A highly effective self-supplying photosensitizer drug for deep-tissue metastatic tumours treatment

A highly effective self-supplying photosensitizer drug for deep-tissue metastatic tumours treatment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A highly effective self-supplying photosensitizer drug for deep-tissue metastatic tumours treatment Zhengze Yu, Hanxiang Li, Mingchao Xia, Yuhang Wang, Hao Zhang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5536918/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Due to the inherent defects of photodynamic therapy (PDT), its application in the treatment of deep-tissue metastatic tumours remains challenging. To extend the applicability of PDT, a novel chemiluminescent photosensitizer, Cy7-EOM, was developed by covalently coupling the photosensitizer Cy7 with a peroxycatechol derivative and encapsulating it within folate-modified and disulfide-containing nano-micelles. Upon targeted delivery and selective release, positive charged Cy7-EOM would target the mitochondria and efficiently generate singlet oxygen ( 1 O 2 ) via intramolecular chemiluminescence resonance energy transfer (CRET) by endogenous H 2 O 2 , directly inducing mitochondrial damage and cell apoptosis, realizing an efficient PDT for deep-tissue metastatic tumours. Remarkably, the covalent linkage between the donor and the acceptor greatly reduces the distance, significantly enhancing CRET efficiency. Moreover, the tumour-specific decomposition of the nano-micelles prevents aggregation-induced quenching and mitigates the diffusion barrier of 1 O 2 , while in normal tissues the integrality of nano-micelles shields the lethal effects of 1 O 2 . This method provides a new strategy for transforming adjuvant photosensitizers into direct therapeutic drugs, with significant potential for clinical application in the treatment of metastatic tumours. Biological sciences/Biotechnology/Nanobiotechnology/Nanoparticles Biological sciences/Biotechnology/Biomaterials/Drug delivery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction In cancer-related mortality, 90% are attributed to cancer metastasis and its associated complications 1-4 . The expected five-year survival rate for patients with metastatic tumours is significantly lower than that of patients with primary tumour 5,6 . Currently, the clinical treatment of metastatic tumours primarily relies on surgical removal followed by systemic chemotherapy, which are often associated with severe side effects and limited therapeutic efficacy 7,8 . Consequently, effective treatment for deep-seated metastatic tumours remains a major challenge, underscoring the urgent need for the development of novel and more efficient therapeutic strategies and drugs for metastatic tumours. PDT consists of three essential components: photosensitizers, excitation light, and oxygen. This process activates photosensitizers through specific wavelength excitation light, leading to energy transfer that produces reactive oxygen species (ROS) 9-11 . Due to its non-invasive nature, high spatiotemporal selectivity, low systemic toxicity, and favorable therapeutic efficacy, PDT has been applied in clinical cancer treatments 12,13 . However, the limited tissue penetration of the excitation light required by traditional photosensitizers restricts PDT to superficial lesions or those accessible by endoscopy, such as oral, skin, esophageal, gastric, and bladder cancers, etc 14-18 . It is not yet feasible to apply PDT to metastatic tumours in deep tissues. Therefore, the development of novel strategies for in situ excited PDT in deep tissues, as well as the expansion of its clinical applications, is of critical importance and urgency. Chemiluminescence is a process in which light is generated through chemical energy excitation during a chemical reaction 19-21 . Because it does not require external excitation light, chemiluminescence can be produced in situ within tumour tissue and has been used to construct CRET-based PDT systems 22-25 . Among commonly used chemiluminescent substances, peroxycatechol derivatives are particularly favored for CRET-mediated PDT due to their high sensitivity, quantum efficiency, and long emission lifetime 26 . However, a critical challenge in CRET-mediated PDT is its low efficiency, which primarily results from the nanoscale self-assembly strategies used to construct probes. As all known, the efficiency of energy transfer is critically dependent on the proximity between the donor and acceptor 27 . In current researches, the photosensitizers and CPPO are typically assembled through non-covalent interactions to form nanoscale probes, which then undergo PDT through intermolecular CRET 28-32 . However, this probe construction strategy results in an uncontrollable and long distance between the energy donor and acceptor, leading to low CRET efficiency 33-36 . Moreover, aggregation caused quenching (ACQ) of photosensitizers within the nanomaterial reduces ROS production efficiency 37-39 . Even more critically, due to the short lifetime of ROSand short diffusion distance, the nanoscale assembly strategy significantly shields and impedes the diffusion of ROS, leading to a significant loss of ROS 40,41 To address the aforementioned challenges, a novel photosensitizer (Cy7-EOM) was developed by covalently linking the FDA-approved near-infrared photosensitizer, Cy7 with the organic compound ethyl chlorooxoacetate (EOM), and then encapsulated Cy7-EOM into folate-modified nano-micelles featuring disulfide bonds via self-assembly (Cy7-EOM SS NMs). Upon targeting metastatic tumours, Cy7-EOM SS NMs will consume the high intracellular levels of reductive glutathione (GSH) and degrade, releasing the photosensitizer Cy7-EOM, which would specifically target mitochondria due to the positive charge. Under conditions of high endogenous intracellular H 2 O 2 , Cy7-EOM can be activated in situ via an intramolecular CRET mechanism. It is noteworthy that, compared to intermolecular CRET, the covalent coupling of the energy donor with the acceptor significantly reduces the distance between them, enabling more efficient CRET and ROS generation. The generated ROS will directly induce mitochondrial dysfunction and subsequent apoptosis of cancer cells. Furthermore, the specific decomposition of the nano-micelles in tumour tissue not only prevents the occurrence of the ACQ phenomenon, but eliminates the shielding interference of ROS diffusion. Combined with GSH depletion, the efficacy of PDT for deep-tissue metastatic tumours has been synergistically enhanced. In normal tissue, the low redox levels cannot activate the photosensitizer and the nano-micelles can remain intact, which can effectively shield the diffusion of ROS and protecting normal tissue from oxidative damage. Thus, an efficient and tumour selective PDT was achieved for the treatment of metastatic tumours. The structure of Cy7-EOM and the nano-micelle Cy7-EOM SS NMs and the details of intramolecular CRET based mitochondria targeted PDT against cancer metastasis are illustrated in Fig. 1. Design, Synthesis and Characterization of the photosensitizer We first designed and synthesized the intramolecular CRET based photosensitizer Cy7-EOM, which consists of two components: 1) a peroxyoxalate group acts as the chemiluminescence substrate which can generate a high-energy intermediate, (i.e. 1,2-dioxetanedione) via interacting with H 2 O 2 and 2) a Cy7 derivative serves as a photosensitizer to capture chemical energy upon deactivation of unstable intermediate for 1 O 2 generation. The final product Cy7-EOM was obtained by covalent coupling of EOM and hydroxylated Cy7 through an esterification reaction. Thus, the designed photosensitizer could be activated specifically in tumour tissue due to the high levels of intracellular H 2 O 2 , and the chemical connection facilitates the proximity between the donor peroxyoxalate and the acceptor Cy7, resulting in efficient intramolecular CRET and self-supplying 1 O 2 generation without the requirement of an external light source. The detailed synthetic route of Cy7-EOM and mechanism of intramolecular CRET based PDT are illustrated in Fig. 1a and b. The designed Cy7 and Cy7-EOM were successfully characterized by nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HR-MS), respectively (Supplementary Fig. 1-6). To achieve targeted delivery and selective release into cancer cells, Cy7-EOM was co-assembled with folate modified and disulfide-containing amphiphilic diblock copolymer (DSPE-SS-PEG:DSPE-PEG-FA=2:1), yielding aqueous nano-micelles (Cy7-EOM SS NMs). As the control group, nano-micelles without disulfide bonds (Cy7-EOM NMs) was prepared via the same methods (DSPE-PEG:DSPE-PEG-FA=2:1) (Fig. 1c). As shown in TEM images, both Cy7-EOM SS NMs and Cy7-EOM NMs displayed spherical morphology with excellent monodispersity (Fig. 2a and Supplementary Fig. 7a). Cy7-EOM NMs had a larger hydrodynamic diameter of 531.2 nm than that of Cy7-EOM SS NMs (259.2 nm) by dynamic light scattering analysis (DLS) and they had similar neutral surface charge by zeta potential measurement (Supplementary Fig. 8, 9). Due to the specific response of disulfide bonds to GSH, nano-micelles containing disulfide bonds will decompose and release the cargos. TEM images displayed obvious morphology change of Cy7-EOM SS NMs after incubation with GSH for 3 h, while the morphology of Cy7-EOM NMs remains intact (Fig. 2b and Supplementary Fig. 7b). Then, the optical properties of Cy7-EOM and relevant nano-micelles were recorded and analyzed. As shown in absorption spectra, the maximum absorption peak of Cy7-EOM was approximately 774 nm, the same with Cy7 parent, while that of Cy7-EOM SS NMs was redshifted to 786 nm (Fig. 2c). Similarly, fluorescence and chemiluminescence spectra demonstrated the maximum emission peak of Cy7-EOM was at 803 nm, while it were redshifted by about 25 nm after forming nano-micelles (Fig. 2d, e). And the FL emission intensity of Cy7-EOM SS NMs was approximately half that of Cy7-EOM at the same concentration, which resulted from the aggregation caused quenching (ACQ). Another control group was set up by preparing nano-micelles without disulfide bonds and loaded with individual Cy7+CPPO (Cy7/CPPO NMs). After incubation with H 2 O 2 , chemiluminescence intensity of Cy7-EOM SS NMs is much higher than that of Cy7/CPPO NMs, revealing more efficient intramolecular CRET than intermolecular CRET (Fig. 2f). Then, a chemiluminescence dynamic experiment of Cy7-EOM and Cy7-EOM SS NMs with different concentration of H 2 O 2 was also carried out. From chemiluminescence images we found that Cy7-EOM has a rapid and concentration-dependent response to H 2 O 2 , suggesting that effective intramolecular CRET could be achieved in the designed photosensitizer molecule, while the response of Cy7-EOM SS NMs to H 2 O 2 showed a lag in time (Fig. 2g). This is mainly due to the shielding effect of the nano-micelle, which hinders the effective contact between Cy7-EOM and H 2 O 2 . Therefore, in the same way, nano-micelle will also hinder the diffusion of low-lifetime ROS produced inside the nanoparticles, leading to the loss of ROS and inefficient PDT. To validate our hypothesis, 1 O 2 generation via CRET in each group was detected by using Singlet Oxygen Sensor Green (SOSG). As shown in fluorescence spectra, the fluorescence intensity of SOSG significantly increased and showed a 5-fold enhancement after H 2 O 2 was added to the solution of Cy7-EOM, indicating the generation of 1 O 2 (Fig. 2h). Moreover, as expected, fluorescent enhancements of SOSG were also observed for Cy7-EOM SS NMs compared to Cy7/CPPO NMs, demonstrating the superior efficiency of 1 O 2 generation through intramolecular CRET to intermolecular CRET (Fig. 2i). In addition, GSH pre-treated Cy7-EOM SS NMs exhibited more 1 O 2 generation than Cy7-EOM SS NMs without GSH treatment, further confirming that ACQ and nano-micelles structure could affect effective utilization of 1 O 2 (Fig. 2j). Furthermore, electron spin resonance (ESR) spectroscopy was also employed for the 1 O 2 analysis by using a radical scavenger, 2,2,6,6-tetramethylpiperidine (TEMP). The appearance of the characteristic peaks in the ESR spectra after the addition of H 2 O 2 demonstrated 1 O 2 generation and the increased intensity of ESR signal in the sample of Cy7-EOM further prove that the release of Cy7-EOM from nano-micelles is beneficial to PDT (Supplementary Fig. 10). Evaluation of Intracellular 1 O 2 generation First, folate mediated cellular uptake performance of Cy7-EOM SS NMs was investigated in 4T1 cells through fluorescence analysis by using confocal laser scanning microscopy (CLSM). As displayed in CLSM images, the fluorescence intensity of 4T1 cells significantly increased over time, indicating effective cellular uptake, and reached a plateau at 6 hours by fluorescence statistical data (Supplementary Fig. 11). To verify the promoting effect of folate on the cellular uptake of Cy7-EOM SS NMs, 4T1 cells were pre-treated with free folate molecules before incubation with Cy7-EOM SS NMs. Confocal images and corresponding statistical data showed that 4T1 cells pre-treated with folate exhibit a weaker red fluorescence signal than those without treatments, which is mainly because the premature binding of folate molecules to folate receptors on the cancer cell membrane hindered the binding of folate in the nanomaterials to folate receptors, thereby interfering with the cell uptake of Cy7-EOM SS NMs (Supplementary Fig. 12). And due to the much higher expression level of folate receptors on cancer cell membrane, nano-micelles are more likely to enter cancer cells than AML-12 cells, mouse normal liver cells (Supplementary Fig. 13). After entering cancer cells, Cy7-EOM SS NMs would degrade under the high concentration of intracellular GSH and release Cy7-EOM. As GSH consumption can not only lead to the release of Cy7-EOM, but also can enhance PDT effect in a synergetic manner, intracellular GSH level was analyzed using a fluorescence probe, 3-naphthalene-dicarboxaldehyde (NDA). CLSM images showed that the fluorescence intensity of NDA in 4T1 cells incubated with Cy7/CPPO NMs, Cy7-EOM NMs or Cy7-EOM SS NMs decreased to different degrees and Cy7-EOM SS NMs performed best in GSH consumption (Supplementary Fig. 14). Then the targeting ability of released Cy7-EOM to mitochondria was evaluated through mitochondrial co-localization experiment by using Mito-Tracker Green, a commercial mitochondrial labeling probe. As shown in confocal images, bright yellow signals were observed, resulting from the superposition of the red signal from Cy7-EOM and green signal from Mito-Tracker Green. The line scanning profiles and the Pearson's correlation coefficient (ρ, calculated to be 0.90) from co-localization scatter plot suggested the excellent targeting ability of the photosensitizer Cy7-EOM to mitochondria (Fig. 3a). Subsequently, Cy7-EOM will react with intracellular H 2 O 2 to generate 1 O 2 through CRET, which was verified by SOSG. CLSM images and corresponding fluorescence quantitative statistics showed that Cy7-EOM NMs has a higher 1 O 2 yield than Cy7/CPPO NMs, providing further evidence for the higher efficiency of intramolecular CRET than intermolecular CRET. Notably, 4T1 cells treated with Cy7-EOM SS NMs exhibit the brightest green fluorescence (Fig. 3b and Supplementary Fig. 15). This is because that, in addition to efficient intramolecular CRET, the release of the Cy7-EOM mitigates the adverse effects of ACQ and the physical barrier against 1 O 2 diffusion. ROS burst and apoptosis mechanism As mitochondria is one of the most important organelles in the cell and approximately 90 % of intracellular ROS are generated in mitochondria, 1 O 2 generation in mitochondria in situ will directly cause mitochondria dysfunction and further a series of cascade reactions, including the drop of mitochondrial membrane potential (MMP), release of cytochrome C, activation of caspase 3, etc., leading to the ROS burst and eventually cell apoptosis. Then, the mechanism of Cy7-EOM SS NMs induced apoptosis was studied in detail. Firstly, JC-1 staining was employed to investigate the change of MMP by CLSM. From confocal images we can see that obvious green fluorescence of JC-1 monomer emerged in 4T1 cells with treatment of Cy7-EOM NMs and Cy7-EOM SS NMs and the highest ratio of green/red fluorescence intensity in the group of Cy7-EOM SS NMs indicated the maximum MMP drop and most serious mitochondrial damage (Fig. 3c and Supplementary Fig. 16). Subsequently, the release of cytochrome C and activation of caspase 3 induced by mitochondria depolarization were then evaluated and confirmed by immunofluorescence staining, which further demonstrated the most severe cell apoptosis induced by Cy7-EOM SS NMs (Supplementary Fig. 17 and 18). Then, the total amount of ROS in 4T1 cells with different treatments was detected by DCFH-DA to verify ROS burst. As displayed in confocal images, the strongest fluorescence intensity was observed in 4T1 cells treated with Cy7-EOM SS NMs, and the sharp rise in ROS concentration verified the domino burst (Fig. 3d and supplementary Fig. 19). And ROS burst induced cell apoptosis was investigated by live/dead cell the calcein acetoxymethyl ester (calcein AM)/propidium iodide (PI) double staining and flow cytometry analysis of AnnexinV-FITC/PI staining. CLSM images and corresponding fluorescence statistical quantization showed that the largest percentage of cell death appeared in the cells with treatment of Cy7-EOM SS NMs (Fig. 4a). Similarly, in flow cytometry data, the proportion of FITC + /PI + 4T1 cells is 81.91%, higher than 66.76 % of 4T1 treated with Cy7-EOM NMs and 23.08 % of 4T1 cells treated with Cy7/CPPO NMs, suggesting its superior PDT efficiency to the others (Fig. 4b). The cytotoxicity of Cy7-EOM SS NMs was also evaluated by methyl thiazolyl tetrazolium (MTT) assay. As shown in Fig. 4c, the viability of 4T1 cells decreased significantly with the increase concentration of Cy7-EOM SS NMs and the viability was as low as 18.44% at concentration of 50 μg/mL, while the viabilities of 4T1 treated with Cy7 NMs, Cy7/CPPO NMs and Cy7-EOM NMs were much higher (86.5%, 76.1% and 32.6%, respectively) (Fig. 4d). By comparison, negligible cell death was observed in AML12 cells with the treatment of Cy7-EOM SS NMs even at high incubation concentrations (Fig. 4e). According to the above results, this significant difference is determined by multiple factors, and the detailed mechanism was illustrated in Fig. 4f. In vivo CL imaging and treatments of metastatic tumours Encouraged by the excellent therapeutic effect on cancer cells and extreme low side effects on normal cells, the anti-tumour efficiency of Cy7-EOM SS NMs was then assessed in vivo . Before that, the ability of nanomaterials to perform targeted imaging and diagnosis was first verified. Mouse models of lung metastases were established by intravenous injection of Luciferase transfected 4T1 cells (4T1-Luc). As shown in fluorescence images, after intravenous injection of Cy7-EOM SS NMs, a clear chemiluminescence signal was observed in the lung. It is worth noting that there is also a chemiluminescence signal in the abdominal cavity. In order to find out the reason, the mouse was euthanized and autopsied and we found that new metastatic tumour sites were observed in the intestines (Fig. 5a and Supplementary Fig. 20). The results revealed that the designed Cy7-EOM SS NMs have distinguished targeting and diagnostic capabilities, even small metastatic tumour sites. Then the anti-tumour effects were evaluated and the detailed treatment schedule is shown in Fig. 5b. The mice were randomly divided into five groups with different treatments of PBS, Cy7 NMs, Cy7/CPPO NMs, Cy7-EOM NMs and Cy7-EON SS NMs at a dosage of 2.5 mg/kg, respectively. Bioluminescence imaging was employed for the evaluation of the therapeutic effects. As shown, the bioluminescence intensity of the mice treated with PBS and Cy7 NMs increased significantly and there was a new metastasis site in the abdominal cavity, indicating rapid tumour progression. By comparison, Cy7/CPPO NMs, Cy7-EOM NMs and Cy7-EOM SS NMs treated mice showed obvious inhibition of tumour growth. Among them, Cy7-EOM SS NMs performed best and the bioluminescence intensity decreased to 9.8 % compared with that in PBS group (Fig. 5c, 5d). In particularly, mice in the groups treated with PBS, Cy7 NMs and Cy7/CPPO NMs shows multiple metastatic tumour sites in the intestines, while a faint bioluminescence signal in the lung and no bioluminescence signal in the abdominal cavity were observed in mice treated with Cy7-EOM SS NMs, demonstrating its great potential as drugs for the treatment of metastasis tumours and even multiple metastatic tumours. After 15 days treatment, the lungs in each group are dissected. Photographs and H&E staining also demonstrated that lungs in groups of PBS, Cy7 NMs, Cy7-EOM NMs were occupied with more metastasis tumour nodules, while almost no metastasis foci could be observed in the Cy7-EOM SS NMs group (Fig. 5g, 5h). In addition, after treated with Cy7-EOM SS NMs, the life span of mice was greatly extended and 60% of mice survived for more than 40 days, while almost all the mice in control group died within 25 days (Fig. 5e). Moreover, the mice weights in each group were monitoring during the treatment and no obvious decrease in body weight further confirmed the low side effects and safety of Cy7-EOM SSNMs (Fig. 5f). Conclusion In this study, to address the current issue of shallow penetration depth of the excitation light in PDT and the challenge of its application in the treatment of metastatic tumours in clinical settings, a chemiluminescence method was adopted as the excitation light to develop an efficient self-supplying photosensitizer molecule Cy7-EOM by covalently coupling the energy donor peroxyphthalazone derivative with the receptor photosensitizer. The covalent coupling of the donor and receptor in the molecular structure significantly enhances the efficiency of PDT. Under high concentrations of H 2 O 2 in the tumour microenvironment, PDT can be autonomously activated in situ, overcoming the limitations of traditional PDT, which suffers from shallow penetration depth and is ineffective against deep tissue metastatic tumours. And Cy7-EOM was encapsulated into a tumour specific response nano-micelle Cy7-EOM SS-NMs-FA for targeted delivery and release. In cancer cells, Cy7-EOM SS-NMs-FA would consume large amounts of reducing GSH, leading to degradation and the release of Cy7-EOM, which specifically targets mitochondria. The generated ROS then act directly on the mitochondria, inducing mitochondrial ROS bursts that trigger cell apoptosis. This approach has been successfully applied to treat breast cancer cells and multiple metastatic tumours in mice, achieving efficient PDT of metastatic tumours and extremely low systemic toxicity. These findings expand the practical application range of photosensitizers and offer new strategies for photodynamic anticancer drugs, with significant potential for clinical translation. Declarations Acknowledgements This work was supported by the Natural Science Foundation of Shandong Province (ZR2020YQ15), National Natural Science Foundation of China (22377113) and Taishan Scholar Program of Shandong Province (tsqn202306103) Author Information 1 College of Chemistry and Chemical Engineering, Qingdao University, Qingdao 266071, P. R. China. Zhengze Yu, Hanxiang Li, Mingchao Xia, Yuhang Wang and Hao Zhang 2 Department of Emergency Medicine, Shandong Provincial Clinical Research Center for Emergency and Critical Care Medicine, Qilu Hospital of Shandong University, Jinan, 250014, P. R. China. Yue Tang 3 Key laboratory of marine drugs, ministry of education; Molecular synthesis center, and School of medicine and pharmacy, Ocean University of China, Qingdao, 266003 P. R. China. Hongyu Wang 4 Laoshan Laboratory, Qingdao, 266237, P. R. China. Bo Tang Corresponding authors 1 Zhengze Yu. E-mail: [email protected] . 2 Yue Tang. E-mail: [email protected] . 3 Hongyu Wang. E-mail: [email protected] . 4 Bo Tang. E-mail: [email protected] . Contributions Z.Y. and H.L. contributed equally to this work. Z.Y. conceived the study. Z.Y. and H.L. designed the experiments. H.L. conducted the chemical syntheses, prepared the nanomaterials and conducted the in vitro characterization. H.L. M.X., Y.W. and H.Z. conducted the cell experiments. Z.Y. and H.L. performed the in vivo experiments. Z.Y., H.L., Y.T., H.W. and B.T. analyzed the data. Z.Y., Y.T., and B.T. drafted the manuscript. Ethics declarations Competing interests The authors declare no conflicts of interest. References Massagué, J., Obenauf, A. Metastatic colonization by circulating tumour cells. Nature 529 , 298-306 (2016). Kiri, S., Ryba, T. Cancer, metastasis, and the epigenome. Mol. Cancer 23 , 154 (2024). 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Ding, D. et al. Ultrabright Organic Dots with Aggregation-Induced Emission Characteristics for Real-Time Two-Photon Intravital Vasculature Imaging. Adv. Mater. 25 , 6083-6088 (2013). Lucky, S. S., Soo, K. C., Zhang, Y. Nanoparticles in Photodynamic Therapy. Chem. Rev. 115 , 1990-2042 (2015). Klaper, M., Fudickar, W., Linker, T. Role of Distance in Singlet Oxygen Applications: A Model System. J. Am. Chem. Soc. 138 , 7024-7029 (2016). Methods Materials Unless otherwise noted, all commercial reagents and solvents were obtained from the commercial provider and used without further purification. All reagents used in experiments were purchased from Aladdin Industrial Corporation (Beijing, China). DSPE-PEG 2000 and DSPE-PEG 2000 -FA were purchased from Ponsure Biological Technology Co. Ltd. (Shanghai, China). DSPE-SS-PEG 2000 was purchased from Qiyue Biological Technology Co. Ltd. (Xi’an, China). Singlet Oxygen Sensor Green Fluorescent Probe (SOSG) was purchased from Maokang Biological Technology Co. Ltd. (Shanghai, China). A naphthalene-2,3-dicarboxaldehyde (NDA) were purchased from Heowns Biochem Technologies, LLC, (Tianjin, China). Calcein-AM/PI Double staining Kit, Mitochondrial Membrane Potential Detection Kit (JC-1) was purchased from G-Clone Biotechnology Co., LTD (Beijing China). 4T1 cells and 4T1-luc cells were purchased from Procell Life Science&Technology Co., Ltd. (Wuhan, China). The mouse normal liver cell line (AML 12) was purchased from Ubigene Biosciences (Guangzhou, China). The water used was Mill-Q secondary ultrapure water (18.2MΩ/cm). Synthesis of Cy7-EOM Synthesis of Compound 1 2,3,-trimethyl-3H indole (5.0 g, 31.5 mmol) and 2-iodoethanol (7.5 g, 44 mmol) was dissolved in MeCN (50 mL). The solution was refluxed under nitrogen for 24 hours. The reaction mixture was cooled to room temperature and product precipitated by the addition of hexane. The purple solid was filtered and dried without further purification. Yield: 90%. Synthesis of Compound 2 To a solution of DMF (20 mL, 273 mmol) in ice-bathed anhydrous DCM (20 mL) under Ar, POCl 3 (17.5 mL, 115 mmol) in anhydrous DCM (5 mL) was added dropwise within 0.5 h. Then, cyclohexanone (5.0 g, 50 mmol) was injected slowly into the above solution. The resulting mixture was stirred vigorously at 80 °C for 3 h, and poured into ice-cold water under stirring to obtain a yellowish precipitation. The solid was filtered off, washed with water, and dried under vacuum to give compound 2 (7.9 g, 91.9%) as a yellowish solid with a fine purity. Synthesis of Cy7 Compound 1 (2.9 mg, 11.0 mmol), Compound 2 (860 mg, 5.0 mmol) and CH 3 COOK(1.1 mg, 11.0 mmol) was dissolved in dry ethanol under nitrogen overnight. After that, the reaction mixture was cooled and a large amount of green precipitation was collected by filtration. The crude precipitation was further purified by column chromatography using CH 2 Cl 2 /MeOH (20:1, v/v) to provide compound Cy7 (2.23 mg, 82%) as a dark green solid. The 1 H NMR spectrum is shown in Supplementary Fig. 1. 1 H NMR (400 MHz, Chloroform-d) δ 8.36-8.36(d, 2H), 7.40-7.34(m, 4H), 7.25-7.20(m, 4H), 6.51-6.48(d, 2H), 4.37-4.35(t, 4H), 4.07-4.04(t, 4H), 2.83-2.80(tH), 2.09(s, 1H), 1.97-1.94(t, 2H), 1.73(s, 12H). The 13 C NMR spectrum is shown in Supplementary Fig. 2. 13 C NMR (400 MHz, Chloroform-d) δ 173.31, 144.68, 142.66, 141.07, 128.86, 127.94, 125.19, 122.09, 111.39, 102.26, 77.36, 77.15, 76.94, 58.76, 49.38, 47.26, 46.12, 28.34, 27.08, 20.87, 8.79, 0.07. Synthesis of Cy7-EOM Cy7 (2.72 mg, 5.0 mmol) and Ethyl oxalyl monochloride (2.3 mg, 15.0 mmol) were mixed in 20 mL of anhydrous DCM and then triethylamine (1 mL) was added to the solution in an ice bath. The mixture was stirred under an argon (Ar) atmosphere for 6 h at room temperature. The solvent was removed by vacuum rotary evaporation and the residue was purified by column chromatography to obtain Required products using CH 2 Cl 2 /MeOH (20:1, v/v) to provide compound Cy7-EOM. The 1 H NMR spectrum is shown in Supplementary Fig. 4. 1 H NMR (400 MHz, DMSO-d6) δ 8.26-8.22(d, 2H), 7.62-7.60(d, 2H), 7.45-7.39(, 4H), 7.29-7.25(2H), 6.43-6.40(2H), 4.29(4H), 3.79(4H), 3.09-3.02(4H), 2.70-3.67(4H), 1.85-1.82(2H), 1.68(12H), 1.21-1.17(6H). The 13 C NMR spectrum is shown in Supplementary Fig. 5. 13 C NMR (101 MHz, CDCl 3 ) δ 173.21, 150.48, 144.63, 142.52, 140.95, 128.82, 127.95, 125.17, 122.06, 111.37, 102.14, 77.42, 77.10, 76.78, 58.59, 49.34, 47.20, 46.51, 29.71, 28.31, 27.25, 20.67, 14.06, 8.83. Preparation of various Nano-micelles (NMs) NMs were fabricated through a thin-film hydration method. For Cy7/CPPO NMs, Cy7 (1.0 mg), CPPO (2.5 mg), DSPE-PEG (4.0 mg) and DSPE-PEG-FA (2.0 mg) were dissolved at 1 mL DCM under ultrasound for 10 min, and then DCM was removed by evaporation.1 mL of deionized water was added and was vigorously stirred overnight at room temperature to afford an aqueous solution of nano-micelles. The solution was dialyzed in deionized water for 24 h. Almost no Cy7 and CPPO were detected in the dialysate by measuring the absorption, indicating a nearly 100 % encapsulation rate. The resulting solution was stored at 4 °C for further use. The same procedure was applied for Cy7 NMs, Cy7-EOM NMs and Cy7-EOM SS NMs: For Cy7 NMs, Cy7 (1.0 mg), DSPE-PEG (4.0 mg) and DSPE-PEG-FA (2.0 mg) were used. For Cy7-EOM NMs, Cy7-EOM (1.0 mg), DSPE-PEG (4.0 mg) and DSPE-PEG-FA (2.0 mg) were used. For Cy7-EOM NMs, Cy7-EOM (1 mg), DSPE-PEG (4.0 mg) and DSPE-PEG-FA (2.0 mg) were used. For Cy7-EOM SS NMs, Cy7-EOM (1.0 mg), DSPE-SS-PEG (4.0 mg) and DSPE-PEG-FA (2.0 mg) were used. The remaining steps are the same as those described above. In subsequent experiments, the amount of Cy7 or Cy7-EOM in the nano-micelles in each group is equivalent, and all the concentration values referred to Cy7 or Cy7-EOM. The chemiluminescent properties of the Cy7-EOM Different concentration of H 2 O 2 (final concentration: 0.1, 1.0, 5.0, 10, 25 and 50 mM) were added to Cy7-EOM and Cy7-EOM SS NMs (0.25 mM), respectively. Time dependent chemiluminescent images were obtained by PerkinElmer IVIS Lumina III. Detection of 1 O 2 by SOSG The 1 O 2 generation was measured using SOSG as an indicator (λex=488 nm, λem=525 nm). For Cy7-EOM, H 2 O 2 (10 mM) was added to a mixed solution of SOSG (1.0 µM) and Cy7-EOM (10 µM). After incubation for 15 min, the fluorescence intensity of SOSG was measured with excitation at 488 nm. A group without H 2 O 2 was set as the control. For Cy7-EOM NMs and Cy7/CPPO NMs, H 2 O 2 (10 mM) was added to a mixed solution of SOSG (1 µM) and Cy7-EOM NMs (or Cy7/CPPO NMs) (10 µM). After incubation for 15 min, the fluorescence intensity of SOSG was measured with excitation at 488 nm. For Cy7-EOM SS NMs, Cy7-EOM SS NMs (10 µM) was pre-treated with GSH (10 µM) for 3 h to destroy the nano-micelles. And the SOSG (1 µM) was added to the above solution followed by adding H 2 O 2 (10 mM). After incubation for 15 min, the fluorescence intensity of SOSG was measured with excitation at 488 nm. Detection of 1 O 2 by ESR H 2 O 2 (10 mM) was added to Cy7-EOM (10 µM) or Cy7-EOM SS NMs (10 µM) solutions containing 2,2,6,6-Tetramethyl-4-piperidone hydrochloride (TEMP, 100mM), respectively. After incubation for 15 min, the produced singlet oxygen was measured by ESR. Cell culture 4T1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10 % fetal bovine serum and 1 % penicillin/streptomycin. 4T1-luc cells were treated with high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum and 1 % penicillin/streptomycin. AML 12 cells were cultured in DMEM medium containing 10 % fetal bovine serum, 0.5 % Insulin-Transferrin-Selenium (ITS-G, 100X), dexamethasone (40 ng/mL) and 1 % penicillin/streptomycin. All the cells were incubated at 37 °C in a humidified atmosphere with 5 % CO 2 . Cytotoxicity test 4T1 cells or AML 12 cells were seeded onto the 96-well microtiter plate and cultured for 24 h in a cell culture incubator, followed by incubation with different concentrations of Cy7-EOM SS NMs (0, 10, 20, 30, 40,50, 60 µg/mL) for 12 hours. Then, the cell culture medium was removed and replaced by 150 µL of MTT solution (0.5 mg/mL). After 4 hours incubation, the MTT solution was removed, and 150 µL of DMSO was added into each well. Subsequently, the 96-well microtiter plate was shaken slightly in the dark to fully dissolve the formazan, and the absorbance at a wavelength of 490 nm was measured. Cell viability is estimated based on the following formula: Cell viability (%) = (OD treatment/OD control) × 100%. Then, the same step was repeated to evaluate the toxicity of different groups (PBS, Cy7 NMs, Cy7/CPPO NMs, Cy7-EOM NMs, Cy7-EOM SS NMs) against 4T1 cells at the same concentration of 50 µg/mL. Intracellular uptake of Cy7-EOM SS NMs by 4T1 cells 4T1 cells were seeded onto a 6-well plate and incubated in a cell incubator for 24 hours. Then, the culture medium was replaced with 1.5 mL medium containing Cy7-EOM SS NMs (50 μg/mL). After incubation for different times (2, 4, 6, 8 h), the cells were washed three times with PBS and then stained with hoechst33342 at 37 °C for 10 minutes. Finally, the fluorescence images were acquired using CLSM (λex=638 nm, λem=760-860 nm). Verify the promoting effect of folate receptor mediated cell uptake 4T1 cells were seeded onto two confocal dishes and incubated in a cell incubator. Then, the culture medium in one group was replaced with 300 µL medium containing folate (100 μg/mL). After incubation for 6 h, Cy7-EOM SS NMs was added to two groups. After further incubating for 6 h, the cells were washed three times with PBS and then stained with hoechst33342 at 37 °C for 10 minutes. Finally, the cells were washed three times with PBS and fluorescence images were acquired using CLSM (λex=638 nm, λem=760-860 nm). Cell uptake of folate modified nano-micelles by 4T1 cells and AML 12 cells 4T1 cells were seeded onto a 6-well plate and incubated in a cell incubator for 24 hours. Then, the culture medium was replaced with 1.5 mL medium containing Cy7-EOM NMs (50 μg/mL). After incubation for 6 h, the cells were washed three times with PBS and then stained with hoechst33342 at 37 °C for 10 minutes. Finally, the fluorescence images were acquired using CLSM (λex=638 nm, λem=760-860 nm). Intracellular GSH consumption The level of intracellular GSH level was measured by using the fluorescent probe 2,3-Naphthalenedicarbaldehyde (NDA). 4T1 cells were seeded onto a 6-well plate and incubated in for 24 hours. Then, the incubate was replaced with 1.5 mL medium containing PBS, Cy7 NMs, Cy7/CPPO NMs Cy7-EOM NMs, Cy7-EOM SS NMs (50 μg/mL). Then, the cells were incubated for another 8 h, followed by washed three times with PBS and stained with NDA for 20 minutes. Finally, fluorescence images were acquired by CLSM (NDA: λex = 405 nm, λem = 470-510 nm.) Intracellular 1 O 2 generation The level of intracellular 1 O 2 was measured by the fluorescent probe SOSG. 4T1 cells were seeded onto a 6-well plate and incubated in a cell incubator for 24 hours. Then, the culture medium was replaced with 1.5 mL medium containing PBS, Cy7 NMs, Cy7/CPPO NMs Cy7-EOM NMs, Cy7-EOM SS NMs (50 μg/mL), and the cells were incubated for an additional 8 h. After incubation, the cells were washed three times with PBS and then stained with SOSG (1 μM) at 37 °C for 30 minutes. Finally, cells were washed three times with PBS and fluorescence images were acquired using CLSM (λex=488 nm, λem=500-560 nm). Detection of mitochondrial transmembrane potential ( ΔΨ m ) : JC-1 fluorescent probe was used to detect the changes of mitochondrial membrane potential. 4T1 cells were seeded onto a confocal dish and incubated for 24 hours. Then, the culture medium was replaced with 1.5 mL medium containing PBS, Cy7 NMs, Cy7/CPPO NMs Cy7-EOM NMs, Cy7-EOM SS NMs (50 μg/mL), and the cells were incubated for an additional 8 h. After that, the cells were washed with PBS and stained with JC-1 at 37 °C for 20 minutes. Then the cells were washed with buffer solution, and subsequently the fluorescence intensity was observed by CLSM. (JC-1 monomer: λex= 488 nm and λem= 515-555 nm; JC-1 aggregates: λex= 561 nm and λem= 580-620 nm). Intracellular ROS burst The level of intracellular ROS was measured by using the fluorescent probe 2’,7’-dichlorofluorescein diacetate (DCFH-DA). 4T1 cells were seeded onto a 6-well plate and incubated in a cell incubator for 24 hours. Then, the culture medium was replaced with 1.5 mL medium containing PBS, Cy7 NMs, Cy7/CPPO NMs Cy7-EOM NMs, Cy7-EOM SS NMs (50 μg/mL), and the cells were incubated for an additional 12 h. Next, the cells were washed three times with PBS and stained with DCFH-DA (10 μM) at 37 °C for 30 minutes. Finally, cells were washed three times with PBS and fluorescence images were acquired using CLSM (λex=488 nm, λem=510-550 nm). Live-dead cell staining experiments 4T1 cells were seeded onto a 6-well plate and incubated in a cell incubator for 24 hours. Then, the culture medium was replaced with 1.5 mL medium containing PBS, Cy7 NMs, Cy7/CPPO NMs Cy7-EOM NMs, Cy7-EOM SS NMs (50 μg/mL). After incubation for 12 h, the cells were washed three times with PBS and then stained with Calcein-AM and PI for 20 mins. Finally, cells were washed three times with PBS and the fluorescence images were acquired by CLSM (Calcein-AM: λex=488 nm, λem=500-550 nm and PI: λex=535 nm, λem=590-660 nm). Apoptosis assay 4T1 cells were seeded onto a 6-well plate and incubated in a cell incubator for 24 hours. Then, the culture medium was replaced with 1.5 mL medium containing PBS, Cy7 NMs, Cy7/CPPO NMs Cy7-EOM NMs, Cy7-EOM SS NMs (50 μg/mL). After incubating for 12 h, cells were collected and stained with Annexin V-FITC/PI. Finally, the cells were analyzed by flow cytometry. Animal Models All animal experiments were approved by Ethics Committee of Medical College of Qingdao University (QDU-AEC-2024109). For all animal experiments, Female BALB/c mice (4-5 weeks old) were housed under normal conditions with 12 hours light and dark cycles and given access to food and water ad libitum. To establish the tumor lung metastasis mouse model, 4T1-luc cells (5 × 10 5 cells per mouse) were injected intravenously into female BALB/c mice. After 4 days, the growth of tumors in mice was monitored by in vivo imaging system. After the successful establishment of the mouse model was confirmed by using the in vivo imaging system, follow-up experiments were performed. Efficacy of Cy7-EOM SS NMs in Lung Metastatic Tumor Model 4T1-luc tumor-bearing mice were randomly divided into five groups (n = 5 per group): (1) PBS alone, (2) Cy7 NMs, (3) Cy7/CPPO NMs, (4) Cy7-EOM NMs, (5) Cy7-EOM SS NMs. The mice were injected intravenously with various drugs with the dosage of 2.5 mg/kg per mouse at 1, 6 and 11 day post injection. The growth of tumors in mice was monitored by in vivo imaging system (Perkin Elmer). The body weight was recorded every three days. After 16 days treatments, all mice were euthanized and the lungs were isolated. The lungs were treated with 4% paraformaldehyde for bouin fixative staining or H&E immunohistochemical analysis. In Vivo Chemiluminescence Imaging of Tumor 4T1-luc tumor-bearing mice were injected intravenously with Cy7-EOM SS NMs (3 mg/mL, 1ml), and luminescence imaging of the mouse tumors was monitored using an in vivo imaging system (Perkin Elmer). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryInformation.docx Supplementary Information Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5536918","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":391335872,"identity":"7c2c74d3-dcfe-4c0d-b43a-5959be9e5e9a","order_by":0,"name":"Zhengze Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtElEQVRIiWNgGAWjYBAC9gbmBhAtx8befoA4LTwHGMFajPl4ziSQpiVxnoSDAZFaJBIbmHn+1KW3STAkMPyo2EasFh623DbpxgOMPWduE9ZiL5HY/ptHgie3TeZAAjNjGxFaILYYSKSzSSQYkKIlwSCBBC08DxsY5xxIMGwDBvJBovzCw558gOHNnzp5+fb2gw9+VBChhUEggYGJB8o+QIR6IOA/wMD4gzilo2AUjIJRMFIBAIqYNbdFIErqAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-8520-8382","institution":"Qingdao University","correspondingAuthor":true,"prefix":"","firstName":"Zhengze","middleName":"","lastName":"Yu","suffix":""},{"id":391335873,"identity":"e11e59ce-ed38-4b0c-93b1-521ae5f9dee0","order_by":1,"name":"Hanxiang Li","email":"","orcid":"","institution":"Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Hanxiang","middleName":"","lastName":"Li","suffix":""},{"id":391335874,"identity":"e897fb13-1734-46b3-89f8-ca13af7bf83e","order_by":2,"name":"Mingchao Xia","email":"","orcid":"","institution":"Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Mingchao","middleName":"","lastName":"Xia","suffix":""},{"id":391335875,"identity":"0a7458a4-fc60-4be4-aed2-980d312a4049","order_by":3,"name":"Yuhang Wang","email":"","orcid":"","institution":"Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Yuhang","middleName":"","lastName":"Wang","suffix":""},{"id":391335876,"identity":"cbaa1715-be4c-4668-9099-40bb9bc87ed4","order_by":4,"name":"Hao Zhang","email":"","orcid":"","institution":"Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Zhang","suffix":""},{"id":391335877,"identity":"5c09a547-f627-4dca-8c2e-955301b2c2e4","order_by":5,"name":"Yue Tang","email":"","orcid":"","institution":"Department of Emergency Medicine, Shandong Provincial Clinical Research Center for Emergency and Critical Care Medicine, Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Tang","suffix":""},{"id":391335878,"identity":"382d0b76-379a-461d-9627-172dea501413","order_by":6,"name":"Hongyu Wang","email":"","orcid":"https://orcid.org/0000-0001-5720-6542","institution":"Ocean University of China","correspondingAuthor":false,"prefix":"","firstName":"Hongyu","middleName":"","lastName":"Wang","suffix":""},{"id":391335879,"identity":"32838fd7-fa8a-4b02-a661-5e1964967518","order_by":7,"name":"Bo Tang","email":"","orcid":"","institution":"Laoshan Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Tang","suffix":""}],"badges":[],"createdAt":"2024-11-27 16:10:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5536918/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5536918/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73853557,"identity":"dd255b48-eaed-4381-a523-f39de5d00474","added_by":"auto","created_at":"2025-01-15 10:07:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":235849,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Synthesis route of Cy7-EOM. Schematic illustration of the CL imaging and PDT based on intramolecular CRET (\u003cstrong\u003eb)\u003c/strong\u003e, Preparation of Cy7-EOM NMs and Cy7-EOM SS NMs and GSH triggered degradation of Cy7-EOM SS NMs (\u003cstrong\u003ec\u003c/strong\u003e), and the details of intramolecular CRET based mitochondria targeted PDT against cancer metastasis (\u003cstrong\u003ed\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5536918/v1/321734974663bd7b8f51b548.png"},{"id":73853559,"identity":"36150acf-21a0-488d-877e-f7f4110be5dc","added_by":"auto","created_at":"2025-01-15 10:07:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":189094,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of Cy 7-EOM and Cy7-EOM SS NMs.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, TEM images of Cy7-EOM SS NMs. \u003cstrong\u003eb\u003c/strong\u003e, TEM images of Cy 7-EOM SS NM after incubation with GSH (10 mM) for 3 h. Scale bars are 400 nm. \u003cstrong\u003ec\u003c/strong\u003e, UV-Vis absorption spectra of Cy7, Cy7-EOM and Cy7-EOM SS NMs. \u003cstrong\u003ed\u003c/strong\u003e, The fluorescence excitation and emission spectra of Cy7-EOM and Cy7-EOM SS NMs. \u003cstrong\u003ee\u003c/strong\u003e, Norrmalized CL spectra of Cy7-EOM and Cy7-EOM SS NMs. \u003cstrong\u003ef\u003c/strong\u003e, Relative CL intensity of Cy7/CPPO NMs and Cy7-EOM SS NMs at the same concentration after the addition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003e(10 mM), respectively. Insets: corresponding CL images. \u003cstrong\u003eg\u003c/strong\u003e, Time-dependent intensity changes in CL signals of Cy7-EOM SS NMs (upper line in each picture) and Cy7-EOM (bottom line in each picture) with different concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (from left to right: 0.1, 1.0, 5.0, 10, 25 and 50 mM). The fluorescence spectra of the SOSG probe for the detection of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e production: \u003cstrong\u003eh\u003c/strong\u003e, Cy7-EOM and Cy7-EOM + H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, \u003cstrong\u003ei\u003c/strong\u003e, Cy7/CPPO NMs + H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and Cy7-EOM SS NMs + H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, \u003cstrong\u003ej\u003c/strong\u003e, Cy7-EOM + H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and GSH pre-treated Cy7-EOM SS NMs + H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Data are reported as the means ± S.D. and analyzed by two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5536918/v1/2f7cbb8dbbae9bfc9381824a.png"},{"id":73854718,"identity":"0f3733f3-cfd9-4d57-89fe-c50a594ef926","added_by":"auto","created_at":"2025-01-15 10:15:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":396948,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitochondrial targeting and ROS generation of the Cy7-EOM SS NMs. a\u003c/strong\u003e, Co-localization experiments of Cy7-EOM SS NMs with Mito Tracker Green labelled Mitochondria. Scale bars are 100 μm. The quantification of fluorescent intensity of the line scanning profiles and fluorescence scatter diagram in the corresponding confocal images. \u003cstrong\u003eb\u003c/strong\u003e, CLSM images of 4T1 cells stained by SOSG (1 μM) after different treatments. Scale bars are 150 μm. \u003cstrong\u003ec\u003c/strong\u003e, CLSM images of 4T1 cells stained by JC-1 (10 μM, red for aggregate and green for monomer) after different treatments. Scale bars are100 μm. \u003cstrong\u003ed\u003c/strong\u003e, CLSM images of 4T1 cells stained by DCFH-DA (10 μM) after different treatments. Scale bars are 150 μm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5536918/v1/f1304ecb42d236108307f17e.png"},{"id":73853562,"identity":"f4a1944e-cf3a-449c-94df-ccefb4b0bd2f","added_by":"auto","created_at":"2025-01-15 10:07:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":230416,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntracellular anticancer efficiency of the Cy7-EOM SS NMs. a, \u003c/strong\u003eLive/dead cell co-staining assays of 4T1 cells after different treatments by using Calcein-AM (10 μM) and PI (10 μM) as indicators, Scale bars are 200 μm. \u003cstrong\u003eb\u003c/strong\u003e, Cell apoptosis assessment with annexin V-FITC/PI by flow cytometry analysis after different treatments. \u003cstrong\u003ec,\u003c/strong\u003e The viabilities of 4T1 cells treated with Cy7-EOM SS NMs with different concentrations. \u003cstrong\u003ed\u003c/strong\u003e, The viabilities of 4T1 cells with different treatments with the concentration of 50 μg/mL. \u003cstrong\u003ee\u003c/strong\u003e, The viabilities of AML 12 cells treated with Cy7-EOM SS NMs with different concentrations. \u003cstrong\u003ef\u003c/strong\u003e, Schematic illustration of the difference in the toxicity of Cy7-EOM SS NMs to 4T1 cells and AML 12cells. Data are reported as the means ± S.D. and analyzed by two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5536918/v1/f9e90f42c0e7968b73d980d2.png"},{"id":73853558,"identity":"51375a33-a44a-4ebe-afa5-1d0b940dc2c8","added_by":"auto","created_at":"2025-01-15 10:07:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":382300,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e anti-tumour effects againstmetastatic tumours. a\u003c/strong\u003e, Chemiluminescent images of mice with intravenous injection of Cy7-EOM SS NMs. Corresponding photograph of the mouse with metastatic tumours in the lung and intestine after dissection (white arrows). \u003cstrong\u003eb\u003c/strong\u003e, The schedule of the therapeutic process. \u003cstrong\u003ec\u003c/strong\u003e, Bioluminescence images of mice receiving different treatments, n = 5. \u003cstrong\u003ed\u003c/strong\u003e, Curves of tumour cell bioluminescence intensity in the lungs of mice after different treatments, n = 5. \u003cstrong\u003ee\u003c/strong\u003e, Survival curves of mice after different treatments n = 5. \u003cstrong\u003ef\u003c/strong\u003e, Body weights of mice in each group during the treatment. \u003cstrong\u003eg\u003c/strong\u003e, Representative lung photographs stained by Bouin fixative solution after different treatments. \u003cstrong\u003eh\u003c/strong\u003e, Representative H\u0026amp;E staining images of the lungs of mice in different groups. Data are reported as the means ± S.D. and analyzed by two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5536918/v1/7fb064e62ed41cbcbaaeae16.png"},{"id":73855504,"identity":"e45b0981-62ab-4619-b007-7d900cecb8c4","added_by":"auto","created_at":"2025-01-15 10:23:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2415006,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5536918/v1/a1c93e3f-ba67-493f-bc9d-eb9ad3411135.pdf"},{"id":73853564,"identity":"764dee39-588a-444f-8325-da7c863ff15a","added_by":"auto","created_at":"2025-01-15 10:07:21","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":60882647,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5536918/v1/4ae28c0931100d8620b3d95f.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"A highly effective self-supplying photosensitizer drug for deep-tissue metastatic tumours treatment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn cancer-related mortality, 90% are attributed to cancer metastasis and its associated complications\u003csup\u003e1-4\u003c/sup\u003e. The\u0026nbsp;expected\u0026nbsp;five-year survival rate for patients with metastatic tumours is significantly lower than that of patients with primary tumour\u003csup\u003e5,6\u003c/sup\u003e.\u0026nbsp;Currently, the clinical treatment of metastatic tumours primarily relies on surgical removal followed by systemic chemotherapy, which are often associated with severe side effects and limited therapeutic efficacy\u003csup\u003e7,8\u003c/sup\u003e. Consequently, effective treatment for deep-seated metastatic tumours remains a major challenge, underscoring the urgent need for the development of novel and more efficient therapeutic strategies and drugs for metastatic tumours.\u0026nbsp;PDT consists of three essential components: photosensitizers, excitation light, and oxygen.\u0026nbsp;This process activates photosensitizers through specific wavelength excitation light, leading to energy transfer that produces reactive oxygen species (ROS)\u003csup\u003e9-11\u003c/sup\u003e.\u0026nbsp;Due to its non-invasive nature, high spatiotemporal selectivity, low systemic toxicity, and favorable therapeutic efficacy, PDT has been applied in clinical cancer treatments\u003csup\u003e12,13\u003c/sup\u003e.\u0026nbsp;However, the limited tissue penetration of the excitation light required by traditional photosensitizers restricts PDT to superficial lesions or those accessible by endoscopy, such as oral, skin, esophageal, gastric, and bladder cancers,\u0026nbsp;etc\u003csup\u003e14-18\u003c/sup\u003e. It is not yet feasible to apply PDT to metastatic tumours in deep tissues.\u0026nbsp;Therefore, the development of novel strategies for\u0026nbsp;in situ excited PDT in deep tissues,\u0026nbsp;as well as the expansion of its clinical applications, is of critical importance and urgency.\u003c/p\u003e\n\u003cp\u003eChemiluminescence is a process in which light is generated through chemical energy excitation during a chemical reaction\u003csup\u003e19-21\u003c/sup\u003e. Because it does not require external excitation light, chemiluminescence can be produced in situ within tumour tissue and has been used to construct CRET-based PDT systems\u003csup\u003e22-25\u003c/sup\u003e. Among commonly used chemiluminescent substances, peroxycatechol derivatives are particularly favored for CRET-mediated PDT due to their high sensitivity, quantum efficiency, and long emission lifetime\u003csup\u003e26\u003c/sup\u003e. However, a critical challenge in CRET-mediated PDT is its low efficiency, which primarily results from the nanoscale self-assembly strategies used to construct probes. As all known, the efficiency of energy transfer is critically dependent on the proximity between the donor and acceptor\u003csup\u003e27\u003c/sup\u003e. In current researches, the photosensitizers and CPPO are typically assembled through non-covalent interactions to form nanoscale probes, which then undergo PDT through intermolecular CRET\u003csup\u003e28-32\u003c/sup\u003e. However, this probe construction strategy results in an uncontrollable and long distance between the energy donor and acceptor, leading to low CRET efficiency\u003csup\u003e33-36\u003c/sup\u003e. Moreover,\u0026nbsp;aggregation caused quenching (ACQ) of photosensitizers within the nanomaterial reduces ROS production efficiency\u003csup\u003e37-39\u003c/sup\u003e.\u0026nbsp;Even more\u0026nbsp;critically, due to the short lifetime of ROSand short diffusion distance, the nanoscale assembly strategy significantly shields and impedes the diffusion of ROS, leading to a significant loss of ROS\u003csup\u003e40,41\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo address the aforementioned challenges, a novel photosensitizer (Cy7-EOM) was developed by covalently linking the FDA-approved near-infrared photosensitizer, Cy7 with the organic compound ethyl chlorooxoacetate (EOM), and then encapsulated Cy7-EOM into folate-modified nano-micelles featuring disulfide bonds via self-assembly (Cy7-EOM SS NMs). Upon targeting metastatic tumours, Cy7-EOM SS NMs will consume the high intracellular levels of reductive glutathione (GSH) and degrade, releasing the photosensitizer Cy7-EOM, which would specifically target mitochondria due to the positive charge. Under conditions of high endogenous intracellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, Cy7-EOM can be activated in situ via an intramolecular CRET mechanism. It is noteworthy that, compared to intermolecular CRET, the covalent coupling of the energy donor with the acceptor significantly reduces the distance between them, enabling more efficient CRET and ROS generation. The generated ROS will directly induce mitochondrial dysfunction and subsequent apoptosis of cancer cells. Furthermore, the specific decomposition of the nano-micelles in tumour tissue not only prevents the occurrence of the ACQ phenomenon, but eliminates the shielding interference of ROS diffusion. Combined with GSH depletion, the efficacy of PDT for deep-tissue metastatic tumours has been synergistically enhanced. In normal tissue, the low redox levels cannot activate the photosensitizer and the nano-micelles can remain intact, which can effectively shield the diffusion of ROS and protecting normal tissue from oxidative damage. Thus, an efficient and tumour selective PDT was achieved for the treatment of metastatic tumours. The structure of Cy7-EOM and the nano-micelle Cy7-EOM SS NMs and the details of intramolecular CRET based mitochondria targeted PDT against cancer metastasis are illustrated in Fig. 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDesign, Synthesis and Characterization of the photosensitizer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe first designed and synthesized the intramolecular CRET based photosensitizer Cy7-EOM, which consists of two components: 1) a peroxyoxalate group acts as the chemiluminescence substrate which can generate a high-energy intermediate, (i.e. 1,2-dioxetanedione) \u003cem\u003evia\u003c/em\u003e interacting with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and 2) a Cy7 derivative serves as a photosensitizer to capture chemical energy upon deactivation of unstable intermediate for \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation. The final product Cy7-EOM was obtained by covalent coupling of EOM and hydroxylated Cy7 through an esterification reaction. Thus, the designed photosensitizer could be activated specifically in tumour tissue due to the high levels of intracellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and the chemical connection facilitates the proximity between the donor peroxyoxalate and the acceptor Cy7, resulting in efficient intramolecular CRET and self-supplying \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation without the requirement of an external light source. The detailed synthetic route of Cy7-EOM and mechanism of intramolecular CRET based PDT are illustrated in Fig. 1a and b. The designed Cy7 and Cy7-EOM were successfully characterized by nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HR-MS), respectively (Supplementary Fig. 1-6). To achieve targeted delivery and selective release into cancer cells, Cy7-EOM was co-assembled with folate modified and disulfide-containing amphiphilic diblock copolymer (DSPE-SS-PEG:DSPE-PEG-FA=2:1), yielding aqueous nano-micelles (Cy7-EOM SS NMs). As the control group, nano-micelles without disulfide bonds (Cy7-EOM NMs) was prepared \u003cem\u003evia\u003c/em\u003e the same methods (DSPE-PEG:DSPE-PEG-FA=2:1) (Fig. 1c). As shown in TEM images, both Cy7-EOM SS NMs and Cy7-EOM NMs displayed spherical morphology with excellent monodispersity (Fig. 2a and Supplementary Fig. 7a). Cy7-EOM NMs had a larger hydrodynamic diameter of 531.2 nm than that of Cy7-EOM SS NMs (259.2 nm) by dynamic light scattering analysis (DLS) and they had similar neutral surface charge by zeta potential measurement (Supplementary Fig. 8, 9). Due to the specific response of disulfide bonds to GSH, nano-micelles containing disulfide bonds will decompose and release the cargos. TEM images displayed obvious morphology change of Cy7-EOM SS NMs after incubation with GSH for 3 h, while the morphology of Cy7-EOM NMs remains intact (Fig. 2b and Supplementary Fig. 7b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThen, the optical properties of Cy7-EOM and relevant nano-micelles were recorded and analyzed. As shown in absorption spectra, the maximum absorption peak of Cy7-EOM was approximately 774 nm, the same with Cy7 parent, while that of Cy7-EOM SS NMs was redshifted to 786 nm (Fig. 2c). Similarly, fluorescence and chemiluminescence spectra demonstrated the maximum emission peak of Cy7-EOM was at 803 nm, while it were redshifted by about 25 nm after forming nano-micelles (Fig. 2d, e). And the FL emission intensity of Cy7-EOM SS NMs was approximately half that of Cy7-EOM at the same concentration, which resulted from the aggregation caused quenching (ACQ). Another control group was set up by preparing nano-micelles without disulfide bonds and loaded with individual Cy7+CPPO (Cy7/CPPO NMs). After incubation with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, chemiluminescence intensity of Cy7-EOM SS NMs is much higher than that of Cy7/CPPO NMs, revealing more efficient intramolecular CRET than intermolecular CRET (Fig. 2f). Then, a chemiluminescence dynamic experiment of Cy7-EOM and Cy7-EOM SS NMs with different concentration of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was also carried out. From chemiluminescence images we found that Cy7-EOM has a rapid and concentration-dependent response to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, suggesting that effective intramolecular CRET could be achieved in the designed photosensitizer molecule, while the response of Cy7-EOM SS NMs to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e showed a lag in time (Fig. 2g). This is mainly due to the shielding effect of the nano-micelle, which hinders the effective contact between Cy7-EOM and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Therefore, in the same way, nano-micelle will also hinder the diffusion of low-lifetime ROS produced inside the nanoparticles, leading to the loss of ROS and inefficient PDT. To validate our hypothesis, \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation \u003cem\u003evia\u003c/em\u003e CRET in each group was detected by using Singlet Oxygen Sensor Green (SOSG). As shown in fluorescence spectra, the fluorescence intensity of SOSG significantly increased and showed a 5-fold enhancement after H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was added to the solution of Cy7-EOM, indicating the generation of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e (Fig. 2h). Moreover, as expected, fluorescent enhancements of SOSG were also observed for Cy7-EOM SS NMs compared to Cy7/CPPO NMs, demonstrating the superior efficiency of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation through intramolecular CRET to intermolecular CRET (Fig. 2i). In addition, GSH pre-treated Cy7-EOM SS NMs exhibited more \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation than Cy7-EOM SS NMs without GSH treatment, further confirming that ACQ and nano-micelles structure could affect effective utilization of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e (Fig. 2j). Furthermore, electron spin resonance (ESR) spectroscopy was also employed for the \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e analysis by using a radical scavenger, 2,2,6,6-tetramethylpiperidine (TEMP). The appearance of the characteristic peaks in the ESR spectra after the addition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e demonstrated \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation and the increased intensity of ESR signal in the sample of Cy7-EOM further prove that the release of Cy7-EOM from nano-micelles is beneficial to PDT (Supplementary Fig. 10).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of Intracellular \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst, folate mediated cellular uptake performance of Cy7-EOM SS NMs was investigated in 4T1 cells through fluorescence analysis by using confocal laser scanning microscopy (CLSM). As displayed in CLSM images, the fluorescence intensity of 4T1 cells significantly increased over time, indicating effective cellular uptake, and reached a plateau at 6 hours by fluorescence statistical data (Supplementary Fig. 11). To verify the promoting effect of folate on the cellular uptake of Cy7-EOM SS NMs, 4T1 cells were pre-treated with free folate molecules before incubation with Cy7-EOM SS NMs. Confocal images and corresponding statistical data showed that 4T1 cells pre-treated with folate exhibit a weaker red fluorescence signal than those without treatments, which is mainly because the premature binding of folate molecules to folate receptors on the cancer cell membrane hindered the binding of folate in the nanomaterials to folate receptors, thereby interfering with the cell uptake of Cy7-EOM SS NMs (Supplementary Fig. 12). And due to the much higher expression level of folate receptors on cancer cell membrane, nano-micelles are more likely to enter cancer cells than AML-12 cells, mouse normal liver cells (Supplementary Fig. 13). After entering cancer cells, Cy7-EOM SS NMs would degrade under the high concentration of intracellular GSH and release Cy7-EOM. As GSH consumption can not only lead to the release of Cy7-EOM, but also can enhance PDT effect in a synergetic manner, intracellular GSH level was analyzed using a fluorescence probe, 3-naphthalene-dicarboxaldehyde (NDA). CLSM images showed that the fluorescence intensity of NDA in 4T1 cells incubated with Cy7/CPPO NMs, Cy7-EOM NMs or Cy7-EOM SS NMs decreased to different degrees and Cy7-EOM SS NMs performed best in GSH consumption (Supplementary Fig. 14). Then the targeting ability of released Cy7-EOM to mitochondria was evaluated through mitochondrial co-localization experiment by using Mito-Tracker Green, a commercial mitochondrial labeling probe. As shown in confocal images, bright yellow signals were observed, resulting from the superposition of the red signal from Cy7-EOM and green signal from Mito-Tracker Green. The line scanning profiles and the Pearson's correlation coefficient (ρ, calculated to be 0.90) from co-localization scatter plot suggested the excellent targeting ability of the photosensitizer Cy7-EOM to mitochondria (Fig. 3a). Subsequently, Cy7-EOM will react with intracellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to generate \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e through CRET, which was verified by SOSG. CLSM images and corresponding fluorescence quantitative statistics showed that Cy7-EOM NMs has a higher \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e yield than Cy7/CPPO NMs, providing further evidence for the higher efficiency of intramolecular CRET than intermolecular CRET. Notably, 4T1 cells treated with Cy7-EOM SS NMs exhibit the brightest green fluorescence (Fig. 3b and Supplementary Fig. 15). This is because that, in addition to efficient intramolecular CRET, the release of the Cy7-EOM mitigates the adverse effects of ACQ and the physical barrier against \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e diffusion.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eROS burst and apoptosis mechanism\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs mitochondria is one of the most important organelles in the cell and approximately 90 % of intracellular ROS are generated in mitochondria, \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation in mitochondria in situ will directly cause mitochondria dysfunction and further a series of cascade reactions, including the drop of mitochondrial membrane potential (MMP), release of cytochrome C, activation of caspase 3, etc., leading to the ROS burst and eventually cell apoptosis. Then, the mechanism of Cy7-EOM SS NMs induced apoptosis was studied in detail. Firstly, JC-1 staining was employed to investigate the change of MMP by CLSM. From confocal images we can see that obvious green fluorescence of JC-1 monomer emerged in 4T1 cells with treatment of Cy7-EOM NMs and Cy7-EOM SS NMs and the highest ratio of green/red fluorescence intensity in the group of Cy7-EOM SS NMs indicated the maximum MMP drop and most serious mitochondrial damage (Fig. 3c and Supplementary Fig. 16). Subsequently, the release of cytochrome C and activation of caspase 3 induced by mitochondria depolarization were then evaluated and confirmed by immunofluorescence staining, which further demonstrated the most severe cell apoptosis induced by Cy7-EOM SS NMs (Supplementary Fig. 17 and 18). Then, the total amount of ROS in 4T1 cells with different treatments was detected by DCFH-DA to verify ROS burst. As displayed in confocal images, the strongest fluorescence intensity was observed in 4T1 cells treated with Cy7-EOM SS NMs, and the sharp rise in ROS concentration verified the domino burst (Fig. 3d and supplementary Fig. 19). And ROS burst induced cell apoptosis was investigated by live/dead cell the calcein acetoxymethyl ester (calcein AM)/propidium iodide (PI) double staining and flow cytometry analysis of AnnexinV-FITC/PI staining. CLSM images and corresponding fluorescence statistical quantization showed that the largest percentage of cell death appeared in the cells with treatment of Cy7-EOM SS NMs (Fig. 4a). Similarly, in flow cytometry data, the proportion of FITC\u003csup\u003e+\u003c/sup\u003e/PI\u003csup\u003e+\u003c/sup\u003e 4T1 cells is 81.91%, higher than 66.76 % of 4T1 treated with Cy7-EOM NMs and 23.08 % of 4T1 cells treated with Cy7/CPPO NMs, suggesting its superior PDT efficiency to the others (Fig. 4b). The cytotoxicity of Cy7-EOM SS NMs was also evaluated by methyl thiazolyl tetrazolium (MTT) assay. As shown in Fig. 4c, the viability of 4T1 cells decreased significantly with the increase concentration of Cy7-EOM SS NMs and the viability was as low as 18.44% at concentration of 50 μg/mL, while the viabilities of 4T1 treated with Cy7 NMs, Cy7/CPPO NMs and Cy7-EOM NMs were much higher (86.5%, 76.1% and 32.6%, respectively) (Fig. 4d). By comparison, negligible cell death was observed in AML12 cells with the treatment of Cy7-EOM SS NMs even at high incubation concentrations (Fig. 4e). According to the above results, this significant difference is determined by multiple factors, and the detailed mechanism was illustrated in Fig. 4f.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;CL imaging and treatments of metastatic tumours\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEncouraged by the excellent therapeutic effect on cancer cells and extreme low side effects on normal cells, the anti-tumour efficiency of Cy7-EOM SS NMs was then assessed \u003cem\u003ein vivo\u003c/em\u003e. Before that, the ability of nanomaterials to perform targeted imaging and diagnosis was first verified. Mouse models of lung metastases were established by intravenous injection of Luciferase transfected 4T1 cells (4T1-Luc). As shown in fluorescence images, after intravenous injection of Cy7-EOM SS NMs, a clear chemiluminescence signal was observed in the lung. It is worth noting that there is also a chemiluminescence signal in the abdominal cavity. In order to find out the reason, the mouse was euthanized and autopsied and we found that new metastatic tumour sites were observed in the intestines (Fig. 5a and Supplementary Fig. 20). The results revealed that the designed Cy7-EOM SS NMs have distinguished targeting and diagnostic capabilities, even small metastatic tumour sites. Then the anti-tumour effects were evaluated and the detailed treatment schedule is shown in Fig. 5b. The mice were randomly divided into five groups with different treatments of PBS, Cy7 NMs, Cy7/CPPO NMs, Cy7-EOM NMs and Cy7-EON SS NMs at a dosage of 2.5 mg/kg, respectively. Bioluminescence imaging was employed for the evaluation of the therapeutic effects. As shown, the bioluminescence intensity of the mice treated with PBS and Cy7 NMs increased significantly and there was a new metastasis site in the abdominal cavity, indicating rapid tumour progression. By comparison, Cy7/CPPO NMs, Cy7-EOM NMs and Cy7-EOM SS NMs treated mice showed obvious inhibition of tumour growth. Among them, Cy7-EOM SS NMs performed best and the bioluminescence intensity decreased to 9.8 % compared with that in PBS group (Fig. 5c, 5d). In particularly, mice in the groups treated with PBS, Cy7 NMs and Cy7/CPPO NMs shows multiple metastatic tumour sites in the intestines, while a faint bioluminescence signal in the lung and no bioluminescence signal in the abdominal cavity were observed in mice treated with Cy7-EOM SS NMs, demonstrating its great potential as drugs for the treatment of metastasis tumours and even multiple metastatic tumours. After 15 days treatment, the lungs in each group are dissected. Photographs and H\u0026amp;E staining also demonstrated that lungs in groups of PBS, Cy7 NMs, Cy7-EOM NMs were occupied with more metastasis tumour nodules, while almost no metastasis foci could be observed in the Cy7-EOM SS NMs group (Fig. 5g, 5h). In addition, after treated with Cy7-EOM SS NMs, the life span of mice was greatly extended and 60% of mice survived for more than 40 days, while almost all the mice in control group died within 25 days (Fig. 5e). Moreover, the mice weights in each group were monitoring during the treatment and no obvious decrease in body weight further confirmed the low side effects and safety of Cy7-EOM SSNMs (Fig. 5f).\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, to address the current issue of shallow penetration depth of the excitation light in PDT and the challenge of its application in the treatment of metastatic tumours in clinical settings, a chemiluminescence method was adopted as the excitation light to develop an efficient self-supplying photosensitizer molecule Cy7-EOM by covalently coupling the energy donor peroxyphthalazone derivative with the receptor photosensitizer. The covalent coupling of the donor and receptor in the molecular structure significantly enhances the efficiency of PDT. Under high concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in the tumour microenvironment, PDT can be autonomously activated in situ, overcoming the limitations of traditional PDT, which suffers from shallow penetration depth and is ineffective against deep tissue metastatic tumours. And Cy7-EOM was encapsulated into a tumour specific response nano-micelle Cy7-EOM SS-NMs-FA for targeted delivery and release. In cancer cells, Cy7-EOM SS-NMs-FA would consume large amounts of reducing GSH, leading to degradation and the release of Cy7-EOM, which specifically targets mitochondria. The generated ROS then act directly on the mitochondria, inducing mitochondrial ROS bursts that trigger cell apoptosis. This approach has been successfully applied to treat breast cancer cells and multiple metastatic tumours in mice, achieving efficient PDT of metastatic tumours and extremely low systemic toxicity. These findings expand the practical application range of photosensitizers and offer new strategies for photodynamic anticancer drugs, with significant potential for clinical translation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of Shandong Province (ZR2020YQ15), National Natural Science Foundation of China (22377113) and Taishan Scholar Program of Shandong Province (tsqn202306103)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eCollege of Chemistry and Chemical Engineering, Qingdao University, Qingdao 266071, P. R. China.\u003c/p\u003e\n\u003cp\u003eZhengze Yu, Hanxiang Li, Mingchao Xia, Yuhang Wang and Hao Zhang\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eDepartment of Emergency Medicine, Shandong Provincial Clinical Research Center for Emergency and Critical Care Medicine, Qilu Hospital of Shandong University, Jinan, 250014, P. R. China.\u003c/p\u003e\n\u003cp\u003eYue Tang\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003eKey laboratory of marine drugs, ministry of education; Molecular synthesis center, and School of medicine and pharmacy, Ocean University of China, Qingdao, 266003 P. R. China.\u003c/p\u003e\n\u003cp\u003eHongyu Wang\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e4\u003c/sup\u003eLaoshan Laboratory, Qingdao, 266237, P. R. China.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBo Tang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eZhengze Yu.\u0026nbsp;E-mail: [email protected].\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eYue Tang. E-mail: [email protected].\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003eHongyu Wang. E-mail: [email protected].\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e4\u003c/sup\u003eBo Tang. E-mail: [email protected].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.Y. and H.L. contributed equally to this work. Z.Y. conceived the study. Z.Y. and H.L. designed the experiments. H.L. conducted the chemical syntheses, prepared the nanomaterials and conducted the \u003cem\u003ein vitro\u003c/em\u003e characterization. H.L. M.X., Y.W. and H.Z. conducted the cell experiments. Z.Y. and H.L. performed the \u003cem\u003ein vivo\u003c/em\u003e experiments. Z.Y., H.L., Y.T., H.W. and B.T. analyzed the data. Z.Y., Y.T., and B.T. drafted the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eMassagu\u0026eacute;, J., Obenauf, A. Metastatic colonization by circulating tumour cells. Nature \u003cstrong\u003e529\u003c/strong\u003e, 298-306 (2016).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKiri, S., Ryba, T. Cancer, metastasis, and the epigenome. Mol. Cancer \u003cstrong\u003e23\u003c/strong\u003e, 154 (2024).\u003c/li\u003e\n \u003cli\u003ePriestley, P. et al. Pan-cancer whole-genome analyses of metastatic solid tumours. 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Role of Distance in Singlet Oxygen Applications: A Model System. J. Am. Chem. Soc. \u003cstrong\u003e138\u003c/strong\u003e, 7024-7029 (2016).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnless otherwise noted, all commercial reagents and solvents were obtained from the commercial provider and used without further purification. All reagents used in experiments were purchased from Aladdin Industrial Corporation (Beijing, China). DSPE-PEG\u003csub\u003e2000\u003c/sub\u003e and DSPE-PEG\u003csub\u003e2000\u003c/sub\u003e-FA were purchased from Ponsure Biological Technology Co. Ltd. (Shanghai, China). DSPE-SS-PEG\u003csub\u003e2000\u003c/sub\u003e was purchased from Qiyue Biological Technology Co. Ltd. (Xi\u0026rsquo;an, China). Singlet Oxygen Sensor Green Fluorescent Probe (SOSG) was purchased from Maokang Biological Technology Co. Ltd. (Shanghai, China). A naphthalene-2,3-dicarboxaldehyde (NDA) were purchased from Heowns Biochem Technologies, LLC, (Tianjin, China). Calcein-AM/PI Double staining Kit, Mitochondrial Membrane Potential Detection Kit (JC-1) was purchased from G-Clone Biotechnology Co., LTD (Beijing China). 4T1 cells and 4T1-luc cells were purchased from Procell Life Science\u0026amp;Technology Co., Ltd. (Wuhan, China). The mouse normal liver cell line (AML 12) was purchased from Ubigene Biosciences (Guangzhou, China). The water used was Mill-Q secondary ultrapure water (18.2M\u0026Omega;/cm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis of Cy7-EOM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis of Compound 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e2,3,-trimethyl-3H indole (5.0 g, 31.5 mmol) and 2-iodoethanol (7.5 g, 44 mmol) was dissolved in MeCN (50 mL). The solution was refluxed under nitrogen for 24 hours. The reaction mixture was cooled to room temperature and product precipitated by the addition of hexane. The purple solid was filtered and dried without further purification. Yield: 90%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis of Compound 2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo a solution of DMF (20 mL, 273 mmol) in ice-bathed anhydrous DCM (20 mL) under Ar, POCl\u003csub\u003e3\u003c/sub\u003e (17.5 mL, 115 mmol) in anhydrous DCM (5 mL) was added dropwise within 0.5 h. Then, cyclohexanone (5.0 g, 50 mmol) was injected slowly into the above solution. The resulting mixture was stirred vigorously at 80 \u0026deg;C for 3 h, and poured into ice-cold water under stirring to obtain a yellowish precipitation. The solid was filtered off, washed with water, and dried under vacuum to give compound 2 (7.9 g, 91.9%) as a yellowish solid with a fine purity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis of Cy7\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompound 1 (2.9 mg, 11.0 mmol), Compound 2 (860 mg, 5.0 mmol) and CH\u003csub\u003e3\u003c/sub\u003eCOOK(1.1 mg, 11.0 mmol) was dissolved in dry ethanol under nitrogen overnight. After that, the reaction mixture was cooled and a large amount of green precipitation was collected by filtration. The crude precipitation was further purified by column chromatography using CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e/MeOH (20:1, v/v) to provide compound Cy7 (2.23 mg, 82%) as a dark green solid. The \u003csup\u003e1\u003c/sup\u003eH NMR spectrum is shown in Supplementary Fig. 1. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, Chloroform-d) \u0026delta; 8.36-8.36(d, 2H), 7.40-7.34(m, 4H), 7.25-7.20(m, 4H), 6.51-6.48(d, 2H), 4.37-4.35(t, 4H), 4.07-4.04(t, 4H), 2.83-2.80(tH), 2.09(s, 1H), 1.97-1.94(t, 2H), 1.73(s, 12H). The \u003csup\u003e13\u003c/sup\u003eC NMR spectrum is shown in Supplementary Fig. 2. \u003csup\u003e13\u003c/sup\u003eC NMR (400 MHz, Chloroform-d) \u0026delta; 173.31, 144.68, 142.66, 141.07, 128.86, 127.94, 125.19, 122.09, 111.39, 102.26, 77.36, 77.15, 76.94, 58.76, 49.38, 47.26, 46.12, 28.34, 27.08, 20.87, 8.79, 0.07.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis of Cy7-EOM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCy7 (2.72 mg, 5.0 mmol) and Ethyl oxalyl monochloride (2.3 mg, 15.0 mmol) were mixed in 20 mL of anhydrous DCM and then triethylamine (1 mL) was added to the solution in an ice bath. The mixture was stirred under an argon (Ar) atmosphere for 6 h at room temperature. The solvent was removed by vacuum rotary evaporation and the residue was purified by column chromatography to obtain Required products using CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e/MeOH (20:1, v/v) to provide compound Cy7-EOM. The \u003csup\u003e1\u003c/sup\u003eH NMR spectrum is shown in Supplementary Fig. 4. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-d6) \u0026delta; 8.26-8.22(d, 2H), 7.62-7.60(d, 2H), 7.45-7.39(, 4H), 7.29-7.25(2H), 6.43-6.40(2H), 4.29(4H), 3.79(4H), 3.09-3.02(4H), 2.70-3.67(4H), 1.85-1.82(2H), 1.68(12H), 1.21-1.17(6H). The \u003csup\u003e13\u003c/sup\u003eC NMR spectrum is shown in Supplementary Fig. 5. \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta; 173.21, 150.48, 144.63, 142.52, 140.95, 128.82, 127.95, 125.17, 122.06, 111.37, 102.14, 77.42, 77.10, 76.78, 58.59, 49.34, 47.20, 46.51, 29.71, 28.31, 27.25, 20.67, 14.06, 8.83.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of various Nano-micelles (NMs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNMs were fabricated through a thin-film hydration method.\u003c/p\u003e\n\u003cp\u003eFor Cy7/CPPO NMs, Cy7 (1.0 mg), CPPO (2.5 mg), DSPE-PEG (4.0 mg) and DSPE-PEG-FA (2.0 mg) were dissolved at 1 mL DCM under ultrasound for 10 min, and then DCM was removed by evaporation.1 mL of deionized water was added and was vigorously stirred overnight at room temperature to afford an aqueous solution of nano-micelles. The solution was dialyzed in deionized water for 24 h. Almost no Cy7 and CPPO were detected in the dialysate by measuring the absorption, indicating a nearly 100 % encapsulation rate. The resulting solution was stored at 4 \u0026deg;C for further use.\u003c/p\u003e\n\u003cp\u003eThe same procedure was applied for Cy7 NMs, Cy7-EOM NMs and Cy7-EOM SS NMs: For Cy7 NMs, Cy7 (1.0 mg), DSPE-PEG (4.0 mg) and DSPE-PEG-FA (2.0 mg) were used. For Cy7-EOM NMs, Cy7-EOM (1.0 mg), DSPE-PEG (4.0 mg) and DSPE-PEG-FA (2.0 mg) were used. For Cy7-EOM NMs, Cy7-EOM (1 mg), DSPE-PEG (4.0 mg) and DSPE-PEG-FA (2.0 mg) were used. For Cy7-EOM SS NMs, Cy7-EOM (1.0 mg), DSPE-SS-PEG (4.0 mg) and DSPE-PEG-FA (2.0 mg) were used. The remaining steps are the same as those described above. In subsequent experiments, the amount of Cy7 or Cy7-EOM in the nano-micelles in each group is equivalent, and all the concentration values referred to Cy7 or Cy7-EOM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe chemiluminescent properties of the Cy7-EOM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferent concentration of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (final concentration: 0.1, 1.0, 5.0, 10, 25 and 50 mM) were added to Cy7-EOM and Cy7-EOM SS NMs (0.25 mM), respectively. Time dependent chemiluminescent images were obtained by PerkinElmer IVIS Lumina III.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e by SOSG\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation was measured using SOSG as an indicator (\u0026lambda;ex=488 nm, \u0026lambda;em=525 nm).\u003c/p\u003e\n\u003cp\u003eFor Cy7-EOM, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (10 mM) was added to a mixed solution of SOSG (1.0 \u0026micro;M) and Cy7-EOM (10 \u0026micro;M). After incubation for 15 min, the fluorescence intensity of SOSG was measured with excitation at 488 nm. A group without H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was set as the control.\u003c/p\u003e\n\u003cp\u003eFor Cy7-EOM NMs and Cy7/CPPO NMs, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (10 mM) was added to a mixed solution of SOSG (1 \u0026micro;M) and Cy7-EOM NMs (or Cy7/CPPO NMs) (10 \u0026micro;M). After incubation for 15 min, the fluorescence intensity of SOSG was measured with excitation at 488 nm.\u003c/p\u003e\n\u003cp\u003eFor Cy7-EOM SS NMs, Cy7-EOM SS NMs (10 \u0026micro;M) was pre-treated with GSH (10 \u0026micro;M) for 3 h to destroy the nano-micelles. And the SOSG (1 \u0026micro;M) was added to the above solution followed by adding H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (10 mM). After incubation for 15 min, the fluorescence intensity of SOSG was measured with excitation at 488 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e by ESR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (10 mM) was added to Cy7-EOM (10 \u0026micro;M) or Cy7-EOM SS NMs (10 \u0026micro;M) solutions containing 2,2,6,6-Tetramethyl-4-piperidone hydrochloride (TEMP, 100mM), respectively. After incubation for 15 min, the produced singlet oxygen was measured by ESR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e4T1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10 % fetal bovine serum and 1 % penicillin/streptomycin. 4T1-luc cells were treated with high-glucose Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) containing 10 % fetal bovine serum and 1 % penicillin/streptomycin. AML 12 cells were cultured in DMEM medium containing 10 % fetal bovine serum, 0.5 % Insulin-Transferrin-Selenium (ITS-G, 100X), dexamethasone (40 ng/mL) and 1 % penicillin/streptomycin. All the cells were incubated at 37 \u0026deg;C in a humidified atmosphere with 5 % CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytotoxicity test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e4T1 cells or AML 12 cells were seeded onto the 96-well microtiter plate and cultured for 24 h in a cell culture incubator, followed by incubation with different concentrations of Cy7-EOM SS NMs (0, 10, 20, 30, 40,50, 60 \u0026micro;g/mL) for 12 hours. Then, the cell culture medium was removed and replaced by 150 \u0026micro;L of MTT solution (0.5 mg/mL). After 4 hours incubation, the MTT solution was removed, and 150 \u0026micro;L of DMSO was added into each well. Subsequently, the 96-well microtiter plate was shaken slightly in the dark to fully dissolve the formazan, and the absorbance at a wavelength of 490 nm was measured. Cell viability is estimated based on the following formula: Cell viability (%) = (OD treatment/OD control) \u0026times; 100%.\u003c/p\u003e\n\u003cp\u003eThen, the same step was repeated to evaluate the toxicity of different groups (PBS, Cy7 NMs, Cy7/CPPO NMs, Cy7-EOM NMs, Cy7-EOM SS NMs) against 4T1 cells at the same concentration of 50 \u0026micro;g/mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntracellular uptake of Cy7-EOM SS NMs by 4T1 cells\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e4T1 cells were seeded onto a 6-well plate and incubated in a cell incubator for 24 hours. Then, the culture medium was replaced with 1.5 mL medium containing Cy7-EOM SS NMs (50 \u0026mu;g/mL). After incubation for different times (2, 4, 6, 8 h), the cells were washed three times with PBS and then stained with hoechst33342 at 37 \u0026deg;C for 10 minutes. Finally, the fluorescence images were acquired using CLSM (\u0026lambda;ex=638 nm, \u0026lambda;em=760-860 nm).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVerify the promoting effect of folate receptor mediated cell uptake\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e4T1 cells were seeded onto two confocal dishes and incubated in a cell incubator. Then, the culture medium in one group was replaced with 300 \u0026micro;L medium containing folate (100 \u0026mu;g/mL). After incubation for 6 h, Cy7-EOM SS NMs was added to two groups. After further incubating for 6 h, the cells were washed three times with PBS and then stained with hoechst33342 at 37 \u0026deg;C for 10 minutes. Finally, the cells were washed three times with PBS and fluorescence images were acquired using CLSM (\u0026lambda;ex=638 nm, \u0026lambda;em=760-860 nm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell uptake of folate modified nano-micelles by 4T1 cells and AML 12 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e4T1 cells were seeded onto a 6-well plate and incubated in a cell incubator for 24 hours. Then, the culture medium was replaced with 1.5 mL medium containing Cy7-EOM NMs (50 \u0026mu;g/mL). After incubation for 6 h, the cells were washed three times with PBS and then stained with hoechst33342 at 37 \u0026deg;C for 10 minutes. Finally, the fluorescence images were acquired using CLSM (\u0026lambda;ex=638 nm, \u0026lambda;em=760-860 nm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntracellular GSH consumption\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe level of intracellular GSH level was measured by using the fluorescent probe 2,3-Naphthalenedicarbaldehyde (NDA). 4T1 cells were seeded onto a 6-well plate and incubated in for 24 hours. Then, the incubate was replaced with 1.5 mL medium containing PBS, Cy7 NMs, Cy7/CPPO NMs Cy7-EOM NMs, Cy7-EOM SS NMs (50 \u0026mu;g/mL). Then, the cells were incubated for another 8 h, followed by washed three times with PBS and stained with NDA for 20 minutes. Finally, fluorescence images were acquired by CLSM (NDA: \u0026lambda;ex = 405 nm, \u0026lambda;em = 470-510 nm.)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntracellular \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe level of intracellular \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e was measured by the fluorescent probe SOSG. 4T1 cells were seeded onto a 6-well plate and incubated in a cell incubator for 24 hours. Then, the culture medium was replaced with 1.5 mL medium containing PBS, Cy7 NMs, Cy7/CPPO NMs Cy7-EOM NMs, Cy7-EOM SS NMs (50 \u0026mu;g/mL), and the cells were incubated for an additional 8 h. After incubation, the cells were washed three times with PBS and then stained with SOSG (1 \u0026mu;M) at 37 \u0026deg;C for 30 minutes. Finally, cells were washed three times with PBS and fluorescence images were acquired using CLSM (\u0026lambda;ex=488 nm, \u0026lambda;em=500-560 nm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of mitochondrial transmembrane potential (\u003cem\u003e\u0026Delta;\u0026Psi;\u003csub\u003em\u003c/sub\u003e\u003c/em\u003e)\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJC-1 fluorescent probe was used to detect the changes of mitochondrial membrane potential. 4T1 cells were seeded onto a confocal dish and incubated for 24 hours. Then, the culture medium was replaced with 1.5 mL medium containing PBS, Cy7 NMs, Cy7/CPPO NMs Cy7-EOM NMs, Cy7-EOM SS NMs (50 \u0026mu;g/mL), and the cells were incubated for an additional 8 h. After that, the cells were washed with PBS and stained with JC-1 at 37 \u0026deg;C for 20 minutes. Then the cells were washed with buffer solution, and subsequently the fluorescence intensity was observed by CLSM. (JC-1 monomer: \u0026lambda;ex= 488 nm and \u0026lambda;em= 515-555 nm; JC-1 aggregates: \u0026lambda;ex= 561 nm and \u0026lambda;em= 580-620 nm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntracellular ROS\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eburst\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe level of intracellular ROS was measured by using the fluorescent probe 2\u0026rsquo;,7\u0026rsquo;-dichlorofluorescein diacetate (DCFH-DA). 4T1 cells were seeded onto a 6-well plate and incubated in a cell incubator for 24 hours. Then, the culture medium was replaced with 1.5 mL medium containing PBS, Cy7 NMs, Cy7/CPPO NMs Cy7-EOM NMs, Cy7-EOM SS NMs (50 \u0026mu;g/mL), and the cells were incubated for an additional 12 h. Next, the cells were washed three times with PBS and stained with DCFH-DA (10 \u0026mu;M) at 37 \u0026deg;C for 30 minutes. Finally, cells were washed three times with PBS and fluorescence images were acquired using CLSM (\u0026lambda;ex=488 nm, \u0026lambda;em=510-550 nm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLive-dead cell staining experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e4T1 cells were seeded onto a 6-well plate and incubated in a cell incubator for 24 hours. Then, the culture medium was replaced with 1.5 mL medium containing PBS, Cy7 NMs, Cy7/CPPO NMs Cy7-EOM NMs, Cy7-EOM SS NMs (50 \u0026mu;g/mL). After incubation for 12 h, the cells were washed three times with PBS and then stained with Calcein-AM and PI for 20 mins. Finally, cells were washed three times with PBS and the fluorescence images were acquired by CLSM (Calcein-AM: \u0026lambda;ex=488 nm, \u0026lambda;em=500-550 nm and PI: \u0026lambda;ex=535 nm, \u0026lambda;em=590-660 nm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApoptosis assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e4T1 cells were seeded onto a 6-well plate and incubated in a cell incubator for 24 hours. Then, the culture medium was replaced with 1.5 mL medium containing PBS, Cy7 NMs, Cy7/CPPO NMs Cy7-EOM NMs, Cy7-EOM SS NMs (50 \u0026mu;g/mL). After incubating for 12 h, cells were collected and stained with Annexin V-FITC/PI. Finally, the cells were analyzed by flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal Models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by Ethics Committee of Medical College of Qingdao University (QDU-AEC-2024109). For all animal experiments, Female BALB/c mice (4-5 weeks old) were housed under normal conditions with 12 hours light and dark cycles and given access to food and water ad libitum. To establish the tumor lung metastasis mouse model, 4T1-luc cells (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per mouse) were injected intravenously into female BALB/c mice. After 4 days, the growth of tumors in mice was monitored by \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003eimaging system. After the successful establishment of the mouse model was confirmed by using the \u003cem\u003ein vivo\u003c/em\u003e imaging system, follow-up experiments were performed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEfficacy of Cy7-EOM SS NMs in Lung Metastatic Tumor Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e4T1-luc tumor-bearing mice were randomly divided into five groups (n\u0026thinsp;=\u0026thinsp;5 per group): (1) PBS alone, (2) Cy7 NMs, (3) Cy7/CPPO NMs, (4) Cy7-EOM NMs, (5) Cy7-EOM SS NMs. The mice were injected intravenously with various drugs with the dosage of 2.5 mg/kg per mouse at 1, 6 and 11 day post injection. The growth of tumors in mice was monitored by\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e imaging system (Perkin Elmer). The body weight was recorded every three days. After 16 days treatments, all mice were euthanized and the lungs were isolated. The lungs were treated with 4% paraformaldehyde for bouin fixative staining or H\u0026amp;E immunohistochemical analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn Vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Chemiluminescence Imaging of Tumor\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e4T1-luc tumor-bearing mice were injected intravenously with Cy7-EOM SS NMs (3 mg/mL, 1ml), and luminescence imaging of the mouse tumors was monitored using an \u003cem\u003ein vivo\u003c/em\u003e imaging system (Perkin Elmer).\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":false,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5536918/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5536918/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDue to the inherent defects of photodynamic therapy (PDT), its application in the treatment of deep-tissue metastatic tumours remains challenging. To extend the applicability of PDT, a novel chemiluminescent photosensitizer, Cy7-EOM, was developed by covalently coupling the photosensitizer Cy7 with a peroxycatechol derivative and encapsulating it within folate-modified and disulfide-containing nano-micelles. Upon targeted delivery and selective release, positive charged Cy7-EOM would target the mitochondria and efficiently generate singlet oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e) via intramolecular chemiluminescence resonance energy transfer (CRET) by endogenous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, directly inducing mitochondrial damage and cell apoptosis, realizing an efficient PDT for deep-tissue metastatic tumours. Remarkably, the covalent linkage between the donor and the acceptor greatly reduces the distance, significantly enhancing CRET efficiency. Moreover, the tumour-specific decomposition of the nano-micelles prevents aggregation-induced quenching and mitigates the diffusion barrier of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e, while in normal tissues the integrality of nano-micelles shields the lethal effects of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e. This method provides a new strategy for transforming adjuvant photosensitizers into direct therapeutic drugs, with significant potential for clinical application in the treatment of metastatic tumours.\u003c/p\u003e","manuscriptTitle":"A highly effective self-supplying photosensitizer drug for deep-tissue metastatic tumours treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-15 10:07:15","doi":"10.21203/rs.3.rs-5536918/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"729598e0-da0f-4452-8143-f682adb65032","owner":[],"postedDate":"January 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":41708411,"name":"Biological sciences/Biotechnology/Nanobiotechnology/Nanoparticles"},{"id":41708412,"name":"Biological sciences/Biotechnology/Biomaterials/Drug delivery"}],"tags":[],"updatedAt":"2025-03-27T19:01:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-15 10:07:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5536918","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5536918","identity":"rs-5536918","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}