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Bio-nanocomplexes impair iron homeostasis to induce non-canonical ferroptosis in cancer cells

Journal of Nanobiotechnology volume 23, Article number: 121 (2025) Cite this article

Abstract

The targeted elevation of the labile iron pool (LIP) represents the most direct and effective strategy to induce ferroptosis in cancer cells. However, the efficiency of increasing LIP to induce ferroptosis via iron supplementation is controversial due to the iron homeostasis between LIP and storage iron pool, leading to poor effects and serious safety concerns. In this study, a bio-nanocomplex named AbDA-Lim, composed of albumin, polydopamine, and limonene, is prepared to promote LIP and induce non-canonical ferroptosis in cancer cells by destroying the iron balance. Albumin avidity drives AbDA-Lim entering cancer cells. Subsequently, the released polydopamine enhances the expression of HMOX1 to degrade haem and facilitate the transformation of Fe (III) to Fe (II). Meanwhile, limonene reduces glutathione (GSH) levels via inhibiting CBS, thereby, triggering the release of Fe (II) into LIP from its GSH-bound storage state. The augmentation of LIP ultimately triggers non-canonical ferroptosis in cancer cells. Furthermore, the photothermal property of polydopamine augments the synergistic anti-tumor efficiency of AbDA-Lim by incorporating photothermal therapy. This study presents a distinctive, cascading, and biotic strategy for promoting LIP non-canonically to induce ferroptosis.

Introduction

Ferrous iron (Fe(II)) triggers lipid peroxidation and generates reactive oxygen species (ROS) via the Fenton reaction, resulting in oxidative damages to cells, termed ferroptosis [1]. Cancer cells typically exhibit a high demand for iron to support rapid growth and metastasis, because iron plays a crucial role in biological processes such as DNA synthesis, cell proliferation and metabolic regulation [2]. In theory, the increased iron availability makes cancer cells more susceptible to ferroptosis [3]. However, cancer cells frequently regulate intracellular labile iron levels and circumvent ferroptosis by modulating the balance of iron uptake, utilization and storage [4]. Iron is predominantly stored in a sequestered form as Fe (III) or bound to GSH to prevent ROS production [5]. Moreover, when Fe (III) is converted into Fe (II), cancer cells utilize reductive GSH to bind with oxidative Fe (II), thus sequestering it in a status resistant to oxidative damage [6]. To initiate the Fenton reaction, Fe (II) must be released from its bound status and enter LIP [7, 8]. Therefore, enhancing LIP is a potent strategy to enhance ferroptosis vulnerability of cancer cells, and disturbing the Fe (II) homeostasis between LIP and storage pool is pivotal to promote LIP [9].

The majority of iron in the body exists as the form of haem [10]. Upon cellular uptake, haem is degraded by haem oxygenase 1 (HMOX1), leading to the transformation of Fe (III) into Fe (II) [7, 11]. Some studies have demonstrated that elevated levels or activity of HMOX1 could significantly augment LIP [12,13,14]. Feng et al. utilized metal-organic frameworks (MOF) to target HMOX1 for the introduction of ferroptosis [15]. However, the role of HMOX1 in ferroptosis is paradoxical, as Fe (II) liberated from haem is typically sequestered within ferritin as Fe (III) or bound to GSH rather than entering LIP [16, 17]. The Fenton reaction is triggered when GSH is decreased and Fe (II) is released from its storage status. Moreover, GSH serves as the primary substrate for glutathione peroxidase 4 (GPX4) in reducing phospholipid hydroperoxides (PLOOH) [18]. Reduced GSH levels impair the reducing activity of GPX4, leading to the accumulation of PLOOH and ferroptosis [19]. Therefore, elevating the LIP requires a coordinated effort to increase Fe (II) levels and release it from the storage iron pool.

To accomplish this objective, we utilize albumin nanotechnology [20, 21] to prepare a bio-nanocomplex named AbDA-Lim, which integrates limonene and polydopamine, initiating ferroptosis by elevating free Fe (II) levels within cancer cells (Scheme 1). Albumin serves as a primary nutrient source for proliferating cancer cells and a main carrier of cancer cells scavenging lipids [22, 23]. Therefore, albumin showed tumor-targeting properties. Through albumin, this nanocomplex can be transported to tumor. Limonene, a naturally compound with documented anti-cancer properties derived from plants, has been utilized in numerous clinical trials for cancer treatment (NCT05525260, NCT01046929, NCT01459172) [24, 25]. Transcriptomic analysis reveals a non-canonical mechanism underlying ferroptosis induced by AbDA-Lim. Polydopamine enhances HMOX1 expression, thereby elevating Fe (II) generation, while limonene reduces cystathionine beta synthase (CBS) level. CBS is a crucial enzyme in the transsulfuration pathway responsible for cystathionine production, a precursor for GSH synthesis [26]. Previous studies have demonstrated that the absence of CBS can lead to iron overload and ferroptosis induction [27, 28]. By inhibiting CBS, AbDA-Lim effectively reduces GSH levels and releases Fe (II) into LIP. Moreover, the high photothermal conversion ability of polydopamine [29] enhances the production of reactive oxygen species (ROS) during AbDA-Lim treatment. Due to the modulating ability of iron homeostasis, cancer cells are less susceptible to ferroptosis [4]. Thereby, the strategy of inducing ferroptosis through iron delivery typically requires a large amount of iron supplementation [30, 31], which can lead to iron overload (serum ferritin ≥ 1000 μg/L) and cause damages to normal tissues [32]. In contrast, AbDA-Lim is a biological nanocomplex to efficiently enhance LIP in a non-canonical manner without iron supply, which is safe. Moreover, cancer cells need more iron, implying that the iron-homeostasis destruction induced by AbDA-Lim would improve ferroptosis susceptibility of cancer cells.

Materials and methods

Materials

Anti-CBS rabbit pAb (14787-1-AP) and anti-HMOX1 rabbit pAb (10701-1-AP) were from Proteintech. Anti-GPX4 mAb (ab252833) was from Abcam. FerrOrange (F734) was obtained from DOJINDO. BODIPYâ„¢ 581/591 C11 (D3861) was purchased from Thermofisher. Reactive Oxygen Species Assay Kit (S0033S) and GSH and GSSG Assay Kit (S0053) were obtained from Beyotime.

Preparation and characterization of AbDA-Lim

AbDA-Lim was prepared using the method previously reported by us [20, 21]. In brief, 100 ml dopamine solution at a concentration of 1 mg/ml was heated and stirred to promote oxidation, forming polydopamine, followed by the addition of 20% albumin to achieve a final concentration of 0.1%. After stirring for 10 min, AbDA was obtained. Subsequently, 1 ml limonene solution (10 mg/ml in ethanol) was added to the mixture, which was then cooled, concentrated by ultrafiltration, and sterilized using a 0.22 μm filter membrane to obtain AbDA-Lim. The limonene concentration was determined using a Shimadzu LC-20 A HPLC system with an Agilent HC-C18(2) column (4.6 × 250 mm, 5 μm) and a mobile phase of 22% ACN:63% MeOH:15% water.

The particle size of AbDA-Lim was measured using Dynamic Light Scattering (DLS). DLS and potential were analyzed with a Brookhaven NanoBrook 90Plus Zeta Particle Size Analyzer. The morphological features of AbDA-Lim were evaluated using a transmission electron microscope (TEM) (JEM-2100 F Field Emission Electron Microscope).

Cell culture

Human SW620, MDA-MB-231, DU145, A549, BxPC3 and HepG2 cancer cell lines, and mouse B16 cancer cell line were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10% serum.

MTT assay

Cells were seeded in a 96-well plate at a density of 1 × 103 cells/well. After overnight adherence, the cells were treated with AbDA-Lim, AbDA, or Lim at different concentrations (0, 0.1, 1, 10, 20, 50, 100, 200, 500, 1000 μM) for 24 h. Followed by replenishment with medium containing0.5%MTT(3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di- phenytetrazoliumromide). About 2 h later, the medium was removed, 150 μl DMSO per well was added. The absorbance at 570 nm was measured using a Molecular Devices M3 reader to assess cell proliferation.

ROS or lipid ROS detection

Cells were seeded in a 6-well plate. The next day, AbDA-Lim, AbDA, or Lim was added for 24 h. Then, the cells were incubated with DCFH-DA or C11-BODIPY (581/591) for 30 min and analyzed using flow cytometry (FCM) or imaged using a confocal laser scanning microscope.

GSH assay

Cells were seeded in a 6-well plate and treated with 50μM AbDA-Lim, AbDA, or Lim. After 24 h, the intracellular GSH level was measured using a GSH and GSSG Assay Kit (S0053, Beyotime, China).

RNA-sequencing

Cells were treated with AbDA-Lim, AbDA, or Lim at a concentration of 50μM. After 24 h, RNA was extracted and sequenced at Annoroad, and the data were processed and analyzed using Galaxy platform [33] (https://usegalaxy.org/) and NetworkAnalyst [34] (https://www.networkanalyst.ca/NetworkAnalyst/).

Fe (II) assay

Cells were treated with AbDA-Lim, AbDA, or Lim at a concentration of 50 μM for 24 h, followed by staining with FerroOrange (F374, Dojindo Laboratories, Japan) and imaging using a FV3000 Confocal Laser Scanning Microscope. Ex/Em = 561 nm/570–620 nm.

Cystathionine (CTH) measurement

Cells were seeded into 10 mm culture dishes and treated with 50 μM AbDA-Lim, AbDA, or Lim. 24 h later, the medium was removed and cells were washed with PBS for 3 times. Cell sediments were treated with liquid nitrogen for 30s, followed by CTH extraction using 0.1% formic acid in water. CTH supernatant was freeze dried and the precipitate was collected for protein measurement.

Sample analysis was performed using an LC-MS/MS 8040 system (Shimadzu Corporation, Japan) equipped with an electrospray ionization source operating in positive ionization mode and multiple reaction monitoring (MRM) to achieve unit resolution.

Aliquots for Cystathionine analysis were reconstituted in a solution of 10% acetonitrile (ACN) with 0.1% formic acid (FA). A volume of 10 μL of the extract was injected onto a reversed-phase UPLC C18 column (2.1 mm × 100 mm × 1.8 μm, Shimadzu Corporation, Japan) maintained at room temperature. Mobile phase A consisted of water containing 0.1% FA, while mobile phase B was ACN. The flow rate was set at 0.25 mL/min, and the elution gradient was as follows: 0% B was initially maintained for 3 min, then linearly increased to 5% B from 3 to 5 min, increased to 100% B from 5 to 10 min, maintained at 100% B for 2 min, followed by equilibration at 0% B for 4.5 min.

The MRM detection parameters were as follows: nebulizer gas flow rate: 3.0 L/min; drying gas flow rate: 15 L/min; desolvation line temperature: 250 °C; heat block temperature: 400 °C. The MRM transitions for Cystathionine involved the precursor ion [M + H] + at 223 m/z and the characteristic product ions at 88 m/z and 134 m/z. Data collection and quantitation were performed using Lab Solutions LCMS Ver.5.89 (Shimadzu Corporation, Japan). Cystathionine standard substance was obtained from Yuanye (S20433, China).

Western blot

Cells were seeded in 6-well plates and incubated overnight. The cells were then treated with 50μM AbDA-Lim, AbDA, or Lim for 24 h. Afterward, the cells were collected and total cellular proteins were extracted using RIPA lysis buffer (P0013B, Beyotime, China) and quantified using the BCA method. 20–30 μg of protein was separated by SDS-PAGE electrophoresis and transferred onto a PVDF membrane. The PVDF membrane was blocked with 5% BSA in TBST at room temperature for 1 h, followed by overnight incubation with primary antibodies at 4 ℃. The next day, the PVDF membrane was washed three times with TBST for 5 min each time, and then incubated with HRP-coupled anti-mouse or rabbit secondary antibodies at room temperature for 1 h. Subsequently, the membrane was washed three times with TBST and visualized using the Ultrasensitive ECL Detection Kit (PK10003, Proteintech, China) and the Tanon 4600 chemiluminescence imaging system. The imaging results were analyzed using Image J software. The HO-1/HMOX1 Rabbit Polyclonal antibody (10701-1-AP), GPX4 Mouse Monoclonal antibody (67763-1-Ig), GAPDH Mouse Monoclonal antibody (60004-1-Ig), and Beta Actin Mouse Monoclonal antibody (60008-1-Ig) were purchased from Proteintech. The CBS Rabbit monoclonal [EPR8579] antibody was obtained from Abcam.

Network pharmacology

SMILES information of dopamine and limonene were extracted from PubChem and inputted into the SwissTargetPrediction (http://www.swisstargetprediction.ch) to obtain the potential target proteins for both compounds. Then, the interacting proteins of HMOX1 and CBS were screened in STRING database (https://cn.string-db.org). Finally, cross-referencing analysis of the interacting proteins with the potential targets of dopamine and limonene were performed.

Animal experiment

40 male nude mice aged 6–8 weeks were used to establish a tumor-bearing mouse model by inoculating SW620 colon cancer cells (2 × 105 cells). The animal experiment was conducted in accordance with the guidelines of the Affidavit of Approval of Animal Ethical and Welfare Committee of Beijing Shijitan Hospital (KYD-2024-0006-001). The tumor-bearing mice were divided into 7 groups: saline group (10 mice), Saline + IR group, AbDA group, AbDA + IR group, Lim group, AbDA-Lim group, and AbDA-Lim + IR group, with 5 mice in each group. The drugs were administered intravenously at a dose of 7.5 mg/kg for Lim. Two hours after injection, the mice were irradiated with 808 nm laser for 10 min at an intensity of 1.0 W/cm2, and the temperature before and after irradiation was recorded. The treatment was performed every 3 days, and the body weight and tumor volume of the mice were recorded. Tumor volume was calculated using the formula: (a × b2)/2, where a is the longest length and b is the shortest length [35]. At the end of the treatment, the mice were anesthetized and sacrificed, and the tumors and other normal tissues were collected for HE staining, CBS and HMOX1 immunohistochemistry, and FerroOrange staining.

Statistical analysis

Data analysis was performed using GraphPad Prism 8.4.3 software, and statistical tests including One-way ANOVA, two-way ANOVA, and paired Student’s t-tests were used for analysis. Specific details can be found in the corresponding figure legends.

Results

Preparation and characterization of AbDA-Lim

The AbDA-Lim nanoparticle was prepared utilizing our albumin-based nanotechnology method [20, 21]. In brief, dopamine was oxidated to form polydopamine, which then crosslinked with albumin (Scheme 1). Upon addition of D-Limonene (Lim), these components were self-assembled into nanocomplexes. Transmission electron microscopy (TEM) imaging showed a homogeneous spherical morphology of AbDA-Lim (Fig. 1a). The diameter of AbDA-Lim in ddH2O ranged from 60 to 100 nm with a zeta potential of -31 mv (Fig. 1b, c).

Scheme 1
scheme 1

Schematic of AbDA-Lim preparation and mechanism to enhance LIP and induce ferroptosis in tumors. Dopamine is first oxidized to polydopamine, which is then encapsulated together with limonene into albumin, forming AbDA-Lim. After AbDA-Lim is taken up by tumor cells, polydopamine limonene are released. Polydopamine increases HMOX1 expression to degrade haem and promote the conversion of Fe (III) to Fe (II). Limonene inhibits CBS expression, resulting in reduced GSH synthesis, thereby promoting the release of Fe (II) from storage into the labile iron pool (LIP). Finally, iron homeostasis is disrupted by AbDA-Lim, leading to ferroptosis in tumor cells

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AbDA-Lim suppresses proliferation of multiple cancer cells

Subsequently, we assessed the anticancer efficacy of AbDA-Lim in a panel of various cancer cell lines, including colon cancer cell SW620, breast cancer cell MDA-MB-231, melanoma cell B16, prostate cancer cell DU145, lung cancer A549, pancreatic cancer cell BxPC3 and hepatic cancer cell HepG2. Polydopamine-albumin conjugates (AbDA) and Lim alone served as controls. Our results demonstrated a concentration-dependent inhibition of proliferation by AbDA-Lim across all cancer cell lines, whereas the anticancer effects of Lim were observed only at a higher concentration, and AbDA exhibited no significant impact on cell proliferation (Fig. 1d-f, Fig. S1a-d). Consistent with our findings, polydopamine has been demonstrated to serve as a biocompatible carrier with minimal toxicity, and the anticancer efficacy of Lim has been extensively validated in various preclinical studies and clinical trials [24, 25].

Fig. 1
figure 1

AbDA-Lim inhibits growth of multiple cancer cells. (a) TEM characterization of the morphological features of AbDA-Lim. Scale bar, 1 μm. (b) Particle size analysis of AbDA-Lim via DLS. (c) Zeta potential of AbDA-Lim. (d-f) Effect of AbDA-Lim on proliferation of SW620, MDA-MB-231 and B16 cancer cells. Cells were subjected to the indicated treatments for 24 h before MTT analysis. n = 6. Two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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AbDA-Lim induces ferroptosis

To evaluate whether the inhibitory effect of AbDA-Lim on cancer cell proliferation is associated with ferroptosis, we initially assessed the content the level of reactive oxygen species (ROS) in cells using flow cytometry (FCM). Our results revealed a significant elevation in ROS levels within the AbDA-Lim-treated group compared to the control (Ctrl) group (Fig. 2a, b). Moreover, both AbDA and Lim individually elicited a modest increase in ROS levels (Fig. 2a, b, Fig. S2a-f), indicating a potential alteration in cellular redox status upon co-administration of polydopamine (DA) and Lim. Subsequently, we examined the intracellular level of GSH, which binds to Fe (II) within LIP and serves as a cofactor for GPX4-mediated lipid peroxidation [36]. Our results revealed a marked reduction in GSH levels following AbDA-Lim treatment across various cancer cell lines, suggesting a potential correlation with Fe (II) levels. Consistent with the observed increase in ferroptosis-related ROS, the GSH/GSSG ratio exhibited a simultaneous decrease. Furthermore, compared to the Ctrl group, the Lim group displayed varying degrees of reduction in both GSH levels and the GSH/GSSG ratio (Fig. 2c, d). Conversely, AbDA did not exert any discernible effect on GSH levels (Fig. 2c, d), suggesting that the influence of AbDA-Lim on GSH levels may primarily stem from Lim. Additionally, the attenuating effect of AbDA-Lim on GSH and the GSH/GSSG ratio was similarly evident in MDA-MB-231 and B16 cells (Fig. S2g-i).

To further validate the induction of ferroptosis by AbDA-Lim, we conducted C11-BODIPY staining on SW620 cells after treatments of Ctrl, AbDA, Lim and AbDA-Lim. FCM and confocal imaging revealed a notable increase in the oxidized form of C11-BODIPY (Fig. 2e-h), indicating lipid peroxidationt induced by the nanoparticles. Similar trends were observed in the AbDA and Lim groups, suggesting that both DA and Lim have the potential to initiate ferroptosis (Fig. 2e-h). Moreover, AbDA-Lim significantly elevated lipid ROS levels in MDA-MB-231, B16, and BxPC3 cells. However, the impact of AbDA and Lim on lipid ROS was less pronounced compared to SW620 cells (Fig. S3). This discrepancy could be attributed to the heterogeneity of tumor cells or the synergistic effect of AbDA and Lim in enhancing lipid ROS production. it may be due to the synergistic effect of AbDA and Lim in promoting lipid ROS. Furthermore, TEM was employed to assess submicroscopic morphological alterations in mitochondria. Following treatment with AbDA-Lim, we observed morphological aberrations in mitochondria, including reduction or absence of cristae and membrane rupture (Fig. 2i). These ultramicroscopic changes align with the characteristic features associated with ferroptosis development [37].

GPX4 is a major inhibitor of lipid reactive oxygen species and a pivotal regulator of ferroptosis [38, 39]. Consequently, we assessed GPX4 expression levels in tumor cells to elucidate its involvement in AbDA-Lim-induced ferroptosis. Western blot analysis revealed a notable decrease in GPX4 protein levels following treatment with AbDA-Lim (Fig. 2j, k), underscoring the nanocomplex’s induction of ferroptosis.

Fig. 2
figure 2

AbDA-Lim induces ferroptosis in cancer cells. (a) FCM analysis for ROS in SW620 cells subjected to indicated treatments for 24 h. (b) Quantification of mean fluorescence intensity (MFI) from (a). mean ± s.e.m. n = 3. One-way ANOVA. (c, d) GSH concentration and GSH/GSSG ratio. mean ± s.e.m. n = 6. One-way ANOVA. (e) FCM analysis for lipid ROS. (f) Quantification of MFI from (e). mean ± s.e.m. n = 3. One-way ANOVA. (g) Confocal imaging for lipid ROS in SW620 cells. (h) Semi-quantification of MFI from (g). mean ± s.e.m. n = 7. One-way ANOVA. (i) Representative TEM images for submicroscopic features of mitochondria. The arrow represents a typical aberrant mitochondrion undergone ferroptosis, including reduction or absence of cristae and membrane rupture. Scale bar, 500 nm. (j) Western blot showing the protein levels of GPX4. (k) Semi-quantification of protein levels from (j). mean ± s.e.m. n = 3. One-way ANOVA. In all the experiments, SW620 cells were subjected to indicated treatments for 24 h

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AbDA-Lim enhances LIP via increasing HMOX1 and inhibiting CBS

To elucidate the mechanism underlying AbDA-Lim-induced ferroptosis, we conducted transcriptomics analysis on SW620 cells treated with AbDA-Lim, AbDA, and Ctrl group. Principal component analysis (PCA) revealed distinct clustering patterns among the Ctrl, AbDA, and AbDA-Lim treatment groups (Fig. 3a). Comparative analysis identified 272 differentially expressed genes in the AbDA-Lim group compared to the Ctrl group (Fig. 3b). Gene set enrichment analysis (GSEA) of these differentially expressed genes revealed a significant enrichment of ferroptosis-related gene signatures in AbDA-Lim-treated cells (Fig. 3d, Fig. S4a), indicating the induction of cellular ferroptosis by AbDA-Lim. Besides, AbDA-Lim also induced genes abundance in peroxisomes (Fig. S4b and c), which promote ferroptosis by synthesizing substances for lipid peroxidation [40]. Notably, among these 272 differentially expressed genes, the most significantly altered gene was HMOX1 (Fig. 3b, e), which promotes the generation and release of Fe (II) into LIP by degrading haem [41]. Transcriptomic comparison between the AbDA-Lim and AbDA groups revealed 4 different genes, including CBS (Fig. 3c, f), a key enzyme involved in cystathionine production for GSH synthesis [42]. CBS also emerged as one of the top genes exhibiting significant differences between the AbDA-Lim and Ctrl groups (Fig. 3b, f). By inhibiting CBS, AbDA-Lim suppressed GSH generation, thereby facilitating the release of Fe (II) from its storage status into the LIP. These differences among AbDA-Lim, AbDA, and Ctrl groups suggest that limonene can inhibit CBS expression, while polydopamine can promote HMOX1 expression. Consequently, the upregulation of HMOX1 facilitates the transformation of haem Fe (III) in Fe (II), while the inhibition of CBS reduces GSH synthesis, thereby accelerating the liberation of Fe (II) into the LIP from its storage status.

To verify the effect of AbDA-Lim on HMOX1 and CBS expression, we performed western blotting to assess the corresponding protein levels. Elevated levels of HMOX1 were observed in the AbDA-Lim and AbDA groups compared to the Ctrl group, while no notable difference was found between the Lim and Ctrl groups (Fig. 3g, h). This suggests that HMOX1 expression can be modulated by polydopamine rather than Lim. As for CBS, a reduction in protein level was found in the Lim and AbDA-Lim groups (Fig. 3i, j), indicating that Lim can inhibit CBS expression. CBS serves as a pivotal enzyme in the transsulfuration pathway, which plays a crucial role in GSH synthesis [26]. The Inhibition of CBS leads to diminished GSH levels [43]. To further validate the inhibitory effect of AbDA-Lim on CBS, the levels of cystathionine, the CBS-catalyzed product, were quantified in cells following different treatments using LC-MS. After 24 h of treatment, both Lim and AbDA-Lim significantly reduced the level of cystathionine compared to the Ctrl and AbDA groups (Fig. 3k), indicating the suppression of the CBS-mediated GSH synthesis pathway. The reduction of GSH leads to a decline in GPX4 activity, thereby promoting the release of Fe (II) from its complex with GSH into the LIP and initiating the Fenton reaction.

Next, to assess the LIP-promoting effect of AbDA-Lim, we measured the intracellular Fe (II) content using a Fe (II) probe, FerroOrange, and confocal laser scanning microscopy (CLSM). Compared to the Ctrl group, cells treated with AbDA, Lim, and AbDA-Lim showed varying degrees of increased fluorescence intensity, while the AbDA-Lim group exhibited an obviously higher mean fluorescence intensity (MFI) compared to AbDA and Lim (Fig. 3l, m). This indicates that AbDA-Lim does indeed increase the level of free Fe (II). These findings indicate that AbDA-Lim uniquely elevates cellular LIP levels by upregulating HMOX1 and inhibiting CBS, representing a non-canonical mechanism for inducing ferroptosis.

To explore how dopamine and limonene affect the expression of HMOX1 and CBS, we retrieved potential binding targets of both compounds from the SwissTargetPrediction database. Among them, the strongest interaction with dopamine was found with Lysine Demethylase 4E (KDM4E), an iron-dependent enzyme [44]. Dopamine could serve as an enzyme inhibitor by preventing the binding of Fe (II) to the active site of KDM4E [45]. The expression of hypoxia-induced genes like HMOX1 is dependent on KDM4E activity [46], and decreased KDM4E activity leads to histone hypermethylation, resulting in increased expression of HMOX1 [47]. This suggests that AbDA-Lim may promote HMOX1 upregulation by inhibiting KDM4E activity. According to SwissTargetPrediction, limonene showed the strongest potential interaction with Peroxisome Proliferator-Activated Receptor Alpha (PPARα) [48]. In consistent with our findings, various studies have proved the activating effect of limonene on PPARα [49] PPARα activations can inhibit VEGF expression [50], which directly regulates CBS expression [51]. Therefore, the inhibitory effect of AbDA-Lim on CBS expression may be related to limonene’s agonistic action on PPARα.

Fig. 3
figure 3

AbDA-Lim induces ferroptosis by promoting HMOX1 and inhibiting CBS expression. (a) Principal-component analysis (PCA) for RNA-seq analysis in SW620 cells treated with Ctrl, AbDA and AbDA-Lim. (b, c) Volcano plots depicting significant differences in gene expression between AbDA-Lim and Ctrl, as well as between AbDA-Lim and AbDA groups. (d) GSEA analysis showing abundance of ferroptosis related gene signatures after AbDA-Lim treatment. (e, f) Relative mRNA levels of HMOX1 and CBS. mean ± s.e.m. n = 3. Paired two-tailed Student’s t-test. (g) Representative Western blot showing expression of HMOX1. (h) Semi-quantification of HMOX1 levels from (g) using GAPDH as control. mean ± s.e.m. n = 3. One-way ANOVA. (i) Representative Western blot showing expression of CBS. (j) Semi-quantification of CBS levels from (i) using GAPDH as control. mean ± s.e.m. n = 3. One-way ANOVA. (k) Cystathionine concentration evaluation using LC-MS/MS. mean ± s.e.m. n = 3. One-way ANOVA. (l) Fluorescence imaging of cells with FerroOrange staining. (m) Semi-quantification of MFI from (l). mean ± s.e.m. n = 6. One-way ANOVA. In all the experiments, SW620 cells were subjected to indicated treatments for 24 h

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AbDA-Lim initiates ferroptosis to suppress tumour growth

We assessed the therapeutic efficacy of AbDA-Lim in SW620 xenograft nude mice. The drug was intravenously administered every three days, with saline, AbDA, and limonene serving as controls (Fig. 4a). At the conclusion of the treatment, the tumor volumes in the AbDA-Lim group were significantly smaller than those in the other groups, indicating a pronounced anti-tumor effect (Fig. 4b, c, Fig. S6a). Tunel staining of tumor tissues revealed varying degrees of positive results in the Lim group and AbDA-Lim group, further confirming the anti-tumor effect of AbDA-Lim (Fig. S5). H&E staining demonstrated that AbDA-Lim did not cause significant damage to normal tissues (Fig. S6b), and there were no apparent changes in mouse body weight (Fig. S6c), suggesting favorable safety of AbDA-Lim. Subsequent ROS staining of tumor tissues showed a significant increase in ROS levels in the AbDA-Lim group, indicating oxidative damage induced by AbDA-Lim (Fig. S7).

In vitro experiments confirmed that AbDA-Lim induces ferroptosis by augmenting LIP. Therefore, we further evaluated the Fe (II) levels in tumors using FerroOrange staining. Compared to the Ctrl group, free Fe (II) levels ascended by 6.53, 5.3, and 18.68 folds in the AbDA, Lim, and AbDA-Lim groups, respectively (Fig. 4d, e). These changes are consistent with the in vitro results. Subsequently, we performed immunohistochemical detection and quantitative analysis of CBS and HMOX1 in tumor tissue sections. AbDA-Lim and AbDA groups showed a 25-fold and 26-fold increase in HMOX1 expression in tumor tissues compared to the Ctrl group, while the Lim group exhibited no distinct change (Fig. 4f, g), suggesting that AbDA-Lim and AbDA enhanced the generation of Fe (II). CBS levels in tumors were reduced by 58.39% and 78.87% in the Lim and AbDA-Lim groups, respectively (Fig. 4h, i), indicating obvious inhibition of CBS expression and a decrease in cystathionine generation. These findings were consistent with the results of RNA-seq and western blotting. In summary, these results indicate that AbDA-Lim induces tumor ferroptosis in a non-canonical manner by inhibiting CBS and increasing HMOX1 levels.

Next, the safety of AbDA-Lim was evaluated via H&E staining on normal tissues from mice subjected to different treatments. The results showed that AbDA-Lim did not cause significant toxicity to normal tissues such as the heart, liver, spleen, lung, and kidney (Fig. S8a). Besides, we further measured the levels of biochemical markers such as AST, ALT, ALP and TBIL in the plasma of mice after AbDA-Lim treatment. The results showed that AbDA-Lim did not cause significantly effect on these biochemical markers compared to the control group (Fig S8b-d), indicating that AbDA-Lim has favorable biocompatibility.

Fig. 4
figure 4

AbDA-Lim induces tumor ferroptosis in mice. (a) Animal experimental design for in vivo research. (b) Tumors from euthanized mice at the end of treatment. (c) Tumor growth kinetics in SW620 implanted mice treated with saline, AbDA, Lim and AbDA-Lim. n = 5. Mean ± s.e.m. Statistical analysis was conducted by two-way ANOVA. (d) Fluorescence imaging for FerroOrange staining in tumors under indicated treatments. (e) Semi-quantification of MFI from (d). mean ± s.e.m. n = 5. One-way ANOVA. (f) Immunohistochemical staining of HMOX1. (g) Semi-quantification results of HMOX1-positive cells. Mean ± s.e.m. n = 5. One-way ANOVA. (h) Immunohistochemical staining of CBS. (i) Semi-quantification results of CBS-positive cells. Mean ± s.e.m. n = 5. One-way ANOVA

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AbDA-Lim serves as a photosensitizer to improve photothermal therapy

Dopamine is a substance with photothermal properties, implying that AbDA-Lim also possesses photothermal effects. To validate this, SW620 tumor-bearing mice were randomly allocated into four groups: Saline, Saline + NIR, AbDA + NIR, and AbDA-Lim + NIR. Following intravenous administration for 6 h, tumors were exposed to an 808 nm laser for 10 min (1.0 W cm− 2), and post-irradiation temperatures were recorded. After irradiation, the temperatures of AbDA and AbDA-Lim rose to 69.8℃ and 72.1℃, respectively, significantly higher than the Saline group (51.9℃) (Fig. 5a, b), indicating the photothermal effects of AbDA and AbDA-Lim. By monitoring changes in tumor volume, we observed a certain inhibitory effect of IR treatment alone on tumors (Fig. 5c, d). The combined application of NIR with AbDA and AbDA-Lim exhibited superior therapeutic effects on tumors. Upon conclusion of the treatment, tumor tissues were collected for H&E staining. As depicted in Fig. 5e, the combined application of AbDA-Lim and IR induced pronounced necrosis in tumors, indicating that AbDA-Lim can serve as a photosensitizer and enhance the sensitivity of tumors to photothermal therapy.

Fig. 5
figure 5

AbDA-Lim improves photothermal therapy. (a, b) Temperature record at the site of mouse tumors after NIR irradiation. Mean ± s.e.m. n = 3. One-way ANOVA. (c) Tumors from euthanized mice at the end of treatment. (d) Tumor growth kinetics in SW620 implanted mice subjected to the indicated treatments. n = 5. Mean ± s.e.m. Two-way ANOVA. (e) H&E staining in tumor tissues. Scale bar, 50 μm

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Discussion

The central component of ferroptosis is Fe (II), and increased iron intake can promote the proliferation of tumor cells, indicating that tumor cells are generally sensitive to ferroptosis. However, this is not straightforward, cancer cells keep an iron homeostasis by storing iron in the form of Fe (III), which needs to be converted into Fe (II) to generate ROS [1]. Furthermore, Fe (II) is sequestered within the cell by forming complexes with GSH, thereby mitigating oxidative damage resulting from the Fenton reaction [7, 8]. Only when GSH levels decrease, Fe (II) is subsequently released from its storage status and enters LIP, triggering the Fenton reaction. Hence, iron homeostasis is the pivotal obstacle to enhancing LIP. In this study, we have developed a bio-inspired nanocomplex termed AbDA-Lim, which destroys iron homeostasis by facilitating the conversion of Fe (III) to Fe (II) and the release of Fe (II) from its storage status into LIP, thereby inducing ferroptosis in tumor cells.

AbDA-Lim comprises three constituents: Albumin, Dopamine, and Limonene. Utilizing our albumin self-assembly technique, spherical nanoparticles with a diameter of approximately 70 nm were formed. Due to the nanoscale size of the drug, it is easier for the nanomedicine to penetrate the cell membrane and reach the depth of the lesion. Albumin is the most abundant protein in blood and the main lipid carrier. Dopamine and limonene are delivered to tumor and synergistically impair iron homeostasis by incorporation into albumin. Limonene, as a natural active ingredient, has demonstrated anti-tumor effects in various clinical trials [24, 25]. Our research further confirmed that AbDA-Lim significantly inhibits the proliferation of various tumor cells, and this tumor-suppressive effect is correlated with the elevation of ROS induced by AbDA-Lim. ROS within cells are byproducts of the Fenton reaction triggered by Fe (II), and we corroborated the induction of ferroptosis in tumor cells by AbDA-Lim through the detection of GSH, lipid ROS, and cellular transmission electron microscopy (TEM).

Through RNA-seq analysis, we observed that AbDA-Lim indeed modulates the expression of ferroptosis-related genes, with the most prominent impact on HMOX1 and CBS. HMOX1 can degrade intracellular haem, thereby releasing Fe (II). Excessive activation of HMOX1 significantly elevates LIP, consequently inducing ferroptosis [52]. Consistent with our findings, numerous studies have also indicated that upregulating HMOX1 can drive cell ferroptosis [53,54,55,56]. Upon comparing the RNA-seq results of Ctrl, AbDA, and AbDA-Lim, we observed that the elevation of HMOX1 induced by AbDA-Lim is primarily attributable to AbDA, whereas AbDA alone did not cause noticeable oxidative damage. Additionally, polydopamine (DA) itself is a widely used and safe carrier. The controversy surrounding the driving role of HMOX1 in ferroptosis can be explained by the storage status of Fe (II). Released Fe (II) can bind and sequester with reducing molecules like GSH, protecting cells from oxidative damage caused by Fe (II) [16, 17]. Therefore, to effectively harness the pro-ferroptosis effects of HMOX1, it is essential to reduce the binding of Fe (II) with the redox-active small molecule GSH.

We find the inhibitory effect of AbDA-Lim on CBS is primarily attributed to Lim. An essential source of intracellular GSH is synthesized through the transsulfuration pathway, in which CBS plays a crucial role [26]. CBS catalyzes the conversion of homocysteine to cystathionine, a precursor for GSH synthesis. Inhibiting CBS can reduce GSH levels [43], thereby driving the release of stored Fe(II) from its complex with GSH, entering the LIP and initiating the Fenton reaction. By enhancing HMOX1 to promote the release of Fe (II) from haem and inhibiting CBS to suppress the entry of Fe (II) into the storage status, AbDA-Lim elevates LIP to induce ferroptosis. Moreover, through the integration of network pharmacology, we further hypothesized that dopamine, released from AbDA-Lim, acts as an inhibitor of KDMs to directly regulate the expression of HMOX1. On the other hand, limonene serves as an agonist of PPARα, which suppresses the expression of CBS.

In consistent with the in vitro findings, animal experiments demonstrate that AbDA-Lim elicits tumor ferroptosis. Immunohistochemistry and FerroOrange staining results further confirm that AbDA-Lim increases Fe (II) flux in LIP by upregulating HMOX1 and inhibiting CBS, thereby driving tumor cell ferroptosis. Additionally, the photothermal effect of polydopamine can further enhance the oxidative damage induced by AbDA-Lim.

Conclusion

In summary, this study introduced a bio-inspired LIP promotor, AbDA-Lim. The nanocomplex composition is straightforward simple. AbDA-Lim impairs iron homeostasis and elevates intracellular Fe (II) flux in cancer cells via a distinctive non-canonical pathway, ultimately instigating ferroptosis in cancer cells without the need for supplementary iron. This bio-nanocomplex provides a novel principle for enhancing iron flux to induce ferroptosis.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

Not applicable.

Funding

This study was supported by National Natural Science Foundation of China (No. 82204293 & 82241054), Scientific and Technological General Program of Beijing Municipal Education Commission of China (No. KM202010025007) and Collaborative Innovation Project of Yangtze River Delta Science and Technology Community (2023CSJZN0800).

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Author notes

  1. Xin Wang and Tianyi Zhang contributed equally to this work.

Authors and Affiliations

  1. Department of General Surgery, Center of Nutrition and Metabolism of Cancer, Beijing Shijitan Hospital, Key Laboratory of Cancer FSMP for State Market Regulation, Capital Medical University, Beijing International Science and Technology Cooperation Base for Cancer Metabolism and Nutrition, Beijing, 100038, China

    Xin Wang & Hanping Shi

  2. State Key Laboratory of Pharmaceutical Biotechnology, Institute of Drug R&D, Jiangsu Key Laboratory for Nano Technology, Medical School, School of Life Science, Nanjing University, Nanjing, 210093, China

    Tianyi Zhang, Shuai Wang, Hong Dong, Yiqiao Hu & Chunyan Yue

  3. Office of International Cooperation and Exchanges, Central South University, Changsha, 410008, China

    Yanning Huang

  4. CAS Key Laboratory of Standardization and Measurement for Nanotechnology, National Center for Nanoscience and Technology, Beijing, 100190, China

    Wenjia Lai

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Contributions

These authors contributed equally. C.Y. conceived the study. C.Y. and Y.H designed the study. X. W., H. S., T. Z., S., W. and Y. H. performed animal experiments. T. Z., S., W. and C.Y. conducted cell growth, immunocytochemistry assays, western blotting, all confocal imaging experiments, RNA-seq and all statistical analyses. T. Z. conducted preparation and characterization of AbDA-Lim and AbDA, flow cytometry analysis. T. Z. and H. D. performed HPLC detection. W.L conducted LC-MS scans. X. W., W. L., Y. H. and C.Y. wrote the manuscript. All authors read and edited the manuscript.

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Correspondence to Wenjia Lai, Yiqiao Hu or Chunyan Yue.

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Wang, X., Zhang, T., Wang, S. et al. Bio-nanocomplexes impair iron homeostasis to induce non-canonical ferroptosis in cancer cells. J Nanobiotechnol 23, 121 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12951-025-03117-3

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