A Singlet Oxygen Reservoir Based on Poly-Pyridone and Porphyrin Nanoscale Metal–Organic Framework for Cancer Therapy

Open AccessCCS ChemistryRESEARCH ARTICLE1 May 2021A Singlet Oxygen Reservoir Based on Poly-Pyridone and Porphyrin Nanoscale Metal–Organic Framework for Cancer Therapy Bo-Ru Xie, Chu-Xin Li, Yun Yu, Jin-Yue Zeng, Ming-Kang Zhang, Xiao-Shuang Wang, Xuan Zeng and Xian-Zheng Zhang Bo-Ru Xie Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072 , Chu-Xin Li Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072 , Yun Yu Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072 , Jin-Yue Zeng Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072 , Ming-Kang Zhang Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072 , Xiao-Shuang Wang Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072 , Xuan Zeng Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072 and Xian-Zheng Zhang *Corresponding author: E-mail Address: [email protected] Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072 https://doi.org/10.31635/ccschem.020.202000201 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail Here, a methacrylate-modified pyridone derivative (mPYR) was loaded into a porphyrin nanoscale metal–organic framework (porphyrin-nMOF). Then, the loaded mPYR was further polymerized to obtain poly-pyridone (poly-mPYR) to form poly-mPYR loaded porphyrin-nMOF, which is designated as PLP and used as a reservoir of singlet oxygen (1O2). It was found that PLP could quickly capture 1O2 in vitro and slowly release 1O2 in vivo to induce cancer cell death. The release of 1O2 was light and oxygen independent, and the entire process did not cause intracellular oxygen consumption. PLP also displayed good therapeutic effect in the treatment of both solid tumor and lung metastasis cancer. This strategy of oxygen- and light-independent 1O2 treatment presents great potential for treating refractory cancer. Also, the form of 1O2 capturing polymer-loaded nMOF expands the biomedical applications of MOFs and polymers, which can be used as a platform for biomedical applications. Download figure Download PowerPoint Introduction Reactive oxygen species (ROS), such as singlet oxygen (1O2), superoxide radical (O2•−), hydrogen peroxide (H2O2), and hydroxyl radical (HO•), have been frequently used in cancer therapy for their robust cytotoxicity and advantages in circumvention of cancer cell drug resistance that usually occurs during traditional chemotherapy.1–3 There are many ways to produce ROS, such as Fenton reaction,4–6 enzymatic reaction,7–9 and photosensitizer (PS) activation.10,11 Among these treatments, photodynamic therapy (PDT) has been considered as a promising therapeutic approach.12,13 In PDT processes, PSs are transported in vivo and activated by light irradiation and then 1O2 is generated for inducing cancer cell apoptosis. Though PDT has been widely studied in cancer research, there are still some limitations, such as limited light penetration depth and tumor hypoxia, that restrict its in vivo efficiency.14–16 Furthermore, the lifetime of 1O2 in water is as short as 3.5 µs.17 The short lifetime of 1O2 will lead to the rapid quenching of ROS generated in PDT procedures, resulting in short effect range of PDT and fast consumption of oxygen. Based on this, 1O2 production of PSs in vivo is unpredictable, resulting in therapeutic effects of PDT usually below expectations. Recently, a number of compounds called endoperoxides (EPOs) were reported for ROS capture and release. These compounds can capture 1O2 to form peroxide bonds intramolecularly through a fast and high yield Diels–Alder reaction.12,18–20 After EPO is produced, it can slowly release 1O2 by a retro-Diels–Alder reaction. This 1O2 release reaction is controlled by the structure of EPO and temperature but does not rely on the presence of enzymes or other intracellular triggers.21 Compared with traditional PDT treatments, after EPO is formed in vitro, the subsequent release of 1O2 in vivo does not depend on light or oxygen concentration. Many kinds of compounds with specific groups can form EPO, such as the derivatives of methylnaphthalene, anthracene, and pyridone.22–24 It was reported that methylnaphthalenes and anthracenes can be used for cancer cell damage.12,25 However, the short lifetime made them unable to perform well in cancer treatment.26 Although some photothermal materials have been used for accelerating ROS release, excessive temperature can also induce damage to normal tissues.12,25 In these EPO generating structures, pyridone and its derivatives show their superiorities, such as mild generating conditions, fast 1O2 capture times, high EPO yields, suitable 1O2 release half-lives, and no side reactions.21,26 Since the formation of EPO relies on trapping 1O2, and the use of EPO in cancer therapy requires sufficient ROS release to produce cytotoxicity, the carriers for EPO should have good 1O2 production capacity and high loading capacity. It seems that porphyrin nanoscale metal–organic frameworks (porphyrin-nMOFs) are suitable to be carriers of EPO. Porphyrin-nMOFs have been used for protein immobilization or drug delivery due to advantages of large surface area, high drug loading rate, and biodegradability.7,9,10,15,27,28 Furthermore, porphyrin-nMOFs also showed good 1O2 yield and had high PSs loading and self-quenching behaviors in PDT.15,29–33 Therefore, porphyrin-MOFs not only act as carriers of EPO, but also act as 1O2 donors for EPO generation. Herein, a methacrylate-modified pyridone derivative (mPYR) was synthesized and then loaded into porphyrin-nMOF. The loaded mPYR in porphyrin-nMOF was further polymerized to make the pyridone polymer (poly-mPYR) loaded porphyrin-nMOF (designated as PLP) (Scheme 1a). As shown in Schemes 1a and 1b, once irradiated under red light, PLP could capture 1O2 in vitro. Owing to the quick 1O2 capture ability of the pyridone structure and the short distance between the pyridone structure and PSs, the 1O2 trapping process took place quickly. After the rapid charging like 1O2 preloaded procedure, PLP could release 1O2 under body temperature conditions in vivo to induce cancer cell death. This in vivo treatment was independent of light and oxygen concentration. The therapeutic potential of PLP was verified by subcutaneous solid tumor and lung metastasis tumor models (Scheme 1c), both of which displayed encouraging outcomes. Scheme 1 | The preparation and treatment mechanism of PLP. (a) Preparation and 1O2 capture process of PLP. (b) Illustration of 1O2 release from PLP + hν for cancer cell treatment. (c) Using PLP + hν for the treatment of solid tumor and lung metastasis models. Download figure Download PowerPoint Experimental Methods Materials Methanol (MeOH), tetrahydrofuran, ethanol, dichloromethane (DCM), trichloromethane (CHCl3), dimethyl sulfoxide (DMSO), triethylamine (TEA, NEt3), sodium bicarbonate (NaHCO3), hydrochloric acid (HClaq), anhydrous sodium sulfate (Na2SO4), N,N′-dimethylformamide (DMF), methyl p-formylbenzoate, pyrrole, propionic acid, azobisisobutyronitrile (AIBN), and potassium carbonate (K2CO3) were purchased from Shanghai Chemical Co, (Shanghai, China). (Hydroxyethyl)methacrylate (HEMA), bromoacetyl bromide, 3-methyl-2(1H)-pyridone, and 1,3-diphenylisobenzofuran (DPBF) were obtained from Sigma-Aldrich Corp. (St. Louis, Missouri, USA). 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was purchased from R&D-SYSTEMS (Minneapolis, Minnesota, USA). Trypsin, fetal bovine serum (FBS), Dulbecco's phosphate buffered saline (PBS), antibiotics (penicillin–streptomycin) solution (10,000 U/mL) penicillin–streptomycin, Roswell Park Memorial Institute-1640 (RMPI-1640), Hoechst 33342, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium-bromide (MTT), Singlet Oxygen Sensor Green, propidium iodide (PI), Annexin V-FITC, LysoTracker Green, and MitoTracker Green were purchased from Invitrogen Corp (Carlsbad, California, USA). Instruments The nuclear magnetic resonance hydrogen (1H NMR) was analyzed by Bruker AVANCE III HD (Bruker, 400 MHz). Electrospray ionization mass spectrometry (ESI-MS) was analyzed by TripleQTOF5600+ (Absciex). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images were observed by a scanning electron microscope (Sigma) and Tecnai G20S-TWIN (Hillsboro, Oregon, USA), respectively. The absorbance was obtained by UV–Vis spectroscopy (Lambda Bio40, Waltham, Massachusetts, USA). Powder X-ray diffraction (PXRD) was measured by Rigaku MiniFlex (Akishima-shi, Tokyo, Japan). The ζ potential and particle size were detected by nano-ZS ZEN3600 (Malvern Instruments, Worcestershire, UK). The cell viability was measured by microplate reader (Model 550; Bio-Rad, Hercules, California, USA). The in vivo imaging results were obtained by IVIS imaging systems (Perkin-Elmer, Waltham, Massachusetts, USA). The EPO generation experiments used a 660 nm He−Ne laser (300 mW). Fluorescence microscopy images were observed by a confocal laser scanning microscopy (CLSM; DMI8, Leica, Germany). Flow cytometry of cell uptake behaviors and cell death was analyzed by BD FACSAria TM III (Bergen, New Jersey, USA). Synthesis of 2-(2-bromoacetoxy)ethyl methacrylate 2-(2-Bromoacetoxy)ethyl methacrylate was synthesized according to a previously reported method.34 HEMA (2.5 g, 2.34 mL, 19.2 mmol) and TEA (2.91 g, 3.99 mL, 28.8 mmol) were added to DCM (25 mL), the solution was stirred under argon atmosphere at 0 °C. Then, bromoacetyl bromide (4.66 g, 2.01 mL, 23.1 mmol, diluted in 10 mL DCM) was added dropwise over a period of 30 min. After addition, the reaction was stirred at room temperature overnight. The mixture solution was diluted with DCM and washed with saturated NaHCO3 aqueous, and then with 1 M HCl aqueous, after which it was dried over anhydrous Na2SO4. The solvent was removed by reduced pressure and purified via column chromatography with DCM and MeOH as eluents. Yield: 1.9 g, 40%. 1H NMR (400 MHz, CDCl3, δ): 6.15 (m, 1H, =CHH), 5.62 (m, 1H, =CHH), 4.43 (m, 2H, –OCH2), 4.39 (m, 2H, CH2O–), 3.87 (s, 2H, –OCH2Br), 1.95 (s, 3H, –CH3). Synthesis of 2-[2-(3-methyl-2-oxo-1,2-dihydropyridin-1-yl)acetoxy]ethyl methacrylate (mPYR) 3-Methyl-2-pyridone (200 mg, 1.83 mmol) and K2CO3 (760 mg, 5.5 mmol) were added to dry DMF (20 mL). Then 2-(2-bromoacetoxy)ethyl methacrylate (520 mg, 2 mmol) was added. The mixture was stirred under argon atmosphere at room temperature overnight. After that, the solvent was removed by reduced pressure and the residue was dissolved in DCM and washed with water and then saturated saline. The organic layer was dried over anhydrous Na2SO4, concentrated in vacuo and purified by column chromatography with DCM and MeOH as eluents. Yield: 160 mg, 31%. 1H NMR (400 MHz, CDCl3, δ): 7.23 (d, 1H, –CH), 7.15 (d, 1H, –NCH), 6.13–6.16 (m, 2H, –CH, –CHH), 5.60 (s, 1H, –CHH), 4.66–4.68 (d, 2H, –CH2), 4.38–4.43 (dd, 4H, –OCH2CH2O–), 2.16 (m, 3H, –CH3), 1.95 (m, 3H, –CH2CCH3). Synthesis of porphyrin-nMOF The 150 nm porphyrin-nMOF was synthesized by dissolving ZrOCl2·8H2O (300 mg), tetrakis(4-carboxyphenyl)porphyrin (75 mg), and benzoic acid (2.8 g) in 50 mL DMF. The solution was stirred at 90 °C for 5 h. After cooling, the precipitate was collected by centrifugation (12,000 rpm, 30 min), washed with DMF, and suspended in DMF for further use. Synthesis and detection of 2-{2-{6-oxo-2,3-dioxa-5-azabicyclo[2.2.2]oct-7-en-5-yl}acetoxy}ethyl methacrylate (EPO) mPYR (100 mg) and porphyrin-nMOF (100 μg) were dissolved in 5 mL CDCl3. This solution was purged with O2 at 0 °C and irradiated with a 660 nm laser at 300 mW. After 1 h, the solution was centrifuged at –10 °C and the clarifying colorless supernatant was used for 1H NMR detection directly. 1H NMR (400 MHz, CDCl3, δ): 6.91 (dd, 1H, –CH), 6.47 (dd, 1H, –CH), 6.14 (s, 1H, –CHH), 5.74 (d, 1H, –CH), 5.62 (s, 1H, –CHH), 4.67–4.71 (d, 1H, –CH2), 4.31–4.44 (m, –4H, –OCH2CH2O–), 3.89–3.95 (d, 1H, –CH2), 1.95 (m, 3H, –CH2CCH3), 1.62 (m, 3H, –CH3). Preparation of PLP Porphyrin-nMOF (10 mg) was first stirred in ethanol overnight. After centrifugation, porphyrin-nMOF was suspended in a solution of mPYR (280 mg, 1 mmol) and AIBN (4.1 mg, 0.025 mmol) in DMF (1 mL). The mixture was stirred at room temperature with argon bubbling for 24 h. Then, the solution was pumped to remove residual oxygen and ventilated with Ar. The solution was sealed carefully, kept in 75 °C and stirred for another 24 h. After that, PLP was collected by centrifugation (12,000 rpm, 30 min) and washed with DMF several times. PLP was suspended in DMF and stored in the shield of light. 1O2 capture ability of mPYR A CHCl3 (or H2O) solution of mPYR (250 µM) and mesotetraphenylporphyrin (TPP; 50 µM) was purged with O2. Then, the solution was irradiated under a 660 nm laser at a power intensity of 100 mW. The absorbance changes were monitored by UV–Vis spectroscopy at different times. 1O2 release ability of mPYR mPYR (250 µM) and PS (TPP or Rose Bengal) 50 µM in DMSO were purged with O2 and irradiated under a 660 nm (or 532 nm for Rose Bengal) laser at a power intensity of 100 mW for 15 min. After that, DPBF (50 µM) was added. The solution was incubated at 37 °C and the absorbance changes were monitored by UV–Vis spectroscopy at different times for 12 h. If the PS was TPP, the same solution was separated to two parts before DPBF added and one of them was used as a baseline. The solution of DPBF (50 µM) or mPYR and PS without light irradiation was used as a control. Cell culture Murine mammary carcinoma (4T1) cells were cultured in RMPI-1640 medium containing 10% FBS and 1% antibiotics (penicillin−streptomycin, 10,000 U/mL) at 37 °C in a humidified atmosphere containing 5% CO2. ROS production and measurement in living cells 4T1 cells were seeded and cultured for 24 h. PLP or porphyrin-nMOF in DMSO was irradiated (660 nm laser, power intensity: 300 mW) for 20 min. Then, the preirradiated PLP (PLP + hν) and preirradiated porphyrin-nMOF (porphyrin-nMOF + hν) were centrifuged and suspended in medium. Next, PLP (200 µg/mL), porphyrin-nMOF + hν (200 µg/mL) or PLP + hν (200 µg/mL), and DCFH-DA (1 μM) were added to the cells at the same time. The cells were incubated in hypoxic atmosphere (1% O2) to imitate the hypoxic tumor microenvironment and avoid oxidation of DCFH by O2. After 8 h, the cells were washed by PBS, and then observed by CLSM. To study the ROS release ability of PLP and PLP + hν overtime, 4T1 cells were incubated with PLP (200 µg/mL) or PLP + hν (200 µg/mL). DCFH-DA (1 μM) was added once the material was added and the cells were cultured in hypoxic atmosphere (1% O2). The fluorescent of DCFH was observed at different times by CLSM. Animal model All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the Animal Experiment Center of Wuhan University (Wuhan, China). All mouse experimental procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of People's Republic of China (Beijing, China). For building solid tumor models, female BALB/c mice were subcutaneously injected 4T1 cells on the right hind leg. For lung metastasis tumor model, female BALB/c mice were intravenously injected 4T1 cells engineering with stable luciferase expression (4T1-luc). More detailed experimental methods are available in the Supporting Information, including separation of poly-mPYR, 1O2 capture and release experiments of poly-mPYR, 1O2 capture and release experiments of PLP, subcellular location observations, cytotoxicity assay, in vivo imaging, antisolid tumor experiments, preparation of macrophage membrane coated PLP ([email protected]) and preirradiated [email protected] (PLP + hν@M), lung metastasis tumor imaging, and lung metastasis tumor inhibition experiments. Results and Discussion Preparation and characterization of PLP We initially synthesized a derivative of pyridone with a methyl group in the three position, and modified it with a methacrylate group to obtain mPYR as the 1O2 accepter ( Supporting Information Figure S1). Although there have been studies using pyridone derivatives as loaders for 1O2, the design defects of the molecular structure have resulted in the rapid release of 1O2, which resulted in limited transport of 1O2 in the body.35,36 Thus, the structural design of mPYR was according to a previous report to ensure its fast 1O2 trapping ability, high retro-Diels–Alder reaction yield, and suitable ROS release speed in water.21 mPYR was characterized by 1H NMR spectra and UV–Vis absorption spectra ( Supporting Information Figures S2–S4). Then, porphyrin-nMOF was synthesized according to a literature report.37 The morphology of porphyrin-nMOF was observed by SEM (Figure 1a) and TEM (Figure 1b), which showed its average size at about 150 nm. As for mPYR loading, we tried to polymerize it in porphyrin-nMOF. MOFs have been utilized in many industrially interesting applications in the polymer field.38,39 They have been combined with polymers either by ligand modification or by polymerizing in the pores and surface. The methods for the formation of polymers in MOFs include free-radical polymerization, reversible addition–fragmentation chain transfer, and atom transfer radical polymerization.38,40,41 In this research, we hope that the polymerization of mPYR in porphyrin-nMOF can increase the loading rate of mPYR, prevent the leakage of pyridone, and shorten the distance between pyridone structure and the porphyrin skeleton to more quickly capture 1O2. Since porphyrin-nMOF is an open-framework structure with channels of 1.85 nm in diameter, the small molecules of mPYR and initiator could easily be adsorbed.37,42 For these reasons, mPYR was loaded into porphyrin-nMOF and polymerized via a free-radical polymerization to obtain PLP. Briefly, porphyrin-nMOF was incubated with a solution of mPYR and initiator AIBN and remained stirring at ambient temperature for 24 h under argon to ensure the infiltration of monomer in the MOF channels. After that, the mixture was polymerized at 75 °C for 24 h. The TEM image of PLP showed that the poly-mPYR was coated outside of the porphyrin-nMOF with a thickness of about 20 nm (Figure 1c). The narrow hydrodynamic size of PLP was 225 nm, which was bigger than porphyrin-nMOF (205 nm, Figure 1d). Compared with the positive charge potential of porphyrin-nMOF (27 mV), the zeta potential of PLP changed to –1.46 mV (Figure 1e), which was because of the ethylene glycol groups in poly-mPYR on the surface of PLP. The change of size and zeta potential of PLP proved the integration of poly-mPYR to some extent. Furthermore, there was no obvious difference in PXRD between porphyrin-nMOF and PLP (Figure 1f), which indicated that the loading and polymerization of mPYR had no influence on the porphyrin-nMOF structure. Figure 1 | Synthesis and characterizations of PLP. (a) SEM image of porphyrin-nMOF. (b) TEM image and magnified TEM image (inserted) of porphyrin-nMOF. (c) TEM image and magnified TEM image (inserted) of PLP. (d) Hydrodynamic size distribution of porphyrin-nMOF and PLP. (e) Zeta potential of porphyrin-nMOF and PLP. (f) PXRD image of porphyrin-nMOF and PLP. (g) AFM-phase image of porphyrin-nMOF. (h) AFM-phase image of PLP. (i) UV–Vis absorption spectra of porphyrin-nMOF, PLP, mPYR, and poly-mPYR separated from PLP. Download figure Download PowerPoint To survey the properties of poly-mPYR in PLP, we used atomic force microscopy (AFM) to observe PLP and porphyrin-nMOF. As shown in two-dimensional (2D) and three-dimensional (3D) height images ( Supporting Information Figures S5 and S6), the corresponding height of PLP was about 150 nm, which was a little higher than porphyrin-nMOF (120 nm, Supporting Information Figures S7 and S8). Furthermore, by comparing phase images of porphyrin-nMOF and PLP (Figures 1g and 1h), there was an obvious poly-mPYR phase coating on the surface and filling in the channels of porphyrin-nMOF. The AFM results proved that poly-mPYR was successfully loaded in porphyrin-nMOF. We also isolated poly-mPYR from PLP and characterized it. The absorption of pyridone structure in the UV–Vis absorption spectra of PLP was hard to find due to the absorption of porphyrin-nMOF (Figure 1i), but the poly-mPYR extracted from PLP showed a UV absorption at 300 nm (Figure 1i), which indicated that poly-mPYR does exist in PLP. After gel permeation chromatography measurements, the number average molecular weight (Mn) of the poly-mPYR in PLP was 3410 Da ( Supporting Information Figure S9). Thermogravimetric analysis (TGA) also confirmed the successful loading of poly-mPYR in PLP, and it also showed that the loading rate of poly-mPYR was 11.73% ( Supporting Information Figure S10). These results prove that mPYR could be successfully loaded and polymerized in porphyrin-nMOF, and the poly-mPYR in PLP had good load efficiency. 1O2 capture and release ability of mPYR and poly-mPYR Since PLP had encapsulated poly-mPYR to act as the 1O2 "reservoir," the 1O2 trapping and releasing ability should be researched. We initially evaluated whether mPYR could capture 1O2 and generate EPO as drawn in Figure 2a. For convenient synthesis and EPO detection, a deuterated chloroform (CDCl3) solution of mPYR (20 mg/mL) with O2 bubbling was prepared, and porphyrin-nMOF (20 μg/mL) was used as the PS to supplied 1O2. After irradiation under a 660 nm laser (300 mW), this solution was centrifuged to separate the nanoscale PS porphyrin-nMOF. The 1H NMR result of the supernatant shows that almost all mPYR had trapped 1O2 and formed peroxide bonds intramolecularly to provide EPO (Figure 2b and Supporting Information Figure S11), which indicated that our mPYR could act as a 1O2 capturer and had a high EPO generation rate. The 1O2 capture process was then characterized by monitoring the UV absorption of mPYR. A solution of mPYR (250 µM) and TPP (50 µM, acted as PS) in CHCl3 was irradiated under a 660 nm laser (100 mW) with O2 bubbling. As shown in Figures 2c and 2d, during light irradiation, the absorbance of mPYR at λ = 296 nm was decreased, which indicated that mPYR captured 1O2 continuously and formed EPO. The EPO generating process was so rapid that the absorbance decreased quickly within 10 min of irradiation, which indicated the excellent 1O2 capture ability of mPYR. The same experiment was also performed in water. As shown in Supporting Information Figures S12 and S13, although the generation speed and yield of EPO were lower than in CHCl3 due to the short lifetime of the generated 1O2 in water ( Supporting Information Figure S14), similar results could also be observed. These phenomena proved that mPYR had the ability to trap 1O2 quickly and efficiently. The process of mPYR capturing 1O2 and the retro-Diels–Alder reaction product after complete release of 1O2 were also detected by ultraperformance liquid chromatography–electrospray ionization (UPLC-ESI) ( Supporting Information Figure S15). After 5 min of light irradiation, mPYR and EPO could be detected in the reaction solution, indicating that the Diels–Alder reaction was in progress ( Supporting Information Figures S15c and S15f). After the irradiaton process, only EPO could be detected, indicating the complete 1O2 capture reaction of mPYR ( Supporting Information Figures S15g and S15h). EPO was then placed at 37 °C for 48 h to allow the retro-Diels–Alder reaction to proceed completely. The subsequent UPLC-ESI results indicated that EPO was completely converted to mPYR ( Supporting Information Figures S15i and S15j). These results proved that mPYR could capture 1O2 by Diels–Alder reaction and release 1O2 by retro-Diels–Alder reaction. The 1O2 capture ability of poly-mPYR was also evaluated by monitoring its UV absorption changes during irradiation in CHCl3 solution. The UV results in Supporting Information Figures S16 and S17 showed that poly-mPYR had similar 1O2 capture ability as mPYR monomer. This indicated that polymerization did not affect the 1O2 capture ability of mPYR. For this reason, PLP loaded with poly-mPYR might also have the ability to capture 1O2. Figure 2 | 1O2 capture and release ability of mPYR and PLP. (a) Schematic illustration of the mechanism of mPYR for 1O2 capture and release. (b) Partial 1H NMR spectrum of mPYR (top) and EPO (bottom). (c) The UV absorption spectra of mPYR (250 µM) in O2 bubbling CHCl3 mixed with TPP (50 µM) after different periods of laser irradiation (660 nm, 100 mW). (d) The corresponding UV absorption changes of mPYR (250 µM) at 296 nm in O2 bubbling CHCl3 mixed with TPP (50 µM) after different periods of laser irradiation (660 nm, 100 mW). (e) The UV–Vis absorption spectra of DPBF (50 µM) at different times in the DMSO solution of mPYR + hν (250 µM). TPP (50 µM) was used as PS in mPYR solution. (f) The UV–Vis absorption changes of DPBF (50 µM) at different times in the DMSO solution of mPYR + hν, mPYR or in the pure DMSO. (g) Time-related DPBF (50 µM) absorption changes in the solution of PLP (50 µg/mL) and porphyrin-nMOF (50 µg/mL) on the response of light irradiation (660 nm, 30 mW/cm2). (h) The UV–Vis absorption changes of DPBF (50 µM) at different periods in the DMSO solution of porphyrin-nMOF + hν, PLP, PLP + hν, or in the pure DMSO. (i) CLSM images of 4T1 cells after treatment with DCFH-DA and (I) PBS, (II) PLP, (III) porphyrin-nMOF + hν, or (IV) PLP + hν. Download figure Download PowerPoint The release of 1O2 from EPO was then indicated by the UV–Vis absorption spectrum based on a 1O2 trap molecular DPBF. DPBF is a commonly used 1O2 detection reagent, which can trap 1O2 and lead to a decrease in absorption.19,25 After the DMSO solution of mPYR (250 µM) and TPP (50 µM) was irradiated (660 nm, 100 mW) under O2 purging, DPBF (50 µM) was added. The mixture solution was incubated at 37 °C and the absorbance of DPBF was monitored at different time intervals. As shown in Figure 2e, after incubation with the preirradiated mPYR (mPYR + hν) solution, the absorbance of DPBF kept decreasing over 12 h, but there was no obvious change in mPYR without irradiation or in blank DPBF solution (Figure 2f). A similar result also appeared when the PS was changed to Rose Bengal ( Supporting Information Figure S18), which indicated that after loading 1O2 in advance, the EPO structure generated from mPYR had an efficient and continuous 1O2 release ability. Similar results also appeared in the 1O2 release test of poly-mPYR ( Supporting Information Figure S19), which indicated that poly-mPYR had good 1O2 release ability consistent with mPYR monomer. 1O2 capture and release ability of PLP Inspired by good 1O2 trapping and release ability of mPYR, we evaluated 1O2 capture and release ability of PLP. Due to the absorption of porphyrin-nMOF at 300 nm, it was difficult to evaluate the generation process of EPO in PLP by monitoring the self-absorption intensity change of the pyridone structure (Figure 1i). For this reason, we used the DMSO solution of PLP (50 µg/mL) and porphyrin-nMOF (50 µg/mL) with DPBF (50 µM) added to evaluate the uncaptured free 1O2 in the solution during light irradiation, so that we could evaluate the 1O2 capture capacity of PLP by measuring the absorption intensity change of DPBF. With light irradiation (660 nm, 30 mW/cm2), the absorption intensity of DPBF was recorded every 5 s. It could be found that the absorption intensity of DPBF in porphyrin-nMOF solution decreased rapidly, demonstrating effective 1O2 production in porphyrin-nMOF solution, while it decreased more slowly in PLP solution (Figure 2g). This was because during light irradiation, the 1O2 generated in the porphyrin-nMOF solution was almost free so that it could be trapped by DPBF. On the contrary, in PLP solution, part of the generated 1O2 was captured by the pyridone structures in PLP, which led to less 1O2 trapped by DBPF and led to less absorption intensity change of DPBF. Therefore, the