A glycosylated AIE-active Fe(III) photosensitizer activated by the tumor microenvironment for synergistic type I photodynamic and chemodynamic therapy

Abstract

Photodynamic therapy (PDT) and chemodynamic therapy (CDT) are both promising cancer treatments to inhibit tumor cells by generating highly cytotoxic reactive oxygen species (ROS). Herein, we report a novel tumor microenvironment (TME) stimulus-responsive water-soluble glycosylated photosensitizer (BT-TPE@Fe-Lac), which can serve as a high-efficiency antitumor agent by combining PDT and CDT, based on the coordination of Fe³⁺ with lactosyl bis(2-pyridylmethyl)amine and an AIE luminogen (benzothiazole-hydroxytetraphenylethene, BT-TPE). BT-TPE@Fe-Lac is stable under physiological conditions and selectively targets HepG2 cells via asialoglycoprotein receptor (ASGPR)-mediated endocytosis. It rapidly dissociates into AIE-active BT-TPE molecules and a lactosyl ferric(III) complex in the acidic lysosomes of cancer cells. Upon exposure to light, BT-TPE produces O₂˙⁻ radicals for type I PDT. The ferric(III) complex is reduced to an Fe(II) complex upon depletion of glutathione, which primes the breakdown of endogenous H₂O₂ within the tumor microenvironment, thus generating highly toxic ˙OH for enhanced CDT. Compared with the monotherapy of PDT or CDT, BT-TPE@Fe-Lac can significantly increase the intracellular ROS levels to induce more tumor cell death under low drug doses and hypoxia-dependent conditions. This strategy leverages the unique properties of the TME to optimize therapeutic outcomes, offering a promising approach for the TME-responsive nanoplatform in advanced cancer therapy.

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Introduction

The occurrence, progression, and metastasis of tumors are highly correlated with the specific tumor microenvironment (TME), which is characterized by overexpression of hydrogen peroxide and glutathione (GSH), low pH, hypoxia, and vascular abnormalities.^1–3 Glutathione is a highly expressed reducing agent in tumor cells and is responsible for eliminating oxidative stress to maintain the intracellular redox balance.^4–6 The consumption of intracellular GSH is considered as an effective additional means of improving treatment outcomes through reactive oxygen species (ROS) generation strategies.^7,8 The tumor microenvironment has been recognized as a significant factor affecting the effectiveness of the aforementioned treatments, and more and more studies are incorporating the tumor microenvironment into the diagnosis and treatment of tumors.⁹

Photodynamic therapy (PDT) has been considered as a novel alternative to traditional oncology treatments with high selectivity, low toxicity, no drug resistance and lower non-invasiveness.^10–14 Although PDT has shown great potential in cancer treatment, many challenges still need to be overcome in practical applications. The hypoxia of the TME seriously restricts the PDT effect of conventional type-II photosensitizers (PSs), which are highly dependent on O₂.^15,16 Type-I photosensitizers predominantly generate superoxide radicals (O₂˙⁻) through a cascade of electron transfer involving excited PSs, adjacent substrates, and molecular oxygen.^17,18 Subsequently, through the superoxide dismutase (SOD)-mediated disproportionation reaction and the subsequent Fenton reaction, O₂˙⁻ is converted into highly cytotoxic hydroxyl radicals (˙OH) and causes oxidative stress.^19–21 However, due to the low concentration of endogenous ferric ions and the presence of peroxidase in tumor cells, most H₂O₂ is decomposed into water and oxygen.²² Thus, the generation of ˙OH is often limited, resulting in reduced therapeutic efficacy. Therefore, it is crucial to develop strategies to improve the controllability and efficiency of ˙OH generation, in order to enhance the therapeutic outcomes of PDT. Chemodynamic therapy (CDT) involves the production of highly reactive ˙OH in the tumor microenvironment through Fenton or Fenton-like reactions with H₂O₂ ^23–25 which provides a method for directly killing cancer cells using endogenous H₂O₂, and has become a new strategy for cancer treatment. So far, many research groups have explored the use of different materials as CDT reagents to trigger cancer cell apoptosis and inhibit tumor growth.^26–31 However, the relatively weak catalytic efficiency of CDT leads to the limited production of ROS. The combination of PDT and CDT will be an effective strategy to increase the generation of ROS.^32,33 From the perspective of synergistic therapy, the combination of PDT and CDT is considered as a breakthrough tactic to achieve superimposed effects while overcoming their respective obstacles. Currently, most studies on CDT/PDT synergistic therapy involve the intricate doping or assembly of photosensitizers and Fenton agents,^34–36 and the resulting nanosystems usually have poor bioavailability due to low water solubility.

Furthermore, to achieve excellent tumor theranostic efficiency, tumor targeted delivery is essential. Due to enhanced permeability and retention (EPR) effects, most of the drug-loaded micelles can be passively accumulated in tumors but often suffer from low efficiency.³⁷ It still remains a great challenge to smartly construct an integrated and actively targeted nanoplatform with the simplest and most biocompatible components to promote multimodal tumor treatment diagnosis. The asialoglycoprotein receptor (ASGPR), which is overexpressed on the surface of liver cells,³⁹ provides an ideal target for hepatocyte-specific delivery,³⁸ and specifically recognizes galactose residues. As a disaccharide composed of galactose and glucose, lactose has gained popularity due to easy preparation, nontoxicity, non-immunogenicity, easy carrying and release of various drugs, excellent water solubility, biocompatibility, and delivery of the target drug to hepatocellular carcinoma cells.

Compared with the aggregation caused quenching (ACQ) of traditional fluorescent dyes, aggregation-induced emission (AIE) has excellent photophysical properties. However, the low water solubility of most AIE fluorescent dyes limits their application scopes in the detection and biosensing fields to a certain extent.⁴⁰ In recent years, our group has developed a series of glycosyl AIE luminogens for bioluminescence imaging, tumor therapy, small molecule detection and other related fields.⁴¹ Based on the above analysis, herein, we constructed a multifunctional photosensitizer BT-TPE@Fe-Lac with TME stimuli-responsive capacities, which is facilely prepared via the coordination of Fe³⁺ with an AIE luminogen (benzothiazole-hydroxytetraphenylethene, BT-TPE) and lactosyl bis(2-pyridylmethyl)amine. The amphiphilic BT-TPE@Fe-Lac could self-assemble into nanoparticles (BT-TPE@Fe-LacNPs) in aqueous solution (Scheme 1a and b). Compared with the typical reported drugs used for PDT/CDT (Table S1†), the designed BT-TPE@Fe-LacNPs are endowed with the following significant characteristics: (i) by convenient and facile synthesis, composites with synergistically enhanced functions can be fabricated utilizing the coordination characteristics of Fe(III), and easy assembly of the glycosylated photosensitizer can be achieved; (ii) lactosyl BT-TPE@Fe-LacNPs improve both aqueous solubility and targeted delivery, and are mainly accumulated in lysosomes through ASGPR-mediated endocytosis, leading to more effective precision therapy; (iii) the fluorescence, photodynamic therapy, and chemodynamic therapy functions are quenched under normal physiological conditions, which can minimize adverse effects on normal tissues; (iv) BT-TPE@Fe-Lac is a TME stimulus-responsive photosensitizer, which rapidly dissociates into AIE-active BT-TPE molecules and a ferric(III) complex in the acidic lysosomes of cancer cells; (v) BT-TPE can produce O₂˙⁻ under light irradiation and realize oxidative damage induced cell death by PDT. Notably, even under severe hypoxic conditions (2% O₂), it still generates sufficient cytotoxicity via type I photoreactions; (vi) the ferric(III) complex is reduced to an Fe(II) complex upon depletion of glutathione, which primes the breakdown of endogenous H₂O₂ within the tumor microenvironment, thus disrupting the redox balance and generating highly toxic ˙OH for enhanced CDT; (vii) compared with the monotherapy of PDT or CDT, BT-TPE@Fe-LacNPs have been shown to remarkably suppress the growth of HepG2 cells through a synergistic combination of CDT and PDT (Scheme 1c), achieving an impressive IC₅₀ value of 1.2 μM. Based on the above descriptions, BT-TPE@Fe-Lac is considered to provide a promising platform for synergistic tumor therapy, facilitating the precise diagnosis and targeted treatment of HepG2 cells. To the best of our knowledge, this is the first report on utilizing dual coordination of Fe³⁺ to construct glycosylated photosensitizer aggregates for tumor microenvironment synergistic therapy.

Scheme 1 (a) Schematic illustration of the molecular engineering. (b) Schematic representation of pH-responsive active targeting nanoparticles with “off/on” function. (c) Mechanism of photodynamic therapy combined with chemodynamic therapy for cancer theranostics.

Results and discussion

Firstly, BT-TPE and Fe-Lac were synthesized and characterized by electrospray ionization mass spectrometry, nuclear magnetic resonance spectroscopy, etc. The polyphenolic structure of BT-TPE can coordinate with the Fe(III) complexes (Fe-Lac) to afford the desired BT-TPE@Fe-Lac. The detailed preparation process of BT-TPE@Fe-Lac is illustrated in Scheme S1 in the ESI.†

The successful assembly of BT-TPE@Fe-Lac into BT-TPE@Fe-LacNPs in aqueous solution was initially characterized by transmission electron microscopy (TEM). The TEM images (Fig. 1a) demonstrated that BT-TPE@Fe-LacNPs are monodisperse with an approximate size of 200 nm. Due to the hydration layer formed on the surface of the NPs, the particle size observed using dynamic light scattering (DLS) is slightly larger, measuring approximately 220 nm (Fig. 1b). An obvious Tyndall effect could be observed in the aqueous solution (10 μM) of BT-TPE@Fe-Lac (Fig. S1†), indicating that abundant nanoparticles exist and form large hydrodynamic particle size micelles. Furthermore, BT-TPE@Fe-LacNPs had an excellent stability in phosphate buffered saline (PBS) for up to 7 days (Fig. S2†). Meanwhile, the TEM elemental mapping showed the uniform distribution of carbon (C), oxygen (O), nitrogen (N), sulfur (S) and iron (Fe) atoms (Fig. 1f). The FTIR spectra (Fig. 1c) showed distinct peaks at around 1650, 2800, and 3500 cm⁻¹, attributed to the stretching vibrations of C=C, C–H, and O–H, respectively. Compared with Fe-Lac, the FTIR spectrum of BT-TPE@Fe-Lac exhibited a low-frequency band at approximately 557 cm⁻¹, corresponding to the Fe–O vibration peak. In addition, X-ray photoelectron spectroscopy (XPS) was carried out to investigate the chemical composition (Fig. 1d). The measured spectrum exhibited five characteristic peaks: C 1s (284.9 eV), N 1s (400.0 eV), O 1s (532.8 eV), S 2p (165.72 eV), and Fe 2p (711.12 eV, 714.62 eV, 724.27 eV, 727.82 eV) (Fig. S3†). Furthermore, the high-resolution XPS spectrum of Fe 2p (Fig. 1e) showed two main peaks located at 714.62 eV and 727.82 eV respectively, corresponding to Fe 2p_3/2 and Fe 2p_1/2, which had been proved to be the typical peaks of Fe³⁺. All these data demonstrated the successful synthesis and self-assembly of BT-TPE@Fe-Lac and the excellent stability, uniformity and potential for the following drug delivery to targeted tumors.

Fig. 1 (a) TEM image of BT-TPE@Fe-Lac (1 μM) at pH 7.4. (b) DLS image of BT-TPE@Fe-Lac (1 μM) at pH 7.4. (c) FTIR spectrum of BT-TPE@Fe-Lac and Fe-Lac. (d) High-resolution X-ray photoelectron spectroscopy spectra of BT-TPE@Fe-Lac. (e) High-resolution Fe 2p spectrum of BT-TPE@Fe-Lac. (f) Elemental TEM mapping of BT-TPE@Fe-Lac with the elements C, N, O, Fe and S.

Fe³⁺ is an excellent electron transfer agent.⁴² When complexed with Fe³⁺, the electrons from BT-TPE are more likely to transfer to the metal Fe in its empty orbitals, which implies that the fluorescence imaging and photosensitization functions of BT-TPE in the BT-TPE@Fe-LacNPs are in the quenched state under neutral conditions. As shown in Fig. S4,† the fluorescence of BT-TPE is dramatically quenched when assembled into BT-TPE@Fe-LacNPs. It is generally believed that lower pH can promote the cleavage of Fe–O bonds. The increased electrostatic repulsion between H⁺ and metal ions in acidic media weakens the coordination between Fe³⁺ and BT-TPE, leading to the disintegration of the complex. As a result, the fluorescence imaging and photosensitization functions of BT-TPE in the BT-TPE@Fe-LacNPs can be restored under acidic conditions (Scheme 1b).

As a designed specific antitumor theranostic agent, the responses of BT-TPE@Fe-LacNPs were evaluated in solution. Mass spectrometry (Fig. S5 and S6†) revealed peaks corresponding to BT-TPE and Fe-Lac after acid addition (PBS solution with pH = 5.4), indicating the successful molecular complexation and acid-promoted dissociation. The images of TEM and DLS (Fig. S7†) demonstrated that BT-TPE@Fe-LacNPs degraded upon incubation at pH 5.4 for 24 hours. Additionally, the fluorescence intensity of the BT-TPE@Fe-LacNPs reliably increases as the pH value decreases (Fig. S8a†). To gain further insight into the stability of the NPs, we quantified their release over time. It was observed that the BT-TPE@Fe-LacNPs remained stable at pH 7.4, and the nanomaterial decomposed at pH 5.4 (Fig. S8b, c and d†). In addition, no significant absorbance change of BT-TPE or BT-TPE@Fe-LacNPs was observed in the presence of light illumination even after 80 minutes, manifesting the excellent photostability (Fig. S9†). Combining these results, the acid stimuli-responsive disassembly feature of BT-TPE@Fe-LacNPs was carefully verified.

It is known that Fe(III) ions can be reduced to Fe(II) upon depletion of glutathione (GSH).⁴³ To investigate this reduction process, BT-TPE@Fe-LacNPs were incubated with high levels of GSH (2 mM) and the Fe(II) chelator phenanthroline, whose coordination was monitored by UV/VIS absorption spectroscopy (Fig. 2a). The color of the solution (Fig. S10c†) and the characteristic absorption band of the Fe(II)-phenanthroline complex at 512 nm increased significantly with increasing concentration of GSH. Meanwhile, the consumption of GSH by BT-TPE@Fe-Lac was investigated using the GSH-specific probe 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) via UV/VIS absorption spectroscopy. As shown in Fig. 2b, the absorption band at 412 nm decreased markedly, suggesting the substantial depletion of GSH.

Fig. 2 (a) Changes in the UV-Vis absorption spectra of the Fe(II) solution at different concentrations from 1 μM to 30 μM with or without GSH (1 mM). (b) Changes in the UV-Vis absorption spectra of the consumption of GSH by different BT-TPE@Fe-Lac concentrations from 1 μM to 4 μM. (c) Electron spin resonance spectra of BT-TPE and BT-TPE@Fe-Lac upon incubation with the O₂˙⁻ scavenger 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) under different conditions. Laser irradiation at 400–800 nm, 40 mW cm⁻². (d) Electron spin resonance spectra of BT-TPE@Fe-Lac upon incubation with the ˙OH scavenger 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) under different conditions. (e) UV-Vis absorption spectra of ABTS in different solutions. (f) Absorbance at 732 nm of ABTS in different solutions.

Next, to evaluate the BT-TPE@Fe-LacNPs’ ability to generate ROS, we examined the production of superoxide anions (O₂˙⁻) and singlet oxygen (¹O₂) using specific indicators: dihydroethidium (DHE) for O₂˙⁻ and 9,10-anthracenediyl-bis(methylene)-dimalonic acid (ABDA) for ¹O₂. As shown in Fig. S11a and b,†BT-TPE@Fe-Lac was a type-I PS, which could generate O₂˙⁻ upon light irradiation at pH 5.4. We further investigated the generation of free radicals by using electron spin resonance (ESR) spectroscopy. Both BT-TPE and BT-TPE@Fe-LacNPs were incubated with the O₂˙⁻ scavenger 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) (Fig. 2c). The mixtures were then exposed to radiation or kept in the dark. No characteristic signal of DMPO-OH (peak integral ratio 1 : 2 : 2 : 1) was detected in either BT-TPE or BT-TPE@Fe-Lac, but DMPO-O₂˙⁻ (peak integral ratio 1 : 1 : 1 : 1) was generated upon irradiation. Further studies explored the ability of BT-TPE@Fe-Lac to decompose hydrogen peroxide (H₂O₂) into ˙OH via the Fenton reaction in the presence of Fe(III). The ESR spectrum revealed a characteristic signal corresponding to the presence of ˙OH (peak integral ratio 1 : 2 : 2 : 1), with the signal intensity increasing under acidic conditions (Fig. 2d). Importantly, BT-TPE alone could not generate ˙OH under these conditions (Fig. S11c†). The ability of BT-TPE@Fe-Lac to decompose H₂O₂ into ˙OH was further confirmed using UV/VIS absorption spectroscopy with the specific probe 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS) (Fig. 2e). In the presence of hydroxyl radicals, colorless ABTS is oxidized to green oxABTS. As shown in Fig. 2f, only in the co-existence of GSH, H₂O₂ and BT-TPE@Fe-Lac, can ABTS be oxidized into the green product (Fig. S10f†). These results indicate that Fe²⁺, generated after acid addition, can react with H₂O₂ through the Fenton reaction to produce hydroxyl radicals. Above all, these properties illustrate the potential of BT-TPE@Fe-LacNPs for effectively enhancing cell death through the activation of oxidative stress signaling pathways upon the consumption of GSH and the generation of reactive oxygen species (˙OH, O₂˙⁻).

In view of the excellent performance of BT-TPE@Fe-LacNPsin vitro, we further explored the applications of BT-TPE@Fe-LacNPs at the cellular level. ASGPR is an asialoglycoprotein receptor overexpressed significantly on the membrane of HepG2 cells,^44–48 which can specifically recognize galactose residues, potentially providing an ideal platform for the uptake of lactose-complexed BT-TPE@Fe-Lac. Thus, we conducted internalization and selective experiments of BT-TPE@Fe-Lac on three different cell types (HepG2, 4T1 and HeLa cells) through confocal laser scanning microscopy (CLSM) and flow cytometry (Fig. 3a and e). As illustrated in Fig. 3a and b, HepG2 cells treated with BT-TPE@Fe-LacNPs emitted obvious fluorescence after 9 h, whereas weak fluorescence was observed in 4T1 or HeLa cells. In addition, the cellular uptake experiment of BT-TPE was also conducted through CLSM and flow cytometry (Fig. 3c and f). Fluorescence imaging revealed weaker fluorescence signals upon incubation with BT-TPE than with BT-TPE@Fe-Lac in HepG2 cells, which were quantified using Image-J (Fig. 3d). Due to the lactose residues in BT-TPE@Fe-Lac as specific ligands for ASGPRs, they facilitate a faster uptake of BT-TPE@Fe-Lac into HepG2 cells compared with BT-TPE. On this basis, the cellular localization ability of BT-TPE@Fe-Lac was further studied by CLSM. As illustrated in Fig. S12,† the green fluorescence from the BT-TPE@Fe-LacNPs inside the cells was well colocalized with the red fluorescence from lysosomes, and the Pearson correlation coefficient was calculated to be 0.85. The above results confirmed that BT-TPE@Fe-LacNPs mainly accumulated in lysosomes after being endocytosed into the cells by ASGPR-mediated recognition.

Fig. 3 (a) Fluorescence images and (b) quantification of different cells at different time points in the presence of BT-TPE@Fe-Lac (1.0 μM). Scale bar: 20 μm. (c) Fluorescence images and (d) quantification of HepG2 cells at different time points in the presence of BT-TPE (1.0 μM) or BT-TPE@Fe-Lac (1.0 μM). Scale bar: 20 μm. (e) Flow cytometry histogram of different cells at different time points in the presence of 2.0 μM BT-TPE@Fe-Lac. Scale bar: 20 μm. (f) Flow cytometry histogram of fluorescence signals in HepG2 cells after different lengths of time of incubation with BT-TPE@Fe-Lac (2.0 μM) or BT-TPE (2.0 μM).

Considering the aforementioned results of ROS generation in aqueous solution, the ability of BT-TPE@Fe-LacNPs to produce ROS in HepG2 cells was evaluated using 2-dichlorodihydrofluorescein-diacetate (DCFH-DA). Upon light exposure treatment, an obvious green fluorescence signal was observed via CLSM imaging, and BT-TPE@Fe-Lac efficiently generated ROS under normoxic and hypoxic conditions (Fig. S13†). Meanwhile, the dihydroethidium (DHE) staining assay was also carried out to detect the generation of intracellular O₂˙⁻. HepG2 cells treated with BT-TPE@Fe-Lac and DHE followed by photoirradiation showed the characteristic red fluorescence, indicating DHE exposure to O₂˙⁻ (Fig. S13†). Furthermore, we evaluated the generation of reactive oxygen species (ROS) by BT-TPE in living cells treated with DCFH-DA and DHE respectively (Fig. 4a and d). It showed that weaker fluorescence signals were obtained upon incubation with BT-TPE than with BT-TPE@Fe-Lac in HepG2 cells, which were quantified using Image-J (Fig. 4b and e). The results revealed a stronger ROS and O₂˙⁻ production capacity of BT-TPE@Fe-LacNPs in comparison with BT-TPE.

Fig. 4 (a) Fluorescence microscopy images of HepG2 cells incubated with PBS, BT-TPE, and BT-TPE@Fe-LacNPs with laser and stained with DCFH-DA (laser irradiation at 400–800 nm, 40 mW cm⁻²). Scale bar: 50 μm. (b) Relative pixel intensity measurements of the green channel obtained from the images of HepG2 cells incubated under different incubation conditions. (c) Intracellular GSH in HepG2 cells treated with BT-TPE@Fe-LacNPs at different concentrations. (d) Fluorescence microscopy images of HepG2 cells incubated with PBS, BT-TPE, and BT-TPE@Fe-LacNPs with laser and stained with DHE (laser irradiation at 400–800 nm, 40 mW cm⁻²). (e) Relative pixel intensity measurements of the red channel obtained from the images of HepG2 cells incubated under different conditions. (f) Intracellular GSH in HepG2 cells treated with BT-TPE (100 μM) or BT-TPE@Fe-LacNPs (100 μM) (the parameter of CLSM: green channel: 500–550 nm, excited at 487 nm; red channel: 570–620 nm, excited at 562 nm).

Based on the fact that GSH in aqueous solution can be consumed during the reduction of Fe(III) to Fe(II) in BT-TPE@Fe-LacNPs, we evaluated the intracellular GSH consumption. As shown in Fig. 4c, the concentration of GSH in HepG2 cells was also reduced with increasing concentration of BT-TPE@Fe-LacNPs. While the treatment with BT-TPE did not influence the GSH concentration, the incubation with BT-TPE@Fe-LacNPs caused the depletion of GSH (Fig. 4f). These findings suggest that BT-TPE@Fe-LacNPs should stimulate glutathione oxidative stress after entering into tumor cells and create conditions for further intracellular Fenton reactions. As a result, BT-TPE@Fe-LacNPs have the potential to disrupt the redox homeostasis of tumor cells.

Considering the ability to produce ROS and consume GSH in living cells, the synergistic inhibitory effect of BT-TPE@Fe-Lac on the growth of HepG2 cells was examined. In detail, the cytotoxicity of BT-TPE@Fe-Lac towards HepG2 cells was assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay (Fig. 5a). Compared to BT-TPE, BT-TPE@Fe-Lac displayed a certain degree of dark toxicity, which we speculated was caused by CDT. Specifically, BT-TPE@Fe-LacNPs were dissociated in the acidic tumor microenvironment and reacted with excess endogenous hydrogen peroxide in tumor cells via the Fenton reaction to generate ˙OH, resulting in cell apoptosis. Notably, both BT-TPE and BT-TPE@Fe-Lac exhibited phototoxicity after light exposure (Fig. 5a). BT-TPE@Fe-LacNPs contributed to the most striking inhibition of cell viability under light treatment. Under normoxic conditions, the BT-TPE@Fe-LacNPs exhibited significant cytotoxicity towards HepG2 cells under white LED light irradiation for 40 min. The half-maximal inhibitory concentration (IC₅₀) for BT-TPE@Fe-Lac was 1.2 μM. Excitingly, even under hypoxic conditions, BT-TPE@Fe-Lac retained its original phototoxicity, indicating its function was independent of O₂ (Fig. S14†). To visualize the therapeutic effect, HepG2 cells were stained with the cell viability probes calcein AM and propidium iodide (PI) after treatment with BT-TPE@Fe-Lac (Fig. S15†). When incubated with BT-TPE@Fe-Lac and exposed to light, the proportion of red fluorescence increased, suggesting most HepG2 cells were inhibited after 20 hours. Compared to BT-TPE@Fe-Lac, only a minimal amount of cell death was observed on exposure to irradiation after being incubated with BT-TPE (Fig. 5c). In addition, the cancer cell apoptosis was further studied using an Annexin V-FITC/propidium iodide dual staining assay. As shown by flow cytometry, BT-TPE@Fe-LacNPs + light treatment produced the highest apoptosis rate of 94.04% (Fig. S16†), which was in good agreement with the cytotoxicity results from the MTT assay. The results suggest that BT-TPE@Fe-LacNPs induced HepG2 cell death through the synergistic effects of type I PDT and CDT. Moreover, we conducted MTT assays (Fig. 5b) and live/dead cell staining experiments (Fig. 5d) respectively to compare the inhibition effects of BT-TPE@Fe-Lac in three types of tumor cells. It revealed that the inhibition effect of BT-TPE@Fe-Lac in HepG2 cells was superior compared with the other two cell types. Overall, these results show that BT-TPE@Fe-LacNPs can induce cell death of HepG2 cells by a combination of PDT and CDT.

Fig. 5 (a) Cell viability of HepG2 cells under different incubation conditions for 50 h in the dark under normoxic conditions. (b) Cell viability of HepG2, 4T1 and HeLa cells incubated with BT-TPE@Fe-LacNPs (1.0 μM) for different lengths of time. (c) Fluorescence microscopy images of HepG2 cells incubated with PBS, BT-TPE, and BT-TPE@Fe-LacNPs with or without laser and stained with calcein-AM/PI (laser irradiation at 400–800 nm, 40 mW cm⁻²). Scale bar: 200 μm. (d) Fluorescence microscopy images of different cells incubated with BT-TPE@Fe-LacNPs with laser and stained with calcein-AM/PI (laser irradiation at 400–800 nm, 40 mW cm⁻²). Scale bar: 100 μm. Cell viabilities of HepG2 cells were determined upon co-incubation with (e) BT-TPE@Fe-Lac (0.5 μM), (f) BT-TPE (0.5 μM), and different inhibitors. Inhibitor Fer-1 (50 μM), Nec-1 (50 μM), 3-MA (100 μM), and z-VAD-fmk (20 μM) (the parameter of CLSM: green channel: 500–550 nm, excited at 487 nm; red channel: 570–620 nm, excited at 562 nm).

The above results made us further explore the mechanism of cell death induced by BT-TPE@Fe-LacNPs. HepG2 cells in a 96-well plate were incubated with various cell death pathway inhibitors: apoptosis inhibitors (z-VAD-fmk), necrosis inhibitors (Nec-1), autophagy inhibitors (3-methyladenine, 3-MA), and ferroptosis inhibitors (ferrostatin-1). After treatment with these inhibitors, the cells were exposed to BT-TPE@Fe-LacNPs and cell viability was assessed using MTT assay (Fig. 5e). The results showed that BT-TPE@Fe-Lac treatment primarily triggered cell death through a combination of apoptosis and a small part of ferroptosis. In contrast, treatment with BT-TPENPs alone induced cell death exclusively through the apoptotic pathway, without ferroptosis (Fig. 5f). These findings highlight that BT-TPE@Fe-Lac is crucial for activating ferroptosis, suggesting a dual mechanism of action involving both apoptosis and ferroptosis, which could have significant implications for therapeutic applications.

Conclusions

In summary, a novel tumor microenvironment-activated nanoplatform (BT-TPE@Fe-Lac) was successfully developed in this study, which integrates an Fe(III) ion, the AIE-active BT-TPE photosensitizer, and a lactose molecule through dual coordination. The nanoplatform was then utilized for combined photodynamic therapy and chemodynamic therapy. BT-TPE@Fe-Lac was stable under physiological conditions and selectively targeted HepG2 cells which overexpressed ASGPR. To our delight, it rapidly dissociated into BT-TPE molecules and a ferric(III) complex in the acidic lysosomes of cancer cells. Upon irradiation, the BT-TPE molecules only generated O₂˙⁻, inducing cell apoptosis primarily. The released ferric(III) complex played a crucial role in depleting intracellular glutathione, thereby amplifying oxidative stress and enhancing the efficacy of ROS-related therapies. Additionally, the ferric(III) complex reacted with excessive H₂O₂ in the tumor microenvironment to produce ˙OH, thereby facilitating the CDT function. In vitro experiments demonstrated that BT-TPE@Fe-Lac exhibits a significant photodynamic and chemodynamic synergistic antitumor effect on HepG2 cells. It is believed that the innovative combination therapy leveraging both PDT and CDT should open up a new strategy for the treatment of cancer tumors, offering a promising approach to enhance therapeutic outcomes through targeted, multi-modal interventions.

Author contributions

Guo-Wen Xing: conceptualization, formal analysis, funding acquisition, investigation, project administration, resources, supervision, visualization, writing – original draft, and writing – review & editing. Gai-Li Feng: data curation, writing – original draft, and writing – review & editing. Wei Zhou: writing – review & editing. Jin-ping Qiao: data curation and writing – review & editing. Guang-Jian Liu: conceptualization, project administration, supervision, validation, and writing – review & editing.

Data availability

All the data associated with the research in this manuscript are available on reasonable request and can be acquired from the corresponding authors.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We are grateful for financial support from the National Natural Science Foundation of China (21977014).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4nr03871a

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