopic N1S1 rat model; (B) T₁-weighted MR images of N1S1 rats at different times post treatment (PBS, TAE, αPD-L1, Syr/IL-2@MPDA + L + TAE, and Syr/IL-2@MPDA + L + αPD-L1 + TAE, n = 5); (C) Body weight and (D) tumor volume of each group. (E) H&E staining of tumor slices collected from each group at Day 2 post-treatment. Scale bar: 200 µm; (F) Immunofluorescence images of CD8⁺ T cells in the tumors of each group. Scale bar: 50 µm.

The variation in mRNA expression in N1S1 tumor tissues with or without treatment was analyzed using transcriptomics to explore the mechanism of Syr/IL-2@MPDA combined with TAE. Principal component analysis (PCA) was performed based on normalized RNA-seq expression data. The first principal component (PC1, 66.81%) and the second principal component (PC2, 20.90%) accounted for most of the transcriptional variation, enabling clear discrimination among treatment groups and demonstrating good intra-group consistency. A Venn diagram (Fig. 6B) showed that a significant number of genes were differentially expressed between the Syr/IL-2@MPDA + L + TAE and PBS groups, among which 642 genes were downregulated, and 245 genes were upregulated in the Syr/IL-2@MPDA + L + TAE group (Fig. 6C). A heatmap was constructed to visualize the differences in expression of 170 genes related to the immune system (Fig. 6D). Several key regulators of the tumor immune microenvironment were identified. Upregulated genes such as Hspa1b, Il23a, P2rx7, Cxcl12, Cd28, Cxcl14, Gzmb, Klrd1 and Skap1 promote antitumor immunity by enhancing immune cell activation, cytokine production, and cytotoxic responses. Downregulated genes, including Il2ra, Arg1, Cd24, Cxcl13, S100a9, Fgl1, Lag3, Ido1, and Ido2, mediate immunosuppressive mechanisms, including T cell exhaustion, Treg cell recruitment, and metabolic suppression of effector lymphocytes. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the DEGs were predominantly involved in immune-related pathways, including antigen processing and presentation, cytokine-cytokine receptor interaction, T cell receptor signaling, JAK-STAT signaling, and natural killer cell-mediated cytotoxicity, indicating enhanced immune activation within the TME. These pathways are closely associated with cytotoxic lymphocyte activation and effector function. Consistent with the KEGG analysis, Gene Ontology (GO) analysis (Fig. 6F) revealed significant overrepresentation of immune-associated biological processes, including immune system processes, innate and adaptive immune responses, and inflammatory responses. Notably, several GO terms directly related to T cell function, including positive regulation of T cell–mediated cytotoxicity, activated T cell proliferation, and T cell differentiation, were significantly enriched, suggesting reinforced CD8⁺ T cell activation and antitumor immune responses after treatment.

Fig. 6.

Transcriptomic analysis of Syr/IL-2@MPDA + L with TAE. (A) PCA of transcriptomic analysis data; (B) Venn diagram of genes detected in N1S1 tumors treated with PBS and Syr/IL-2@MPDA + L + TAE; (C) Volcano plot of differentially expressed genes in the PBS and Syr/IL-2@MPDA + L + TAE groups; (D) Heatmap of differential genes in the PBS and Syr/IL-2@MPDA + L + TAE groups; (E) KEGG enrichment analysis and (F) GO enrichment analysis of genes related to immune system processes.

4. Conclusions

ICB therapy and TAE are widely adopted as treatment for HCC in clinical practice, but their therapeutic efficacy is limited by the tumor’s immunosuppressive environment and low responses of “cold” tumors and other factors. Although it has been reported that TAE induces immune cell recruitment in TME, TAE-induced lactate accumulation and hypoxia lead to immune cell exhaustion, which eventually results in tumor recurrence and metastasis. Herein, a potent immune nano-agent for enhanced TAE and ICB therapy was prepared by incorporating the T-cell growth factor IL-2 and the MCT inhibitor Syr into MPDA. The biocompatible MPDA not only featured MPDA mesopores for Syr encapsulation and Ni²⁺ chelation for His-tagged IL-2 adsorption but also induced photothermal effects and promoted DC maturation and enhanced T cell function. Moreover, the photothermal effects induced by MPDA turned “cold” tumors “hot” and enhanced the sensitivity to ICB therapy.

Meanwhile, Syr acted as an MCT inhibitor and alleviated TME acidity exacerbated by TAE treatment, thereby promoting CD8⁺ T cell function and reducing Treg cell infiltration into tumors. Moreover, IL-2 function and signaling in antitumor immunity were rescued by Syr-mediated blockade of lactate efflux. As a result, Syr/IL-2@MPDA displayed comprehensive therapeutic effects against HCC when combined with αPD-L1 and TAE.

Overall, this study demonstrates that rational regulation of embolization-induced metabolic dysregulation represents an effective strategy to overcome the immune suppression following TAE. By integrating locoregional embolization, lactate modulation, cytokine delivery, and photothermal immune activation within a single platform, this work provides a mechanistically grounded and potentially translatable approach for improving combinational immunotherapy for advanced HCC.

CRediT authorship contribution statement

Fengkai Qiu: Writing – original draft, Funding acquisition, Formal analysis, Data curation, Conceptualization. Xiaoxiao Chen: Writing – original draft, Funding acquisition, Formal analysis, Data curation. Qianhui He: Writing – original draft, Formal analysis, Data curation. Kai Li: Formal analysis, Data curation. Xinlong Chai: Formal analysis, Data curation. Shiji Fang: Investigation, Formal analysis. Fazong Wu: Investigation, Data curation. Dengke Zhang: Investigation, Data curation. Liyun Zheng: Investigation, Data curation. Zhongwei Zhao: Investigation, Funding acquisition, Formal analysis. Minjiang Chen: Supervision, Investigation. Jin Su: Writing – review & editing, Supervision, Conceptualization. Jiansong Ji: Writing – review & editing, Supervision, Funding acquisition. Gaofeng Shu: Writing – review & editing, Supervision, Formal analysis, Conceptualization.

Conflicts of interest

The authors declare that there is no conflicts of interest.