CaCO₃-assisted engineering of NIR-II phototheranostics enables photothermally enhanced ferroptosis in cancer through synergistically depleting intracellular glutathione

Abstract

Ferroptosis, characterized by iron-dependent lipid peroxidation, represents a promising therapeutic target for cancer treatment. Strategies that disrupt intracellular antioxidant systems to induce ferroptosis in cancer cells have been extensively explored. Herein, we developed a pH-responsive phototheranostic agent (designated as FSB₄Ca NPs) by encapsulating conjugated boron dipyrromethene tetramers (B4) within ferric ion-sulfasalazine metallo-network polymer-coated calcium carbonate hollow nanoparticles. Sulfasalazine, a known ferroptosis inducer that inhibits System X_c⁻-mediated cysteine influx, synergizes with ferric ion-driven glutathione (GSH) depletion to collectively amplify intracellular lipid peroxidation. In addition to serving as a second near-infrared (NIR-II) fluorophore for tracking the in vivo distribution of FSB₄Ca NPs, B4 mediates a photothermal effect that significantly enhances lipid peroxidation induction by boosting the Fenton catalytic activity of ferrous ions. Combined with localized 915-nm laser irradiation, intravenously administered FSB₄Ca NPs achieved substantial tumor suppression in mouse models, with a complete remission rate of 80%. This study establishes a facile strategy for developing long-circulating NIR-II phototheranostic agents with self-amplified lipid peroxidation induction capacity, enabling photothermally augmented ferroptosis for cancer therapy.

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New concepts

In this work, we have prepared a pH-responsive phototheranostic nanoformulation by encapsulating NIR-II fluorophore B4 in Fe-SAS metallo-network polymer-coated CaCO₃ nanoparticles for NIR-II imaging-guided cancer therapy. The obtained FSB₄Ca NPs showed effective ferroptosis-inducing ability in cancer cells via concurrent enhancement of Fenton reaction-mediated ⋅OH production, SAS-suppressed GSH generation, and ferric ion-mediated GSH depletion. Together with the superior photothermal heating efficacy of B4, which reinforces the Fenton reaction, FSB₄Ca NPs synergized with external 915 nm laser exposure to kill cancer cells. In vivo NIR-II imaging confirmed high tumor accumulation of the FSB₄Ca NPs, leading to significant suppression of tumor growth, particularly when combined with tumor-localized 915 nm laser irradiation. This study highlights a rational chemical engineering strategy for constructing NIR-II phototheranostics to induce potent ferroptosis in cancer cells through photothermally self-amplified lipid peroxidation induction.

1. Introduction

Ferroptosis is a newly identified programmed cell death pathway characterized by the accumulation of iron-dependent intracellular lipid peroxidation (LPO).¹ Due to its unique cell killing mechanism, which differs from that of conventional chemotherapeutics and other modalities, ferroptosis has emerged as an alternative cancer therapeutic target,² particularly promising for hard-to-treat tumors, such as drug-resistant ones. Over the past decade, numerous strategies have been developed to induce ferroptosis, either by directly promoting the peroxidation of polyunsaturated fatty acids^3,4 or downregulating antioxidant systems.^5–7 For instance, various nanoparticle formulations incorporating lipoxidase⁸ or Fenton catalysts^9,10 have been rationally designed to initiate lipid peroxidation chain reactions and eventually induce ferroptosis in cancer cells. In addition, diverse small-molecule inhibitors targeting key intracellular components, such as glutathione peroxidase 4 (GPX4) and system X_c⁻ (a cystine/glutamate antiporter system), have also been proven effective in inducing ferroptosis in relevant clinical trials (NCT00098254, NCT06080841). Therefore, rationally combining different ferroptosis-inducing agents represents a promising strategy for triggering potent ferroptosis.¹¹

As an essential intracellular antioxidant, glutathione (GSH) has been proven to be capable of directly quenching reactive oxygen species and functioning as a cofactor of GPX4 to catalytically scavenge lipid peroxides, thereby protecting cancer cells from ferroptosis.¹² Given the strong capacity of the intracellular labile iron pool to promote the initiation and propagation of lipid peroxidation via Fenton catalysis, increasing this pool has been established as a practical strategy for inducing ferroptosis.^13,14 Several recent studies have demonstrated that nanoformulations containing ferric ions can simultaneously increase the intracellular labile iron pool and scavenge GSH and other antioxidants, thereby sustaining the Fenton reaction by facilitating the reduction of ferric ions to ferrous ones.^15–17 Owing to the inherent ability of nanoparticles to encapsulate and deliver diverse therapeutic agents precisely and in a targeted manner,^18–20 diverse ferric ion-containing nanoformulations have been rationally designed to induce ferroptosis synergistically in cancer cells.²¹

CaCO₃ has been extensively explored for tumor acidity-responsive drug delivery due to its superior biocompatibility, versatile ion/molecule loading capacity, and pH-dependent dissociation behavior.²² In addition to directly absorbing therapeutic molecules and ions via different mechanisms, CaCO₃ nanoparticles are also utilized as a template to guide the synthesis of various pH-responsive nanomedicines.²³ In this study, uniform amorphous CaCO₃ nanoparticles were first coated with a thin metallo-network polymer layer composed of ferric ions and sulfasalazine (SAS), a small-molecule inhibitor that targets system X_c⁻, which mediates the exchange of glutamate and cystine. The resultant FSCa hollow nanocomposites were loaded with an NIR-II fluorophore, boron dipyrromethene tetramer (B4), which exhibits excellent photothermal conversion efficiency, and then surface-modified with a lipid bilayer using a two-step liposome formation process. The yielded FSB₄Ca NPs exhibited efficient GSH scavenging and Fenton catalytic capacities, the latter of which was significantly enhanced upon 915 nm laser irradiation, thereby synergistically suppressing tumor cell growth via ferroptosis induction. Through recording the NIR-II fluorescence, the intravenously injected FSB₄Ca NPs showed high accumulation at tumor sites, and a complete tumor regression rate of 80% in CT26 murine tumor models, together with tumor-localized 915 nm laser irradiation. Thus, this study highlights that FSB₄Ca NPs are an efficient phototheranostic platform that enables NIR-II fluorescence imaging-guided precise tumor treatment via photothermally enhanced ferroptosis (Scheme 1).

Scheme 1 A schematic diagram illustrating the tumor inhibitory mechanism of FSB₄Ca NPs-mediated photothermal therapy. Under the guidance of NIR-II fluorescence imaging, the intravenously injected FSB₄Ca NPs accumulate effectively at the tumor sites and induce potent ferroptosis of tumor cells through simultaneous SAS-suppressed GSH generation, ferric ion-mediated GSH scavenging, and enhanced Fenton reaction-mediated ·OH production, the latter of which could be further augmented through 915-nm laser irradiation-mediated heating.

2. Results and discussion

2.1. Preparation and characterization of FSB₄Ca NPs

CaCO₃ nanoparticles with an average diameter of 105.7 nm were first synthesized through the previously developed gas diffusion method.^24,25 Then, leveraging the strong coordination affinity between ferric ions and the phenolic and carboxyl groups of SAS, the as-synthesized CaCO₃ nanoparticles were coated with a thin Fe-SAS metallo-network polymer (MNP) layer via a surface-protected etching process (Fig. 1a). The obtained FSCa NPs exhibited a uniform hollow structure under transmission electron microscopy (TEM) imaging (Fig. 1b). The formation of a hollow structure occurs because the Fe-SAS MNP layer formed on the surface of the CaCO₃ nanoparticles attenuates their etching by Fe³⁺, ultimately leaving a partially etched CaCO₃. High-angle annular dark-field scanning TEM (HAADF-STEM) imaging revealed that the element signals of C, O, Ca, S and Fe were homogenously distributed throughout the nanoparticles (Fig. 1c). The presence of these elements in the FSCa NPs was further validated by their energy dispersive spectrum (EDS, Fig. 1d). These results collectively demonstrate the successful preparation of FSCa NPs.

Fig. 1 Preparation and characterization of the FSB₄Ca NPs. (a) A schematic diagram showing the synthesis process of the FSB₄Ca NPs via a CaCO₃ surface-protected etching process. (b) Representative TEM images of the CaCO₃ nanoparticles and FSCa NPs. (c) STEM images showing the elemental distributions of Ca, Fe, S, N and O. (d) EDS spectrum of the FSCa NPs. (e) DLS size distribution behaviors of the FSB₄Ca NPs, FSCa NPs and naked CaCO₃. (f) UV-vis-NIR spectra of the CaCO₃, SAS and FSCa NPs. (g) Photoluminescence spectrum of the FSB₄Ca NPs in PBS. (h) DLS size evolution behaviors of the FSB₄Ca NPs in different physiological solutions. Data are presented as mean ± SD.

Then, B4 molecules were loaded into the FSCa NPs via simply stirring a mixture of FSCa NPs and B4 in DMF. The obtained FSB₄Ca NPs were then sequentially coated with 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) and other commercial lipids to endow them with excellent physiological stability. Meanwhile, pristine CaCO₃ and FSCa NPs were coated via the same method and utilized as the control in the following experiments. Dynamic light scattering (DLS) measurement showed that the diameter of the FSB₄Ca NPs was 141.8 nm, slightly larger than the 105.7 nm and 122.4 nm for CaCO₃ and FSCa NPs, respectively (Fig. 1e). The typical absorption peak of SAS at 365 nm clearly emerged in the UV-vis spectrum of the FSCa NPs (Fig. 1f). The FSB₄Ca NPs showed bright NIR-II fluorescence emission peaking at 1100 nm under the excitation of an 808 nm laser (Fig. 1h), demonstrating the successful loading of B4. Meanwhile, the encapsulation contents of SAS, Fe³⁺ and B4 were determined to be approximately 4.0%, 4.7% and 2.2%, respectively, by measuring their UV-Vis typical peaks at 365 nm and 540 nm for SAS and B4, and using inductively coupled plasma-optical emission spectroscopy (ICP-OES) for Fe (Fig. S1, SI). Using a bicinchoninic acid (BCA) assay, the FSB₄Ca NPs showed negligible protein absorption, comparable to neutral liposomes and significantly lower than cationic liposomes after 2 h in 5% fetal bovine serum (FBS) solution (Fig. S2). In addition, the FSB₄Ca NPs showed consistent DLS size distribution profiles after incubation in water, phosphate-buffered saline (PBS), saline, and cell culture medium supplemented with 10% FBS for up to 24 h (Fig. 1h). Furthermore, the FSB₄Ca NPs also showed consistent side distribution profiles after 14 days of storage in water at 4 °C in the dark, illustrating their excellent long-term stability (Fig. S3).

TEM imaging then showed that the FSB₄Ca NPs rapidly dissociated in acidic solutions (pH 6.5 and 5.5), mimicking the acidic extracellular and intracellular microenvironments (Fig. S4). We therefore investigated the pH-dependent release profiles of Fe³⁺ and SAS. Approximately ∼80.4% Fe³⁺ was released from the FSB₄Ca NPs after incubation at pH 5.5 for 4 h, compared to only ∼28.9% and 9.1% released after incubation at pH 6.5 and 7.4, respectively (Fig. 2a). A similar pH-responsive release profile was observed for SAS (Fig. 2b). We then assessed the Fenton catalytic activity of the FSB₄Ca NPs by monitoring hydroxyl group (⋅OH) generation using a methylene blue (MB) degradation test. The FSB₄Ca NPs demonstrated pH-dependent catalytic activity, with the most effective MB degradation occurring at pH 5.5 (Fig. 2c). Furthermore, the addition of GSH (1 mM) significantly enhanced the Fenton catalytic ability of the FSCa NPs across all pH conditions. This enhancement can be attributed to the reduction of ferric ions released from the nanoparticles to ferrous ions, which exhibit higher Fenton catalytic efficacy. These results indicate that the FSCa NPs function as a bifunctional ROS nanoamplifier through concurrently promoting the conversion of H₂O₂ to highly reactive ⋅OH and facilitating sustained depletion of intracellular GSH and other antioxidants.

Fig. 2 Drug release, Fenton catalytic and photothermal profiles of the FSB₄Ca NPs. (a) and (b) The Fe³⁺ (a) and SAS (b) release profiles of FSB₄Ca NPs incubated in PBS of different pH value. (c) UV-vis-NIR spectra change of an MB solution containing H₂O₂ (1 mM) after incubation with FSCa NPs after different treatments. (d) The temperature change curves and corresponding IR thermal imaging of the FSB₄Ca NPs at different B4 concentrations when irradiated with a 915 nm laser (0.3 W cm⁻², 5 min). (e) The temperature change curves of FSB₄Ca NP solutions in one heating/cooling cycle (blue) and the linear time versus −ln θ dated from the cooling period (red). (f) The temperature change curves of the FSB₄Ca NP solution during 5 repeated heating/cooling cycles. Data are presented as mean ± SD.

Owing to the excellent photothermal conversion ability of B4 molecules, we further evaluated the photothermal conversion ability of the FSB₄Ca NPs. Using an infrared thermal camera, the temperature increase rate of the FSB₄Ca NP solution under 915-nm laser irradiation was positively correlated with the B4 concentration (Fig. 2d). In addition, based on a previously established method,²⁶ the photothermal conversion efficiency (PCE) of the FSB₄Ca NPs was quantified to be ∼ 51.6% (Fig. 2e). The FSB₄Ca NPs also maintained consistent photothermal heating rates over five consecutive heating–cooling cycles under 915 nm laser irradiation, demonstrating high photothermal stability (Fig. 2f). Furthermore, external 915-nm laser exposure (0.3 W cm⁻², 15 min) markedly enhanced the MB degradation capability of the FSB₄Ca NPs (B4 = 10 µg mL⁻¹) in the presence of GSH (1 mM) (Fig. S5). These results together demonstrate the superior photothermally enhanced Fenton catalytic activity of the FSB₄Ca NPs, which could be attributed to the fact that higher temperature might speed up the GSH-mediated reduction of Fe³⁺ and Fe²⁺-catalyzed Fenton reactions according to the Arrhenius equation.²⁷

2.2. Photothermally enhanced ferroptosis induction by FSB₄Ca NPs

We then investigated the cellular profiles of the FSB₄Ca NPs. By recording the fluorescence of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD), noncovalently inserted into the lipid bilayer of FSB₄Ca NPs, CT26 murine cancer cells exhibited time-dependent engulfment of FSB₄Ca NPs as indicated by the gradually increased intracellular DiD fluorescence via confocal microscopy (Fig. 3a). Given that SAS can block system X_c⁻-mediated glutamate efflux, we then investigated the extracellular glutamate levels of CT26 cells treated with FSCa NPs by using a commercial assay kit. The results showed that FSCa NP incubation (Fe³⁺ = 10 µM, SAS = 50 µg mL⁻¹, 6 h) markedly reduced glutamate secretion into the cell culture medium compared to SAS treatment alone (Fig. 3b), probably due to the enhanced cellular uptake of FSCa NPs. Consequently, CT26 cells treated with FSCa NPs (Fe³⁺ = 10 µM, SAS = 50 µg mL⁻¹) exhibited apparent time-dependent intracellular GSH depletion (Fig. S6). A comparative study further indicated that the FSCa NPs exhibited more effective intracellular GSH depletion (Fig. 3c) when compared to treatments with Fe³⁺ alone, SAS alone, and Fe³⁺ + SAS, attributable to the concurrent System X_c⁻ inhibition and ferric ion-reduction mediated GSH consumption.

Fig. 3 In vitro cellular experiments of the FSB₄Ca NPs. (a) Confocal images of CT26 cells after incubation with DiD-labeled FSCa NPs at the designated time points. (b) and (c) Extracellular glutamate (b) and intracellular GSH (c) level of the cells after different treatments (n = 3). (d) Cell viability of CT26 cells treated with Fe, SAS, FSB₄Ca NPs and FSB₄Ca NPs plus 43 °C at different concentrations of Fe and SAS (n = 5). (e) Live/dead staining images of CT26 cells treated with the control, Fe, SAS, FSCa NPs and FSCa NPs plus laser irradiation. (f) and (g) Representative confocal images (f) and flow cytometry analysis (g) of CT26 cells after different treatments as indicated before being stained with BODIPY-C11. Data are presented as mean ± SD.

We next evaluated the ability of the FSB₄Ca NPs to induce ferroptosis. It was revealed that the FSB₄Ca NPs exhibited significant cytotoxicity toward CT26 cells in a concentration-dependent manner (Fig. 3d). In contrast, ferric ions and CaCO₃ alone at equivalent concentrations exhibited negligible cytotoxicity (Fig. S7), while SAS alone induced moderate cytotoxicity at the corresponding concentrations (Fig. S8). The FSCa NPs also effectively induced B16 melanoma cell death, but exhibited markedly attenuated cytotoxicity to human umbilical vein endothelial cells (HUVECs) (Fig. S9), collectively implying their selective tumor suppression efficacy. Furthermore, due to the temperature-dependent Fenton reaction, treatment with FSB₄Ca NPs for 6 h followed by a consecutive heating at 43 °C for 15 minutes resulted in enhanced cytotoxicity. The photothermally enhanced cytotoxicity of the FSB₄Ca NPs was further confirmed by a live/dead cell dual staining assay (Fig. 3e).

We then evaluated the capacity of the FSB₄Ca NPs to induce intracellular lipid peroxidation, a hallmark of ferroptosis, with the BODIPY^TM 581/591 C11 fluorescent probe via confocal microscopy and flow cytometry (Fig. 3f and g). CT26 cells incubated with FSCa NPs (Fe³⁺ = 10 µM, SAS = 50 µg mL⁻¹, 6 h) at 43 °C exhibited the highest level of intracellular lipid peroxidation among all treatments. Moreover, this temperature-dependent lipid peroxidation was almost completely suppressed by cotreatment with ferrostatin-1 (Fer-1, 100 µM), a known ferroptosis inhibitor. Furthermore, CT26 cells treated with FSCa NPs exhibited significantly lower GPX4 activity compared to those with other treatments via a commercial GPX4 activity detection kit (Fig. S10). These findings collectively prove that the FSB₄Ca NPs are capable of inducing ferroptosis in cancer cells in a temperature-dependent manner.

2.3. In vivo pharmacokinetic profiles of the FSB₄Ca NPs

We then investigated the in vivo pharmacokinetic behaviors of the FSB₄Ca NPs (Fig. 4a). By monitoring the NIR-II fluorescence intensity in the tumor regions of CT26 tumor-bearing mice post intravenous injection of FSB₄Ca NPs (B4 = 0.5 mg kg⁻¹), the FSB₄Ca NPs showed time-dependent tumor accumulation, which peaked at 12 h post injection (p.i.) and remained high until 24 h p.i. (Fig. 4b and c). Subsequent ex vivo NIR-II fluorescence imaging showed that tumors harvested at 24 h p.i. exhibited the highest fluorescence intensity compared to major organs collected at the same time point (Fig. 4d). Additionally, we further semi-quantitatively analyzed the pharmacokinetic profiles of the FSB₄Ca NPs by measuring the fluorescence intensity of DiD molecules. Based on the DiD fluorescence in lysed blood samples collected from the tumor-bearing mice at designated time points, the FSCa NPs displayed a comparable blood circulation profile fitting a two-compartment model (Fig. 4e), with the first and second half-life calculated to be 0.55 ± 0.02 h and 4.18 ± 0.46 h. Meanwhile, the tumor accumulation efficacy of the FSCa NPs at 24 p.i. was quantified to be 11%ID g⁻¹, but lower than 21.6%ID g⁻¹ and 19.2%ID g⁻¹ in the liver and spleen, respectively (Fig. 4f). These results collectively demonstrate the prolonged blood circulation and efficient tumor accumulation capacities of the FSB₄Ca NPs.

Fig. 4 In vivo NIR-II pharmacokinetic profiles of the FSB₄Ca NPs. (a) A schematic diagram of the experimental schedule. (b) NIR-II FL imaging of CT26 tumor-bearing mice post injection of FSB₄Ca NPs at the designated time points. (c) Relative FL intensity of the tumor masses taken from (b). (d) Ex vivo FL imaging of the main organs and tumors collected from CT26 tumor-bearing mice 24 h post injection of FSB₄Ca NPs. (e) Blood circulation profiles of FSB₄Ca NPs in CT26 tumor-bearing mice by recording the DiD FL intensity of lysed blood samples collected at the designated time points. (f) Biodistribution profiles of FSB₄Ca NPs in the homogenized organs and tumors harvested from CT26 tumor-bearing mice at 24 h p.i. by recording the DiD fluorescence intensity (n = 3). Data are presented as mean ± SD.

2.4. In vivo photothermally enhanced antitumor efficacy of FSB₄Ca NPs

We first investigated the photothermal performance of the FSB₄Ca NPs in CT26 tumor-bearing Balb/c mice by recording the tumor temperature increase behaviors with an infrared thermal camera. It was shown that CT26 tumors in mice with intravenous injection of FSB₄Ca NPs (B4 = 1 mg kg⁻¹) and subjected to tumor-localized 915 nm laser exposure (0.3 W cm⁻², 15 min) exhibited a rapid temperature increase to ∼45 °C (Fig. 5b and c). In contrast, tumors in mice receiving the same laser exposure alone exhibited a minimal temperature increase (∼35 °C), indicating the superior photothermal heating efficacy of the FSB₄Ca NPs in vivo.

Fig. 5 In vivo FSB₄Ca NP-mediated photothermal therapy. (a) A schematic diagram of the therapeutic process of FSB₄Ca NP-assisted PTT on CT26 tumor-bearing mice. (b) and (c) Temperature change curves (b) and corresponding IR thermal images (c) of tumors from control CT26 tumor-bearing mice and mice treated with FSB₄Ca NPs. (d)–(g) Average (d) and individual (e) tumor growth curves, mobility-free survival curves (f) and average body weight change curves (g) of mice after different treatments as indicated. Data are presented as mean ± SD.

We then evaluated the photothermal therapeutic efficacy of the FSB₄Ca NPs in CT26 tumor-bearing mice. Five groups of mice (∼100 mm³) received the following treatments: (1) control; (2) 915-nm laser exposure; (3) SAS injection; (4) FSB₄Ca NP injection; (5) FSB₄Ca NP injection plus 915-nm laser exposure (Fig. 5a). The injection doses of SAS and B4 were 1 mg kg⁻¹ and 0.5 mg kg⁻¹, respectively. Mice in groups 2 & 5 were subjected to 915 nm laser irradiation at a power density of 0.3 W cm⁻² for 15 min, 24 h after injection. It was uncovered that the combined treatment of FSB₄Ca NP injection and 915-nm laser exposure led to the most effective suppression of tumor growth (Fig. 5d and e), with 4 of the 5 mice in this group achieving complete tumor regression (Fig. 5f). Although FSB₄Ca NP treatment alone showed a limited suppression effect on tumor growth, this group still reached a 50% survival rate according to the survival rate curve. In contrast, SAS or 915-nm laser exposure alone showed minimal inhibitory effects on tumor growth and survival. In addition, all of these treatments showed negligible body weight change in the mice throughout the study (Fig. 5g). These results demonstrate the excellent photothermally enhanced therapeutic efficacy and biocompatibility of the FSB₄Ca NPs.

2.5. In vivo biocompatibility assessment of FSB₄Ca NP-assisted photothermal therapy

We evaluated the biocompatibility of FSB₄Ca NP-mediated PTT in CT26 tumor-bearing mice via biochemical blood tests and complete blood panel analysis. Two groups of CT26 tumor-bearing mice (n = 3) that received intravenous injection of PBS and FSB₄Ca NPs and were subjected to PTT as mentioned above were sacrificed 14 days post treatment with whole blood and sera collected for corresponding assessment. It was shown that FSB₄Ca NP-mediated PTT at the tested dose did not induce significant alterations in hepatic or renal function markers, including alanine aminotransferase (ALT), urea (UREA), white blood cell (WBC), neutrophil count (Neu), lymphocyte count (Lym), monocyte count (Mon), basophil count (Bas), red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width-coefficient of variation (RDW-CV), red cell distribution width-standard deviation (RDW-SD), mean platelet volume (MPV), and platelet distribution width (PDW, Fig. S11). These findings further validate the excellent biocompatibility of FSB₄Ca NP-mediated PTT.

3. Conclusion

In this work, we prepared a pH-responsive phototheranostic nanoformulation by encapsulating NIR-II fluorophore B4 into Fe-SAS metallo-network polymer-coated CaCO₃ nanoparticles for NIR-II imaging-guided cancer therapy. The obtained FSB₄Ca NPs showed effective ferroptosis-inducing ability in cancer cells via concurrent enhancement of Fenton reaction-mediated ⋅OH production, SAS-suppressed GSH generation, and ferric ion-mediated GSH depletion. Together with the superior photothermal heating efficacy of B4, which reinforces the Fenton reaction, the FSB₄Ca NPs synergized with external 915 nm laser exposure to kill cancer cells. In vivo NIR-II imaging confirmed high tumor accumulation of the FSB₄Ca NPs, leading to significant suppression of tumor growth, particularly when combined with tumor-localized 915 nm laser irradiation. This study highlights a rational chemical engineering strategy for constructing NIR-II phototheranostics to induce potent ferroptosis in cancer cells through photothermally self-amplified lipid peroxidation induction.

4. Experimental section

4.1. Materials

Calcium chloride (CaCl₂), ferric chloride (FeCl₃·6H₂O), and ammonia bicarbonate (NH₄HCO₃) were obtained from Sinopharm Chemical Reagent Co. Sulfasalazine was purchased from MedChem Express. Polyvinyl pyrrolidone (PVP) and the Pierce™ BCA detection kit were purchased from Sigma-Aldrich. 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt) (DOPA) was obtained from Avanti Lipids Polar, Inc, and other lipids used for nanoparticle preparation were purchased from Xi’an Ruixi Biological Technology Co., Ltd. Methylene blue was purchased from Aladdin. B4 molecules were synthesized and characterized according to our previously developed method.²⁸ Methylene blue trihydrate was purchased from J & K Chemical Co.

Cell culture medium and penicillin/streptomycin were purchased from Cytiva, and fetal bovine serum (FBS) was obtained from Inner Mongolia Opcel Biotechnology Co., Ltd. Cell culture dishes (Tissue culture treated, 715001) were purchased from NEST Biotechnology. Red blood cell lysis buffer (R1010) and the reduced glutathione (GSH) content assay kit (BC1175) were purchased from Beijing Solarbio Science & Technology Co., Ltd. BODIPY-C11 probe was purchased from ThermoFisher Scientific China Co., Ltd. The calcein-AM/PI double stain kit (abs50056) was purchased from Absin. The GPX4 detection kit (S0056) was purchased from Beyotime.

4.2. Synthesis and surface modification of the FSB₄Ca NPs

Amorphous CaCO₃ nanoparticles were first synthesized through our previously developed gas diffusion method.^23,24 Then, the CaCO₃ ethanol solution (2 mL, 3 mg mL⁻¹) was first mixed with the SAS ethanol solution (1 mL, 1 mg mL⁻¹) and PVP (100 µL, 200 mg mL⁻¹). After being stirred for 5 min, the reaction mixture was mixed with FeCl₃·6H₂O ethanol solution (200 µL, 0.09 M) and stirred for another 2 h. The obtained FSCa NPs were collected and purified with ethanol three times by centrifugation (14 800 rpm, 21 100 × g, 5 min). Then, the FSCa NPs ethanol solution (5 mL, 4 mg mL⁻¹) was mixed with B4 DMF solution (2 mg mL⁻¹) and stirred for 2 h before being collected via centrifugation (14 800 rpm, 21 100 × g, 10 min). After that, the obtained FSB₄Ca NPs were sequentially coated with DOPA, DPPC, cholesterol and DSPE-PEG_5k to yield PEGylated FSB₄Ca NPs via our previously developed method.²⁹ The liposomes were synthesized with the same lipid formulation, while the positively charged liposomes were synthesized with the addition of DOTAP.

4.3. Characterization of the FSB₄Ca NPs

The microscopic morphology of the amorphous CaCO₃ and FSB₄Ca NPs was observed under a transmission electron microscope (TEM, Tecnai F20, FEI). The UV-vis spectra and size distribution profiles of different formulations were measured with a UV-vis spectrometer (GENESYM™ 10S, Thermo) and a Malvern Zetasizer (Nano ZS90 Malvern), respectively. The NIR-II fluorescence emission profile was recorded with the Series II 900/1700 In Vivo Imaging System (Suzhou NIR-Optics Co., Ltd., China). The content of Fe³⁺ was quantified with an ICP-MS (Aurora M90, Bruker, Germany).

To evaluate the pH-dependent drug release profile of the FSB₄Ca NPs, the nanoparticles were incubated in different solutions at pH 7.4, 6.5, and 5.5. The solution was collected via centrifugation (14 800 rpm, 10 min) and then resuspended at the designated time points. The Fe and SAS content was then measured by ICP and the characteristic absorbance was recorded at 365 nm.

To test the Fenton reaction catalytic efficacy, FSCa NPs (SAS = 50 µg mL⁻¹, Fe³⁺ = 10 µM) were precultured in GSH solution (1 mM) for 1 h and then added into a solution containing H₂O₂ (1 mM) and MB (15 µg mL⁻¹) at pH 7.4, 6.5 or 5.5 and incubated at room temperature for 2 h (43 °C group was incubated in a 43 °C oven). The absorption spectra of the solutions were measured with UV-vis-NIR spectroscopy at determined time points.

Photothermal properties were recorded using an infrared thermal camera and calculated by the standard protocol.²⁶

4.4. In vitro cell experiments

CT26 murine colon cells were cultured in complete Roswell Park Memorial Institute (RPMI) 1640 culture medium containing 10% FBS and 1% penicillin/streptomycin under the standard culture conditions (37 °C, 5% CO₂).

The cellular internalization behavior of the DiD-labeled FSB₄Ca NPs was evaluated by recording the DiD fluorescence in CT26 cells at designated time intervals via confocal microscopy (Zeiss LSM 800) according to a previously used method.³⁰ The cytotoxicity of the FSB₄Ca NPs at both 37 °C and 43 °C for designated time intervals was evaluated in CT26 cells via the standard MTT assay and calcein-AM/PI dual staining assay according to the vendors’ experimental procedures. The ability of FSCa NPs to induce intracellular lipid peroxidation under the designated experimental conditions was evaluated with BODIPY-C11 dyes and analyzed via both confocal microscopy and flow cytometry (BD, AccuriTM C6 Plus). The intracellular GSH level and extracellular Glu level of the CT26 cells with the designated treatments were detected using the respective commercial detection kits.

4.5. In vivo animal experiments

All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Soochow University and approved by the Animal Ethics Committee of Soochow University (SYXK(Su) 2021-0073). Female Balb/c mice used in this study were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. A CT26 tumor model was established by subcutaneously injecting CT26 cells on the right back of each Balb/c mouse. The tumor accumulation profiles of FSB₄Ca NPs intravenously injected into the CT26 tumor-bearing mice were evaluated by recording their NIR-II fluorescence in the tumor regions under a Series II 900/1700 In Vivo Imaging System (Suzhou NIR-Optics Co., Ltd., China). Ex vivo NIR-II fluorescence imaging of the main organs and tumors harvested at 24 h post injection was performed to evaluate the in vivo distribution profile of the FSB₄Ca NPs using the same equipment. The semiquantitative in vivo blood circulation and distribution profiles of the FSB₄Ca NPs were also quantified by recording the fluorescence intensity of DiD tags according to a previously used protocol.²⁹ To evaluate the therapeutic responses of the FSB₄Ca NPs, 5 groups of CT26 tumor-bearing mice received the designated treatments. The length and width of each tumor were recorded using a digital caliper, and tumor volume was calculated according to the standard formula: tumor volume = length × width × width/2. The body weight was measured by using a digital balance, and tumor volume and body weight were measured every 2 days. When the tumor volume reached 1500 mm³, the mice were immediately considered to be dead and sacrificed.

Author contributions

Juxin Gao performed subject conceptualization, experimental methodology, result validation, formal analysis and manuscript writing. Liangzhu Feng performed conceptualization, supervision and financial support. Zhuang Liu performed supervision. Hengze Ding, Qinghua Wu, Yuhang Hu, Yifan Yan, Minming Chen, Chunjie Wang performed methodology and manuscript review. All the authors read, revised and reviewed the final manuscript.

Conflicts of interest

There are no conflicts to declare.

Data availability

All data generated or analyzed during this study are included in this published article.

Acknowledgements

This work was partially supported by the National Research Programs from the Ministry of Science and Technology (MOST) of China (2021YFF0701800), the National Natural Science Foundation of China (T2321005, 52032008, and 32322046), the Collaborative Innovation Center of Suzhou Nano Science and Technology, the Suzhou Key Laboratory of Nanotechnology and Biomedicine, and the 111 Program from the Ministry of Education of China. The authors also thank the website app.Biorender.com for the assistance in creating the figure illustrations.

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