MaterialsAll commercially available starting compounds and solvents used in the research, except additionally stated, were purchased from Adamas-beta without further purification.Synthesis of mcm-1A 20 × 60 mm Pyrex tube, loaded with 0.02 mmol BPY (8.40 mg), Zn-TBPP (20.80 mg), 0.8 mL n-Butanol, 0.8 mL 1,2-Dichlorobenzene, 30 μL aniline and 30 μL benzaldehyde, underwent 20 min of sonication, followed by the addition of 0.4 mL 6 M acetic acid. Subsequently, the tube was flash frozen at 77 K, evacuated to 50 mTorr, flame sealed, and heated at 120 °C for 72 h. The resulting powder was filtered, DMF washed until the filtrate was colorless, then washed with 5 × 5 mL THF, and finally purified in a Soxhlet with acetone for 12 h. The dark green product was vacuum dried at room temperature overnight. Yield: 85%. Elemental analysis of mcm-1: Calcd. for C188N20Zn2H108: C, 81.39%; N, 10.10%; Zn, 4.62%; H, 3.89%.Synthesis of mcm-2The synthesis was carried out by utilizing the same protocol with a mixture of 0.02 mmol QPTA (8.36 mg), 0.02 mmol Zn-TBPP (20.80 mg), 0.5 mL n-Butanol, 0.5 mL 1,2-Dichlorobenzene, 30 μL aniline, 30 μL benzaldehyde and 0.2 mL aqueous acetic acid (6 M). Finally, the dark green product was evacuated at room temperature under vacuum overnight. Yield: 78%. Elemental analysis of mcm-2: Calcd. for C192N16Zn2H112: C, 83.24%; N, 8.09%; Zn, 4.62%; H, 4.05%.Synthesis of mcm-3The synthesis was carried out by utilizing the same protocol with a mixture of 0.02 mmol BPY (8.40 mg), 0.02 mmol H-TBPP (19.56 mg), 0.5 mL n-Butanol, 0.5 mL 1,2-Dichlorobenzene, 30 μL aniline, 30 μL benzaldehyde and 0.2 mL aqueous acetic acid (6 M). Finally, the maroon product was evacuated at room temperature under vacuum overnight. Yield: 75%. Elemental analysis of mcm-3: Calcd. for C188N20H112: C, 85.20%; N, 10.57%; H, 4.23%.Synthesis of mcm-4The synthesis was carried out by utilizing the same protocol with a mixture of 0.02 mmol QPTA (8.36 mg), 0.02 mmol H-TBPP (19.56 mg), 0.8 mL n-Butanol, 0.8 mL 1,2-Dichlorobenzene, 30 μL aniline, 30 μL benzaldehyde and 0.4 mL aqueous acetic acid (6 M). Finally, the maroon product was evacuated at room temperature under vacuum overnight. Yield: 79%. Elemental analysis of mcm-4: Calcd. for C192N16H116: C, 87.14%; N, 8.47%; H, 4.39%.Photocatalytic H2
production
5 mg of photocatalyst was carefully dispersed in 25 mL of H2O with 0.1 M ascorbic acid acting as a sacrificial agent. This solution was then placed into a sealed quartz tube setup. A Pt co-catalyst (3 wt%) was photo-deposited onto the photocatalyst using H2PtCl6. Before the photocatalytic evaluation, the system was purged with Ar to remove air. A 300 W Xe lamp (Beijing Perfectlight Technology Co., Ltd., Microsolar300) served as the light source, and a 420 nm or 700 nm cutoff filter facilitated visible light irradiation (780 nm > λ > 420 nm or 780 nm > λ > 700 nm). Hydrogen production was measured by gas chromatography. Post-collection and water rinsing, the cyclic photocatalytic tests were repeated, adhering to the same procedure.Apparent quantum yields measurementThe measurement of apparent quantum yields (AQY) for hydrogen evolution followed the photocatalytic hydrogen evolution protocol, with the addition of band-pass filters at 420 nm, 600 nm, and 700 nm on the Xe lamp. The irradiance was quantified using an optical photodiode power meter (ILT 950 Spectroradiometer). Then, the AQY was calculated by following the equation:$${{{\rm{AQY}}}}=\frac{2\times {N}_{e}}{{N}_{p}}\times 100\%=\frac{2\times M\times {N}_{A}\times h\times c}{S\times P\times t\times \lambda }\times 100\%$$Where, Ne is the number of generated electrons, Np is the number of incident photons, M is the yield of H2 molecules (mol), NA is Avogadro constant (6.022 × 1023 mol−1), h is the Planck constant (6.626 × 10−34 J·s), c is the speed of light (3 × 108 m s−1), S is the irradiation area (cm2), P is the intensity of irradiation light (W cm−2), t is the photoreaction time (s), λ is the wavelength of the incident monochromatic light (m).Photocurrents and photoelectrochemical measurementsWith an electrochemical workstation (CHI660E, CHI Instruments, Shanghai, China), we evaluated the Mott-Schottky plots, photocurrent responses, and electrochemical impedance spectra of the catalysts. Illumination was provided by a 300 W Xe lamp, and a 0.5 M Na2SO4 solution at pH 6.5 was used as the electrolyte for photocurrent tests. The reference electrode was Ag/AgCl, and a platinum wire served as the counter electrode. A 10 μL solution with 5 wt% Nafion, 0.5 mL of anhydrous ethanol, and 2 mg of photocatalyst was sonicated for an hour to create a homogenous suspension. Then, 200 μL of this mixture was spread onto an ITO glass slide and left to dry at ambient temperature. Conversion of potential vs. Ag/AgCl to RHE potential was performed using the following equation:$${{{\rm{E}}}}({{{\rm{VS}}}}.\,{{{\rm{RHE}}}})={{{\rm{E}}}}({{{\rm{VS}}}}.\,{{{\rm{Ag}}}}/{{{\rm{AgCl}}}})+0.197\,{{{\rm{V}}}}+0.0591*{{{\rm{pH}}}}$$DFT calculationGeometry optimizations and frequency calculations utilized the hybrid B3LYP functional with the 6-31 G(d) basis set for C, H, and N, and the LANL2DZ basis set for Zn atoms, all performed with the Gaussian 03 software. The model, derived from a simulated COFs structure, had its dangling bonds capped with hydrogens. Post full optimization, frequency calculations were executed at the same computational level. The electronic energies were refined by single point calculations using the expanded 6-31++ G(d,p) basis set for nonmetal atoms. Visualization of the HOMO and LUMO molecular orbitals was achieved with Multiwfn and VMD software48.Calculation of the photothermal conversion efficiencyThe 200 μL solution of mcm-1 at a concentration of 100 μg mL−1 was irradiated with a 660 nm near-infrared laser at an intensity of 1 W cm−2, provided by Beijing Laserwave Optoelectronics Technology Co., Ltd., for a duration of 5 min, after which it was allowed to cool to ambient temperature. Temperature fluctuations throughout the process were captured in real time using an infrared camera, the FOTRIC 225 s from China. The PCE (η) was determined using the relevant formulas:$${{{\rm{\eta }}}}=\frac{{hS}({T}_{max }-{T}_{{surr}})-{Q}_{s}}{I(1-{10}^{-{A}_{660}})}$$
(1)
$${hS}=\frac{{mC}}{{\tau }_{s}}$$
(2)
$$t=-{\tau }_{s}ln(\theta )$$
(3)
$$\theta=\frac{T-{T}_{{surr}}}{{T}_{max }-{T}_{{surr}}}$$
(4)
The heat transfer coefficient is denoted by h; S represents the surface area of the container. Qs accounts for the heat dissipation from the laser, mediated by both the solvent and the container. The laser power I is 1 W, equivalent to the product of power density and the irradiation area. The absorbance of the mcm-1 solution at 660 nm is given by A660. The mass of the photoactive solution m is 0.2 g, and C is its specific heat capacity, valued at 4.2 J g−1 K−1. The sample system time constant τs is ascertained through linear regression of the temperature cooling time against a specific equation, referred to as Eq. 3 in the text. θ is a dimensionless driving force temperature parameter. Tmax and TSurr correspond to the maximum steady-state temperature and the surrounding environmental temperature, respectively.Calculation of the singlet oxygen (1O2) quantum yield (ΦΔ)The 1O2 quantum yield of mcm-COF was ascertained using DPBF (1,3-diphenylisobenzofuran) as the indicator for 1O2, with indocyanine green (ICG) serving as a reference standard, known to have a quantum yield of ΦΔ = 0.077. The DPBF absorbance at 415 nm was standardized to approximately 1.0 in DMSO, while the absorbance of mcm-COFs or ICG was set to the range of 0.2 to 0.3. Upon irradiation at 660 nm for various durations, the absorbance spectra were documented. The quantum yield of 1O2 was then computed using the formula presented in Eq. 5:$${\varPhi }_{\varDelta {sam}}={\varPhi }_{\varDelta {std}}\left(\frac{{m}_{{sam}}}{{m}_{{std}}}\right)\left(\frac{{F}_{{std}}}{{F}_{{sam}}}\right)$$
(5)
Where “sam” and “std” denote the “mcm-COFs” and “ICG”, respectively. “m” corresponds to the slope of absorbance change curve of DPBF at 415 nm, F = 1 − 10−O.D. (O.D. is the absorbance of the solution at 660 nm).Photothermal propertiesThe ROS generation efficiency of four COF samples was evaluated using DCFH-DA as an indicator. The COF samples were suspended in a PBS solution at a concentration of 100 μg mL−1, followed by the addition of DCFH-DA to a final concentration of 10 μM. Upon exposure to a 660 nm laser at an intensity of 1 W cm−2, the resulting fluorescence was monitored at various intervals with an F7000 spectrofluorometer from Hitachi, capturing the emission spectra.Detection of ROS in solvents200 μL of COF samples in varying concentrations were irradiated using a 660 nm near-infrared laser sourced from Beijing Laserwave Optoelectronics Technology Co., Ltd. The temperature variations were captured in real time using the FOTRIC 225 s infrared camera.Cell and animalsThe 4T1 mouse breast cancer cell line was sourced from the National Collection of Authenticated Cell Cultures in Shanghai, China, and maintained in culture medium comprising RPMI-1640 supplemented with 1% penicillin-streptomycin and 10% certified FBS, all from Gibco. The 4T1 cell line, cataloged as CRL-2539, was additionally procured from Wuhan Procell Life Science & Technology, while the 4T1-Luc cell line was acquired from Xiamen Immocell Biotechnology Co., Ltd., with its original source being ATCC. Healthy female BALB/c nude mice, 4–6 weeks old and ~15 g in weight, were selected for the study and obtained from the Model Animal Research Center (MARC) of Nanjing University (Nanjing, China), due to the predominance of mammary cancers in females. These mice were kept in an SPF-grade facility with a 12-hour light/dark cycle, ambient temperature ranging from 20 to 26 °C, and humidity levels stabilized between 40% and 70%. Conducting all animal experiments in accordance with the guidelines approved by the Animal Ethical and Welfare Committee of Nanjing University, the research was carried out under the oversight of protocol number IACUC-2405005.Cytotoxicity assay4T1 cells (3 × 103) were initially cultured in RPMI-1640 medium overnight. Subsequently, the medium was replaced with COF materials at varying concentrations (0, 25, 50, 75 and 100 μg mL−1), and subjected to 660 nm laser at an intensity of 1 W cm−2 for 5 min. After 24 h incubation, the viability of the 4T1 cells was evaluated using the MTT assay.In vitro cellular uptake4T1 cells were meticulously seeded onto round coverslips at the base of a 12-well plate and incubated at 37 °C for 12 h. Subsequently, they were subjected to COF samples at a concentration of 100 μg mL−1 for durations between 0 to 24 h, after which the cells were assessed using flow cytometry. In preparation for cell imaging, the 4T1 cells were first treated with mcm-1, followed by staining with Mito Tracker Green at 0.5 μM and Hoechst 33342 at 1 μM for 30 min. The cells were then extensively washed with PBS, a minimum of three times, prior to fixation with a 4% paraformaldehyde solution for 20 min. The prepared samples were examined under CLSM, utilizing excitation wavelengths of 405 nm with an emission bandpass of 410–500 nm, 488 nm with an emission bandpass of 550–650 nm, and 633 nm with an emission bandpass of 650–750 nm.Detection of ROS in cellsThe 4T1 cells were treated with COF samples at 100 μg mL−1 for 6 h. Specific samples were treated with NAC at 100 μM for 30 min, followed by irradiation with a 660 nm laser at 1 W cm−2. Subsequently, the cells were stained with DCFH-DA at 10 μM for 30 min and then observed using CLSM.Analysis of mitochondrial membrane potential4T1 cells were evenly spread on round coverslips in 12-well plates and incubated at 37 °C for 12 h. After 6 h treatment with 100 μg mL−1 COF samples, some were exposed to 660 nm laser irradiation at 1 W cm−2 for 5 min. The JC-1 kit was employed to evaluate mitochondrial membrane potential, with samples analyzed by flow cytometry and CLSM. JC-1 monomers were identified with excitation at 514 nm and emission at 529 nm, whereas aggregates were detected with excitation at 585 nm and emission at 590 nm.Mitochondrial morphology assays4T1 cells were treated with mcm-1 at a concentration of 100 μg mL−1 for 24 h. After irradiation with a 660 nm laser at 1 W cm−2 for 5 min, the cells were incubated for an additional 6 h, then washed three times with PBS and fixed in 4% paraformaldehyde for 1 h. The mitochondrial morphology was examined using TEM. The cells were initially fixed in 0.1 M sodium cacodylate buffer (pH 7.4) containing 2.5% glutaraldehyde, followed by post-fixation in 2% aqueous osmium tetroxide and dehydration in an ethanol/propylene oxide mixture. Subsequently, the samples were embedded in Epon and incubated at 60 °C for 24 h (Merck, Darmstadt, Germany). Prior to TEM analysis, ultrathin sections were prepared and stained with 0.5% uranyl acetate for 30 min, followed by 3% lead citrate for 7 min.Intracellular detection of GPX4 and TFR14T1 cells were treated overnight with different samples. Following treatment, 2 × 103 cells were fixed in 4% paraformaldehyde and infiltrated with 0.5% Triton X-100 (BS084, Biosharp). They were then incubated in 3% BSA (Biosharp BS114) at room temperature with (a) an anti-GPX4 antibody diluted 1:200 (CY6959, Abways) followed by goat anti-rabbit Cy3 diluted 1:300 (AB0133, Abways), or (b) an anti-TFR1 antibody diluted 1:200 (CY6618, Abways) followed by goat anti-rabbit IgG Alexa Fluor 488 diluted 1:400 (AB0141, Abways), both incubations lasting 1 h. The cells were finally counterstained with DAPI (P0131, Beyotime Biotechnology) and examined using an SP8 confocal microscope from Leica.Western blotProteins were lysed with 30 min in buffer provided by Beyotime and rinsed with PBS. Protein concentrations were assessed using the BCA assay kit from the same manufacturer, loading a standard volume of 30 μg per well. Separation of proteins was achieved through polyacrylamide gel electrophoresis with Epizyme reagents, and the resolved proteins were transferred onto a PVDF membrane from Beyotime. Non-specific antigens were blocked with 5% skimmed milk for 2 h, after which immunoblotting was performed with different antibodies, including glutathione peroxidase 4 (1:1000 dilution, CY6959, Abways), transferrin receptor (1:1000 dilution, CY6618, Abways), and GAPDH (1:5000 dilution, 60004-1-Ig, Proteintech).Measurement of lipid ROSCells from each experimental group were rinsed and immersed in 1 mL of PBS with 0.5 μM C11-BODIPY (581/591) dye (D3861, Invitrogen) at 37 °C for 60 min. Subsequently, the alterations in fluorescence signals were evaluated using confocal microscopy.Animal experimentsHealthy female BALB/c nude mice, 4 to 6 weeks old and approximately 15 g in weight, were procured from Model Animal Research Center (MARC) of Nanjing University (Nanjing, China). The experimental procedures were strictly in line with the Institutional Animal Care and Use Committee (IACUC-2405005) guidelines at Nanjing University.In vivo treatmentTo establish a 4T1 hormonal mouse model, healthy female BALB/c nude mice aged 4–6 weeks were subcutaneously injected into 4T1 cells (1 × 106 cells in PBS) in the right hind leg. Once tumors reached a volume of 100 mm3, the mice were randomly divided into four groups (n = 5): (1) PBS, (2) PBS + Laser, (3) mcm-1, and (4) mcm-1 + Laser. mcm-1 was dosed at 10 mg kg−1. Treatments were applied three times. Body weight and tumor volume (calculated as volume[V] = length × width2/2) were monitored every two days for 12 days of treatment. At the end of treatment, mice were euthanized, and tumors along with major organs were harvested from each group for analysis via H&E staining. To observe apoptosis after different treatments, TUNEL and Ki67 staining were additionally applied. Immunohistochemical staining for GPX4 and TFR1 was performed to evaluate the expression levels of GPX4 versus TFR1 in the tumor tissues among the different groups.Reporting summaryFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Cairo pentagon tessellated covalent organic frameworks with mcm topology for near-infrared phototherapy
