Near infrared II excitation conjugated polymers-based phototheranostic nanoplatform for hyperthermia-enhanced ferroptosis and immunotherapy

Abstract

Ferroptosis has the potential to induce immunogenic cell death (ICD) and remodel the immunosuppressive tumor microenvironment (TME). However, intracellular antioxidant glutathione (GSH) inhibits the efficacy of ferroptosis. Consequently, enhancing iron ion uptake while simultaneously depleting GSH presents a promising strategy to improve ferroptosis efficacy. Herein, we have developed phenylboronic acid-modified near infrared II photothermal therapy (NIR-II PTT) conjugated polymers DPP-CPDT-PBA (DCP), which co-load Fe²⁺ and GSH scavenger cisplatin (Pt), and encapsulate them within an amphiphilic DSPE-mPEG₂₀₀₀ to finally construct nanoplatform DCP@Pt@Fe. The formulated DCP@Pt@Fe exhibits outstanding NIR-II photothermal property and NIR-II imaging tracking capability. The thermo- and acidic TME-responsive release of Pt and Fe²⁺ synergistically induces ferroptosis in cancer cells through Pt-mediated GSH consumption by forming stable Pt-GSH complexes and Fe²⁺-driven reactive oxygen species (ROS) generation via NIR-II PTT-enhanced Fenton reactions. Notably, ferroptotic tumor cells exhibit high immunogenicity, reprogram tumor-associated macrophage polarization, promote dendritic cell maturation, mobilize T cell activation, and significantly reverse the immunosuppressive TME. Simultaneously, interferon-γ (IFN-γ) produced by tumor-infiltrating immune cells inhibits SLC7A11 expression in cancer cells, which contributes to the ferroptosis therapeutic efficacy. Consequently, our nanoplatform DCP@Pt@Fe effectively eradicates ovarian cancer via synergistic NIR-II-PTT/ferroptosis/immunotherapy, providing an exciting new avenue for therapeutic interventions.

Graphical abstract

Highlights

• •DCP possesses excellent NIR-II PTT activity, NIR-II imaging capacity, and controllable co-loading property for Fe²⁺ and Pt.
• •DCP@Pt@Fe boosts ferroptosis via photothermal-enhanced Fe²⁺-driven Fenton reactions and Pt-mediated glutathione consumption.
• •DCP@Pt@Fe reverses immunosuppressive TME by triggering potent immunogenic cell death and eliciting robust antitumor immunity.

1. Introduction

Ovarian cancer stands as the deadliest malignancy in women, with over 200,000 deaths globally each year [1]. The conventional therapeutic approach for ovarian cancer typically involves surgical resection combined with chemotherapy based on platinum compounds. Since the early 2000s, the rapid progress in immunotherapy has fundamentally transformed the therapeutic landscape for various malignancies [2,3]. Although therapeutic modalities for ovarian cancer have significantly advanced, the 5-year overall survival remains merely 51 %, predominately attributable to both the low immunogenicity of cancer cells and the immunosuppressive microenvironment within tumor tissues [4,5]. Specifically, ovarian cancer cells tend to exhibit low immunogenicity that facilitate their evasion from immune surveillance [6,7]. Moreover, chemokines secreted by cancer cells, such as macrophage chemoattractant protein-1 (MCP-1), recruit tumor-associated macrophages (TAMs) into the tumor microenvironment (TME) and predispose their polarization toward the pro-tumoral M2 phenotype [[8], [9], [10]]. Thus, improving cancer cell immunogenicity and reversing the immunosuppressive microenvironment represent a promising strategy for ovarian cancer treatment.

As a novel regulated cell death, ferroptosis, driven by iron-mediated lipid peroxidation, has garnered significant attention in cancer treatment [[11], [12], [13]]. In addition, the peroxidation cascades during ferroptosis promote exposure of damage-associated molecular patterns (DAMPs), potentially triggering immunogenic cell death (ICD) [14]. These signals contribute to enhance tumor immunogenicity, facilitate recognition of tumor antigens by antigen presenting cells (APCs), mobilize infiltration of T cells, ultimately initiate robust immune responses [15,16]. Encouragingly, recent studies have unveiled that IFN-γ secreted by tumor-infiltrating immune cells exerts direct inhibition on the cystine/glutamate transporter (System Xc⁻), thereby impeding intracellular glutathione (GSH) synthesis and sensitizing cancer cells to ferroptosis [17,18]. Collectively, induction ferroptosis in cancer cells harbors substantial promise for enhancing anti-tumoral immunity and thoroughly eradicating tumors.

Currently, the strategic incorporation of iron ions to induce ferroptosis is increasingly employed in nanomedicine [[19], [20], [21]]. The enhancement of intracellular Fe²⁺ uptake can catalyze H₂O₂ through Fenton reactions to generate potent reactive oxygen species (ROS), thus causing ferroptosis. However, cancer cells are equipped with a sophisticated antioxidant system that contributes to their resistance against ferroptosis. GSH, a powerful endogenous antioxidant, collaborates with glutathione peroxidase 4 (GPX4) to effectively scavenge ROS and prevent the onset of ferroptosis. Herein, the depletion of GSH profoundly impairs the antioxidant capacity, resulting in an accumulation of ROS and lipid peroxides (LPO) [22]. Cisplatin (Pt), the first-line therapeutic drug for ovarian cancer treatment, orchestrates the formation of stable Pt-GSH complexes by coordination with the thiol group of GSH, resulting in substantial GSH consumption and provoking ferroptotic cascades in cancer cells [[23], [24], [25], [26], [27]]. Therefore, the synergistic integration of Pt and Fe²⁺ can considerably augment ferroptosis in cancer cells through a bidirectional mechanism, holding profound implications for cancer therapy.

Moreover, hyperthermia generated by photothermal therapy (PTT) disrupts the cell membrane to increase the iron influx and enhances the efficacy of Fenton reactions, thereby promoting ferroptosis efficacy in cancer cells [28,29]. More importantly, ROS and LPO can efficiently degrade heat shock proteins (HSPs), an intrinsic self-protection mechanism against thermal damage, thereby significantly improving the specificity and effectiveness of PTT [28,30]. Therefore, the combination of ferroptosis and PTT establishes a positive feedback loop that acts as a powerful accelerator for effective tumor eradication. Notably, compared to near infrared I (NIR-I, 650–900 nm) light, near infrared II (NIR-II, 900–1700 nm) light is particularly suitable for PTT due to its lower scattering and deeper penetration capacity, enabling precise energy delivery to deep-seated tumors while minimizing off-target effects [[31], [32], [33]]. Among various NIR-II excitation photothermal agents, conjugated polymers exhibit remarkable advantages of excellent biocompatibility and facile processability, and hence, they have garnered significant attention in NIR-II PTT researches [34]. Therefore, it is imperative to engineer a conjugated polymers-based nanoplatform that concurrently integrates both ferroptosis and NIR-II PTT to elicit robust anti-tumoral effects.

In this study, we have designed an innovative NIR-II excitation and pH-sensitive multifunctional nanoplatform (DCP@Pt@Fe) for NIR-II fluorescence imaging (NIR-II FL)-guided synergistic NIR-II PTT/ferroptosis/immunotherapy. This advanced nanoplatform demonstrated potent anti-tumoral effects and strong immunostimulatory capabilities (Scheme 1). The DCP@Pt@Fe was meticulously constructed by integrating the phenylboronic acid (PBA)-functionalized NIR-II conjugated polymers DPP-CPDT-PBA (DCP), Pt, Fe²⁺, and the amphiphilic PEGylated phospholipid DSPE-mPEG₂₀₀₀. DSPE-mPEG₂₀₀₀ is widely recognized as an effective modifier to enhance the biocompatibility of nanoparticles [35]. Pt and Fe²⁺ were intricately conjugated to DCP within the micelle core via highly reactive donor-acceptor coordination with the PBA group, and efficient binding affinity with backbone sulfur and nitrogen atoms, respectively. This formulated nanoplatform exhibited attractive NIR-II photothermal property and NIR-II imaging tracking capability. NIR-II laser and acidic microenvironment promoted the degradation of nanoplatform and the subsequent release of Pt and Fe²⁺, which facilitated ferroptosis in cancer cells through Pt-mediated GSH consumption and Fe²⁺-driven ROS generation. Combined RNA-sequencing (RNA-seq) analyses further validated the high immunogenicity and robust immunostimulatory capacity of ferroptotic ID8 cells. Mechanistically, ID8 cells undergoing ferroptosis released DAMPs, reprogramed TAMs to the M1 status, promoted maturation of DCs, and mobilized T cell infiltration, ultimately reversing the immunosuppressive microenvironment. Concurrently, IFN-γ released from immune cells attenuated SLC7A11 expression in cancer cells, sensitizing them to ferroptosis. Together, this study compellingly emphasizes the great potential of combined NIR-II PTT/ferroptosis/immunotherapy for the treatment of ovarian cancer.

Scheme 1.

Schematic diagram for DCP@Pt@Fe construction and its anti-tumoral mechanisms via synergistic NIR-II PTT/ferroptosis/immunotherapy.

2. Results and discussion

2.1. Preparation and characterization of DCP@Pt@Fe

The synthetic route of DPP-CPDT-PBA was depicted in Scheme 2. The NIR-II conjugated polymers (DPP-CPDT) were firstly synthesized by Stille coupling polymerization between strong electron acceptor (DPP) and an electron donor (CPDT). And then DPP-CPDT were grafted with phenylboronic acid (PBA) via post-functionalization to obtain conjugated polymer DPP-CPDT-PBA. The chemical structures of DPP-CPDT and DPP-CPDT-PBA were verified using ¹H NMR (Figs. S1 and S2) and Fourier transform infrared spectroscopy (FTIR) (Fig. S3). In addition, the number average molecular weight (Mn) and the polydispersity index (PDI) of DPP-CPDT-PBA were tested by gel permeation chromatography (GPC). As shown in Fig. S4, the Mn and PDI values of DPP-CPDT-PBA were 21 kDa and 1.67, respectively. The PBA groups in the side chain of DPP-CPDT-PBA (DCP) endow this conjugated polymer with efficient conjugation affinity for cisplatin (Pt), via highly reactive donor–acceptor coordination. Meanwhile, the sulfur and nitrogen atoms on DCP conjugated backbone have efficient binding affinity with ferrous ions [36]. As shown in Fig. 1A, self-assembly of DCP, Pt, Fe²⁺, and amphiphilic PEGylated phospholipid DSPE-mPEG₂₀₀₀ was achieved through conventional precipitation, affording multifunctional phototheranostic nanoparticles (named DCP@Pt@Fe).

Scheme 2.

Synthesis route of the NIR-II conjugated polymers DPP-CPDT-PBA (DCP).

Fig. 1.

Characterization of DCP@Pt@Fe. (A) Diagram illustrating the synthesis process of DCP@Pt@Fe. (B) Hydrodynamic diameter of DCP@Pt@Fe measured by DLS. (C) TEM images of DCP@Pt@Fe. (D) TEM-EDS element mapping images of DCP@Pt@Fe. (E) Normalized absorption spectra of DCP@Pt@Fe. (F) Normalized NIR-II fluorescence spectra of DCP@Pt@Fe. (G–H) NIR-II fluorescence image (G) and fluorescence intensity (H) of DCP@Pt@Fe in vitro (n = 3). (I) Photothermal response curves of DCP@Pt@Fe at varying concentration levels (100, 50, and 25 μg mL⁻¹) exposed to 1064 nm laser (1.0 W cm⁻²) (n = 3). (J) Photothermal stability for DCP@Pt@Fe during 5 irradiation cycles (n = 3). (K–L) The cumulative release profiles of Pt (K) and Fe (L) in DCP@Pt@Fe under different conditions (n = 3). (M) The generation of ·OH by DCP@Pt@Fe under different conditions detected by ESR. Data are represented as mean ± SD.

Dynamic light scattering (DLS) revealed that DCP@Pt@Fe exhibited a homogeneous particle distribution with the hydrodynamic diameter (D_h) of 83.30 nm (Fig. 1B). DLS of DCP, DCP@Pt and DCP@Fe also indicated good dispersion (Fig. S5). As evidenced in Fig. 1C, DCP@Pt@Fe displayed spherical shape, with the average diameter of 90 nm, corroborating the findings from DLS assessments. The D_h of DCP@Pt@Fe exhibited no significant alterations in PBS following 15 days of storage at 4 °C (Fig. S6). In addition, the diameter of DCP@Pt@Fe remained consistent in both PBS and 10 % FBS at 37 °C for 24 h (Fig. S7), further confirming its favorable dispersion and colloidal stability. Energy dispersive X-ray element mapping (EDS) illustrated the homogeneous dispersion of N, O, S, Fe and Pt elements throughout DCP@Pt@Fe nanoparticles, demonstrating successful loading of Pt and Fe within the composite structure (Fig. 1D). The ultraviolet (UV) spectra of DCP, DCP@Pt, DCP@Fe, and DCP@Pt@Fe further confirmed co-loading of Pt and Fe in the DCP@Pt@Fe nanoplatform (Fig. S8). Moreover, the loading efficiency of Pt and Fe in DCP@Pt@Fe was calculated to be 4.68 % and 8.34 %, respectively, based on standard curves established through inductively coupled plasma-mass spectrometry (ICP-MS) (Fig. S9). In addition, DCP@Pt@Fe displayed significant absorption in the range of 800–1200 nm (Fig. 1E), and intense NIR-II fluorescence centered at 1060 nm with a broad emission tail that extended up to 1400 nm (Fig. 1F). The fluorescence quantum yield of DCP@Pt@Fe was precisely determined to be 0.87 % (Fig. S10). Notably, the fluorescence intensity showed a positive correlation with the concentration of DCP@Pt@Fe (Fig. 1G and H). The photothermal properties of DCP, DCP@Pt, DCP@Fe and DCP@Pt@Fe were systematically evaluated under 1064 nm laser illumination at an intensity of 1.0 W cm⁻² for 5 min. The results showed that incorporation of Pt and Fe exerted a negligible effect on the photothermal performance of the NPs (Fig. S11). The temperature of the aqueous solution elevated in a concentration-dependent manner (Fig. 1I; Figure S11A-S11C). Specifically, 100 μg mL⁻¹ DCP@Pt@Fe surged to 53.4 °C within 5 min irradiation (Fig. 1I; Figure S12). The photothermal conversion efficiency (η) of DCP@Pt@Fe was calculated as 65.12 % (Fig. S13), which was comparable to those of DCP, DCP@Pt and DCP@Fe, at 62.54 %, 63.76 % and 64.56 % respectively (Figure S11D-S11F). Furthermore, the photothermal stability of DCP, DCP@Pt, DCP@Fe and DCP@Pt@Fe was meticulously assessed by five cycles of on-off irradiation at 1064 nm, demonstrating remarkable thermal consistency with temperature fluctuations below 1 °C (Fig. 1J; Figure S11G-S11I).

We further investigated the thermo- and acid-responsive release profiles of DCP@Pt@Fe utilizing ICP-MS under various conditions. As shown in Fig. 1K, there was negligible release of Pt after 24 h at pH 7.4, indicating DCP@Pt@Fe remains stable under neutral conditions. In contrast, the release rate of Pt significantly increased at pH 5.5, implying an accelerated dissociation of the nanoparticles in acidic environments. Moreover, under 1064 nm laser irradiation, over 70 % of Pt was released at pH 5.5, demonstrating that hyperthermia could further promote the leakage of DCP@Pt@Fe. The release profile of Fe from DCP@Pt@Fe exhibited a pattern similar to that observed for Pt (Fig. 1L). These results substantiated that hyperthermia and acid conditions synergistically facilitated the decomposition of DCP@Pt@Fe. Fenton reactions are crucial for inducing ferroptosis in cancer cells. Herein, we assessed the catalytic capacity of DCP@Pt@Fe to decompose H₂O₂ into reactive hydroxyl radicals (·OH). Electron spin resonance (ESR) tests revealed distinct ·OH signals in the presence of DCP@Pt@Fe and H₂O₂. Notably, 1064 nm laser irradiation markedly augmented these signals (Fig. 1M), suggesting that hyperthermia accelerated the efficiency of Fenton reactions. These above findings support the view that DCP@Pt@Fe maintains stable within normal tissues with a neutral pH, while dissociates drastically and generates ·OH within the acidic TME under 1064 nm laser irradiation.

2.2. DCP@Pt@Fe induces cancer cell ferroptosis in vitro

Initially, the anticancer efficacy of DCP@Pt@Fe at diverse concentrations (0, 6.25, 12.5, 25, 50 or 100 μg mL⁻¹) was assessed in ID8 cells using the CCK-8 assay (Fig. 2A). The findings revealed that treatment with DCP alone exerted no considerable cytotoxicity across all tested concentrations, as confirmed by over 95 % cell viability. Conversely, when combined with 1064 nm laser irradiation (1.0 W cm⁻², 5 min), DCP treatment (100 μg mL⁻¹) induced significant cell death, with cell viability decreasing to 59.25 %, underscoring the effective cancer-killing activity mediated by NIR-II laser-inspired PTT. The DCP@Pt group and DCP@Fe group displayed reduced cell viabilities of 58.64 % and 56.38 %, respectively, at 100 μg mL⁻¹. Notably, ID8 cells treated with the integrated nanoplatform DCP@Pt@Fe exhibited significantly lower cell viability than those exposed to the combination of DCP@Pt and DCP@Fe. Most impressively, when DCP@Pt@Fe (100 μg mL⁻¹) was combined with NIR-II laser irradiation (1.0 W cm⁻², 5 min), cell viability dramatically decreased to 15.82 %. Subsequently, the nanoparticle concentration of 100 μg mL⁻¹ and the 1064 nm laser at 1.0 W cm⁻² (5 min) were selected for subsequent in vitro investigations. Calcein-AM/PI dual staining assays further confirmed that ID8 cells treated with DCP@Pt@Fe exhibited more red fluorescence-labeled dead cells than those in the DCP@Pt + DCP@Fe group. Maximum cytotoxicity occurred in the DCP@Pt@Fe + L group, with the highest dead cell ratio exceeding 90 % (Fig. 2B and C). These findings indicate that the therapeutic performance of the integrated nanoplatform DCP@Pt@Fe is superior to the combinational regimen of DCP@Pt and DCP@Fe, which could be attributed to its pharmacokinetic consistency and synergistic advantages. Moreover, the combination of DCP@Pt@Fe with NIR-II laser irradiation markedly enhances anticancer potency through the co-delivery of Pt and Fe and in situ NIR-II PTT.

Fig. 2.

DCP@Pt@Fe induces cancer cell ferroptosis in vitro. (A) Viability of ID8 cells exposed to DCP, DCP@Pt, DCP@Fe, DCP@Pt + DCP@Fe, DCP@Pt@Fe at different concentrations (0, 6.25, 12.5, 25, 50 or 100 μg mL⁻¹) with or without 1064 nm laser illumination (1.0 W cm⁻², 5 min) (n = 3). (B–C) Calcein/PI staining (B) and PI⁺ cell proportion (C) of ID8 treated with different nanoparticles (100 μg mL⁻¹) with or without 1064 nm laser irradiation (1.0 W cm⁻², 5 min) (n = 3). (D–E) Intracellular Fe²⁺ staining using FerroOrange (D) and relative fluorescence intensity (E) in ID8 cells (n = 3). (F) GSH contents of ID8 cells treated with different interventions (n = 3). (G–H) Intracellular ROS levels (G) and quantification (H) in ID8 cells detected by flow cytometry using DCFH-DA (n = 3). (I–J) Intracellular LPO levels (I) and quantification (J) in ID8 cells detected by flow cytometry using BODIPY™ 581/591C11 (n = 3). (K) MDA contents of ID8 cells treated with different interventions (n = 3). (L) Protein expression of GPX4 and HSP70 in ID8 cells detected by Western blot. (M) Schematic illustration of ferroptosis induction by DCP@Pt@Fe. Data are represented as mean ± SD.

Ferroptosis is an iron ion-driven regulated cell death. Therefore, adequate intracellular Fe²⁺ is imperative for the initiation of ferroptosis [13,37]. As shown in Fig. 2D and E, the red fluorescence of Fe²⁺ was faint in ID8 cells treated with PBS, DCP, DCP + L or DCP@Pt. The intracellular Fe²⁺ levels greatly elevated in the DCP@Fe, DCP@Pt + DCP@Fe and DCP@Pt@Fe groups, whereas the DCP@Pt@Fe + L group displayed the most pronounced red fluorescence, indicating the highest content of intracellular Fe²⁺ accumulation, which may be attributed to NIR-II laser irradiation enhancing Fe²⁺ release. GSH serves as a powerful endogenous antioxidant and possesses the capability to scavenge ROS to avoid ferroptosis [22]. Hence, GSH depletion impairs antioxidant capacity, leading to an accumulation of LPO, thereby facilitating ferroptosis in cancer cells. We further evaluated GSH-depleting capacity of DCP@Pt@Fe. Due to the redox imbalance induced by PTT [[38], [39], [40], [41]], GSH levels decreased in the DCP + L group. A reduction in GSH content was observed in ID8 cells treated with DCP@Pt, owing to Pt-GSH complex formation through coordination between Pt and thiol group of GSH [[24], [25], [26]]. DCP@Fe treatment also diminished GSH levels due to Fe²⁺-induced Fenton reactions. Notably, there has a substantial GSH consumption in ID8 cells treated with DCP@Pt@Fe and 1064 nm laser, revealing the robust GSH-depleting efficacy (Fig. 2F).

The accumulation of LPO induced by excessive ROS is the hallmark of ferroptosis. Typically, the LPO cascade processes involve 3 stages. Initially, ROS attack cellular polyunsaturated fatty acids, leading to the generation of lipid free radicals. Next, with the assistance of oxygen, lipid free radicals transform into lipid peroxy radicals. Finally, lipid peroxy radicals break down to yield reactive aldehydes, such as malondialdehyde (MDA) [42]. As illustrated in Fig. 2G and H, ROS level was extremely low in the PBS and DCP groups. Following treatment with DCP + L, DCP@Pt, DCP@Fe, DCP@Pt + DCP@Fe or DCP@Pt@Fe, ID8 cells exhibited a marked elevation in ROS level. Under 1064 nm laser irradiation, DCP@Pt@Fe further enhanced ROS production in ID8 cells. Subsequently, LPO generation in ID8 cells was monitored using the LPO-specific probe, BODIPY™ 581/591C11. The results showed that PBS and DCP-treated cells displayed minimal LPO content, while significantly increased levels of LPO were observed in those treated with DCP + L, DCP@Pt, DCP@Fe, DCP@Pt + DCP@Fe and DCP@Pt@Fe. Notably, cells in the DCP@Pt@Fe + L group exhibited the greatest extent of LPO accumulation, which was attributed to synergistic effects of PTT and Fenton reactions (Fig. 2I and J). In addition, quantification of MDA, the lipid peroxidation byproducts, indicated that cells in the DCP@Pt@Fe + L group produced superior amounts of MDA in comparison to all other groups (Fig. 2K). Moreover, the expression of GPX4, an essential inhibitor of ferroptosis, was markedly suppressed in cells subjected to DCP@Pt@Fe and NIR-II laser irradiation (Fig. 2L; Figure S14A). The above results confirm that under NIR-II laser irradiation, DCP@Pt@Fe provokes significant LPO accumulation and triggers efficient ferroptosis.

Moreover, heat shock proteins (HSPs), a crucial component of the cellular self-protection system, are activated by hyperthermia and greatly impair the effectiveness of PTT. Fortunately, ROS and LPO have demonstrated the ability to degrade HSPs. LPO produce aldehydes that facilitate cross-linking with protein residues to compromise their structural integrity and biological function [43]. In addition, ROS can also engage in reactions with proteins to form similar cross-links [28,44]. Therefore, we investigated the modulatory effect of ferroptosis on PTT by assessing HSP70 protein expression in ID8 cells. As shown in Fig. 2L and Fig. S14B, cells treated with DCP + L exhibited markedly elevated levels of HSP70, indicating hyperthermia indeed enhanced HSP70 expression. While under 1064 nm laser irradiation, DCP@Pt@Fe effectively neutralized HSP70 expression, confirming the cleavage action exerted by ROS and LPO on HSP70 during ferroptosis. These results suggest that ferroptosis can suppress the expression of HSPs, offering a potential strategy to boost PTT efficacy. The above findings provide substantial evidence that under NIR-II laser irradiation, DCP@Pt@Fe effectively consumes GSH and generates ROS, thereby inducing ferroptosis in cancer cells. Moreover, ferroptosis and PTT are mutually reinforced, synergistically enhancing anti-tumoral efficacy (Fig. 2M).

2.3. DCP@Pt@Fe elicits immunogenicity of cancer cells and modulates immunity in vitro

To comprehensively explore the genomic changes, RNA-seq was conducted to map the genomic landscape in ID8 cells treated with DCP@Pt@Fe (100 μg mL⁻¹) and 1064 nm laser (1.0 W cm⁻², 5 min). The density distribution plot and box plot of gene expression supported the reliability of the RNA-seq data (Figs. S15 and S16). The principal component analysis (PCA) and correlation analysis indicated appropriate concordance among within-group deviations (Figs. S17 and S18). We established the criteria of FDR <0.05 and Fold change ≥1.5 to identify differentially expressed genes (DEGs). Totally, 3704 DEGs were detected, among which 1875 genes exhibited up-regulation, while 1829 genes demonstrated down-regulation in the DCP@Pt@Fe + L group (Fig. 3A; Fig. S19). The Gene Ontology (GO) results showed that DEGs were prominently involved in processes closely associated with ferroptosis, including cell survival (DNA replication, cell cycle, single-strand DNA helicase activity, DNA replication origin binding), heat stress (HSP70 protein binding, heat shock protein binding, response to heat), iron metabolism (cellular iron ion homeostasis, ion channel inhibitor activity), GSH metabolism (glutathione metabolic process, glutathione biosynthetic process, glutathione transferase activity, glutathione-cysteine ligase activity), redox homeostasis (oxidoreductase activity, aldehyde oxidase activity), and lipid metabolism (unsaturated fatty acid biosynthetic process, cellular lipid metabolic process) (Fig. 3B). Gene Set Enrichment Analysis (GSEA) results also revealed that processes related to DNA replication, cell cycle and mismatch repair were inhibited (Figure S20-S22), while processes linked to glutathione metabolism, oxidoreductase activity and cellular lipid metabolic process were activated (Figure S23-25). GSEA and Kyoto Encyclopedia of Genes and Genomes (KEGG) results further confirmed the activation of ferroptosis (Fig. 3C and D). In addition, KEGG results elucidated that DEGs were significantly enriched in the p53 signaling pathway, which is crucial for reinforcing ferroptosis (Fig. 3D) [45]. The RNA-seq results further confirm that DCP@Pt@Fe effectively induces ferroptosis in ID8 cells under NIR-II laser irradiation.

Fig. 3.

DCP@Pt@Fe elicits immunogenicity of cancer cells and modulates immunity in vitro. (A) Volcano plot showing DEGs in ID8 cells treated with PBS or DCP@Pt@Fe (100 μg mL⁻¹) + Laser (1064 nm, 1.0 W cm⁻², 5 min). (B) GO enrichment analysis of DEGs in ID8 cells. (C) GSEA results of Ferroptosis. (D) KEGG analysis of DEGs. (E) Heat map of DEGs involved in Ferroptosis, Glutathione metabolism, Peroxisome, and Antigen processing and presentation. (F) Immunofluorescence staining for CRT and HMGB1 in ID8 cells. (G) Relative fluorescence intensity of CRT in ID8 cells (n = 3). (H) Relative fluorescence intensity of HMGB1 in ID8 cells (n = 3). (I) Schematic illustration showing DCP@Pt@Fe-induced ICD. Data are represented as mean ± SD.

Cancer cells generally escape immune surveillance through reducing the expression of key molecules required for antigen presentation [7]. Ferroptotic cells exhibit high immunogenicity wherein tumor-associated antigens are easily recognized and captured by APCs [14]. Surprisingly, the antigen processing and presentation pathway was identified in ID8 cells treated with DCP@Pt@Fe and 1064 nm laser (Fig. 3D). GO enrichment analysis indicated that cytokine biosynthetic process was upregulated (Fig. 3B). KEGG analysis also revealed enriched pathways related to immunity (TGF-beta signaling pathway, NF-kappa B signaling pathway, TNF signaling pathway) and cellular communication (adherens junction, ECM-receptor interaction, tight junction) (Fig. 3D). Notably, a substantial elevation was observed in the expression of immune-activator molecules, such as Hspa8, Hspa4, Hsp90aa1, Cd74, Tap1, Hspa1b, and Hspa1a (Fig. 3E), suggesting an enhanced immunogenicity of ID8 cells after treatment with DCP@Pt@Fe and 1064 nm laser. These findings suggest that under NIR-II laser irradiation, DCP@Pt@Fe triggers ICD and augments immunogenicity of ID8 cells.

Cancer cells undergoing ferroptosis have been demonstrated to elicit robust ICD effects, which are essential for initiating immune responses against tumor progression [14]. Based on the RNA-seq results, we further validated the ICD and immunoregulatory effects of ID8 cells treated with DCP@Pt@Fe and NIR-II laser. ICD is characterized by the exposure of DAMPs, including CRT and HMGB1 [[46], [47], [48]]. As illustrated in Fig. 3F and G, the green fluorescence of CRT was hardly detectable in PBS and DCP groups. Cells in DCP + L, DCP@Pt, DCP@Fe and DCP@Pt@Fe groups displayed obvious green fluorescence, indicating CRT exposure. DCP@Pt@Fe and 1064 nm laser-treated cells exhibited the highest level of CRT expression on their membranes. Similarly, DCP@Pt@Fe and 1064 nm laser treatment triggered the strongest release of HMGB1 from dying cell nuclei (Fig. 3F and H), showing a robust ICD induction effect.

Next, we established an in vitro co-culture system comprising murine macrophage RAW264.7 and ID8 cells to investigate the immunomodulatory capacity. TAMs are among the predominant tumoral infiltrating immune cell populations and are typically categorized into two functionally distinct subtypes, known as classically activated M1 macrophages (M1-TAMs) and alternatively activated M2 macrophages (M2-TAMs). Usually, M1-TAMs recognize and engulf cancer cells, exerting anti-tumoral capabilities. Conversely, M2-TAMs are involved in immunosuppression and angiogenesis, contributing to tumor progression. Therefore, induction of macrophage M1 polarization promises to be an effective strategy for tumor elimination [49,50]. As shown in Fig. S26, the proportion of M1-TAMs (F4/80⁺CD86⁺) was only 4.43 % and 4.41 % in PBS and DCP groups, respectively. DCP + L, DCP@Pt, DCP@Fe or DCP@Pt@Fe treatment significantly elevated M1-TAMs proportion to 37.97 %, 37.67 %, 38.70 % and 57.60 %, respectively. Notably, the DCP@Pt@Fe + L group had the highest proportion of M1-TAMs, up to 74.37 %, fully exhibiting a significant capacity to promote macrophage M1 polarization. The concentration of nitric oxide (NO) was well consistent with the result of M1-TAMs (Fig. S27). These findings present compelling evidence that under NIR-II laser irradiation, DCP@Pt@Fe nanoparticles proficiently induce ferroptosis and enhance the immunogenicity of ID8 cells, thereby modulating the anti-tumoral immunity (Fig. 3I). This dual mechanism establishes a solid foundation for cascaded NIR-II PTT/ferroptosis/immunotherapy in ovarian cancer treatment in vivo.

2.4. Biodistribution and photothermal properties of DCP@Pt@Fe in tumor-bearing mice

Given the excellent anti-tumoral and subsequent immune modulation capabilities of DCP@Pt@Fe + L in vitro, the in vivo therapeutic efficacy was further investigated. The biodistribution profile of DCP@Pt@Fe was firstly evaluated in an ID8 tumor-bearing mouse model. Fig. 4A and B shows that NIR-II FL signals of DCP@Pt@Fe at the tumor site can be obviously observed 6 h post tail vein injection. These signals progressively increased over time, and peaked at 24 h after injection. At this time point, the NIR-II FL intensity was 8.15-fold higher than that prior to injection, indicating efficient accumulation of DCP@Pt@Fe in tumors via the enhanced permeability and retention (EPR) effects. Therefore, the best time of laser irradiation was determined to be 24 h after intravenous injection. Meanwhile, NIR-II fluorescence imaging of excised tumor and major organs was performed 24 h post-injection. The ex vivo results showed that the fluorescence signals in the tumor were significant, further indicating successful accumulation in the tumor site. Due to their metabolic action, DCP@Pt@Fe also exhibited higher accumulation in the liver and spleen, but lower deposition in the heart, lung and kidney (Fig. S28). Additionally, the contents of Pt and Fe in tumor and major organs were quantified 24 h post-injection using ICP-MS, and the results were consistent with those observed in the NIR-II fluorescence imaging (Fig. S29). Furthermore, the photothermal properties of DCP@Pt@Fe were systematically assessed 24 h post intravenous injection. Under 1064 nm laser irradiation (1.0 W cm⁻²), real-time infrared thermography documented a gradual temperature increase at the tumor site, with a peak of 53.7 °C at approximately 5 min. While no significant temperature increase was observed in tumor-bearing mice treated with PBS (Fig. 4C and D). These findings indicate that DCP@Pt@Fe is capable of accumulating at the tumor site and exhibit favorable photothermal properties, offering great promise for in vivo tumor treatment.

Fig. 4.

Biodistribution and photothermal properties of DCP@Pt@Fe in tumor-bearing mice. (A) NIR-II fluorescence imaging in ID8 tumor-bearing mice (red circle) after intravenous injection with DCP@Pt@Fe (ex: 808 nm, 1064 nm LP filter). (B) Variation curves of FL intensity at the tumor region within 36 h (n = 3). (C) NIR-II thermograms in tumor-bearing mice (white circle) upon 1064 nm laser exposure (1.0 W cm⁻²). (D) Temperature change profile at the tumor region within 5 min (n = 3). Data are represented as mean ± SD. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

2.5. DCP@Pt@Fe promotes ferroptosis of cancer cells and potentiates tumor regression in vivo

Motivated by its remarkable in vitro cytotoxicity, tumor-targeting performance and desirable photothermal properties, we further investigated the anti-tumoral potential of DCP@Pt@Fe in an ID8 tumor-bearing mouse model. Fig. 5A illustrates the entire process of murine model construction, treatment regimen implementation and therapeutic efficacy evaluation. 7 days following ID8 cell inoculation, animals were randomly assigned to six experimental groups: (I) PBS, (II) DCP + L, (III) DCP@Pt, (IV) DCP@Fe, (V) DCP@Pt@Fe, (VI) DCP@Pt@Fe + L, and corresponding treatments were administered. Specifically, the tumor volume in the PBS group continued to increase sharply, while the DCP + L, DCP@Pt, DCP@Fe and DCP@Pt@Fe groups exhibited a relatively moderate increase. Compared with these above groups, the DCP@Pt@Fe + L group demonstrated superior tumor regression, as evidenced by a gradual reduction in tumor volume (Fig. 5B and C). Fig. 5D and E presents the images and weights of tumor tissues resected from mice post therapeutic intervention, which revealed a trend consistent with that of tumor volume. Similarly, among all groups, DCP@Pt@Fe + L treatment caused the most severe damage to tumor tissues evaluated by H&E and TUNEL assays (Fig. 5F and G), confirming its strongest anti-tumoral efficiency. In addition, the expression of GPX4 within tumors was significantly reduced in mice treated with DCP@Pt@Fe and NIR-II laser (Fig. S30). Furthermore, substantial fluorescence was detected in the DCP@Pt@Fe + L group by flow cytometry after labeling with BODIPY™ 581/591C11, further proving the superior anti-tumoral activity attributed to LPO accumulation-induced ferroptosis (Fig. 5H and I).

Fig. 5.

DCP@Pt@Fe promotes ferroptosis of cancer cells and potentiates tumor regression in vivo. (A) Schematic diagram illustrating establishment and intervention of the ID8 tumor-bearing mouse model. (B) The volume of tumors in mice following various interventions, including (I) PBS; (II) intravenous injection with DCP on days 0 and 2, followed by exposure to a laser with 1064 nm (1.0 W cm⁻², 5 min) on days 1 and 3; (III) intravenous injection with DCP@Pt on days 0 and 2; (IV) intravenous injection with DCP@Fe on days 0 and 2; (V) intravenous injection with DCP@Pt@Fe on days 0 and 2; (VI) intravenous injection with DCP@Pt@Fe on days 0 and 2, followed by exposure to a laser with 1064 nm (1.0 W cm⁻², 5 min) on days 1 and 3. (C) Curves represent tumor growth throughout the whole therapeutic period (n = 5). (D) Digital photos of ex vivo tumors. (E) The weight of ex vivo tumors (n = 5). (F) H&E and TUNEL staining images from tumor tissues. (G) Relative fluorescence intensity of TUNEL in tumors (n = 3). (H–I) LPO levels (H) and quantification (I) in tumor cells detected by flow cytometry using BODIPY™ 581/591C11 (n = 3). (J) Immunohistochemistry staining of CRT and HMGB1 in tumor sections. Data are represented as mean ± SD.

We further investigated the level of DAMPs in tumor tissues following different treatment modalities in vivo. Based on CRT staining images and semiquantitative analysis, CRT exposure increased by 2.41-, 2.70-, 2.96- and 4.91-fold in the DCP + L, DCP@Pt, DCP@Fe and DCP@Pt@Fe groups respectively, compared with that in the PBS group. Tumors treated with DCP@Pt@Fe and 1064 nm laser exhibited the most amount of CRT exposure, with 7.23-fold greater than the PBS group. Additionally, the release of HMGB1 from nuclei in tumor tissues was observed as similar to the in vitro data (Fig. 5J; Figure S31). CRT exposure and HMGB1 release of tumor tissues have proven that DCP@Pt@Fe and 1064 nm laser treatment can effectively enhance the ICD effect in vivo. The enhanced immunogenicity of tumor cells paved the way for the subsequent immunity activation. In addition, the mice maintained their body weight without significant changes throughout the treatment process (Fig. S32). Meanwhile, serum biochemical analysis and H&E staining of major organs also demonstrated the high tissue compatibility and biosafety of DCP@Pt@Fe nanoparticles (Figs. S33 and S34). It is ascribed to the cancer-specific ROS generation and externally regulated PTT.

2.6. DCP@Pt@Fe boosts anti-tumoral immunity in vivo

ICD, in which tumor cells are converted from non-immunogenic to immunogenic, mediates the anti-tumoral immune response. The released DAMPs perform as immunostimulatory signals upon cell death, enhance recognition of tumor antigens by APCs, and initiate anti-tumoral immune response [51,52]. Inspired by the prominent ICD induction effect of DCP@Pt@Fe under NIR-II laser illumination, the immune status was further assessed in vivo. TAMs constitute the most potent APCs in the TME and are crucial for modulating immunity. M1-TAMs typically exert anti-tumoral functions, mediating cytotoxicity to eliminate tumor cells; while M2-TAMs facilitate tumor growth through inhibiting T cell activity and promoting angiogenic processes. Both M1 and M2 macrophages possess a high degree of plasticity, enabling them to interconvert in response to TME changes or therapeutic interventions [53,54]. Herein, we evaluated the proportion of M1 and M2-TAMs in the tumor tissues. Flow cytometry analysis revealed that the percentage of M1-TAMs (CD45⁺CD11b⁺F4/80⁺CD86⁺) was only 19.93 % in the PBS group, while M1-TAMs proportion significantly elevated to 32.27 %, 32.73 %, 34.50 % and 41.80 % in the DCP + L, DCP@Pt, DCP@Fe and DCP@Pt@Fe groups respectively (Fig. 6A and B). Notably, the DCP@Pt@Fe + L treatment induced the highest proportion of M1-TAMs, up to 52.53 %. Furthermore, after treatment with DCP@Pt@Fe and NIR-II laser, the ratio of M1-TAMs/M2-TAMs robustly elevated from 1.39 % to 13.82 %, which was approximately 10-fold higher compared to the PBS group (Fig. S35). These results suggest that macrophages in the tumor tissue tend to be M1 polarized following treatment with DCP@Pt@Fe and NIR-II laser, thus reversing the immunosuppressive microenvironment.

Fig. 6.

DCP@Pt@Fe boosts anti-tumoral immunity in vivo. (A–B) Flow cytometry plots and proportion of mature DCs (CD80⁺CD86⁺) after gating on CD45⁺CD11c⁺ cells in TDLNs (n = 3). (C–D) Flow cytometry plots and proportion of CD4⁺ T cells (CD3⁺CD4⁺) after gating on CD45⁺ cells in the spleen (n = 3). (E–F) Flow cytometry plots and proportion of CD8⁺ T cells (CD3⁺CD8⁺) after gating on CD45⁺ cells in the spleen (n = 3). (G–H) Flow cytometry plots and proportion of CD4⁺ T cells (CD3⁺CD4⁺) after gating on CD45⁺ cells in tumors (n = 3). (I–J) Flow cytometry plots and proportion of CD8⁺ T cells (CD3⁺CD8⁺) after gating on CD45⁺ cells in tumors (n = 3). (K–L) Flow cytometry plots and proportion of M1 macrophages (CD86⁺) after gating on CD45⁺CD11b⁺F4/80⁺ cells in tumors (n = 3). (M) IFN-γ contents in tumors after different treatments (n = 3). (N) Immunohistochemistry staining of SLC7A11 in tumor sections after different treatments. (O) Schematic illustration showing immune responses induced by DCP@Pt@Fe in vivo. Data are represented as mean ± SD.

DCs represent another critical type of APCs, playing an indispensable role in sustaining T cell-mediated immune responses [[55], [56], [57]]. When tumor cells succumb to ICD, immature DCs (iDCs) phagocytose tumor debris and cleave captured antigens into peptide epitopes. As they migrate to tumor-draining lymph nodes (TDLNs), these DCs mature and present antigenic peptides to T cells, orchestrating effector T cells to destroy tumors [58,59]. Therefore, the maturation of DCs in the TDLNs was detected. Compared with the PBS group, the proportion of mature DCs (CD45⁺CD11c⁺CD80⁺CD86⁺) in TDLNs significantly increased from 30.03 % to 46.93 % in the DCP@Pt@Fe + L group (Fig. 6C and D). To assess DCs-mediated immunological activation, the infiltration of T lymphocytes within splenic tissue was examined. In comparison to the PBS group, the DCP@Pt@Fe + L group showed a 6.41-fold increase in the proportion of CD4⁺ T cells (CD45⁺CD3⁺CD4⁺) (Fig. 6E and F). Similarly, the percentage of CD8⁺ T cells (CD45⁺CD3⁺CD8⁺) elevated 1.59-, 1.72-, 1.73- and 3.25-fold in the DCP + L, DCP@Pt, DCP@Fe and DCP@Pt@Fe groups respectively, and robustly elevated 6.88-fold in the DCP@Pt@Fe + L group (Fig. 6G and H). Thus, DCP@Pt@Fe and NIR-II laser treatment triggers robust systemic immune responses in mice, providing a foundation for subsequent anti-tumoral immunity. Thereafter, the infiltration of T lymphocytes in tumors was further investigated. The proportion of CD4⁺ T cells elevated from 3.24 % to 11.07 % in DCP@Pt@Fe + L group compared with PBS group (Fig. 6I and J). Notably, CD8⁺ T cell populations dramatically expanded to 6.29 % in the DCP@Pt@Fe + L group, which exhibited 4.19-times increase compared to the PBS group (Fig. 6K and L), indicating that the combination of DCP@Pt@Fe and NIR-II laser treatment is conducive to promoting the expansion of T-cell clones. Furthermore, serum concentrations of inflammatory mediators such as IFN-γ and TNF-α were also determined. In comparison to other groups, IFN-γ and TNF-α levels were distinctly elevated in the DCP@Pt@Fe + L group (Fig. S36). The above findings suggest that the cooperative effect of ferroptosis and NIR-II PTT effectively activates anti-tumoral immunity and enhances the infiltration of T lymphocytes at tumor sites.

Previous studies have revealed that intra-tumoral secretion of IFN-γ by the tumor infiltrating immune cells can trigger ferroptosis of the cancer cells. Mechanistically, IFN-γ directly suppresses system Xc⁻ expression, restrains tumor cell cystine uptake, hinders the intracellular GSH synthesis, disrupts redox homeostasis, and thus triggers ferroptosis. Additionally, the expression level of system Xc⁻ has been proved to be negatively correlated with cancer patient outcomes [17]. Encouragingly, compared with the PBS group, the IFN-γ level was 1.75-, 1.77-, 1.75- and 2.40-fold higher in the DCP + L, DCP@Pt, DCP@Fe and DCP@Pt@Fe groups respectively, and dramatically elevated 3.31-fold in the DCP@Pt@Fe + L group (Fig. 6M). The level of SLC7A11, a functional subunit of system Xc⁻, was further estimated within tumor tissues. As shown in Fig. 6N and Fig. S37, DCP@Pt@Fe + L treatment potently inhibited the expression of endogenous SLC7A11, which sensitized tumor cells to ferroptosis. To further elucidate the role of IFN-γ in inhibiting SLC7A11 expression, we utilized IFN-γ recombinant protein to treat ID8 cells and subsequently assessed the expression levels of SLC7A11. Fig. S38 explicitly showed that IFN-γ treatment significantly reduced the protein expression of SLC7A11. These findings suggest that tumor infiltrating immune cells sensitize tumor cells to ferroptosis through secreting IFN-γ to inhibit SLC7A11 expression.

Collectively, the above results demonstrate that DCP@Pt@Fe with NIR-II laser irradiation effectively induces ferroptosis of tumor cells and augments cellular immunogenicity. This process reprograms TAMs toward an M1 phenotype, promotes DCs maturation, and facilitates T cell infiltration, thereby activating robust anti-tumoral immune response, and reversing the immunosuppressive microenvironment. Meanwhile, IFN-γ secreted by tumor-infiltrating immune cells inhibits system Xc⁻ expression, and sensitizes tumor cells to ferroptosis. Ultimately, the cascaded anti-tumoral therapy can be achieved by synergistic reinforcement of both ferroptosis and immune response (Fig. 6O).

3. Materials and methods

3.1. Preparation of DCP@Pt@Fe NPs

Initially, 1 mg of DCP was dissolved in 1 mL of methanol, 1 mg of cisplatin was dissolved in 1 mL of water, and 15 mg of DSPE-mPEG₂₀₀₀ was dissolved in 10 mL of water. Subsequently, the DCP and cisplatin solution were swiftly injected into the DSPE-mPEG₂₀₀₀ aqueous solution, followed by sonication for 3 min. Once nanoprecipitation occurred, methanol was eliminated using rotary evaporation under vacuum conditions at 40 °C. The resulting aqueous solution was filtered through a polyether sulfone (PES) syringe filter (0.8 μm, Micropore) to remove contaminants and large particles. Then, the solution was washed three times using a 30 K centrifugal filtration unit (Microwell) and centrifuged at 2500 rpm for 10 min. The final DCP@Pt NPs solution was stored at 4 °C for later use.

To minimize oxidation of Fe²⁺, we synthesized DCP@Pt@Fe via a sequential loading strategy of Pt prior to Fe²⁺, drawing on previously published studies [[60], [61], [62]]. Specifically, 0.5 mL of freshly prepared 0.1 M ferrous sulfate solution was added dropwise to 1.0 mL of the pre-fabricated DCP@Pt solution. The mixture was continuously stirred overnight, after which excess ferrous ions were removed through ultra-centrifugation at 2500 rpm for 25 min, followed by three rinses with water. The purified DCP@Pt@Fe NPs were then diluted, filtered through a syringe-driven filter, and the stock solution was obtained through ultra-centrifugation.

3.2. Cell culture

Murine-derived ovarian cancer cells ID8 were cultivated in DMEM medium containing 10 % fetal bovine serum and 1 % penicillin-streptomycin in a humidified 5 % CO₂ environment at 37 °C.

3.3. Calcein/PI staining

ID8 cells were seeded in 12-well plates and subjected to various treatments: (I) PBS, (II) DCP (100 μg mL⁻¹), (III) DCP (100 μg mL⁻¹) with 1064 nm laser irradiation (1.0 W cm⁻², 5 min), (IV) DCP@Pt (100 μg mL⁻¹), (V) DCP@Fe (100 μg mL⁻¹), (VI) DCP@Pt (100 μg mL⁻¹) and DCP@Fe (100 μg mL⁻¹), (VII) DCP@Pt@Fe (100 μg mL⁻¹), (VIII) DCP@Pt@Fe (100 μg mL⁻¹) with 1064 nm laser irradiation (1.0 W cm⁻², 5 min). Following 4 h of intervention, groups (III) and (VIII) received 1064 nm laser irradiation (1.0 W cm⁻²) for 5 min. After an additional 20 h incubation, live/dead cell staining was performed using Calcein AM and PI (37 °C, 30 min). Cellular imaging was conducted using a confocal microscope (ZEISS LSM 710, Germany).

3.4. Detection of intracellular Fe²⁺

Intracellular Fe²⁺ levels were assessed using the FerroOrange probe. ID8 cells receiving 24-h treatments were stained with 1 μM of FerroOrange for 30 min at 37 °C in the dark, and imaged via a confocal microscope (ZEISS LSM 710, Germany).

3.5. Measurement of ROS and LPO

Following 24 h of diverse treatments, ID8 cells were harvested. For the detection of intracellular ROS, cells were incubated with 10 μM of DCFH-DA solution for 30 min at 37 °C in dark. For the detection of intracellular lipid ROS, cells were incubated with 5 μM of BODIPY™ 581/591C11 solution for 60 min at 37 °C in dark. Fluorescence intensity measurements were performed on a flow cytometer (BDFACSMelody, USA).

3.6. Detection of macrophage polarization in vitro

To evaluate ICD-mediated macrophage polarization, pretreated ID8 cells were co-cultured with murine macrophage RAW264.7 cells in a transwell system for 24 h. RAW264.7 cells were then harvested and immunophenotyped using anti-F4/80-PE and anti-CD86-PE-Cy7 antibodies for 30 min at 4 °C (Table S1). Following Stain Buffer (FBS) washes, cells were analyzed by a flow cytometer (BD FACSMelody). Concurrently, culture supernatants were analyzed for NO concentration using a commercial assay kit (Beyotime, China).

3.7. Transcriptomic sequencing and analysis

ID8 cells treated with either PBS or DCP@Pt@Fe + Laser (100 μg mL⁻¹, 1064 nm laser irradiation with 1.0 W cm⁻² for 5 min) were harvested for transcriptomic analysis. Total RNA was isolated by Trizol reagent (Invitrogen, USA) for sequencing (Biomarker Technologies, Beijing, China). DEGs were identified using thresholds of Fold change ≥1.5 and FDR <0.05. Analyses including PCA, GO, KEGG and GSEA were carried out via BMKCloud (www.biocloud.net).

3.8. Animal experiments

Female C57BL/6 mice (6 weeks old) were obtained from Beijing HFK Bio-Technology.co.,LTD and housed under specific pathogen-free conditions. The animal experiments were authorized by the Ethics Committee of Zhengzhou University (2023-KY-0433-001). All of the animal studies complied with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

To establish tumor-bearing mice, 5 X 10⁶ ID8 cells were subcutaneously injected into the right flank of mice. Once tumors reached 80–100 mm³, mice were randomized into 6 groups: (I) PBS, (II) DCP (2 mg mL⁻¹, 150 μL) with 1064 nm laser irradiation (1.0 W cm⁻², 5 min), (III) DCP@Pt (2 mg mL⁻¹, 150 μL), (IV) DCP@Fe (2 mg mL⁻¹, 150 μL), (V) DCP@Pt@Fe (2 mg mL⁻¹, 150 μL), (VI) DCP@Pt@Fe (2 mg mL⁻¹, 150 μL) with 1064 nm laser irradiation (1.0 W cm⁻², 5 min). Nanoparticles were intravenously administered to mice. At 24 h post-injection, groups (II) and (VI) received 1064 nm laser irradiation (1.0 W cm⁻²) for 5 min. Tumor volume was monitored daily according to the formula: V = 0.5 X L X W². L and W represent the long and transverse diameters of the tumor respectively. After 9 days, the mice were euthanized for collection of tumors, serum and major organs.

3.9. Evaluation of immune response in vivo

To assess the maturation of DCs in TDLNs, lymph nodes were grinded and filtered through a 70 μm strainer to obtain the single cell suspension. These cells were first incubated with Fixable Viability Stain 780 (FVS780) to rule out dead cells, following by staining with anti-CD45-FITC, anti-CD11c-PE, anti-CD80-APC and anti-CD86-PE-Cy7 (Table S2).

To evaluate the proportion of T cells in the spleen, mouse spleens were processed similarly to obtain the single cell suspension. After FVS780 viability staining, cells were labeled with anti-CD45-FITC, anti-CD3-APC, anti-CD4-PE, anti-CD8-PerCP-Cy5.5 (Table S2).

To determine the proportion of T cells and macrophages in tumor tissues, tumor tissues were enzymatically digested with DNase I, Collagenase IV and hyaluronidase, and then filtered through a 70 μm strainer to obtain the single cell suspension. After FVS780 staining, cells were incubated with anti-CD45-FITC, anti-CD3-APC, anti-CD4-PE, anti-CD8-PerCP-Cy5.5, anti-CD11b-PerCP-Cy5.5, anti-F4/80-PE, anti-CD86-PE-Cy7, anti-CD206-APC (Table S2). All samples were analyzed using a flow cytometer (BD FACSMelody, USA).

IFN-γ and TNF-α levels were measured using the commercial ELISA kits (NeoBioscience, China) according to the manufacturer's protocol.

3.10. Statistical analysis

Statistical analyses were conducted using GraphPad Prism 7.0 software. Experiments were independently repeated at least 3 times, with results expressed as mean ± standard deviation (SD). one-way analysis of variance (ANOVA) was employed for multi-group comparisons. Significance levels were denoted as: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

4. Conclusion

In summary, a novel NIR-II thermo- and pH-sensitive nanoplatform (DCP@Pt@Fe) is meticulously constructed by utilizing conjugated polymers DCP to encapsulate Pt and Fe²⁺ for photothermal-enhanced ferroptosis and immunotherapy of ovarian cancer. The prepared nanoparticles exhibit outstanding NIR-II photothermal performance, NIR-II imaging tracking capacity, and controllable release property within the acidic TME. Our DCP@Pt@Fe obviously accumulates at the tumor sites and displays superior anti-tumoral activity. Notably, ferroptotic ID8 cells possess high immunogenicity, reprogram TAMs toward M1 phenotype, promote DCs maturation, and mobilize T cell activation, significantly reversing the immunosuppressive TME. Meantime, IFN-γ secreted by immune cells inhibits SLC7A11 expression in cancer cells, sensitizing them to ferroptosis. Together, this warrants our nanoplatform DCP@Pt@Fe to thoroughly eradicate ovarian cancer through synergistic NIR-II PTT/ferroptosis/immunotherapy, providing an exciting new avenue to the treatment of ovarian cancer.

CRediT authorship contribution statement

Yingying Chen: Writing – original draft, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization. Ying Sun: Resources, Methodology. Yuning Qiu: Software, Formal analysis. Mingfei Li: Methodology, Investigation. Pengfei Chen: Methodology, Conceptualization. Bijun Li: Visualization, Software. Xintong Cai: Visualization, Software. Pengfei Sun: Resources, Methodology, Conceptualization. Daifeng Li: Writing – review & editing, Validation, Funding acquisition, Formal analysis, Conceptualization. Ruixia Guo: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

Funding

This work was supported by the National Natural Science Foundation of China (82273229, 82102588), Clinical Medical Scientist of Henan Academy of Innovations in Medical Science (HNCMS20240059), Young Elite Scientists Sponsorship Program by CAST (2023QNRC001), Natural Science Foundation of Henan Province (232300421050, 242301420068), Henan Province Science and Technology Research Key Projects (SBGJ202302074), Henan Medical Science and Technology Research Program (LHGJ20240261).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Data availability

Data will be made available on request.