Disrupting redox homeostasis for tumor therapy based on PDT/chemo/ferroptosis therapeutic hybrid liposomes

Abstract

Synergistic photodynamic therapy (PDT) with other therapeutic modalities can enhance the therapeutic efficacy of tumor treatment and reduce the adverse effects associated with drug leakage and off-target accumulation. However, shaping combined strategies for synergistic therapy remains challenging. Herein, we developed versatile hybrid liposomes self-assembled from Ce6–lipid conjugates and loaded with the chemo drug doxorubicin (DOX) and ferroptosis inducer Fe₃O₄ nanoparticles for synergistic PDT/chemo/ferroptosis therapy. Abundant ROS are generated by PDT upon 650 nm light irradiation, Fe₃O₄-mediated Fenton reaction, and DOX-induced apoptosis. Furthermore, amplifying oxidative stress in cancer cells to disrupt cellular redox homeostasis could accelerate tumor cell death through oxidative damage to lipids, proteins, and DNA. Overall, this work highlights liposome-based therapeutic nanoformulations, thus offering a breakthrough redox homeostasis-based synergistic PDT/chemo/ferroptosis therapy for lung cancer.

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Introduction

Photodynamic therapy (PDT) involves specific excitation wavelengths, oxygen, and photosensitizers, and non-invasive ROS-based tumor therapeutic modalities.^1–3 However, PDT as a monotherapy often suffers from incomplete tumor ablation due to limited light penetration in tissue, hypoxia induced insufficient oxygen supply in solid tumors, challenges in treating metastases, and resistance to the oxidative escape mechanism of the tumor microenvironment (e.g., PDT-induced DNA damage response, which in turn upregulates glutathione peroxidase 4 (GPX4)).^4,5 To date, numerous reports have demonstrated that combined therapy holds great promise for tumor treatment.^6–8 Notably, there are multiple approaches to disrupt intracellular redox homeostasis during antitumor therapy.⁹

Intracellular redox homeostasis refers to the dynamic balance between intracellular redox substances and is vital in maintaining normal physiological processes, e.g., cell growth, metabolism, differentiation, aging, and programmed death.^10,11 This redox homeostasis is also suitable for tumor cells but is maintained at a higher level. Inspired by these overexpressed oxidizing species (H₂O₂) and reducing species (GSH), tremendous efforts have been devoted to achieving controllable drug release^12–14 and redox dyshomeostasis-induced therapeutic strategies. Conversely, amplifying oxidative stress to disrupt cellular redox homeostasis could accelerate tumor cell death through oxidative damage to lipids, proteins, and DNA.^15,16

Combining chemotherapy with PDT is an effective therapeutic strategy for redox dyshomeostasis^17,18 because many chemotherapy drugs can increase the ROS (such as H₂O₂) level in tumor cells. For instance, the FDA-approved doxorubicin (DOX) could rapidly induce oxygen into ROS, including H₂O₂, by a single-electron addition to the quinone moiety of ring C in the DOX molecule to form semiquinone.^19,20 Therefore, the combination of PDT with DOX in a nanoplatform has shown great potential for oxidative damage to tumor cells via ROS accumulation.

Furthermore, converting H₂O₂ into highly toxic ROS, such as hydroxyl radicals (˙OH), would be a better strategy to amplify antitumor effects. Fortunately, the intracellular Fenton reaction is H₂O₂-dependent, and Fe²⁺/Fe³⁺ mediates ˙OH generation.^21,22 The Fenton or Fenton-like-based reagents were recently explored to boost this reaction and then induce cell apoptosis for antitumor therapy.^23,24 In addition, amplified oxide stress causes ferroptosis—a newly discovered iron-dependent, non-apoptotic, and programmed cell death pathway.^25,26 Ferroptosis is characterized by the accumulation of lipid peroxides (LPOs), the downregulation of GPX4, the vacuolation of mitochondria, and the disappearance of their ridges.^27,28 Emerging evidence suggests that related inducers could potentially trigger ferroptosis for antitumor therapy, particularly for eradicating the resistance to traditional monotherapies. Consequently, combining the PDT/chemo/ferroptosis therapeutic strategies exhibits good potential in boosting the intracellular ROS level and breaking redox homeostasis, thus significantly strengthening the antitumor effect.

Herein, we developed Ce6–lipid stabilized DOX and Fe₃O₄ nanoparticle (NP)-based hybrid liposomes for PDT/chemo/ferroptosis synergistic tumor therapy. Synthetic Ce6–lipid, as one of the backbones of liposomes, could effectively prevent the release of Ce6 from hybrid liposomes and avoid the decrease in single oxygen efficiency caused by Ce6 aggregation. The released DOX molecules could anchor the DNA pairs, inducing cell apoptosis and increasing intracellular ROS. Subsequently, Fe₃O₄ NPs could release ferrous ions under acidic TME and convert a high level of H₂O₂ into ˙OH to induce ferroptosis. This study provides a promising synergistic strategy for regulating cellular redox homeostasis and enhancing the therapeutic efficacy of lung cancer (Scheme 1).

Scheme 1 Schematic representation of (a) the hybrid liposomes and (b) their ROS dyshomeostasis mechanism of antitumor action.

Results and discussion

Liposome characterization

To synthesize Ce6–lipid stabilized DOX and Fe₃O₄ hybrid liposomes, various components, including Ce6–lipid and water-soluble Fe₃O₄, were prepared. Briefly, the photosensitizer Ce6 was conjugated to the lyso-PC to form a Ce6–lipid molecule via the ester linkages, and its chemical structure was confirmed by ¹H NMR (Fig. S1a†). It could be incorporated into liposomes as a framework through self-assembly due to the hydrophobic nature of Ce6–lipid. The as-obtained Lipo-Ce6 liposomes could be excited by a specific laser to perform PDT. Thus, to obtain the optimal doping ratio of Ce6–lipid, various Ce6–lipid-based (Lipo-Ce6) liposomes were prepared via thin film hydration. Dynamic light scattering (DLS) results in Fig. S1b† show that a similar size (∼130 nm) and narrow PDI of Lipo-Ce6 liposomes could be detected among the different doping levels of Ce6–lipid. Because cytotoxic singlet oxygen yield is critical for PDT, the singlet oxygen production profiles of the Ce6–lipid-based liposomes were evaluated using a DPBF probe. As shown in Fig. S1c,† with the increase in irradiation time, the absorption spectra intensity of DPBF at 417 nm gradually decreases upon 650 nm light irradiation, indicating that the resulting singlet oxygen consumed the DPBF molecules. A Ce6–lipid content higher than 9% cannot further increase the singlet oxygen yield, implying that aggregation-induced quenching occurs. Thus, the optimal amount of Ce6–lipid is 9% (w/w, Ce6 ≈ 5%). Next, to prepare the Lipo@Fe₃O₄ liposomes, a similar film hydration method was performed using the aqueous Fe₃O₄ NP solution. Before that, the aqueous soluble Fe₃O₄ NPs were obtained using the NOBF₄-treated method²⁹ to remove the surface-capped oleic acid ligands of Fe₃O₄ NPs. After centrifugation, the Fe₃O₄ NPs were transferred from the oil phase (cyclohexane) to the aqueous solution. Afterward, a chemotherapeutic agent (DOX) was loaded into the Lipo-Ce6 liposomes using an (NH₄)₂SO₄ gradient method³⁰ to prepare Lipo-Ce6@DOX liposomes. Subsequently, the Lipo-Ce6@Fe₃O₄–DOX hybrid liposomes were prepared by applying a successive hydration process.

Furthermore, successful encapsulation was confirmed by transmission electron microscopy (TEM) assay. It can be observed that the Fe₃O₄ NPs were packed within the liposomes with high contrast (Fig. 1a). The encapsulation of DOX was further confirmed by comparing UV-Vis absorption spectra before and after introducing the DOX solution (Fig. 1b), and the drug loading efficiency (DLE) of DOX is 70%, where DLE = (weight of loaded drug/weight of input drug) × 100%, drug loading capacity (DLC) is 9.2%, and DLC = (weight of loaded drug/weight of polymer + drug used) × 100%. Those loaded solely with DOX (Lipo-DOX) and Lipo@Fe₃O₄ liposomes were also studied by DLS with 106.6 ± 3.48 nm and 110.06 ± 5.2 nm, along with zeta potentials of −3.2 mV and −9.08 mV, respectively (Fig. 1c and d). These results confirm the successful preparation of various liposomes.

Fig. 1 (a) TEM image of hybrid liposomes, scale bar: 200 nm. (b) UV-vis spectra of liposomes (Lipo-Ce6@Fe₃O₄–DOX, Lipo-Ce6, Lipo@DOX and Lipo@Fe₃O₄) and free DOX. (c) Hydrodynamic diameter and (d) zeta potential of various liposomes (Lipo-Ce6, Lipo@DOX, Lipo@Fe₃O₄, and Lipo-Ce6@Fe₃O₄). (e) Payload release profiles. (f) ROS generation of hybrid liposomes after 650 nm light irradiation with DPBF as a probe.

Evaluation of payload release profiles and singlet oxygen detection

To investigate the DOX release profiles, hybrid liposomes of Lipo-Ce6@Fe₃O₄–DOX were performed in a pH 7.4 buffer medium. The cumulative drug release properties in Fig. 1e demonstrated the DOX-released profiles from the hybrid liposomes within 24 h. A rapid release of DOX was observed in the first 4 h. Then, the DOX release rate decreased, and the cumulative release rate was ∼63%. To avoid the decrease in singlet oxygen efficiency caused by the aggregation of free Ce6 molecules, similarly, we further studied the Ce6 release profiles. Fortunately, unlike DOX, no noticeable characteristic Ce6 absorption spectrum was detected at around 650 nm, indicating that Ce6–lipid molecules were stable in the matrix and not released from hybrid liposomes under the same conditions because they were anchored in the liposome skeleton (Fig. S2†). These results suggest that the hybrid liposomes could maintain stable singlet oxygen efficiency upon 650 nm irradiation. Next, DPBF molecules were employed as a singlet oxygen probe to evaluate the singlet oxygen generation level. As shown in Fig. 1f, after increasing the light exposure time upon 650 nm excitation, the decreased DPBF absorbance at 417 nm indicates effective singlet oxygen generation.

Cellular uptake and in vitro antitumor effects

The endocytosis behavior of Lipo-Ce6@ Fe₃O₄–DOX hybrid liposome was then studied in A549 cells, significantly influencing the phototherapy effects. Fluorescence microscopy was used to evaluate intracellular uptake. The red emission of Ce6 was observed around the nuclei stained by DAPI (blue emission) and within the cell membrane stained by DiO over time (the relative intensity from 1 to 30 min is 15.06, 128.03, and 106.65%, quantitative statistics, as shown in Fig. S3,† indicating that the hybrid liposomes were efficiently taken up in A549 cells within 10 min of incubation (Fig. 2)).

Fig. 2 Intracellular uptake profile of hybrid liposomes at different times. Scale bar = 50 μm. Corresponding quantitative statistics are shown in Fig. S3.†

Next, hybrid liposome-mediated PDT/chemo/ferroptosis synergistic antitumor effects were studied. The CCK8 assay was used to assess the combined therapy effects after various nanoformulations of liposome treatment (Fig. 3). To better demonstrate the therapeutic effects of different treatment methods, the PDT effect related to light dose (time-dependent) was first studied. After the treatment of Lipo-Ce6 liposomes under 650 nm light irradiation (100 mW cm⁻²), the relationship between exposure time and cytotoxicity is shown in Fig. 3a. Cell viability declined rapidly when A549 cells were exposed to the 650 nm light. The cell viability was about 50% after 20 s of irradiation and significantly decreased upon 40 s of irradiation. After confirming the PDT effects in vitro, we further evaluated the dose-dependent effect of DOX and Fe₃O₄ on cell viability in Lipo@DOX and Lipo@Fe₃O₄ liposomes, respectively. With encapsulated DOX over 24 h incubation, only slight cytotoxicity was observed. However, obvious cytotoxicity was observed with free DOX at the same concentration (Fig. S4a†), as it has higher bioavailability and can diffuse freely into cells compared to the DOX encapsulated and retained in liposomes.³¹ In addition, a dose-dependent manner of Lipo@Fe₃O₄ liposomes showed that cell viability decreased to 47.5% at 400 μg mL⁻¹, exhibiting slightly stronger toxicity than Fe₃O₄ simultaneously (Fig. S4b†). Therefore, the dose-dependent Lipo-Ce6@Fe₃O₄–DOX liposomes (determined by Ce6 concentration) with or without 650 nm light irradiation (100 mW cm⁻², 40 s) were investigated. As shown in Fig. 3d, the combined treatment group shows a synergistic effect compared to Lipo-Ce6 + Light and Lipo-Ce6@Fe₃O₄–DOX without light groups. The combination index (CI) was further calculated between PDT and Fe₃O₄/DOX (CI < 1.0) according to Chou and Talalay's principle,^32,33 namely, lower doses achieved greater antitumor effects, indicating better efficacy of Lipo-Ce6@Fe₃O₄–DOX liposomes in inducing cell damage. Flow cytometry with annexin V-FITC and PI staining supported the cellular killing mechanism. As shown in Fig. 3e, the Lipo-Ce6 induced an early apoptosis rate of up to 36.4% compared to the PBS control group. After treatment with Lipo-Ce6@Fe₃O₄–DOX under the same irradiation conditions, the introduction of DOX/Fe₃O₄ further increased cell apoptosis, suggesting that PDT and DOX-induced apoptosis achieved significant therapeutic efficacy because Fe₃O₄ was mainly a ferroptosis inducer.

Fig. 3 Cell viability of A549 cells after treatment with (a) Lipo-Ce6 at 1.5 μg mL⁻¹ and exposure to 650 nm light over time; (b) Lipo@DOX at various concentrations (0, 0.5, 1, 2 and 4 μg mL⁻¹); (c) Lipo@Fe₃O₄ at various concentrations (0, 50, 100, 200 and 400 μg mL⁻¹); (d) Ce6-based liposomes at various concentrations (0, 0.25, 0.5, 1.0 and 2.0 μg mL⁻¹) and (e) apoptosis analysis of A549 cells after different treatments (PBS, Lipo-Ce6 and Lipo-Ce6@Fe₃O₄–DOX), followed by staining with annexin V/PI and analysis using flow cytometry.

To further investigate the PDT-induced cell damage mechanism in vitro, the Ce6-treated cells were irradiated with 650 nm light for RNAseq analysis. We performed RNA-seq analyses on wt and ROS (Ce6-mediated ¹O₂)-treated cells. There was a total of 1124 differentially regulated genes (fold change >2, p-value ≤ 0.01), of which 622 genes were up-regulated, and 502 genes were down-regulated upon PDT treatment (Fig. 4a). The number of genes changing is summarized, as presented in Table 1 of ESI.†

Fig. 4 (a) Volcano plot of genes differentially expressed in ROS compared to WT. Red dots represent genes with a log2 fold change of >±1 and p-value of ≤0.01. Functional annotation analysis of differentially expressed genes using metascape.³⁴ (b) KEGG analysis of upregulated gene pathways. (c) KEGG analysis of downregulated gene pathways. (d) Heatmap of expression of representative ferroptosis genes after PDT.

As shown in Fig. 4b, gene pathway analyses for the upregulated genes showed that they were enriched for pathways, such as protein processing in the endoplasmic reticulum and ferroptosis. The enriched gene pathways for the downregulated genes almost exclusively involve metabolic processes, such as folate biosynthesis and phenylalanine metabolism. In addition, it shows that PDT can regulate the ferroptosis process through up-regulated related genes (Fig. 4c). Furthermore, as shown in Fig. 4d, a significant subset of ferroptosis genes, including PTGS2 and ACSL4, was dysregulated in PDT-treated cells, suggesting that ROS-mediated transcriptome changes carry some lipid metabolism of ferroptosis.

In addition, to boost the ferroptosis of A549 cells, the Fe₃O₄ NPs as ferroptosis inducers were employed and loaded within liposomes (Lipo-Fe₃O₄) for synergetic therapy. We used a Liperfluo probe to detect the accumulated LPO in vitro because LPO accumulation was considered a typical hallmark of ferroptosis. As shown in Fig. 5, no apparent green fluorescence was detected in the PBS group, while the intracellular green fluorescence intensity was enhanced after treatment with Lipo-Ce6@Fe₃O₄–DOX hybrid liposomes; particularly, when exposed to a 650 nm light, it showed further fluorescence intensity boost. Moreover, when the cells were pretreated with deferoxamine (DFO, an iron chelator), the inhibition of LPO accumulation (extremely weak green emission) confirmed the occurrence of ferroptosis. These results verified that combining hybrid liposomes and 650 nm light could induce ferroptosis in A549 cells.

Fig. 5 Detection of intracellular LPO of A549 cells after various treatments. (a) PBS, (b) Lipo-Ce6@Fe₃O₄–DOX, (c) Lipo-Ce6@Fe₃O₄–DOX and (d) pretreated with DFO. Scale bar: 100 μm. The corresponding quantitative statistics are shown in Fig. S5.†

Furthermore, as can be observed from Fig. 6b, the mRNA level of GPX4 demonstrated that the GPX4 expression was down-regulated over time and significantly deactivated after 3 h of incubation, indicating that ferroptosis can be evoked by Fe₃O₄-based liposome.³⁵ Moreover, the dose-dependent results suggested that the concentration of Fe₃O₄ over 50 μg mL⁻¹ significantly deactivated the GPX4 within 6 h (Fig. 6a). As shown in Fig. S6a and b,† the western blot results further confirmed that Fe₃O₄ could induce the ferroptosis of A549 cells, which is consistent with a previous report.²³ Fig. 6c and d show the expression level of GPX4, which was down-regulated after treatment with Lipo-Ce6@Fe₃O₄–DOX NPs. After PDT treatment, GPX4 expression was further suppressed because of enhanced ROS production.

Fig. 6 (a) Dose dependence of Fe₃O₄ on the mRNA level of GPX4. (b) Time-dependent mRNA level after treatment with 200 μg mL⁻¹ of Fe₃O₄. (c) Western blot analysis and relative expression of GPX4 after treatment with Lipo-Ce6@Fe₃O₄–DOX hybrid liposomes (NPs), hybrid liposome + light (NPs + L) and hybrid liposome + DFO (NPs + DFO), and (d) corresponding quantified proteins. n = 3.

In vivo antitumor effects

Next, the antitumor efficacy in vivo was evaluated in Lewis lung carcinoma (LLC) tumor-bearing mice. Different formulations were i.v. injected and treated with various methods, including PBS, 650 nm light only, hybrid liposomes, and hybrid liposomes + 650 nm light (100 mW cm⁻² for 6 min irradiation). As shown in Fig. 7, the hybrid liposomes with or without 650 nm laser irradiation exhibited tumor growth suppression capability compared to the PBS and 650 nm light-only group (Fig. 7a), indicating the effectiveness of our synthesized hybrid liposomes in antitumor therapy. The hybrid liposomes + 650 nm light group induced the most significant inhibition effect, evidenced by the lowest tumor growth rate (Fig. 7b) due to the synergistic light-induced PDT and chemo/ferroptosis (dark). Notably, no significant changes in the body weight of the mice were observed during the treatment (Fig. 7b). Hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assays showed remarkable necrosis of tumor cells in the hybrid liposomes with a 650 nm light irradiation group, and negligible damage to normal organs was determined (Fig. S6†), suggesting the high safety of this liposome-based delivery system.

Fig. 7 In vivo antitumor effects of hybrid micelles. (a) Representative digital photos of tumor tissues from the different groups (PBS, 650 nm laser, Lipo-Ce6@Fe₃O₄–DOX (NPs), and Lipo-Ce6@Fe₃O₄–DOX + 650 nm laser (NPs + L)). (b) Tumor growth curves of mice after various treatments. (c) Body weight of mice after various treatments. (d) H&E and TUNEL staining images of tumor tissues from the different groups on the 14^th day. Scale bar: 100 μm. The data are shown as mean ± standard deviation (s.d.) of three independent experiments, and ***p < 0.001 and *p < 0.05.

Conclusions

In summary, we developed hybrid liposomes of Lipo-Ce6@Fe₃O₄–DOX for PDT/ferroptosis/chemotherapy of tumors. The lipid-Ce6 in the framework of liposome prevents the Ce6 leakage from off-target and aggregate-induced quenching, ensuring long-term stability. The RNAseq analysis indicates that PDT could induce A549 cell apoptosis and ferroptosis, which synergize with a dose-dependent intracellular ROS inducer of DOX and a ferroptosis inducer of Fe₃O₄ NPs, showing effective cancer cell inhibition through improved ROS generation efficiency. The in vivo results further confirmed the effectiveness of hybrid liposomes in antitumor cancer therapy. We propose a breaking redox homeostasis strategy to address a broad spectrum of tumor therapy challenges.

Materials and methods

Preparation of liposomes

Lyso-Pc–Ce6 conjugates (Ce6–lipid) were synthesized by coupling Lyso-Pc with Ce6 molecules via an acylation reaction under the catalysis of EDCI and DMAP. The blank Ce6–lipid stabilized liposomes were prepared by applying thin-film hydration methods with lecithin :  DSPE-PEG : cholesterol : Ce6–lipid (8.5 : 1.5 : 2 : x%, x = 1, 3, 6, 9 and 12, w/w). First, the mixture was fully dissolved in 3 mL of chloroform; then, the solvent was removed by rotary evaporation. After that, 1 mL DI-water was added to the formed film and hydrated at 45–50 °C for about 30 min to form liposomes. Similarly, the Fe₃O₄-only liposomes with Ce6-free were prepared. The optimal ratio of Ce6–lipid in the liposome was determined by singlet oxygen yield upon 650 nm light irradiation. pH-induced hydration methods prepared the DOX-only liposomes (Ce6-free). Typically, 2 mL of NH₄SO₄ solution (123 mM) was added and hydrated at 45–50 °C for 15 min. The as-obtained Lipo@DOX was purified through a 220 nm filter, then transferred to a dialysis bag, and dialyzed against PBS buffer for 30 min. Afterward, the DOX was added to the above solution with 1 mg mL⁻¹ and rehydrated for 30 min at 65 °C. The product was purified through a 220 nm filter and collected after re-dialysis. Similarly, a successive hydration approach was developed to co-encapsulate DOX and Fe₃O₄ NPs. The size and zeta potential of the formulations were characterized using Zetasizer NanoZS nanopositioners (Malvern), and their morphologies were recorded using a JEM-1200EX transmission electron microscope (JEOL, USA). Finally, the DOX-loading efficiency was measured by monitoring the absorption spectrum at 490 nm after dialysis of the hybrid liposomes.

Singlet oxygen detection

DPBF, as an indicator, was used to evaluate the singlet oxygen yield under 650 nm light irradiation in different Ce6–lipid doping levels of liposomes. Typically, 5 μL of DPBF (2 mg mL⁻¹) in ethanol was added to the various formulations and irradiated with 650 nm at 50 mW cm⁻². After determining the irradiation time, the DPBF consumption profiles were monitored by applying a spectrophotometer to record the absorption spectrum at 417 nm.

Payload release profiles in vitro

We investigated the payload release profiles from liposomes using the dialysis method.¹ Briefly, 1 mL liposomes were transferred to a dialysis bag (MW = 3500 cut-off) and immersed in a flask containing 100 mL PBS solution (pH = 7.4) under gentle stirring. Then, a 1 mL release medium containing DOX/Ce6–lipid molecules was removed at the determined time intervals, and the absorption spectrum was monitored at 490 nm or 650 nm using a spectrophotometer. The amount of DOX released from the liposomes was calculated using the following equation: release% = C_release/C_total × 100%.

Cellular experiments

Cell uptake and viability assay. A549 human lung cancer cells were cultured in a 25 mL culture flask with RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin–streptomycin for 24 h. The cells were maintained at 37 °C and 5% CO₂.
Cell viability analysis. A549 cells were incubated with various formulations for 4 h, washed twice with PBS, and subjected to 650 nm laser irradiation or no laser irradiation. A CCK8 assay was used to measure cell viability. In addition, the spectrophotometric absorbance was measured at 450 nm using a microplate reader.
Cell uptake. A549 cells were seeded in glass bottom culture dishes. Afterwards, the Ce6-based liposomes were added to the dishes after 12 h of cell adherence and further incubated for 10 min and 30 min at 37 °C and 5% CO₂, respectively. Then, the cells were fixed with 4% paraformaldehyde for 15 min, washed twice with PBS, and stained with DAPI staining solution (diluted 2000×, Beyotime, China) for 5 min and a DiO kit (1×, Beyotime, China) for 45 min. The fluorescence intensity was observed by confocal microscopy (CSi2, Nikon, Japan).

RNA-seq analysis

To examine the function of Ce6-mediated ROS (¹O₂) at the biological function, the Ce6-treated A549 cells with and without 650 nm light irradiation (100 mW cm⁻² for 10 s) were tested. Total RNA from A549 cells was extracted using the RNAiso Plus (9109, Takara, Tokyo, Japan) and sent to Biomarker Technologies Co., Ltd. (Beijing, China) for RNA-seq analysis. Reads were mapped to the GRCh38 genome using HISAT2 (v2.1.0).³⁶ Transcripts were counted with the R package summarizeOverlaps (https://www.r-project.org/). Differential expression analysis was performed with DESeq2.³⁷ Genes with a false discovery rate ≤ 0.01 and a fold change ≥ 2 were selected.

Flow cytometry assay

For the flow cytometry analysis, the cells were trypsinized and stained with annexin V-FITC and propidium iodide (Annexin V-FITC Apoptosis Staining/Detection Kit) to measure the cells experiencing apoptosis. A549 cells were cultured in 6-well plates and then treated with different reagents for 4 h, and each parallel was then exposed to laser irradiation for 15 s at a density of 100 mW cm⁻². Cells were harvested after 24 h, washed twice with PBS, and resuspended in 50 μL of binding buffer. 5 μL of annexin-V-FITC and 5 μL of PI were added to cells and then incubated for 15 min at room temperature. 400 μL of binding buffer was added to each tube and analyzed by flow cytometry.

Western blotting

A549 cells were disrupted in RIPA lysis buffer. Protein samples were separated through SDS-PAGE and transferred to PVDF membranes for immunoblotting, and the membranes were incubated with specific antibodies targeting GPX4 and GAPDH at 4 °C overnight. Blots were incubated with the corresponding secondary antibodies for 1 h at room temperature and visualized using ECL.

In vivo antitumor therapy

All animal experiments were approved by the Institutional Ethical Committee of Animal Experimentation of the First Hospital of Jilin University, and they were performed strictly compliant with “The National Regulation of China for Care and Use of Laboratory Animals”. C57BL/6 mice (6–8 weeks, ∼20 g) were purchased from Liaoning Changsheng Biotechnology Company. The mice were inoculated by subcutaneous injection of LLC cells (1 × 10⁶) into the right flank of the mice. After the tumor volumes reached ∼100 mm³, the mice were randomly divided into 4 groups (n = 5). The tumor volumes were calculated using the formula [(L × W²)/2], where length (L) represents the larger tumor diameter and width (W) represents the smaller one. The mice were i.v. injected with PBS (200 μL) and hybrid liposomes (200 μL, 0.108 mg mL⁻¹). After 4 h and 12 h, the 650 nm light only (200 μL PBS) and hybrid micelle groups were exposed to 650 nm light at 100 mW cm⁻² for 6 min, respectively. Afterward, tumor growth and body weight were monitored every 2 days, and the mice were sacrificed after 14 d. The tumor and various organs (heart, liver, spleen, lung, and kidney) were excised to fix in 10% neutral buffered formalin and stained via hematoxylin and eosin (H&E) for pathological analysis.

Statistical analysis

All data are expressed as the mean ± standard deviation (SD), and the significance of differences among groups was evaluated with Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001).

Data availability

The data supporting this article have been included as part of the ESI.†

Author contributions

Y. C. and S. H. conceived and designed the experiments. Y. H., H. L., Y. Z., H. C., Q. L. and X. L. performed the experiments. Y. H., H. L., H. C., Q. L., D. C. and Y. C. analyzed the data and prepared the manuscript. All authors contributed to manuscript writing and discussions.

Conflicts of interest

There are no conflicting interests of the authors.

Acknowledgements

This research was financially supported by the National Natural Science Foundation of China (Grant No. 62075217, 62305329), Project of Science and Technology Agency, Jilin Province (20210101148JC, 20210101289JC, 20230508104RC and 20230402039GH), Changchun Science and Technology Bureau (Grants 23GZZ06), the China Postdoctoral Science Foundation (Grants 2023M733432, 2023M741350) and Medical Special Project of Jilin Provincial Department of Finance (JLSWSRCZX2021-054).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra03361b

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