English

• Cancer is a serious global health issue, and due to its secrecy of occurrence and high mortality rate, a large number of people suffer from cancer and lose their lives every year [1,2]. The treatment of cancer remains a huge challenge, and exploring effective and safe cancer treatment methods is of great significance. Carbon monoxide (CO) is a well-known endogenous gas signaling molecule that participates in a variety of life activities and plays a signal transduction role in living organisms [3,4]. Although CO is a toxic gas molecule, recent studies have shown that it has good therapeutic effects on many diseases, especially in tumor treatment, showing excellent anti-tumor performance [5–7]. Notably, cancer patients often develop multidrug resistance (MDR) during treatment, CO therapies can mitigate or reverse MDR via specific mechanisms [6]. Consequently, the use of CO for tumor therapy has received increasing attention [8–13]. However, CO can easily bind to hemoglobin in the body to produce high toxicity, making it very difficult for CO to safely and effectively reach the cancer site. Therefore, how to accurately and controllably supply CO at the cancer site is a major challenge for the clinical application of CO in cancer treatment.

  Due to the lethality of CO gas, it is very difficult to directly administer CO gas to patients for cancer treatment [14]. Thus, the development and use of CO-releasing molecules (CORMs) instead of CO gas has emerged. Although many metal carbonyl complexes have been developed and widely used as CORMs, their CO release is difficult to control. For example, CORM-3, a commercial CO donor, decomposes when it meets water and releases CO immediately and uncontrollably [15]. In addition, the toxicity of heavy metals also hinders the application of these metal carbonyl complexes in cancer treatment [16–19]. More recently, photo-controlled metal-free fluorescent CORMs have received increasing attention. This type of CORMs not only can solve the problem of metal toxicity, but also release CO through light control, and track the CO release process through fluorescence changes [20–23]. However, most of the reported photo-controlled metal-free fluorescent CORMs show short wavelengths and quenched fluorescence during the release of CO, making it difficult to track the release process of CO in vivo. In addition, most of the reported CORMS have not been studied for cancer therapy (Table S1 in Supporting information) [24].

  In this work, we report a novel metal-free visible light-triggered CORM (COR-XAC, Scheme 1) which shows near-infrared (NIR) fluorescence changes during the release of CO and can be used for self-monitoring of CO delivery and effective cancer treatment. COR-XAC is composed of hydroxylflavone and oxanthracene, forming a large conjugated system with NIR fluorescence at 760 nm. COR-XAC adopts the structure of hydroxyflavone because such compounds have good photo-controlled CO release performance [25–33]. When exposed to 580 nm light, COR-XAC undergoes photolysis, releasing CO and producing another compound i-COR-XAC with NIR fluorescence at 690 nm. This makes the CO release process accompanied by ratiometric NIR fluorescence changes so it can be accurately self-monitored. Moreover, the NIR fluorescence has deep tissue penetration and low biological background fluorescence interference [34–37], which enables COR-XAC to track the CO release in vivo. Our results show that the release of CO by COR-XAC not only can be controlled by visible light and tracked by ratiometric fluorescence in cells and in vivo, but also demonstrates significant inhibitory effects on the proliferation of cancer tissues and exhibits good anti-tumor effects.

  Scheme 1

  

  Scheme 1.  NIR fluorescent CORM (COR-XAC) with ratiometric fluorescence changes for carbon monoxide release and tumor therapy.

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  Scheme S1 shows the synthesis process of COR-XAC. Briefly, compound 3 was first prepared using compounds 1 and 2, and then it was condensed with compound 4 to afford COR-XAC. The structure of COR-XAC and compound 3 were characterized by nuclear magnetic resonance (NMR), mass spectrometry (MS), and high-resolution mass spectrometry (HRMS) (Figs. S1–S7 in Supporting information).

  Firstly, the CO release performance of COR-XAC under 580 nm irradiation in phosphate buffered saline (PBS) (10 mmol/L, 30% MeCN, pH 7.4) was investigated. As shown in Fig. 1, before irradiation, the COR-XAC solution shows a maximum absorption peak at 551 nm and emits NIR fluorescence at 760 nm (Fig. 1A). However, when the solution was irradiated with 580 nm light, the fluorescence at 760 nm steadily attenuated, while a novel emission peak progressively emerged at 690 nm, giving obvious ratiometric fluorescence changes (Fig. 1B). Meanwhile, the absorption of COR-XAC at 551 nm gradually decreased until it reached the plateau after being illuminated for 50 min (Fig. S8a in Supporting information). The half-life of COR-XAC was calculated to be about 18 min by fitting the absorption changes at 551 nm (Fig. S8b in Supporting information). In addition, as shown in Fig. S9 (Supporting information), COR-XAC can release CO over a broad pH range with significant fluorescence signal changes, suggesting that it has potential for application in biological systems.

  Figure 1

  

  Figure 1.  (A) The ultraviolet–visible spectroscopy (UV–vis) and fluorescence spectra of COR-XAC (10 µmol/L). (B) Fluorescence spectral changes of COR-XAC (10 µmol/L) after being irradiated (580 nm, 2 mW/cm²) for 60 min, and each data was collected after every 10 min of irradiation. For fluorescence measurements, λ_ex = 560 nm. (C) The released CO from the irradiated COR-XAC solution was identified by Probe 1 (10 µmol/L Probe 1 and 10 µmol/L PdCl₂. λ_ex = 493 nm). (D) The release of CO in the irradiated COR-XAC solution was determined by myoglobin assay.

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  The chemical stability of COR-XAC was then investigated. As shown in Fig. S10 (Supporting information), except for the 580 nm light radiation, when a variety of analytes (metal ions, amino acids, anions, and biothiols) coexist with COR-XAC, only weak fluorescence fluctuations were generated at 760 nm, while no obvious fluorescence changes were observed at 690 nm, indicating that COR-XAC is stable for these analytes and exhibits the uniqueness of photo-controlled CO release. Moreover, in the presence of these analytes, a significant ratiometric fluorescence signal change could still be generated after illuminating the solution of COR-XAC (Fig. S11 in Supporting information), indicating that these substances have no obvious effect on the ability of COR-XAC to release CO, which provides a possibility for the release of CO in complex living systems.

  To verify the CO release of COR-XAC under light irradiation, two methods of CO detection were used. Firstly, a known fluorescent CO probe, Probe 1 was used, which can generate a strong fluorescence signal at ~525 nm in the presence of CO [38]. As shown in Fig. 1C, Probe 1 solution and the mixture of Probe 1 and COR-XAC did not produce obvious fluorescence, however, when the light-exposed COR-XAC solution was treated with Probe 1, an obviously enhanced fluorescence signal was observed at 525 nm, indicating that CO was released from the light irradiated COR-XAC solution. Secondly, myoglobin (Mb) assay was also used. This assay utilizes the principle that deoxymyoglobin (deoxy-Mb) can bind with CO and convert into carbon monoxide myoglobin (MbCO) [39,40]. Deoxy-Mb exhibits a prominent absorption at 552 nm, while MbCO shows two absorption peaks at 543 nm and 580 nm. As shown in Fig. 1D, when deoxy-Mb was added to the light-irradiated COR-XAC solution, the absorption peak of deoxy-Mb disappeared, and the two absorption peaks of MbCO were generated. These results show that COR-XAC releases CO after irradiation. According to previous reports on 3-hydroxylflavone-based CORMs [25–33], another photolysis product i-COR-XAC (Scheme S2 in Supporting information) should be generated during the release of CO. This was proved by the mass analyses of the light-irradiated COR-XAC solution (Figs. S12 and S13 in Supporting information).

  Before exploring the capacity of COR-XAC to release CO in cells, we first evaluated the cytotoxicity of COR-XAC and i-COR-XAC. As shown in Fig. S14 (Supporting information), even if the concentration of COR-XAC (under dark conditions) and i-COR-XAC reached 50 µmol/L, the cells showed a high survival rate after incubation for 24 h, indicating that both of them have low cytotoxicity. Based on this, we then conducted cell imaging of COR-XAC to explore its ability to release CO at the cellular level and monitored the CO release through the Probe 1 system (Fig. 2). Incubation of cells with Probe 1 alone resulted in no apparent fluorescence signals. However, under dark conditions, cells incubated with COR-XAC for 30 min at 37 ℃ displayed strong red fluorescence (D1), while the yellow channel (pseudo color) showed weak fluorescence (C1). In addition, the green channel indicated by Probe 1 (B1) showed very weak fluorescence. This indicates that COR-XAC released a small amount of CO during fluorescence imaging due to laser irradiation (excitation). Next, the cells underwent irradiation using 580 nm light for 30 and 60 min, respectively. One can see that, the red channel fluorescence slowly dropped (D2-D3), while that of the yellow and green channels rose steadily over time (C2-C3, B2-B3 respectively). Moreover, the ratio imaging of the yellow channel and red channel intuitively presents the ratiometric fluorescence changes (F1-F3). This indicates that COR-XAC is not only capable of delivering CO into living cells but also can self-report the release of CO through its changing ratiometric fluorescence.

  Figure 2

  

  Figure 2.  The release of CO from COR-XAC (10 µmol/L) in HeLa cells monitored by itself fluorescence change and Probe 1. (A–F) Cell blank. (A1–F1, A2–F2, and A3–F3) Cells were pretreated with COR-XAC (10 µmol/L) for 30 min, and then irradiated with light for 0, 30, and 60 min, respectively, and finally incubated with Probe 1 for another 30 min. (A–A3) Bright field. (B–B3) Green channel images, λ_em = 500–550 nm, λ_ex = 488 nm. (C–C3) Yellow channel images, λ_em = 600–700 nm, λ_ex = 561 nm. (D–D3) Red channel images, λ_em = 730–800 nm, λ_ex = 561 nm. (E–E3) Merges of the yellow and red channel. (F–F3) The ratio of the yellow and red channels. Scale bar: 25 µm. (G–I) Quantified fluorescence for the green, yellow, and red channels, respectively. Data are presented as mean ± standard deviation (SD) (n = 3).

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  What is more, to prove that COR-XAC has excellent photo-controlled and accurate CO release performance in cells, we randomly select an area in the cell field of view and directly irradiate it with a confocal laser. As shown in Fig. 3, at the initial stage of imaging, the red fluorescence was strong and the yellow fluorescence was weak (A-A1). After irradiation, the fluorescence in the red channel decreased rapidly (B1-D1), and that of the yellow channel increased rapidly (B-D). It can be observed that there is a significant difference in fluorescence signal between the cells in the illuminated area (the yellow box) and the unilluminated cells surrounding (E-E1), and this can be observed more clearly with the ratio imaging (A2-E2). This indicates that COR-XAC can accurately and controllably release CO within cells.

  Figure 3

  

  Figure 3.  Confocal imaging of CO release by COR-XAC (10 µmol/L) in selected HeLa cells (in the yellow dotted box) under 561 nm laser irradiation for different times. (A–E) Yellow channel images (λ_em = 600–700 nm, λ_ex = 561 nm, 12 mW). (A1–E1) Red channel images (λ_em = 730–800 nm, λ_ex = 561 nm, 12 mW). (A2–E2) The ratio of the yellow channel and red channel. For A–A2 and E–E2, scale bars are 25 µm; for B–B2, C–C2, and D–D2, scale bars are 10 µm. (F) Bright field image of E. (F1) Bright field image of the selected cells. Scale bar: 10 µm. (G, H) Quantified fluorescence for the yellow and red channels, respectively. Data are presented as mean ± SD (n = 3).

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  It has been reported that CO has a regulatory effect on cell proliferation, and high concentrations of CO have a good inhibitory effect on the growth of tumor cells [6–13]. Thus, we proceeded to delve deeper into the influence of CO released by COR-XAC on cell proliferation, and the MTT method was used. As shown in Fig. 4A, with the prolongation of illumination duration and the escalating concentration of COR-XAC, the cell survival rate decreased rapidly (to ~27.5% after 24 h), while as shown in Fig. S14a (Supporting information), the cell survival rate of COR-XAC-treated cells under dark was very high (about 94.1% after 24 h with 50 µmol/L COR-XAC), and the released product i-COR-XAC only had a small cell inhibitory effect (Fig. S14b in Supporting information). Moreover, as shown in Figs. S15 and S16 (Supporting information), COR-XAC did not produce significant reactive oxygen species in solution or cells under lighting conditions. These results indicated that the main reason for inhibiting the proliferation of cancer cells was the CO released by COR-XAC, and the higher the concentration of CO released, the greater the cytotoxicity. The apoptosis of cancer cells caused by CO was further studied through Annexin V-FITC/PI double staining and flow cytometry (Fig. 4B and Fig. S17 in Supporting information). Compared with blank cells (0.76%), the proportion of late apoptotic cells in COR-XAC-treated cells was significantly increased to 89.80% and 96.92% after 30 and 60 min of light exposure, respectively. In addition, only 7.45% of cells in the i-COR-XAC treatment group experienced late apoptosis. These results indicate that CO released by COR-XAC can induce significant late apoptosis of cancer cells, thereby inhibiting their proliferation. A living/dead cell staining experiment was also conducted with the calcein AM/propidium iodide (PI) staining kit. As shown in Fig. 4C, the COR-XAC with light-treated cells showed obvious red fluorescence after exposure to light, indicating that the cells were in a dead state (dead cell marker probe PI showed red fluorescence, and live cell probe calcein AM showed green fluorescence), and the cell density was also very low. The blank cell group only had green fluorescence, and the cell density was much higher than that in the COR-XAC + light group. In addition, the COR-XAC and i-COR-XAC groups mainly showed green fluorescence, and the cell density and cell survival rate were higher than that of the experimental group, indicating that these two compounds had little effect on cell proliferation. These results show that the CO released by COR-XAC has a good inhibitory effect on cancer cells, which makes it have potential in cancer treatment.

  Figure 4

  

  Figure 4.  (A) Percentage of survival of HeLa cells treated with different concentrations of COR-XAC and different irradiation times after incubation for 24 h at 37 ℃. (B) Flow cytometry results of cell apoptosis of 20 µmol/L COR-XAC, 20 µmol/L i-COR-XAC, and 20 µmol/L COR-XAC with 30 and 60 min irradiation treated cells, respectively. Q1-LL: viable cells, Q1-LR: early apoptosis, Q1-UR: late apoptosis, Q1-UL: primary necrosis. For an enlarged view, see Fig. S17. (C) Live/dead cell staining using calcein-AM (1 µmol/L) and propidium iodide (PI, 10 µmol/L). HeLa cells were treated with (a–d) PBS, (a1–d1) 20 µmol/L COR-XAC, (a2–d2) 20 µmol/L i-COR-XAC, and (a3–d3) 20 µmol/L COR-XAC + light (580 nm, 2 mW, 60 min). PI imaging: λ_em = 600–650 nm. Calcein-AM imaging: λ_em = 500–560 nm. λ_ex = 488 nm for both. Scale bar: 40 µm. Data are presented as mean ± SD (n = 3).

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  Since CO has a significant inhibitory effect on tumor cells, the tumor mouse model was established for further study. First, the imaging and CO release properties of COR-XAC in tumor models were investigated. As shown in Figs. 5A, A1 and A2, if only Probe 1 was injected in situ into the tumor area (blank group), almost no fluorescence was observed. However, upon injecting COR-XAC into the tumorous area, a strong fluorescence signal was observed at 790 nm (Fig. 5B2), and only weak fluorescence was observed at 670 nm (Fig. 5B1). Subsequently, when the COR-XAC-treated tumor area was illuminated for 30 and 60 min, respectively, the fluorescence at 790 nm gradually decreased, while that at 670 nm gradually enhanced. This process was also checked by Probe 1, which showed that the fluorescence at 520 nm gradually enhanced (Figs. 5C–E). These results indicate that COR-XAC released CO in the tumor under light irradiation, and this CO-releasing process was accompanied by ratiometric fluorescence changes. Although the CO release of COR-XAC is triggered by 580 nm light, which has a relatively shallow tissue penetration depth, it is still suitable for subcutaneous tumors. In addition, to explore the metabolic process of COR-XAC after photolysis, the mice were dissected 48 h later, and fluorescence imaging was performed on the organs and tumor tissues. As shown in Figs. 5F and G, strong fluorescence still can be observed in the tumor when detected at 670 and 790 nm, indicating that COR-XAC can stay in the tumor for a long time, which is beneficial for cancer therapy. We also conducted a real-time evaluation of the residence time of COR-XAC at the tumor site through the fluorescence of COR-XAC. As shown in Fig. S18, after the injection of COR-XAC in the tumor area and under the condition of hiding light, the tumor area showed significant fluorescence at 790 nm within 48 h, further indicating that COR-XAC shows a long residence time in tumors.

  Figure 5

  

  Figure 5.  4T1 tumor was imaged with COR-XAC. (A–A2) Tumor images after incubation of Probe 1 with 60 min irradiation (580 nm) as control. (B–B2) Tumor images after incubation of COR-XAC (100 µL, 200 µmol/L in 20% DMSO) and Probe 1 without light. (C–C2, D–D2) Tumor images after incubation of COR-XAC (100 µL, 200 µmol/L in 20% DMSO), and then incubated with Probe 1, followed by 580 nm irradiation for 30 min and 60 min, respectively. (E–E2) The intensity of fluorescence in A–D, A1–D1, and A2–D2, respectively. (F) Dissected organ images at 520, 670, and 790 nm. (G) Quantification of fluorescence intensity of organs at 520, 670, and 790 nm. 1–6 on the X-axis of G: spleen, heart, lung, cancer tissue, liver, and kidney, respectively. Fluorescent images were collected at 670 and 790 nm (λ_ex = 560 nm, for COR-XAC) and 520 nm (λ_ex = 490 nm, for Probe 1), respectively. Data are presented as mean ± SD (n = 3).

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  Based on the excellent CO-releasing performance of COR-XAC in the tumor model, the therapeutic effect of COR-XAC on living tumors was then explored. All animal experiments were approved by the Animal Ethical Experiments Committee of Central China Normal University. Fig. 6A shows the operation procedure and Fig. 6B shows the tumor proliferation after 12 days of treatment under different conditions. One can see that the PBS + light, COR-XAC (no light irradiation), and i-COR-XAC treatment groups showed almost no tumor inhibition effect. Compared with these groups, the tumor size of the COR-XAC + light group after treatment was significantly smaller, indicating that the COR-XAC + light group showed a significant anti-tumor effect. Besides, as shown in Figs. 6C and D, the tumor mass and tumor volume curve of each group showed that the COR-XAC + light group exhibited an obvious cancer inhibition effect, which was consistent with the trend of tumor size. In addition, during the treatment period, the weight of mice slowly increased (Fig. 6E), indicating that the treatment process had little effect on the growth of mice. After 12 days of treatment, the internal organs of mice were pathologically analyzed by hematoxylin and eosin (H&E) staining, and the treatment group and the PBS group showed no significant difference (Fig. S19 in Supporting information), indicating that the treatment with COR-XAC was safe. Next, H&E staining and Ki67 immunofluorescence (Ki67 was highly expressed in highly active tumor cells) were further used to evaluate the cancer inhibition effect. The cancer tissues in the COR-XAC + light group showed a significant decrease in cell viability, while the other control groups showed high expression of Ki67 (Figs. 6F and G), which indicated that the CO released by COR-XAC had a significant inhibitory effect on tumor tissue growth. All these results indicate that COR-XAC plus light illumination has promising application prospects for cancer treatment.

  Figure 6

  

  Figure 6.  (A) The treatment regimen for 4T1-tumor-bearing mice with COR-XAC. (B) Representative photographs of dissected tumors in the PBS + light group, COR-XAC group, i-COR-XAC group, and COR-XAC + light group, respectively. (C) Tumor weight after different groups of treatments (n = 4). P < 0.05, **P < 0.01. (D) Tumor growth curve. (E) Mice weight changes over time. (F) H&E staining (scale bar: 200 µm) and Ki67 immunofluorescence (scale bar: 400 µm). (G) The amount of Ki67 expression positive rate of 4T1-tumor-bearing mice after treatment under different conditions. Data are presented as mean ± SD (n = 4).

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  In summary, a novel photo-controlled metal-free NIR fluorescent CORM COR-XAC, derived from the integration of hydroxylflavone and oxanthracene, was reported in this work. Under visible light irradiation, COR-XAC releases CO with NIR ratiometric fluorescence (Em = 690 nm/760 nm) changes. This NIR ratiometric fluorescence change can provide high spatiotemporal self-reporting and self-monitoring of its CO release process in the living system. With these properties, COR-XAC has been successfully used to controllably release CO in cells and in vivo with self-reporting fluorescence changes. Moreover, the results show that CO released by COR-XAC has a significant inhibitory effect on tumor proliferation. Overall, this work not only provides a new CORM but also offers a feasible solution for achieving gas therapy of tumors.

  Declaration of competing interest

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

  CRediT authorship contribution statement

  Shengyi Gong: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Guoqiang Feng: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

  Acknowledgments

  This work was supported by the National Natural Science Foundation of China (Nos. 22077044 and 21672080), the Natural Science Foundation of Hubei Province (No. 2022CFA033), and the funding from Wuhan Institute of Photochemistry and Technology (No. GHY2023KF008).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2024.110409.

  
  
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• 

  Scheme 1  NIR fluorescent CORM (COR-XAC) with ratiometric fluorescence changes for carbon monoxide release and tumor therapy.

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  Figure 1  (A) The ultraviolet–visible spectroscopy (UV–vis) and fluorescence spectra of COR-XAC (10 µmol/L). (B) Fluorescence spectral changes of COR-XAC (10 µmol/L) after being irradiated (580 nm, 2 mW/cm²) for 60 min, and each data was collected after every 10 min of irradiation. For fluorescence measurements, λ_ex = 560 nm. (C) The released CO from the irradiated COR-XAC solution was identified by Probe 1 (10 µmol/L Probe 1 and 10 µmol/L PdCl₂. λ_ex = 493 nm). (D) The release of CO in the irradiated COR-XAC solution was determined by myoglobin assay.

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  Figure 2  The release of CO from COR-XAC (10 µmol/L) in HeLa cells monitored by itself fluorescence change and Probe 1. (A–F) Cell blank. (A1–F1, A2–F2, and A3–F3) Cells were pretreated with COR-XAC (10 µmol/L) for 30 min, and then irradiated with light for 0, 30, and 60 min, respectively, and finally incubated with Probe 1 for another 30 min. (A–A3) Bright field. (B–B3) Green channel images, λ_em = 500–550 nm, λ_ex = 488 nm. (C–C3) Yellow channel images, λ_em = 600–700 nm, λ_ex = 561 nm. (D–D3) Red channel images, λ_em = 730–800 nm, λ_ex = 561 nm. (E–E3) Merges of the yellow and red channel. (F–F3) The ratio of the yellow and red channels. Scale bar: 25 µm. (G–I) Quantified fluorescence for the green, yellow, and red channels, respectively. Data are presented as mean ± standard deviation (SD) (n = 3).

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  Figure 3  Confocal imaging of CO release by COR-XAC (10 µmol/L) in selected HeLa cells (in the yellow dotted box) under 561 nm laser irradiation for different times. (A–E) Yellow channel images (λ_em = 600–700 nm, λ_ex = 561 nm, 12 mW). (A1–E1) Red channel images (λ_em = 730–800 nm, λ_ex = 561 nm, 12 mW). (A2–E2) The ratio of the yellow channel and red channel. For A–A2 and E–E2, scale bars are 25 µm; for B–B2, C–C2, and D–D2, scale bars are 10 µm. (F) Bright field image of E. (F1) Bright field image of the selected cells. Scale bar: 10 µm. (G, H) Quantified fluorescence for the yellow and red channels, respectively. Data are presented as mean ± SD (n = 3).

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  Figure 4  (A) Percentage of survival of HeLa cells treated with different concentrations of COR-XAC and different irradiation times after incubation for 24 h at 37 ℃. (B) Flow cytometry results of cell apoptosis of 20 µmol/L COR-XAC, 20 µmol/L i-COR-XAC, and 20 µmol/L COR-XAC with 30 and 60 min irradiation treated cells, respectively. Q1-LL: viable cells, Q1-LR: early apoptosis, Q1-UR: late apoptosis, Q1-UL: primary necrosis. For an enlarged view, see Fig. S17. (C) Live/dead cell staining using calcein-AM (1 µmol/L) and propidium iodide (PI, 10 µmol/L). HeLa cells were treated with (a–d) PBS, (a1–d1) 20 µmol/L COR-XAC, (a2–d2) 20 µmol/L i-COR-XAC, and (a3–d3) 20 µmol/L COR-XAC + light (580 nm, 2 mW, 60 min). PI imaging: λ_em = 600–650 nm. Calcein-AM imaging: λ_em = 500–560 nm. λ_ex = 488 nm for both. Scale bar: 40 µm. Data are presented as mean ± SD (n = 3).

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  Figure 5  4T1 tumor was imaged with COR-XAC. (A–A2) Tumor images after incubation of Probe 1 with 60 min irradiation (580 nm) as control. (B–B2) Tumor images after incubation of COR-XAC (100 µL, 200 µmol/L in 20% DMSO) and Probe 1 without light. (C–C2, D–D2) Tumor images after incubation of COR-XAC (100 µL, 200 µmol/L in 20% DMSO), and then incubated with Probe 1, followed by 580 nm irradiation for 30 min and 60 min, respectively. (E–E2) The intensity of fluorescence in A–D, A1–D1, and A2–D2, respectively. (F) Dissected organ images at 520, 670, and 790 nm. (G) Quantification of fluorescence intensity of organs at 520, 670, and 790 nm. 1–6 on the X-axis of G: spleen, heart, lung, cancer tissue, liver, and kidney, respectively. Fluorescent images were collected at 670 and 790 nm (λ_ex = 560 nm, for COR-XAC) and 520 nm (λ_ex = 490 nm, for Probe 1), respectively. Data are presented as mean ± SD (n = 3).

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  Figure 6  (A) The treatment regimen for 4T1-tumor-bearing mice with COR-XAC. (B) Representative photographs of dissected tumors in the PBS + light group, COR-XAC group, i-COR-XAC group, and COR-XAC + light group, respectively. (C) Tumor weight after different groups of treatments (n = 4). P < 0.05, **P < 0.01. (D) Tumor growth curve. (E) Mice weight changes over time. (F) H&E staining (scale bar: 200 µm) and Ki67 immunofluorescence (scale bar: 400 µm). (G) The amount of Ki67 expression positive rate of 4T1-tumor-bearing mice after treatment under different conditions. Data are presented as mean ± SD (n = 4).

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