Rationally Designed Thiazole-Based COF with Multiphoton Activity for Drug Delivery and Synergistic Therapy of Deep-Seated Tumors

Rationally Designed Thiazole-Based COF with Multiphoton Activity for Drug Delivery and Synergistic Therapy of Deep-Seated Tumors | 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 Research Article Rationally Designed Thiazole-Based COF with Multiphoton Activity for Drug Delivery and Synergistic Therapy of Deep-Seated Tumors Shuting Li, Yan Liu, Ganlin Dong, Yahuan Wang, Yaonan Li, Liefeng Hu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7635511/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Feb, 2026 Read the published version in Journal of Nanobiotechnology → Version 1 posted 10 You are reading this latest preprint version Abstract Multiphoton photosensitizers activated by near-infrared light have attracted considerable attention in the fields of homogeneous photocatalysis, optical imaging, and phototherapy because of their negligible interference, low dilapidation, and deep penetration. Nonetheless, most multiphoton photosensitizers still face issues such as poor long-range ordering, strong π-π stacking hindrances, and potential metal toxicity. In this study, a rationally designed thiazole-based covalent organic framework (DT-COF) with multiphoton activity was efficiently harvested via one-step preparation from inactive small molecules—namely, dithiooxamide and 1,3,5-triformylbenzene. Theoretical calculation and characterization tests revealed that the good performances of deeper multiphoton fluorescence and photodynamic therapy benefited from the donor-π-acceptor configuration, highly ordered long-range structure, weakened π-π stacking interaction between the layers, and good nonlinear optical properties. Furthermore, tirapazamine (TPZ), a hypoxia-activated chemotherapeutic drug, was encapsulated into DT-COF pores with anastomosing dimensions (encapsulation efficiency = 75%). The DT-COF targeted the mitochondria and delivered TPZ to the nucleus. Additionally, the DT-COF consumed oxygen to generate 1 O 2 , promoting TPZ activation and realizing a combination of photodynamic and chemotherapeutic anti-tumor therapy. This study provides a new strategy for the development of multiphoton photosensitizers and demonstrates their potential in cancer theranostics. Multiphoton Covalent-organic framework Drug delivery Photodynamic-chemotherapeutic therapy Deep-seated tumor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Photodynamic therapy (PDT), a promising photo-induced therapeutic strategy approved for clinical use, has shed new light on millions of patients with tumors [ 1 – 3 ]. Compared to conventional therapeutic strategies used clinically, such as radiotherapy, chemotherapy, and surgery, PDT offers unique advantages such as minimal invasiveness, high spatial selectivity, and low side effects [ 4 , 5 ]. The key to high-performance PDT lies in the use of photosensitizers that are effectively activated by specific light exposure to generate cytotoxic singlet oxygen ( 1 O 2 ) [ 6 , 7 ]. As contrast agents, photosensitizers determine the accuracy and tissue depth of fluorescent bioimaging [ 8 , 9 ]. Currently, the excitation wavelengths of most photosensitizers, such as porphyrin, BODIPY dyes, and their derivatives, is still limited to 600–700 nm [ 10 , 11 ]. Moreover, the relatively short wavelength of the excitation light is largely scattered by tissues, resulting in low penetration depth and treatment efficiency [ 12 ]. To resolve these issues, multiphotonic materials can be used, which emit individual photons by absorbing multiple photons at long wavelengths. Multiphoton ( n ≥ 3) photosensitizers activated by near-infrared light—such as small molecules, organometallic complexes, and photosensitive chromophore-integrated nanomaterials—can be used to perform deep-tissue PDT and spatial imaging [ 13 – 16 ]. Nevertheless, their clinical applications remain limited due to high dark toxicity, low stability, and insufficient multiphoton activity. The covalent organic framework (COF), an emerging class of crystalline porous polymers manufactured by conjugating molecules through covalent bonds, is a promising material for biomedical applications, including drug delivery, bioimaging, and disease prevention and treatment, owing to their adjustable pores, good biocompatibility and stability, and compositional and structural tunability [ 17 – 19 ]. The properties of COF materials can be precisely modulated by the atomic insertion of functional units. The periodically extensible framework structure also endows the COF with a confined molecular space for photoelectronic transport and host–guest interactions, thereby demonstrating great potential for theoretically guided photosensitizer design and targeted applications [ 20 , 21 ]. For instance, in 2019, Deng et al. [ 22 ] reported two COFs (COF-808 and COF-909) with excellent photodynamic capacity under 630 nm laser irradiation, which were linked via inactive and inexpensive small molecules. The two COFs exhibited two-dimensional (2D) π-conjugation, and their framework structure also facilitated the diffusion of oxygen and reactive oxygen species (ROS) to enhance therapeutic efficacy. Additionally, the COFs diminished the π-π stacking interaction between the layers and enhanced the multiphoton cross-section to improve their activity. In 2020, Zhang et al. [ 23 ] designed a benzothiadiazole-based COF with high dipole values and two-photon cross-section using the donor-π-acceptor-π-donor (D-π-A-π-D) functional unit as the building block, which led to good near-infrared two-photon-induced fluorescence features. Therefore, engineered COFs have great potential as near-infrared light-excited multiphoton photosensitizers for efficient deep tumor imaging and PDT. Thiazole-derived organic chromophores have recently shown great potential for application in multiphoton excitation owing to their unique charge distribution [ 24 – 26 ]. In the present study (Scheme 1 ), we designed and synthesized a new type of thiazolothiazole-based COF (DT-COF) nanowire with D-A configurations for multiphoton-excited tumor imaging and PDT. The DT-COF was efficiently harvested via a one-step catalyst-free condensation reaction using two inactive and inexpensive small molecules—namely, dithiooxamide (DTO) and 1,3,5-triformylbenzene (TFB)—which achieved good three-photon performance in deep-seated tumor imaging and therapy. Mechanically, the satisfactory results may be attributed mainly to the long-range ordering of the closed-frame structure, polarization enhancement (10.5 times) of the charge distribution caused by the closed D-A configuration of the chromophore, and rigid COF structure-mediated enhancement of the dipole moment (6.3 times) and hyperpolarizability (10.5 times), leading to an increased cross-section of multiphoton induction. In addition, the one-dimensional (1D) pore size of the DT-COF was suitable for the efficient encapsulation of tirapazamine (TPZ), a hypoxia-activated chemotherapeutic drug, with encapsulation rates up to 76.2%. Not only did the proton effect of the thiazole moiety in DT-COF allow it to target the mitochondria, but also the microenvironment of tumor cells stimulated TPZ release and delivery into the nucleus. Furthermore, DT-COF-mediated PDT further depleted oxygen in hypoxic tumors to activate TPZ-induced chemotherapy, resulting in a synergistic oncolytic effect. This study sheds new light on the design of photoactive materials with novel structures and rational applications. Results and discussion Structural performance assessment of DT-COF The engineered thiazolothiazole-based DT-COF was obtained from the Hantzsch thiazole synthesis with some modifications [ 27 ], which involved the polymerization of an aldehyde group and a thioamide group by a catalyst-free one-pot method. Briefly, the yellow solid powder of DT-COF was synthesized in a reaction bottle at solvent circulation temperature for 6 hours by feeding 1.5:1 of DTO and TFB (Fig. 1 a), the yield was as high as 78.6% compared to other COF materials reported in the literature [ 28 , 29 ]. The DT-COF structure was confirmed using powder X-ray diffraction (PXRD), which indicated that the DT-COF had a crystalline structure (Fig. 1 b). The PXRD pattern simulated using Materials Studio (2020 version) suggested that the structure of DT-COF was consistent with the AB stacking mode. Pawley refinement showed negligible differences between the simulated and experimental PXRD patterns. DT-COF was assigned to the space group P1 (AB) with parameters of a = 17.474 Å, b = 17.471 Å, c = 7.707 Å, α = 103.31°, β = 104.71°, γ = 119.97°, and residuals Rwp = 3.45% and Rp = 2.73%. The c-axis parameters for the AB stacking mode are consistent with the 3.9 Å layer spacing date (Fig. 1 c). Based on theoretical inferences, compared with the traditional solvothermal synthesis method, which requires high temperature, high pressure, and harsh conditions, this facile strategy allows COF synthesis to be more sustainable. Furthermore, the multiphoton activity of the DT-COF was preliminarily evaluated using density functional theory calculations. The energy difference between the highest occupied molecular orbital and the lowest unoccupied molecular orbital of the DT-COF structural molecule was calculated to be 3.40 eV, with a dipole moment of 25.9 (Fig. 1 d). However, the energy gap and dipole moment of the DT-COF unit molecule were 3.95 eV and 4.1, respectively, indicating a significant reduction in electron delocalization behavior of the molecular unit compared to the closed ring structure of COF. Moreover, the band structure and density of states of DT-COF were supported (Fig. 1 e and 1 f) with an E g of 1.01 eV, confirming the good electron delocalization ability of DT-COF. Moreover, the hyperpolarizability β of DT-COF and unitary molecules were 24.047 × 10 3 esu and 2.295 × 10 3 esu, respectively, which were calculated by gaussian 09 with the basis set of PBE/6-311 + + G(d,p). This result suggested that DT-COF provided a fundamental platform for nonlinear optical activity 30,31 . In addition, thiazolothiazole-based DT-COF could avoid π-π stacking interactions and achieve mitochondrial-targeting ability for near-infrared light-induced fluorescence imaging as well as PDT. Characterization of DT-COF Furthermore, the morphology of the DT-COF was characterized using transmission electron microscopy (TEM). As shown in Fig. 2 a and 2 b, DT-COF was in the form of a bar shape, and its lattice structure could be clearly observed using high-resolution TEM, further confirming the crystallinity of the DT-COF. The average hydrated particle size of the DT-COF measured by dynamic light scattering (DLS) was approximately 170 nm and showed a normal distribution curve, indicating good dispersion and uniformity (Fig. 2 c). Meanwhile, the zeta potential was − 16.3 ± 1.1 mV, which would enhance the stability [ 32 , 33 ]. The formation of DT-COF was also detected by scanning electron microscopy and surface scan mapping of the distribution of different elements (Fig. 2 d and Fig. S1 ), which showed that the structure was consistent with TEM and contained C, N, and S. The thickness of the DT-COF was also measured to be approximately 15 nm using atomic force microscopy (Fig. S2). Moreover, the structure of the DT-COF was verified using Fourier Transform Infrared Spectroscopy (FTIR). As shown in Fig. 2 e, the stretching vibration peaks of -C-S-C- and -C = N- were newly generated in DT-COF, and the stretching vibration peaks of -C = O-, -CH, -SH, and -NH 2 belonging to TFB and DTO were significantly reduced or even disappeared, indicating that DT-COF was successfully synthesized and that its structure was correct [ 34 , 35 ]. N 2 adsorption and desorption experiments were carried out to investigate the surface area and pore size of the DT-COF. As shown in Fig. 2 f and 2 g, the surface area of DT-COF was calculated to be 141.1 m 2 g − 1 , and the narrow pore width distribution was centered at 1.4 nm, which is in agreement with the theoretical value. These results validate the structural accuracy of the DT-COF. As shown in Fig. 2 h and 2 i, the X-ray photoelectron spectroscopy (XPS) was performed on DT-COF to confirm its structure. The XPS survey spectra clearly indicate that DT-COF was composed of C, N, S, and O. The high-resolution S 2p spectrum of DT-COF was deconvoluted into two peaks at ~ 163.8 and ~ 165.0 eV, which were assigned to S 2p 3/2 and S 2p 1/2 , respectively. This result suggests that S reacts to form a thiazole ring, which is in agreement with other thiazole ring results reported in the literature 36 . In addition, the high-resolution C 1s and N 1s spectra indicated the presence of C-S-C, C-N-C, C = N, N = C, N-C, and N = C-S, confirming the successful formation of thiazole rings (Fig. S3). In addition, the solid-state nuclear magnetic resonance spectra (Fig. 2 j) confirmed the generation of sp2 carbons (~ 150 and 168 ppm). Carbonyl peaks attributed to unreacted aldehyde and thioamide groups were also observed at 192.1 and 35.4 ppm. The XRD patterns of DT-COF did not change significantly after 24 h of co-incubation in different environments (aqueous solution with pH 4, DMEM, and hypoxic conditions), indicating that DT-COF had good stability for subsequent application studies (Fig. S4). Multiphoton activities of DT-COF Subsequently, the photophysical and chemical properties of DT-COF were investigated. The absorption peaks of DT-COF were observed at approximately 360 nm for both the solid powder and aqueous solution (Fig. 3 a). Given the D-A configuration and high dipole moment of DT-COF, its nonlinear optical (NOL) performance was further evaluated. As shown in Fig. 3 b and 3 c, the DT-COF solution exhibited efficient two-photon action (2PA) and 3PA cross-sectional features (600 mW, 80 MHz, and 140 fs). The 2PA cross-section was relatively high at 720 nm and 800 nm, reaching approximately 411 and 386 GM, respectively. The 3PA cross-section reached a maximum of 2.98 × 10 − 80 cm 6 s 2 per photo 2 at 1100 nm and remained 2.24 × 10 − 80 cm 6 s 2 per photo 2 around 1200 nm. These results suggest that DT-COF exhibits good NOL properties and is a promising novel candidate for multiphoton bioimaging and PDT [ 37 ]. Hence, the multiphoton fluorescence and photodynamic capacity of the DT-COF were further evaluated. As shown in Fig. 3 d, the fluorescence emission peak of DT-COF activated by the 404 nm laser was at approximately 550 nm, and the fluorescence intensity gradually increased with the input power of the laser. Additionally, the fluorescence emission was gradually red-shifted with increasing DT-COF concentration (Fig. S5a), and the fluorescence intensity was significantly reduced at a concentration of up to 1000 µg mL − 1 due to aggregation. The results implied that DT-COF had good charge-transfer ability and π-π stacking effect. As shown in Fig. S5b, the logarithm of the fluorescence intensity exhibits a linear dependence on the logarithm of the laser input power (slope = 7.64). As shown in Fig. 3 e and 3 f, the DT-COF aqueous solution exhibited good up-conversion fluorescence emission and power density dependence when excited by 808 nm and 1250 nm lasers at different power densities (100–800 mW), demonstrating the two-photon fluorescence (2PF) and three-photon fluorescence (3PF) characteristics of DT-COF. The logarithmic values of both the fluorescence intensity and laser input power density showed a linear relationship with slopes of 3.82 and 2.3, respectively (Fig. S5c and S5d). Given the good charge transfer and multiphoton activity of DT-COF, its photodynamic ability was measured using electron spin resonance (ESR) and the 1 O 2 indicator 1,3-diphenylisobenzofuran (DPBF) after irradiation with a 200 mW cm − 2 808 nm laser. As shown in Fig. 3 g and 3 h, the signal peaks of 2,2,6,6-tetramethylpiperidine and 5,5-dimethyl-1-pyrroline-N-oxide used as the trapping agents for ESR analysis showed that DT-COF significantly produced ROS upon 808 nm laser excitation in a concentration-dependent manner, whereas no signal was produced under dark conditions. These results suggest that the DT-COF has great potential for use in two-photon laser-activated PDT. In addition, the absorption peak of DPBF also decreased with an increase in the 808 nm laser irradiation time (Fig. S6), while the absorption peaks of DPBF and DT-COF alone irradiated by the 808 nm laser were almost unchanged. The detailed change values that exhibited a significant difference are shown in Fig. 3 i. These results demonstrate the good multiphoton activity of DT-COF and the photodynamic activity of 808 nm (2PF) laser excitation. Preparation and characterization of TPZ@DT-COF The consumption of O 2 during PDT leads to further elevation of hypoxia in inadequately oxygenated tumors. TPZ, a hypoxia-activated chemotherapeutic drug, was loaded into the DT-COF for the synergistic therapeutic effects of PDT and chemotherapy. As shown in Fig. 4 a, the theoretical pore size of the DT-COF was 1.48 nm, and the tested size was approximately 1.4 nm, which is in good agreement with the size of TPZ (Fig. 4 b). Therefore, we envisaged that the DT-COF could effectively encapsulate TPZ. As shown in Fig. 4 c, TPZ-loaded DT-COF (TPZ@DT-COF) was obtained after 24 h of stirring and several washes with ethanol, which increased the hydrated particle size to approximately 206 nm and changed the zeta potential to 0.20 ± 0.03 mV. As shown in Fig. 4 d, TPZ@DT-COF in the DMF solution exhibited the characteristic UV-Vis absorption peaks of DT-COF and TPZ, indicating the successful loading of TPZ. Furthermore, the characteristic absorption peak at 460 nm for gradient concentration of TPZ (7.8–250 µg mL − 1 ) was used for quantitative loading analysis (Fig. 4 e), and the absorption intensity showed a linear positive correlation with concentration (Fig. S7). Significantly, based on the characteristic absorption peak of TPZ in the supernatant after centrifugation of the TPZ@DT-COF aqueous solution, the encapsulated content (EC) and encapsulated efficiency (EE) of TPZ were calculated from Eq. 1 as 43.3% ± 3.3% and 76.2% ± 4.4%, respectively (Fig. 4 f). The inset shows the color change from yellow to red in the nanosystem after TPZ loading. In addition, we selected doxorubicin (DOX) as the control drug loading model with the dimensions of length × width × height as 16.51 Å × 12.56 Å × 9.78 Å, which was larger than the pore size of DT-COF. As shown in Fig. S8, the EC and EE of DOX were significantly reduced to 23.5% ± 1.1% and 33.7% ± 3.7%, respectively. The solution shown in the inset is lighter in color. These results indicated that DT-COF and TPZ possess synergistic sizes and anti-tumor mechanisms to achieve good drug delivery and tumor therapeutic effects. Furthermore, the release behavior of TPZ under different pH conditions was investigated. As shown in Fig. 4 g, the cumulative release of TPZ was pH-dependent. The TPZ release rate was 16.98 ± 1.99% after 96 h of incubation in healthy physiological pH condition (pH = 7.4), whereas the release rate was significantly elevated in the slightly acidic environment of tumor tissues, reaching 70.39 ± 3.28%, 85.35 ± 3.2%, and 89.09 ± 4.82% at pH 6.5, 5.0, and 4.0, respectively. To better understand the pH-responsive release behavior of TPZ@DT-COF, the Ritger–Peppas equation was used to analyze release kinetics. The fitting parameters for the kinetic constant (k) and release exponent (n) are presented in Table S1 . The n values for TPZ release were all < 0.45, suggesting that the release mechanism for TPZ was Fickian diffusion. After incubation at pH 7.4, the morphology of the samples was observed using TEM (Fig. 4 h), which showed that DT-COF could responsively release the drug in the slightly acidic environment of the tumor with little change in morphology. In addition, the stability of TPZ@DT-COF was characterized by variations in its particle size in different environments. As depicted in Fig. 4 i, TPZ@DT-COF was co-incubated in water, PBS, and DMEM containing 10% FBS for 7 days to assess its stability for transportation, storage, and physiological applications. The results showed that the particle size remained stable throughout the 7 days. Drug delivery and multiphoton performance evaluation of TPZ@DT-COF in vitro Next, we evaluated the drug delivery, multiphoton activity-mediated fluorescence imaging, and ROS generation effects of DT-COF in vitro. As shown in Fig. 5 a, the fluorescence intensities of the 1PF, 2PF, and 3PF channels increased gradually with increasing incubation time, with cyan, green, and red fluorescence indicating 1PF, 2PF, and 3PF, respectively. The cell positions of the fluorescence channels and the bright field (BF) channel completely overlapped with each other, suggesting effective cellular internalization and good multiphoton fluorescence imaging performance of the DT-COF. Semi-quantitative data on fluorescence intensity also showed good time dependence and was greatest at 2PF (Fig. 5 b). Furthermore, the multiphoton fluorescence imaging depth of DT-COF was explored using laser confocal layer-by-layer scanning. As shown in Fig. 5 c, the 3PF group exhibited deeper fluorescence emission with the strongest fluorescence at a depth of 70 µm, while the strongest fluorescence at a depth of 50 µm in the 2PF group, indicating that the three-photon laser penetrated to a deeper depth to excite DT-COF. These results demonstrate that DT-COF has the potential for multiphoton fluorescence imaging in deep tissues and can be used as an imaging contrast agent. Furthermore, the targeted delivery effects of the TPZ@DT-COF nanosystem were evaluated based on the mitochondrial-targeting ability of thiazoles reported in our previous work [ 38 ]. As shown in Fig. 5 d, the bright violet fluorescence attributed to DT-COF was excited by a two-photon laser and overlapped with the red fluorescence of Mito-tracker. In contrast, the green fluorescence of the DT-COF was distributed in the nuclei of cells localized by DAPI, which may have resulted from the microenvironment-driven release of TPZ in tumor cells. The results of fluorescence co-localization analyses showed a high degree of overlap between DT-COF and mitochondria, as well as between TPZ and the nucleus (Fig. 5 e). This delivery effect is in good agreement with the corresponding therapeutic pathways of TPZ and DT-COF, resulting in precise delivery and effective therapeutic enhancement. To rigorously demonstrate the mitochondria-targeting ability of the DT-COF, we utilized the mitochondria-targeting molecule triphenylphosphine to block the mitochondria. As shown in Fig. S9, the fluorescence of DT-COF incompletely overlapped with that of the mitochondrial green fluorescent probe (MitoTracker). The enrichment of mitochondria was significantly reduced, and a shift in the channel position was clearly detected by co-localization analysis, indicating that DT-COF exhibited a target-dependent property. The cellular uptake of TPZ@DT-COF after different times (1–24 h) of co-culturing with 4T1 cells was quantified using flow cytometry. As shown in Fig. 5 f, the cellular uptake increased gradually with increasing incubation time, reaching 98.8% and 34.7% at 24 h, as determined by fluorescence quantification of DT-COF and TPZ, respectively. Subsequently, the photodynamic ability of DT-COF was evaluated by detecting the generation of 1 O 2 using DCFH-DA after irradiating 4T1 cells with 200 mW cm − 2 808 nm laser for 5 min. As shown in Fig. 5 g, the control groups without added DT-COF showed no significant green fluorescence under either dark or light conditions in a normoxic environment, and no significant green fluorescence was observed in the DT-COF group alone, whereas the DT-COF group produced obvious green fluorescence after 808 nm laser irradiation. This phenomenon indicates that DT-COF can be activated by the two-photon 808 nm laser to efficiently produce 1 O 2 , rather than by DT-COF or the 808 nm laser irradiation itself. We also compared the generation of 1 O 2 under the same conditions in a hypoxic environment (Fig. 5 h), which was consistent with the conclusions drawn in a normoxic environment. However, the intensity and cell volume of the green fluorescence produced by DT-COF after 808 nm laser irradiation under hypoxic conditions were significantly reduced, suggesting that hypoxia limited the photodynamic performance. Antitumor effects of TPZ@DT-COF in vitro The cytotoxicity of DT-COF against tumor cell of 4T1 and normal cells (L929 and MCF-10A) was assessed using the methyl thiazolyl tetrazolium (MTT) assay. As depicted in Fig. 6 a and Fig. S10a, the cell viability after 24 h incubation with DT-COF was still over 95% even at the maximum concentration of 500 µg mL − 1 , suggesting good biocompatibility of DT-COF for further biological applications. Moreover, the cell viability was above 90%, even after 48 and 72 h of co-incubation with different concentrations of DT-COF (Fig. S10b). Oxygen deprivation limited the photodynamic performance of DT-COF, whereas TPZ@DT-COF offered excellent delivery and therapeutic potential. The therapeutic effects of the chemotherapy and photodynamic-chemotherapy combinations of TPZ@DT-COF in normoxic and hypoxic environments were evaluated. As shown in Fig. 6 b and 6 c, the decrease in cell viability was concentration-dependent, and the cell killing efficiency was greatest at a concentration of 50 µg mL − 1 TPZ@DT-COF (21.6 µg mL − 1 for TPZ). Moreover, TPZ@DT-COF exhibited good chemotherapeutic efficacy in rapidly proliferating 4T1 cells and enhanced chemotherapeutic efficacy under hypoxic conditions. The cell viability decreased from about 50% to 40% at a final concentration of 50 µg mL − 1 , which demonstrated the advantages of the hypoxia-activated drug TPZ in hypoxic tumor therapy. Two-photon PDT with 808 nm laser excitation effectively improved the therapeutic efficiency of TPZ@DT-COF in 4T1 cells, and the enhancement effect was more significant in the normoxic environment. These results suggest that the TPZ@DT-COF nanosystem exhibits good synergy between two-photon PDT and hypoxia-activated chemotherapy. The killing effect of TPZ@DT-COF on 4T1 cells was directly observed in vitro by double staining with calcein AM (CA)/propidium iodide (PI). As shown in Fig. 6 d, live and dead cells were stained with green and red fluorescence, respectively. Almost all 4T1 cells in the untreated control and 808 nm laser or DT-COF treatment groups alone under normoxia and hypoxia were stained green, and the red fluorescence of PI was negligible, indicating that neither the 808 nm laser nor the DT-COF killed 4T1 cells. In contrast, DT-COF plus 808 nm laser irradiation (DT-COF + L) resulted in a significant increase in the number of red fluorescent cells in both normoxic environments, caused by the two-photon photodynamic activity of DT-COF. The TPZ@DT-COF + L group exhibited a significant killing effect in normoxic and hypoxic environments, with a marked increase in red fluorescent cells, indicating a synergistic therapeutic effect. To quantitatively evaluate the apoptotic effect of different treatment groups (1–9) on tumor cells, the apoptotic reagent annexin-V/PI was used and detected by flow cytometry. As shown in Fig. 6 e, the percentages of live and dead cells in each group exhibited a trend consistent with the above results. These results demonstrated that DT-COF exhibited good biocompatibility and photodynamic capacity and could effectively synergize with TPZ to enhance the 4T1 treatment efficiency in vitro. Evaluation of the antitumor efficacy of TPZ@DT-COF in vivo Having proven the good delivery effects and efficient photodynamic chemotherapy outcomes of TPZ@DT-COF in vitro, we evaluated its anti-tumor effects in vivo. Primarily, TPZ@DT-COF exhibited good stability and biocompatibility for biological applications because the nanosystem did not cause hemolysis (Fig. S10c). To further investigate the distribution and metabolism in tumor tissues and verify the performance and potential in practical applications, we utilized the 1PF properties of DT-COF in an in vivo imaging system (IVIS) to monitor its distribution and metabolism in mice (Fig. 7 a and 7 b). Within 8 hours of tail vein injection of DT-COF, the fluorescence intensity at the tumor reached a maximum, and then the fluorescence intensity gradually decreased, and even could not be observed due to the limited fluorescence penetration distance. The visual analysis of distribution and metabolism through isolated major organs (Heart, Liver, Spleen, Lung and Kidney) and tumor tissues at the end of the monitoring procedure showed that DT-COF was metabolized mainly by the liver and kidney. Quantitative fluorescence analysis also showed the same distribution and metabolic trends (Fig. S11a and S11b). In addition, when the tumor volumes reached 80–100 mm 3 , a single intratumoral injection was administered to subcutaneous 4T1 tumor-bearing mice to evaluate the oncological treatment and multiphoton imaging effects of TPZ@DT-COF. As shown in Fig. 7 c, 7 d and 7 f, neither PBS + L nor DT-COF treatment showed any antitumor efficacy, the tumour volume growth curves showed rapid increases, reaching relative tumor volumes of 10.97 ± 2.00 and 10.38 ± 2.32 at 15 days, respectively. While DT-COF + L, TPZ@DT-COF and TPZ@DT-COF + L groups led to a rapid and prominent tumor inhibition, distinguished markedly from other treatments. Besides, TPZ@DT-COF + L group-mediated photodynamic-chemotherapy combination strategy effectively enhanced tumor volume growth inhibition compared to the chemotherapy or PDT alone groups (TPZ@DT-COF or DT-COF + L). Tumor weight and morphology of isolated tumors also provided a clear indication of the tumor suppression effects consisted with the above results. The weight monitoring during the treatment period also showed no abnormal changes, indicating that the nano-formulation possessed good biocompatibility (Fig. S12). The survival rate of mice during the 35-day treatment period also indicated that the synergistic treatment effect of the TPZ@DT-COF + L group significantly prolonged the survival period (Fig. 7 e). The penetration depth of DT-COF fluorescence-induced by 1PF, 2PF and 3PF laser exposure was assessed through tumor tissues. The mice were executed once the tumor volume exceeded 1000 mm 3 . At the same depth of 80 µm, almost no fluorescence emission from group 1PF was noted, the 2PF and 3PF groups had significant fluorescence emission, and the 3PF group possessed the largest fluorescence emission area (Fig. 7 g), indicating that DT-COF-mediated multiphoton fluorescence exhibited deeper tissue imaging capability. In addition, the penetration depths of 1PF, 2PF and 3PF of DT-COF were also examined by layer-by-layer scanning. As shown in Fig. S13, the strongest intensity depth of 1PF was at 30 µm, the fluorescence intensity of 2PF was higher at 40 and 60 µm, and 3PF reached 100 and 120 µm, which suggested that the development of MPPSs exhibited the potential to break the limitation of the light penetration depth. Then, the therapeutic effects were studied by the histopathological analysis of tumor tissues. As depicted in Fig. 7 h, Hematoxylin and eosin (H&E) staining showed intact tumor tissue without significant abnormalities and lesions in the PBS and DT-COF groups, whereas the other three groups showed extensive tissue necrosis. It was clear from the tightness of the tumor tissues in the DT-COF + L, TPZ@DT-COF and TPZ@DT-COF + L groups became loose and necrosis. Notably, H&E of major organs in all groups of mice showed no abnormalities, and the biomarkers such as ALT, AST, CRE and BUN were detected and all were within the normal range and showed no abnormalities (Fig. S14 and Fig. S15a-S15d), indicating the long-term safety of the treatment in mice. Meanwhile, the immunofluorescent staining of TUNEL revealed results consistent with those of H&E staining (Fig. 7 i). The apoptotic and necrotic cells in the TPZ@DT-COF + L group were stained with the greatest intensity and area of green fluorescence, reflecting the superiority of combined chemo-photodynamic therapy for tumor treatment. These results fully demonstrated that the nanosystem exhibited good drug delivery and theranostic performance and showed great potential for anti-tumor applications. In addition, we also explored the in vivo distribution of DT-COF using its single-photon fluorescence properties. Firstly, all indicators in the whole blood analysis were within the normal range compared with the control group (Fig. S15e). These results indicated that BT-nHOFs exhibited good blood safety and did not destroy blood cells and cause obvious blood abnormalities. Acute toxicity experiments at high doses (25 mg kg − 1 ) also showed that DT-COF and TPZ@DT-COF did not cause significant damage to major organs in mice (Fig. S16), suggesting that DT-COF exhibited potential for clinical application. Conclusions In this study, we efficiently constructed DT-COF with strong multiphoton activity, utilizing thiazolothiazole as the nonlinear optical unit, whose framework structure extended the π-electron system, attenuated π-π stacking, and enhanced charge transfer. The DT-COF not only exhibited multiphoton fluorescence imaging and PDT but also specifically and efficiently encapsulated TPZ, a hypoxia-activated chemotherapeutic drug. The pore size of DT-COF matched the size of TPZ, as well as the cascade of anti-tumor mechanisms between DT-COF and TPZ. In addition, the nanosystem exhibited excellent subcellular organelle-targeted delivery performance with precise anti-tumor effects. The anti-tumor results in vitro and in vivo showed deep penetration depth (80 µm) for multiphoton fluorescence imaging and enhanced photodynamic-chemotherapeutic therapy. This work presents a promising design guideline for drug delivery and combined therapy and sheds new light on the theranostics of deep-seated tumors. Histopathological analysis Hematoxylin and eosin (H&E) and immunofluorescent staining of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) were performed following the manufacturer's instructions to evaluate histological therapy of the tumor, livers, kidneys, spleens, lungs, and hearts of mice. The prepared slices of tumor, livers, kidneys, spleens, lungs, and hearts were visualized under an inverted fluorescence microscope. Statistical analysis and reproducibility The experimental data are presented as means ± standard deviation (means ± SD). The statistical analyses were conducted by two-tailed Student's t-test or one-way analysis of variance. Survival curves were analyzed by using the log-rank (Mantel-Cox) test. Statistical significance was defined as * P < 0.05, ** P < 0.01, and *** P < 0.001, no significance (ns). Each experiment in this study was designed to use the minimum number of animals required to obtain informative results. Tumor-bearing mice were randomized before treatment. No data were excluded from the analysis. Experimental Section Materials 1,3,5-triformylbenzene (TFB) and dithioacetamide (DTO) were obtained from Tengqian Biotechnology Co., Ltd. (Shanghai, China). 1,3-diphenylisobenzofuran (DPBF), 2,2,6,6-tetramethylpiperidine (TEMP) and N,N-dimethylformamide (DMF) were purchased from Energy Chemicals (Shanghai, China) without other purification. Other chemicals were obtained from Sinopharm Chemical Reagent (SCR) and used as received. tirapazamine (TPZ) was synthesized according to previous literature 39 . 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 2′,7′-Dichlorofluorescein diacetate (DCFH-DA), 4′,6-diamidino-2-phenyl-indole (DAPI), calcein-AM, doxorubicin hydrochloride and propidium iodide (PI) cell apoptosis kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Gibco Life Technologies (USA). All cell lines used in this study were obtained from the Chinese Type Culture Collection (Wuhan University). The animal experiments were completed at Huazhong Agricultural University. All animal experiments conformed to the guidelines of the Chinese Regulations for the Administration of Affairs Concerning Experimental Animals and were approved by the Institutional Animal Care and Use Committee of Huazhong Agricultural University, China (HZAUMO-2023–0311). Synthesis of DT-COF To synthesize DT-COF nanowires, TFB (0.4 mmol, 1.0 equiv.) and DTO (0.6 mmol, 1.5 equiv.) were added into 10 mL of DMF after 1 h of nitrogen blowdown. The reaction mixture was stirred vigorously at 157°C for 6 h. After cooling to room temperature, the reaction mixture was then filtered. The yellow precipitate was collected and washed 3 times with DMF, then dispersed in a phosphate buffer solution (PBS) for further use. Preparation of TPZ@DT-COF To encapsulate the TPZ into the DT-COF, 5 mg of DT-COF dispersed in PBS was co-mixed with 5 mg of TPZ in PBS solution co-solubilized with DMSO and then stirred for 24 h at room temperature. The mixed solution was washed by centrifugation until the supernatant was free of TPZ. The loading of doxorubicin hydrochloride was done by stirring in PBS solution and in the same steps as described above. Characterization of DT-COF and TPZ@DT-COF Morphology and elemental distribution of DT-COF were assessed by scanning electron microscope (SEM; Nano Surface Division, Bruker, USA) and transmission electron microscopy (TEM; Hitachi HT7700, Japan) at 200 kV. Particle size and zeta potential of DT-COF and TPZ@DT-COF were analyzed with Zetasizer Nano-S90 (Malvern Instruments, UK). The photophysical and chemical properties of DT-COF were detected by UV-Vis and UV-Vis-NIF spectrometers (UV-2600 and 3700 DUV, Shimadzu). The loading of TPZ and Doxorubicin hydrochloride was visualized by applying a UV-vis detector (UV-2600, Shimadzu). To verify the macropores and mesopores in DT-COF, Brunauer-Emmett-Teller (BET) surface area analysis was measured with a Micromeritics ASAP 2420 analyzer. Powder X-ray diffraction (PXRD) was performed using a Rigaku MiniFlex600 Focus Powder Diffractometer with Cu Kα line focused radiation. Confocal laser scanning microscope images were acquired with a Nikon AX and an Olympus FVMPE-RS equipped with the femtosecond laser. Density functional theory calculations Geometric optimization of the position of the unit of DT-COF and the closed DT-COF, as well as calculation of HOMO and LUMO were using the Dmol 3 module in the Materials Studio software. All calculations were performed using the generalized gradient approximation (GGA) with the Perdew–Burke–Ernzerh (PBE) of functional in conjunction with the double number plus polarization function (DNP). In addition, the dipole moment was calculated by Chem3D (PerkinElmer, version 19.0.0.22). Encapsulated content and encapsulated efficiency To determine the encapsulated content (EC) and encapsulated efficiency (EE) of TPZ and doxorubicin hydrochloride, the standard curves of TPZ and doxorubicin hydrochloride were constructed based on their UV-vis absorption. EC and EE were respectively calculated as follows: EC% = The weight of TPZ or doxorubicin hydrochloride in DT-COF∕The total weight of nanosystem, EE% = The weight of loaded TPZ or doxorubicin hydrochloride in DT-COF∕The feed weight of TPZ or doxorubicin hydrochloride, Eq. 1. 2PF spectroscopy and two-photon absorption (2PA) cross-section 2PF spectra were obtained by a femtosecond laser pulse and a Ti: sapphire system (650–820 nm, 80 MHz, 140 fs) as the light source. The concentration of DT-COF was 100 µg mL − 1 . The reference sample is Fluorescein with a concentration of 1.0 × 10 − 3 M. 2PA cross-section was calculated by using the following equation: $$\:\sigma\:={\sigma\:}_{\text{r}\text{e}\text{f}}\frac{{{\varnothing}}_{\text{r}\text{e}\text{f}}{\text{c}}_{ref}{\text{n}}_{ref}F}{{\varnothing}\text{c}\text{n}{F}_{\text{r}\text{e}\text{f}}}$$ Here, ref stands for reference sample, δ is the two-photon absorption cross-section, Ф is fluorescence quantum yield, c is the concentration of the sample, n is the refractive index, and F is two-photon fluorescence integral area. The value of the two-photon absorption cross-section of the reference sample is derived from the literature [ 40 ]. 3PF spectroscopy and 3PA cross-section 3PF spectra were obtained by Coherent Astrella + TOPAS Prime (1050–1300 nm, 1 kHz, 120 fs) as the light source. The reference sample is rhodamine 6G (1.0 × 10 − 3 M). The concentration of DT-COF was 100 µg mL − 1 . The three-photon absorption cross-section was calculated by using the following equation: $$\:\sigma\:=\frac{{\gamma\:}}{{N}_{A}{d}_{0}{10}^{-3}}\times\:{\left(\frac{hc}{\lambda\:}\right)}^{2}$$ Here, γ is the three-photon absorption coefficient, λ is the wavelength of the incident light, N A is the Avogadro constant, and d 0 is the concentration of the sample (0.1 mg mL − 1 ) [ 41 ]. Evaluation of singlet oxygen generation DPBF was used as an indicator to study the singlet oxygen ( 1 O 2 ) generation measuring the attenuation of DPBF absorbance at 425 nm by UV-vis spectrophotometer. 8 µL DMSO solution of DPBF (2.7 mg mL-1) was mixed with an aqueous solution of DT-COF, (1 mL, 100 µg mL − 1 ). Under 808 nm laser irradiation, the absorbance changes of DPBF at 280 nm were recorded at regular time points. Next, to detect the generation of intracellular 1 O 2 , DCFH-DA was used as the indicator with inverted fluorescence microscope imaging. Briefly, 4T1 cells were seeded in 6-well plates for 24 h and then incubated with the medium containing PBS, PBS + Light, DT-COF and DT-COF + Light (in hypoxia or normoxia conditions) for another 4 h. For hypoxia conditions, the Oxygen content was regulated at 1% with N 2 . Subsequently, the cells were treated with 10 µM DCFH-DA for 30 min before observation. Cell culture and cellular uptake The mouse fibroblast cells (L929) and mouse breast cancer cells (4T1) were cultured in DMEM medium with the addition of 10% FBS, and 1% penicillin/streptomycin in an atmosphere containing 5% CO 2 at 37°C to evaluate the biocompatibility and the therapeutic effects of DT-COF and TPZ@DT-COF. To evaluate the cellular uptake, 4T1 cells were incubated with DT-COF or TPZ@DT-COF at 37°C at different times. After washing with PBS, cells were collected and analyzed by CLSM and flow cytometry. For multiphoton fluorescence imaging, 100 µg mL − 1 DT-COF was co-cultured with 4T1 cells for different times and the imaging effects of 1PF, 2PF and 3PF in the 500–600 nm range were monitored using 405 nm, 808 nm and 1250 nm excitation light at 0.2 W cm − 2 , respectively. In addition, the mitochondrial targeting and nuclear delivery capabilities of TPZ@DT-COF were detected by fluorescent probe co-localization analysis. In vitro cellular cytotoxicity 4T1 cell line was cultured at a density of 10 4 cells per well in 96-well plates under normoxic or hypoxic conditions, respectively, for 24 h. A fresh culture medium containing serial concentrations of TPZ@DT-COF was added to each well to incubate with cells for another 24 h. The cells in the PDT group were exposed to an 808 nm laser at the power density of 200 mW cm − 2 for 5 min. The cells in the chemotherapy group without light exposure remained in the dark. After being further incubated for another 24 h, and then the fresh culture medium containing 5 mg mL − 1 MTT was added to incubate for another 4 h to generate formazan crystals. The purple formazan crystals in each well were dissolved by 150 µL DMSO and the absorbance was detected on a microplate reader at a wavelength of 490 nm. In addition, cell apoptosis was further visualized by an inverted fluorescence microscope with Calcein-AM/PI double staining. Generally, the cells were seeded in a 24-well plate and treated in the same way mentioned before, the concentration of DT-COF and TPZ@DT-COF was 15 µg mL − 1 and 25 µg mL − 1 (TPZ content was 10.8 µg mL − 1 ). After being washed with PBS twice, the cells were stained with Calcein-AM (green fluorescence, live cells) and PI (red fluorescence, dead cells) for 15 min. Finally, the culture medium was replaced with fresh PBS before microscope observation. Hemolysis assay As for the hemolysis assay, whole blood of mice was collected and washed using PBS to obtain red blood cells (RBCs), which were further mixed with water, PBS, 125, 250, and 500 µg mL − 1 DT-COF. All samples were incubated at 37°C for 6 hours, and the supernatants were centrifuged to test for UV absorption at 450 nm to assess the rate of hemolysis. All samples were incubated at 37°C for 6 h and centrifuged to obtain supernatant for hemolysis detection. Tumor model The right lower limb of mice was subcutaneously inoculated with 1×10 6 4T1 cells to establish a hypoxic tumor model. The tumor volume was calculated as V = d 2 × D∕2 (d and D: the shortest point and longest point of the tumor, respectively). In vivo multiphoton fluorescence imaging study after intratumoral injection When the tumors reached about 80–100 mm 3 , DT-COF was administrated intratumorally at an equivalent dose (10 mg kg − 1 ). Then, solid tumors were then dissected and subjected to multiphoton fluorescence imaging of deep tissues. In vivo antitumor effect after intratumoral injection Mice were randomly divided into 4 groups (n = 5) with treatments of PBS + L, DT-COF, DT-COF + L, TPZ@DT-COF, and TPZ@DT-COF + L (10 mg kg − 1 based on DT-COF). Two hours after intratumoral administration, the treatment groups used for laser activation were irradiated continuously for 5 minutes using a 200 mW cm − 2 808 nm laser, and the tumor volumes and body weights were recorded every two days. At the end day of the treatment, mice were euthanized, and the tumors in different groups were collected for final observation. The histological study was performed with hematoxylin/eosin (H&E) staining and immunofluorescent staining of TUNEL on tumors. Declarations Supporting Information The online version contains supplementary material available at https://doi.org/10.1186/****** . Ethics approval and consent to participate All animal experiments conformed to the guidelines of the Chinese Regulations for the Administration of Affairs Concerning Experimental Animals and were approved by the Institutional Animal Care and Use Committee of Huazhong Agricultural University, China (HZAUMO-2023-0311). Competing interests The authors declare no competing interests. Author Contribution S.L. and L.H. conceived the project idea, analyzed the data, and wrote the paper. S.L., Y.L. and G.D. prepared and characterized the nanosystem. L.H. and Y.W. performed the cell and animal experiments. All authors read and edited the manuscript. Acknowledgments This work was financially supported by the following funding: Hubei Provincial Natural Science Foundation of China (Grant No. 2025AFB070), State Key Laboratory of New Textile Materials and Advanced Processing (FZ20230024). Data Availability All data are available in the main text, supporting information, and are also on request from the corresponding author. References Hopper C. Photodynamic therapy: a clinical reality in the treatment of cancer. Lancet Oncol. 2000;1:12–219. Karges J. Clinical development of metal complexes as photosensitizers for photodynamic therapy of cancer. Angew Chem Int Edit. 2022;61:e202112236. Lv L, Fan B, Ji X, Liu Y, Chen T, Li Y, Gao X, Chen P, Tang B, Chen G. From the clinical perspective of photodynamic therapy and photothermal therapy: structure-activity-practice. Coordin Chem Rev. 2024;507:215733. Pham TC, Nguyen VN, Choi Y, Lee S, Yoon J. Recent strategies to develop innovative photosensitizers for enhanced photodynamic therapy. Chem Rev. 2021;121:13454–619. Li M, Xu Y, Peng X, Kim JS. From low to no O 2 -dependent hypoxia photodynamic therapy (hPDT): a new perspective. Acc Chem Res. 2022;55:3253–64. Li Y, Du L, Li F, Deng Z, Zeng S. Intelligent nanotransducer for deep-tumor hypoxia modulation and enhanced dual-photosensitizer photodynamic therapy. ACS Appl Mater Interfaces. 2022;14:14944–52. Zhao X, Liu J, Fan J, Chao H, Peng X. Recent progress in photosensitizers for overcoming the challenges of photodynamic therapy: from molecular design to application. Chem Soc Rev. 2021;50:4185–219. Li Z, Lu J, Li X. Recent progress in thermally activated delayed fluorescence photosensitizers for photodynamic therapy. Chem Eur J. 2024;30:e202401001. Chen K, He P, Wang Z, Tang BZ. A feasible strategy of fabricating type I photosensitizer for photodynamic therapy in cancer cells and pathogens. ACS Nano. 2021;15:7735–43. Wang J, Gong Q, Jiao L, Hao E. Research advances in BODIPY-assembled supramolecular photosensitizers for photodynamic therapy. Coordin Chem Rev. 2023;496:215367. Tavakkoli Yaraki M, Liu B, Tan YN. Emerging strategies in enhancing singlet oxygen generation of nano-photosensitizers toward advanced phototherapy. Nano-micro lett. 2022;14:123. Chen J, Zhou Q, Cao W. Multifunctional porphyrin-based sonosensitizers for sonodynamic therapy. Adv Funct Mater. 2024;34:2405844. Juvekar V, Lee DJ, Park TG, Samanta R, Kasar P, Kim C, Rotermund F, Kim HM. Two-photon excitation photosensitizers for photodynamic therapy: From small-molecules to nano-complex systems. Coordin Chem Rev. 2024;506:215711. Cao H, Fang B, Liu J, Shen Y, Shen J, Xiang P, Zhou Q, De Souza SC, Li D, Tian Y, Luo L, Zhang Z, Tian X. Photodynamic therapy: photodynamic therapy directed by three-photon active rigid plane organic photosensitizer. Adv Healthc Mater. 2021;10:2001489. Xu Z, Jiang Y, Shen Y, Tang L, Hu Z, Lin G, Law WC, Ma M, Dong B, Yong KT, Xu G. A biocompatible photosensitizer with a high intersystem crossing efficiency for precise two-photon photodynamic therapy. Mater Horiz. 2022;9:1283–92. Tian S, He J, Lyu D, Li S, Xu QH. Aggregation enhanced photoactivity of photosensitizer conjugated metal nanoparticles for multimodal imaging and synergistic phototherapy below skin tolerance threshold. Nano Today. 2022;45:101534. Deng X, Zhao R, Tang Y, Yi M, Wang D, Lin W, Wang G. FeS 2 @COF based nanocarrier for photothermal-enhanced chemodynamic/thermodynamic tumor therapy and immunotherapy via reprograming tumor-associated macrophages. J Nanobiotechnol. 2024;22:711. Ma J, Shu T, Sun Y, Zhou X, Ren C, Su L, Zhang X. Luminescent covalent organic frameworks for biosensing and bioimaging applications. Small. 2022;18:2103516. Li S, Zhang K, Ma Z, Zhang W, Song Z, Wang W, Han H. Biomimetic nanoplatelets to target delivery hirudin for site-specific photothermal/photodynamic thrombolysis and preventing venous thrombus formation. Small. 2022;18:2203184. Seki S, Paitandi RP, Choi W, Ghosh S, Tanaka T. Electron transport over 2D molecular materials and assemblies. Acc Chem Res. 2024;57:2665–77. Zhou Y, Jing S, Liu S, Shen X, Cai L, Zhu C, Zhao Y, Pang M. Double-activation of mitochondrial permeability transition pore opening via calcium overload and reactive oxygen species for cancer therapy. J Nanobiotechnol. 2022;20:188. Zhang L, Wang S, Zhou Y, Wang C, Zhang XZ, Deng H. Covalent organic frameworks as favorable constructs for photodynamic therapy. Angew Chem Int Edit. 2019;58:14213–8. Zeng JY, Wang XS, Xie BR, Li MJ, Zhang XZ. Covalent organic framework for improving near-infrared light induced fluorescence imaging through two‐photon induction. Angew Chem Int Edit. 2020;59:10087–94. Schmidt K, Leclercq A, Zojer E, Lawson PV, Jang SH, Barlow S, Jen A, K-Y, Marder SR, Brédas JL. Order of magnitude effects of thiazole regioisomerism on the near-IR two‐photon cross‐sections of dipolar chromophores. Adv Funct Mater. 2008;18:794–801. Beverina L, Fu J, Leclercq A, Zojer E, Pacher P, Barlow S, Van Stryland EW, Hagan DJ, Brédas J-L, Marder SR. Two-photon absorption at telecommunications wavelengths in a dipolar chromophore with a pyrrole auxiliary donor and thiazole auxiliary acceptor. J Am Chem Soc. 2005;127:7282–3. Kang G, Nasiri Avanaki K, Mosquera MA, Burdick RK, Villabona-Monsalve JP, Goodson T III, Schatz GC. Efficient modeling of organic chromophores for entangled two-photon absorption. J Am Chem Soc. 2020;142:10446–58. Bramley SE, Dupplin V, Goberdhan DG, Meakins GD. The hantzsch thiazole synthesis under acidic conditions: change of regioselectivity. J Chem Soc. 1987; 639–43. Wu CJ, Li XY, Li TR, Shao MZ, Niu LJ, Lu XF, Kan JL, Gen Y, Dong YB. Natural sunlight photocatalytic synthesis of benzoxazole-bridged covalent organic framework for photocatalysis. J Am Chem Soc. 2022;144:18750–5. El-Kaderi HM, Hunt JR, Mendoza-Cortés JL, Côté AP, Taylor RE, O'Keeffe M, Yaghi OM. Designed synthesis of 3D covalent organic frameworks. Science. 2007;316:268–72. Li B, Lu X, Tian Y, Li D. Embedding multiphoton active units within metal-organic frameworks for turning on high-order multiphoton excited fluorescence for bioimaging. Angew Chem Int Edit. 2022;61:e202206755. Matito E, Solà M. The role of electronic delocalization in transition metal complexes from the electron localization function and the quantum theory of atoms in molecules viewpoints. Coordin Chem Rev. 2009;253:647–65. Zou Q, Abbas M, Zhao L, Li S, Shen G, Yan X. Biological photothermal nanodots based on self-assembly of peptide-porphyrin conjugates for antitumor therapy. J Am Chem Soc. 2017;139:1921–7. Hu L, Xu Y, Zhao Y, Mei Z, Xiong C, Xiao J, Zhang J, Tian J. Supramolecular nanovesicles with in-situ switchable photothermal/photodynamic effects for precisely controllable cancer phototherapy. Chem Eng J. 2023;476:146829. Anbazhagan R, Krishnamoorthi R, Kumaresan S, Thankachan D, Van DTT, Wang JS, Tsai HC. Benzobisthiazole-bridged white fluorescent emitting covalent organic framework for simultaneous mercury detection and removal. React Funct Polym. 2021;169:105083. Zhu X, Tian C, Jin T, Wang J, Mahurin SM, Mei W, Xiong Y, Hu J, Feng X, Liu H, Dai S. Thiazolothiazole-linked porous organic polymers. Chem Commun. 2014;50:15055–8. Hou Y, Zhou P, Liu F, Tong K, Lu Y, Li Z, Liang J, Tong M. Rigid covalent organic frameworks with thiazole linkage to boost oxygen activation for photocatalytic water purification. Nat Commun. 2024;15:7350. Feng Z, Zhu T, Wang L, Yuan T, Jiang Y, Tian X, Tian Y, Zhang Q. Coordination-regulated terpyridine-Mn(II) complexes for photodynamic therapy guided by multiphoton fluorescence/magnetic resonance imaging. Inorg Chem. 2022;61:12652–61. Hu L, Cao J, Lv Y. A mitochondria-targeted biocompatible photothermal nanoscale hydrogen-bonded organic framework for effective multimodal-imaging guided breast cancer therapy. Chem Eng J. 2024;492:152270. Shi X, Mandel SM, Platz MS. On the mechanism of reaction of radicals with tirapazamine. J Am Chem Soc. 2007;129:4542–50. Zhang Q, Lu X, Wang H, Tian X, Wang A, Zhou H, Wu J, Tian Y. A benzoic acid terpyridine-based cyclometalated iridium(III) complex as a two-photon fluorescence probe for imaging nuclear histidine. Chem Commun. 2018;54:3771–4. Feng Z, Li D, Zhang M, Shao T, Shen Y, Tian X, Zhang Q, Li S, Wu J, Tian Y. Enhanced three-photon activity triggered by the AIE behaviour of a novel terpyridine-based Zn(II) complex bearing a thiophene bridge. Chem Sci. 2019;10:7228–32. Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupportingInformation.doc SC1.png Scheme 1 Schematic illustration of the DT-COF with multiphoton activity for drug delivery and anti-tumor applications. (i) Preparation of TPZ@DT-COF composite nanomaterial and anti-tumor mechanisms. (ii) Illustration of the multiphoton activities of DT-COF mediating the fluorescence emission and photodynamic ability. Cite Share Download PDF Status: Published Journal Publication published 21 Feb, 2026 Read the published version in Journal of Nanobiotechnology → Version 1 posted Editorial decision: Revision requested 02 Dec, 2025 Reviews received at journal 26 Nov, 2025 Reviewers agreed at journal 25 Nov, 2025 Reviewers agreed at journal 22 Nov, 2025 Reviews received at journal 03 Nov, 2025 Reviewers agreed at journal 29 Oct, 2025 Reviewers invited by journal 27 Oct, 2025 Editor assigned by journal 19 Sep, 2025 Submission checks completed at journal 17 Sep, 2025 First submitted to journal 17 Sep, 2025 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7635511","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":539508524,"identity":"50ed1910-9e81-4e63-ae32-539ebcd47700","order_by":0,"name":"Shuting Li","email":"","orcid":"","institution":"Wuhan Business University","correspondingAuthor":false,"prefix":"","firstName":"Shuting","middleName":"","lastName":"Li","suffix":""},{"id":539508525,"identity":"6606b90e-39b8-4796-8911-22af1123f7a9","order_by":1,"name":"Yan Liu","email":"","orcid":"","institution":"Wuhan Textile University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Liu","suffix":""},{"id":539508526,"identity":"948d312f-ecfa-4146-a1a5-da5c1c2aef7a","order_by":2,"name":"Ganlin Dong","email":"","orcid":"","institution":"Wuhan Textile University","correspondingAuthor":false,"prefix":"","firstName":"Ganlin","middleName":"","lastName":"Dong","suffix":""},{"id":539508527,"identity":"0f8e4ede-8fa1-42e6-93d6-49316fa0d47b","order_by":3,"name":"Yahuan Wang","email":"","orcid":"","institution":"Wuhan Textile University","correspondingAuthor":false,"prefix":"","firstName":"Yahuan","middleName":"","lastName":"Wang","suffix":""},{"id":539508528,"identity":"bc6cdec1-6d17-47a8-bc49-be7fb9fac375","order_by":4,"name":"Yaonan Li","email":"","orcid":"","institution":"Wuhan Business University","correspondingAuthor":false,"prefix":"","firstName":"Yaonan","middleName":"","lastName":"Li","suffix":""},{"id":539508529,"identity":"3dd4c910-1b72-46ac-bf68-cee22f173aaf","order_by":5,"name":"Liefeng Hu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApElEQVRIiWNgGAWjYBACPuYzDAwfIGwD4rSwseUwMM6AqCZBCzMPiVp4j0nbtv1JbGBv3ibBUHOHGC18adK5bQaJDTzHyiQYjj0jQot8j5l07jagFokcMwnGhsPE2MJjJm0J0iL/hhQtjGBbeIjXYmzZ+8/YuI0nrdgi4RgRWvjZeAxv/DgjJ9vPfnjjjQ81RGhBWAciEkjQMApGwSgYBaMADwAAF3otJcVI9uIAAAAASUVORK5CYII=","orcid":"","institution":"Wuhan Textile University","correspondingAuthor":true,"prefix":"","firstName":"Liefeng","middleName":"","lastName":"Hu","suffix":""},{"id":539508530,"identity":"93d2847f-b34a-44bf-a049-12a214713c32","order_by":6,"name":"Guanqing Yang","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Guanqing","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-09-17 04:23:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7635511/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7635511/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12951-026-04188-6","type":"published","date":"2026-02-21T15:57:52+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":95322074,"identity":"35ae0da1-d9d8-492a-9916-41e5ad1f7cff","added_by":"auto","created_at":"2025-11-06 16:52:07","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":13744391,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/6cdc8cdbe11b24da8e01f364.docx"},{"id":95322058,"identity":"f444f050-7280-4c49-acb7-9493e40edae7","added_by":"auto","created_at":"2025-11-06 16:52:06","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8049,"visible":true,"origin":"","legend":"","description":"","filename":"852a812f3f2d433389e096686028c478.json","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/171b4e39fed55cb27f21528c.json"},{"id":95523728,"identity":"fdb6fb7b-ed97-4c5c-81b6-70ed8bd12bbd","added_by":"auto","created_at":"2025-11-10 10:00:26","extension":"doc","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7513600,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.doc","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/3248b4e81589951eb6869bfc.doc"},{"id":95322063,"identity":"8c80cfbd-7cf8-4600-965f-a0e1c4867563","added_by":"auto","created_at":"2025-11-06 16:52:07","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":133360,"visible":true,"origin":"","legend":"","description":"","filename":"852a812f3f2d433389e096686028c4781enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/7be4189ffd261574eee6fe20.xml"},{"id":95523854,"identity":"885cdbbf-9519-4cc5-9c7c-f1fc5653c392","added_by":"auto","created_at":"2025-11-10 10:01:15","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2450694,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/a7cde92960ba1080939c7f81.jpeg"},{"id":95523714,"identity":"e3b64af8-e117-4a24-8aac-9087f8bf5ad1","added_by":"auto","created_at":"2025-11-10 10:00:17","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1589710,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/53d8ab263261af5dddc6c2bc.jpeg"},{"id":95322068,"identity":"04889ed9-bb5b-4df8-b960-a012d50d76a2","added_by":"auto","created_at":"2025-11-06 16:52:07","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1124872,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/dffd7c684c5bcab02ec100ba.jpeg"},{"id":95524033,"identity":"62a6bbac-8ddf-4a4c-8bc0-9d3b5bd4e17a","added_by":"auto","created_at":"2025-11-10 10:02:00","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":516866,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/fc2ba5b55ea339c8280090b7.jpeg"},{"id":95524533,"identity":"fe2892dd-edf1-4747-b095-019b37f08d6b","added_by":"auto","created_at":"2025-11-10 10:02:53","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":743380,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/e714f0df28524e3f642b69e4.jpeg"},{"id":95524199,"identity":"b24322ce-c103-47cf-b348-b3d74f7a7746","added_by":"auto","created_at":"2025-11-10 10:02:28","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3488444,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/ff54dc93d7f47d2e7184fd2f.jpeg"},{"id":95322078,"identity":"76acccd5-c973-48be-885e-d58465ac819b","added_by":"auto","created_at":"2025-11-06 16:52:07","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2745992,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/4f8d7e017e86e0a6455c46fd.jpeg"},{"id":95523589,"identity":"27366004-eb47-4a97-946f-105f9f85f4e8","added_by":"auto","created_at":"2025-11-10 09:58:45","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1008428,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/2ff1c29ea7161d4462c2ab15.jpeg"},{"id":95322076,"identity":"56ac4c67-d874-4c20-93db-13e146e9f248","added_by":"auto","created_at":"2025-11-06 16:52:07","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":301279,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/57992c04506e720d70828202.png"},{"id":95524592,"identity":"0b903455-ee4b-4bd7-b8a5-0d256b930969","added_by":"auto","created_at":"2025-11-10 10:02:59","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":169155,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/e0d65bc676718722dfe2cddf.png"},{"id":95524401,"identity":"3769fe02-d170-4d2a-8edf-a4fc9d16ac26","added_by":"auto","created_at":"2025-11-10 10:02:43","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":141058,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/15de53f919b6eaeab0390747.png"},{"id":95322064,"identity":"08a6522b-d87d-4286-8c39-c91d707eeafb","added_by":"auto","created_at":"2025-11-06 16:52:07","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":45738,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/5f669f0423b92baaad2ad51a.png"},{"id":95322083,"identity":"263ee902-5715-4764-acb2-996906c1040e","added_by":"auto","created_at":"2025-11-06 16:52:07","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":78482,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/c64015a5152806e50be732f6.png"},{"id":95322081,"identity":"c9fc0b11-5336-4dd4-887e-f860ca4d2588","added_by":"auto","created_at":"2025-11-06 16:52:07","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":270633,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/032a64b96976840e096f9506.png"},{"id":95523752,"identity":"11ad3225-1e63-4d15-b8c0-726b32b9646b","added_by":"auto","created_at":"2025-11-10 10:00:34","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":337504,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/1e550cc4a2715558a4bb07f9.png"},{"id":95322084,"identity":"5be83d46-1af5-4efd-ba7c-cce3cd6a4313","added_by":"auto","created_at":"2025-11-06 16:52:07","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":133905,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/65453e43678776bf16afc3ed.png"},{"id":95322086,"identity":"c11fe725-af38-4863-84d3-34d50944b4fb","added_by":"auto","created_at":"2025-11-06 16:52:07","extension":"xml","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":132077,"visible":true,"origin":"","legend":"","description":"","filename":"852a812f3f2d433389e096686028c4781structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/c9a016b560ec4e44f43d3661.xml"},{"id":95523709,"identity":"f683bfcf-b296-4351-bea7-81b93ab20fc3","added_by":"auto","created_at":"2025-11-10 10:00:16","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":138736,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/501cf40f3c74540536f7c60d.html"},{"id":95322057,"identity":"a885bc97-0c81-46a5-8be6-4a356609121f","added_by":"auto","created_at":"2025-11-06 16:52:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4759571,"visible":true,"origin":"","legend":"\u003cp\u003ePreparation and structural property analysis of DT-COF. (\u003cstrong\u003ea\u003c/strong\u003e) Schematic reaction diagram of DT-COF and insertion of physical powder from a single reaction batch. (\u003cstrong\u003eb\u003c/strong\u003e) Experimental and simulated XRD patterns of DT-COF as well as its Rietveld refinement result. (\u003cstrong\u003ec\u003c/strong\u003e) The model diagram of DT-COF with AB stacking from top and side views. (\u003cstrong\u003ed\u003c/strong\u003e) Energy gap calculated from the HOMO and LUMO orbital energies of DT-COF structure and unit molecule of DT-COF. (\u003cstrong\u003ee\u003c/strong\u003e) The band structure and f) density of states of DT-COF.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/d8f1f8ecec7f6d289b874fb5.png"},{"id":95524006,"identity":"43c1b131-0ced-4293-bd57-aef230d1ad7c","added_by":"auto","created_at":"2025-11-10 10:01:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3278552,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of DT-COF. (\u003cstrong\u003ea\u003c/strong\u003e) The typical TEM images of DT-COF morphology and (\u003cstrong\u003eb\u003c/strong\u003e) enlarged lattice structure. (\u003cstrong\u003ec\u003c/strong\u003e) Hydrodynamic diameters and zeta potential of DT-COF in water. (\u003cstrong\u003ed\u003c/strong\u003e) The TEM image of DT-COF morphology and elemental mapping (C, N and S), Scale bar = 200 µm. (\u003cstrong\u003ee\u003c/strong\u003e) FTIR spectra of DT-COF, TFB and DTO labeled with their respective characteristic peaks. (\u003cstrong\u003ef\u003c/strong\u003e) N\u003csub\u003e2\u003c/sub\u003e adsorption and desorption isotherms of DT-COF. (\u003cstrong\u003eg\u003c/strong\u003e) Pore size distribution of DT-COF. (\u003cstrong\u003eh\u003c/strong\u003e) XPS survey spectra and (\u003cstrong\u003ei\u003c/strong\u003e) S 2p high-resolution spectra of DT-COF. (\u003cstrong\u003ej\u003c/strong\u003e) Solid state \u003csup\u003e13\u003c/sup\u003eC NMR of DT-COF.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/5b0bc2f33f2671e28fa47904.png"},{"id":95523797,"identity":"ac47b6ae-d913-4ac3-afa7-0b3e5d350522","added_by":"auto","created_at":"2025-11-10 10:00:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1536416,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of multiphoton activity. (\u003cstrong\u003ea\u003c/strong\u003e) UV-vis absorption spectra of DT-COF in solid state and aqueous solution. (\u003cstrong\u003eb\u003c/strong\u003e) The two-photon absorption and (\u003cstrong\u003ec\u003c/strong\u003e) three-photon absorption cross-section spectra of DT-COF in water (100 µg mL\u003csup\u003e−1\u003c/sup\u003e). (\u003cstrong\u003ed-f\u003c/strong\u003e) One photon, two-photon and three-photon excited fluorescence emission of DT-COF upon 404 nm, 808 nm and 1250 nm laser excitation, respectively. (\u003cstrong\u003eg\u003c/strong\u003e) ESR signals of DT-COF with different concentrations trapped by TEMP (20 μL) with and without light irradiation (808 nm, 200 mW cm\u003csup\u003e-2\u003c/sup\u003e). (\u003cstrong\u003eh\u003c/strong\u003e) ESR signals of DT-COF (200 μg/mL, aqueous solution) trapped by DMPO (30 μL) with and without 200 mW cm\u003csup\u003e-2\u003c/sup\u003e 808 nm laser irradiation. (\u003cstrong\u003ei)\u003c/strong\u003e The variation curves of absorption values of DPBF at 280 nm after 808 nm laser irradiation at different time points.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/fb16a24f45927327fc67fd11.png"},{"id":95322059,"identity":"fc89d232-5d26-4a6c-be33-c2848de9a1ab","added_by":"auto","created_at":"2025-11-06 16:52:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2211948,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of drug loading performance of DT-COF. (\u003cstrong\u003ea\u003c/strong\u003e) Theoretical size of DT-COF pores. (\u003cstrong\u003eb\u003c/strong\u003e) The calculated size of TPZ molecule. (\u003cstrong\u003ec\u003c/strong\u003e) Hydrodynamic size and zeta potential of DT-COF in water. (\u003cstrong\u003ed\u003c/strong\u003e) UV-Vis absorption spectra of TPZ, DT-COF and TPZ@DT-COF in DMF. (\u003cstrong\u003ee\u003c/strong\u003e) UV-Vis absorption spectra of TPZ and TPZ@DT-COF centrifuged aqueous solutions at different concentrations (7.8-250 μg mL\u003csup\u003e-1\u003c/sup\u003e) in water. (\u003cstrong\u003ef\u003c/strong\u003e) The EC and EE of TPZ in TPZ@DT-COF nanosystem. (\u003cstrong\u003eg\u003c/strong\u003e) Cumulative release profiles of TPZ from TPZ@DT-COF nanosystem in different pH conditions. (\u003cstrong\u003eh\u003c/strong\u003e) Morphology of TPZ@DT-COF after 96 hours of release under different pH conditions (pH 7.4 and 4.0). (\u003cstrong\u003ei\u003c/strong\u003e) The changes in particle size of TPZ@DT-COF incubated under different solution conditions for 7 days. Data are presented as mean values +/− S.D., and P values are calculated by two-tailed Student's t-test. *P \u0026lt; 0.05, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/37783c95aafcbaf0f040714a.png"},{"id":95524383,"identity":"eb30bcd8-b44e-4f54-aa1d-e2f5f317a0fd","added_by":"auto","created_at":"2025-11-10 10:02:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":10970840,"visible":true,"origin":"","legend":"\u003cp\u003eMultiphoton activity and drug delivery studies in vitro. (\u003cstrong\u003ea\u003c/strong\u003e) CLSM images of multiphoton fluorescence imaging of DT-COF after co-incubation with 4T1 cells for different times. (\u003cstrong\u003eb\u003c/strong\u003e) Semi-quantification of intracellular multiphoton fluorescence intensity. (\u003cstrong\u003ec\u003c/strong\u003e) The depth of cell penetration by two-photon fluorescence and three-photon fluorescence. (\u003cstrong\u003ed\u003c/strong\u003e) Nucleus- and mitochondria-targeted co-localization assessment of TPZ@DT-COF. (\u003cstrong\u003ee\u003c/strong\u003e) Positional analysis of nucleus and mitochondria co-localization. (\u003cstrong\u003ef\u003c/strong\u003e) The cellular uptake of TPZ@DT-COF at different time points was measured using flow cytometry. (\u003cstrong\u003eg\u003c/strong\u003e) Detection of ROS production by DT-COF in normoxic and hypoxic environments using DCFH-DA. Scale bar = 50 µm. (\u003cstrong\u003eh\u003c/strong\u003e) The comparison of fluorescence intensity of DT-COF in normoxic and hypoxic environments after 808 nm laser irradiation (100 mW cm\u003csup\u003e-2\u003c/sup\u003e). Data are presented as mean values +/− S.D., and P values are calculated by two-tailed Student's t-test. ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/76443adaa3222dfb804b1c7a.png"},{"id":95524407,"identity":"7a13c15c-7ea9-4d37-ad3a-d5e9337c0c27","added_by":"auto","created_at":"2025-11-10 10:02:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":7686258,"visible":true,"origin":"","legend":"\u003cp\u003eAnti-tumor effects in vitro. (\u003cstrong\u003ea\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eCell viability of 4T1 cells incubated with increasing DT-COF (0-500 μg mL\u003csup\u003e-1\u003c/sup\u003e) under normoxia and hypoxia. (\u003cstrong\u003eb\u003c/strong\u003e) The cytotoxicity of TPZ@DT-COF with and without 808 nm laser irradiation of 4T1 cells under normoxic and (\u003cstrong\u003ec\u003c/strong\u003e) hypoxic conditions. (\u003cstrong\u003ed\u003c/strong\u003e) The representative fluorescent images of 4T1 cells co-stained by CA and PI after various experimental conditions (groups of 1-9 denoted Blank, laser irradiation alone, DT-COF under normoxia and hypoxia, DT-COF plus laser irradiation, TPZ@DT-COF and TPZ@DT-COF plus laser irradiation under normoxia, TPZ@DT-COF and TPZ@DT-COF plus laser irradiation under hypoxia) as indicated on images, scale bar = 100 μm. (\u003cstrong\u003ee\u003c/strong\u003e) Semi-quantitative percentage of red fluorescence of dead cells to total fluorescence intensity. Data are presented as mean values +/− S.D., and P values are calculated by two-tailed Student's t-test. **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ns = no significance.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/7b58e7ac48264ae4216780fc.png"},{"id":95322072,"identity":"169b0790-0f88-4195-8eae-7db434efdcb4","added_by":"auto","created_at":"2025-11-06 16:52:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3438380,"visible":true,"origin":"","legend":"\u003cp\u003eAntitumor effects in vivo. a Fluorescence imaging at different time points in vivo. The white oval indicates tumor tissue. b Quantitative analysis of fluorescence intensity in tumor tissue at different time points (n = 3). c Tumor growth curves for each group after intratumoral injection. d Tumor weights \u003cem\u003eex vivo\u003c/em\u003e in each group at the end of treatment. e Survival of mice in different treatment groups over 35 days. f Representative tumor images. g 1PF, 2PF and 3PF at 80 µm of tumor tissue. Scale bar = 100 µm. h H\u0026amp;E staining images of tumor slices for each group after 15 days of treatments. Scale bar = 50 µm. i Optical microscopic images of tumor slices stained with DAPI and TUNEL. Scale bar = 100 µm. Data are presented as mean values +/− S.D., and P values are calculated by two-tailed Student's t-test. *P \u0026lt; 0.05, ***P \u0026lt; 0.001, ns = no significance.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/58482bc767cf95b80346fdc7.png"},{"id":103251211,"identity":"0d5db7d1-fa38-453a-bc5b-1f5e5b8b8514","added_by":"auto","created_at":"2026-02-23 16:06:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":31322397,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/748c03a6-4b79-4f7c-a417-90d6ad84ea76.pdf"},{"id":95523878,"identity":"493ce705-487f-477c-a538-0492336cb3b4","added_by":"auto","created_at":"2025-11-10 10:01:22","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7513600,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.doc","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/f374cf7668ca3f6c1745a5eb.doc"},{"id":95322061,"identity":"986b2d30-62ac-42ac-a9e1-671c6bfb7030","added_by":"auto","created_at":"2025-11-06 16:52:07","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":9020369,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003e Schematic illustration of the DT-COF with multiphoton activity for drug delivery and anti-tumor applications. (\u003cstrong\u003ei)\u003c/strong\u003e Preparation of TPZ@DT-COF composite nanomaterial and anti-tumor mechanisms. (\u003cstrong\u003eii)\u003c/strong\u003e Illustration of the multiphoton activities of DT-COF mediating the fluorescence emission and photodynamic ability.\u003c/p\u003e","description":"","filename":"SC1.png","url":"https://assets-eu.researchsquare.com/files/rs-7635511/v1/73dbf335a908be4751aacc93.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Rationally Designed Thiazole-Based COF with Multiphoton Activity for Drug Delivery and Synergistic Therapy of Deep-Seated Tumors","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePhotodynamic therapy (PDT), a promising photo-induced therapeutic strategy approved for clinical use, has shed new light on millions of patients with tumors [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Compared to conventional therapeutic strategies used clinically, such as radiotherapy, chemotherapy, and surgery, PDT offers unique advantages such as minimal invasiveness, high spatial selectivity, and low side effects [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The key to high-performance PDT lies in the use of photosensitizers that are effectively activated by specific light exposure to generate cytotoxic singlet oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. As contrast agents, photosensitizers determine the accuracy and tissue depth of fluorescent bioimaging [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Currently, the excitation wavelengths of most photosensitizers, such as porphyrin, BODIPY dyes, and their derivatives, is still limited to 600\u0026ndash;700 nm [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Moreover, the relatively short wavelength of the excitation light is largely scattered by tissues, resulting in low penetration depth and treatment efficiency [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. To resolve these issues, multiphotonic materials can be used, which emit individual photons by absorbing multiple photons at long wavelengths. Multiphoton (\u003cem\u003en\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;3) photosensitizers activated by near-infrared light\u0026mdash;such as small molecules, organometallic complexes, and photosensitive chromophore-integrated nanomaterials\u0026mdash;can be used to perform deep-tissue PDT and spatial imaging [\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Nevertheless, their clinical applications remain limited due to high dark toxicity, low stability, and insufficient multiphoton activity.\u003c/p\u003e\u003cp\u003eThe covalent organic framework (COF), an emerging class of crystalline porous polymers manufactured by conjugating molecules through covalent bonds, is a promising material for biomedical applications, including drug delivery, bioimaging, and disease prevention and treatment, owing to their adjustable pores, good biocompatibility and stability, and compositional and structural tunability [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The properties of COF materials can be precisely modulated by the atomic insertion of functional units. The periodically extensible framework structure also endows the COF with a confined molecular space for photoelectronic transport and host\u0026ndash;guest interactions, thereby demonstrating great potential for theoretically guided photosensitizer design and targeted applications [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. For instance, in 2019, Deng et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] reported two COFs (COF-808 and COF-909) with excellent photodynamic capacity under 630 nm laser irradiation, which were linked via inactive and inexpensive small molecules. The two COFs exhibited two-dimensional (2D) π-conjugation, and their framework structure also facilitated the diffusion of oxygen and reactive oxygen species (ROS) to enhance therapeutic efficacy. Additionally, the COFs diminished the π-π stacking interaction between the layers and enhanced the multiphoton cross-section to improve their activity. In 2020, Zhang et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] designed a benzothiadiazole-based COF with high dipole values and two-photon cross-section using the donor-π-acceptor-π-donor (D-π-A-π-D) functional unit as the building block, which led to good near-infrared two-photon-induced fluorescence features. Therefore, engineered COFs have great potential as near-infrared light-excited multiphoton photosensitizers for efficient deep tumor imaging and PDT.\u003c/p\u003e\u003cp\u003eThiazole-derived organic chromophores have recently shown great potential for application in multiphoton excitation owing to their unique charge distribution [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the present study (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), we designed and synthesized a new type of thiazolothiazole-based COF (DT-COF) nanowire with D-A configurations for multiphoton-excited tumor imaging and PDT. The DT-COF was efficiently harvested via a one-step catalyst-free condensation reaction using two inactive and inexpensive small molecules\u0026mdash;namely, dithiooxamide (DTO) and 1,3,5-triformylbenzene (TFB)\u0026mdash;which achieved good three-photon performance in deep-seated tumor imaging and therapy. Mechanically, the satisfactory results may be attributed mainly to the long-range ordering of the closed-frame structure, polarization enhancement (10.5 times) of the charge distribution caused by the closed D-A configuration of the chromophore, and rigid COF structure-mediated enhancement of the dipole moment (6.3 times) and hyperpolarizability (10.5 times), leading to an increased cross-section of multiphoton induction. In addition, the one-dimensional (1D) pore size of the DT-COF was suitable for the efficient encapsulation of tirapazamine (TPZ), a hypoxia-activated chemotherapeutic drug, with encapsulation rates up to 76.2%. Not only did the proton effect of the thiazole moiety in DT-COF allow it to target the mitochondria, but also the microenvironment of tumor cells stimulated TPZ release and delivery into the nucleus. Furthermore, DT-COF-mediated PDT further depleted oxygen in hypoxic tumors to activate TPZ-induced chemotherapy, resulting in a synergistic oncolytic effect. This study sheds new light on the design of photoactive materials with novel structures and rational applications.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStructural performance assessment of DT-COF\u003c/h2\u003e\u003cp\u003eThe engineered thiazolothiazole-based DT-COF was obtained from the Hantzsch thiazole synthesis with some modifications [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], which involved the polymerization of an aldehyde group and a thioamide group by a catalyst-free one-pot method. Briefly, the yellow solid powder of DT-COF was synthesized in a reaction bottle at solvent circulation temperature for 6 hours by feeding 1.5:1 of DTO and TFB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), the yield was as high as 78.6% compared to other COF materials reported in the literature [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The DT-COF structure was confirmed using powder X-ray diffraction (PXRD), which indicated that the DT-COF had a crystalline structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The PXRD pattern simulated using Materials Studio (2020 version) suggested that the structure of DT-COF was consistent with the AB stacking mode. Pawley refinement showed negligible differences between the simulated and experimental PXRD patterns. DT-COF was assigned to the space group \u003cem\u003eP1\u003c/em\u003e (AB) with parameters of a\u0026thinsp;=\u0026thinsp;17.474 \u0026Aring;, b\u0026thinsp;=\u0026thinsp;17.471 \u0026Aring;, c\u0026thinsp;=\u0026thinsp;7.707 \u0026Aring;, α\u0026thinsp;=\u0026thinsp;103.31\u0026deg;, β\u0026thinsp;=\u0026thinsp;104.71\u0026deg;, γ\u0026thinsp;=\u0026thinsp;119.97\u0026deg;, and residuals Rwp\u0026thinsp;=\u0026thinsp;3.45% and Rp\u0026thinsp;=\u0026thinsp;2.73%. The c-axis parameters for the AB stacking mode are consistent with the 3.9 \u0026Aring; layer spacing date (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Based on theoretical inferences, compared with the traditional solvothermal synthesis method, which requires high temperature, high pressure, and harsh conditions, this facile strategy allows COF synthesis to be more sustainable. Furthermore, the multiphoton activity of the DT-COF was preliminarily evaluated using density functional theory calculations. The energy difference between the highest occupied molecular orbital and the lowest unoccupied molecular orbital of the DT-COF structural molecule was calculated to be 3.40 eV, with a dipole moment of 25.9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). However, the energy gap and dipole moment of the DT-COF unit molecule were 3.95 eV and 4.1, respectively, indicating a significant reduction in electron delocalization behavior of the molecular unit compared to the closed ring structure of COF. Moreover, the band structure and density of states of DT-COF were supported (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef) with an \u003cem\u003eE\u003c/em\u003eg of 1.01 eV, confirming the good electron delocalization ability of DT-COF. Moreover, the hyperpolarizability β of DT-COF and unitary molecules were 24.047 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e esu and 2.295 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e esu, respectively, which were calculated by gaussian 09 with the basis set of PBE/6-311\u0026thinsp;+\u0026thinsp;+\u0026thinsp;G(d,p). This result suggested that DT-COF provided a fundamental platform for nonlinear optical activity\u003csup\u003e30,31\u003c/sup\u003e. In addition, thiazolothiazole-based DT-COF could avoid π-π stacking interactions and achieve mitochondrial-targeting ability for near-infrared light-induced fluorescence imaging as well as PDT.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCharacterization of DT-COF\u003c/h3\u003e\n\u003cp\u003eFurthermore, the morphology of the DT-COF was characterized using transmission electron microscopy (TEM). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, DT-COF was in the form of a bar shape, and its lattice structure could be clearly observed using high-resolution TEM, further confirming the crystallinity of the DT-COF. The average hydrated particle size of the DT-COF measured by dynamic light scattering (DLS) was approximately 170 nm and showed a normal distribution curve, indicating good dispersion and uniformity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Meanwhile, the zeta potential was \u0026minus;\u0026thinsp;16.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 mV, which would enhance the stability [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The formation of DT-COF was also detected by scanning electron microscopy and surface scan mapping of the distribution of different elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), which showed that the structure was consistent with TEM and contained C, N, and S. The thickness of the DT-COF was also measured to be approximately 15 nm using atomic force microscopy (Fig. S2). Moreover, the structure of the DT-COF was verified using Fourier Transform Infrared Spectroscopy (FTIR). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, the stretching vibration peaks of -C-S-C- and -C\u0026thinsp;=\u0026thinsp;N- were newly generated in DT-COF, and the stretching vibration peaks of -C\u0026thinsp;=\u0026thinsp;O-, -CH, -SH, and -NH\u003csub\u003e2\u003c/sub\u003e belonging to TFB and DTO were significantly reduced or even disappeared, indicating that DT-COF was successfully synthesized and that its structure was correct [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. N\u003csub\u003e2\u003c/sub\u003e adsorption and desorption experiments were carried out to investigate the surface area and pore size of the DT-COF. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg, the surface area of DT-COF was calculated to be 141.1 m\u003csup\u003e2\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and the narrow pore width distribution was centered at 1.4 nm, which is in agreement with the theoretical value. These results validate the structural accuracy of the DT-COF. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei, the X-ray photoelectron spectroscopy (XPS) was performed on DT-COF to confirm its structure. The XPS survey spectra clearly indicate that DT-COF was composed of C, N, S, and O. The high-resolution S 2p spectrum of DT-COF was deconvoluted into two peaks at ~\u0026thinsp;163.8 and ~\u0026thinsp;165.0 eV, which were assigned to S 2p\u003csub\u003e3/2\u003c/sub\u003e and S 2p\u003csub\u003e1/2\u003c/sub\u003e, respectively. This result suggests that S reacts to form a thiazole ring, which is in agreement with other thiazole ring results reported in the literature\u003csup\u003e36\u003c/sup\u003e. In addition, the high-resolution C 1s and N 1s spectra indicated the presence of C-S-C, C-N-C, C\u0026thinsp;=\u0026thinsp;N, N\u0026thinsp;=\u0026thinsp;C, N-C, and N\u0026thinsp;=\u0026thinsp;C-S, confirming the successful formation of thiazole rings (Fig. S3). In addition, the solid-state nuclear magnetic resonance spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej) confirmed the generation of sp2 carbons (~\u0026thinsp;150 and 168 ppm). Carbonyl peaks attributed to unreacted aldehyde and thioamide groups were also observed at 192.1 and 35.4 ppm. The XRD patterns of DT-COF did not change significantly after 24 h of co-incubation in different environments (aqueous solution with pH 4, DMEM, and hypoxic conditions), indicating that DT-COF had good stability for subsequent application studies (Fig. S4).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eMultiphoton activities of DT-COF\u003c/h3\u003e\n\u003cp\u003eSubsequently, the photophysical and chemical properties of DT-COF were investigated. The absorption peaks of DT-COF were observed at approximately 360 nm for both the solid powder and aqueous solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Given the D-A configuration and high dipole moment of DT-COF, its nonlinear optical (NOL) performance was further evaluated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, the DT-COF solution exhibited efficient two-photon action (2PA) and 3PA cross-sectional features (600 mW, 80 MHz, and 140 fs). The 2PA cross-section was relatively high at 720 nm and 800 nm, reaching approximately 411 and 386 GM, respectively. The 3PA cross-section reached a maximum of 2.98 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;80\u003c/sup\u003e cm\u003csup\u003e6\u003c/sup\u003e s\u003csup\u003e2\u003c/sup\u003e per photo\u003csup\u003e2\u003c/sup\u003e at 1100 nm and remained 2.24 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;80\u003c/sup\u003e cm\u003csup\u003e6\u003c/sup\u003e s\u003csup\u003e2\u003c/sup\u003e per photo\u003csup\u003e2\u003c/sup\u003e around 1200 nm. These results suggest that DT-COF exhibits good NOL properties and is a promising novel candidate for multiphoton bioimaging and PDT [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Hence, the multiphoton fluorescence and photodynamic capacity of the DT-COF were further evaluated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, the fluorescence emission peak of DT-COF activated by the 404 nm laser was at approximately 550 nm, and the fluorescence intensity gradually increased with the input power of the laser. Additionally, the fluorescence emission was gradually red-shifted with increasing DT-COF concentration (Fig. S5a), and the fluorescence intensity was significantly reduced at a concentration of up to 1000 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e due to aggregation. The results implied that DT-COF had good charge-transfer ability and π-π stacking effect. As shown in Fig. S5b, the logarithm of the fluorescence intensity exhibits a linear dependence on the logarithm of the laser input power (slope\u0026thinsp;=\u0026thinsp;7.64). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, the DT-COF aqueous solution exhibited good up-conversion fluorescence emission and power density dependence when excited by 808 nm and 1250 nm lasers at different power densities (100\u0026ndash;800 mW), demonstrating the two-photon fluorescence (2PF) and three-photon fluorescence (3PF) characteristics of DT-COF. The logarithmic values of both the fluorescence intensity and laser input power density showed a linear relationship with slopes of 3.82 and 2.3, respectively (Fig. S5c and S5d).\u003c/p\u003e\u003cp\u003eGiven the good charge transfer and multiphoton activity of DT-COF, its photodynamic ability was measured using electron spin resonance (ESR) and the \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e indicator 1,3-diphenylisobenzofuran (DPBF) after irradiation with a 200 mW cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e 808 nm laser. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh, the signal peaks of 2,2,6,6-tetramethylpiperidine and 5,5-dimethyl-1-pyrroline-N-oxide used as the trapping agents for ESR analysis showed that DT-COF significantly produced ROS upon 808 nm laser excitation in a concentration-dependent manner, whereas no signal was produced under dark conditions. These results suggest that the DT-COF has great potential for use in two-photon laser-activated PDT. In addition, the absorption peak of DPBF also decreased with an increase in the 808 nm laser irradiation time (Fig. S6), while the absorption peaks of DPBF and DT-COF alone irradiated by the 808 nm laser were almost unchanged. The detailed change values that exhibited a significant difference are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei. These results demonstrate the good multiphoton activity of DT-COF and the photodynamic activity of 808 nm (2PF) laser excitation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003ePreparation and characterization of TPZ@DT-COF\u003c/h3\u003e\n\u003cp\u003eThe consumption of O\u003csub\u003e2\u003c/sub\u003e during PDT leads to further elevation of hypoxia in inadequately oxygenated tumors. TPZ, a hypoxia-activated chemotherapeutic drug, was loaded into the DT-COF for the synergistic therapeutic effects of PDT and chemotherapy. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, the theoretical pore size of the DT-COF was 1.48 nm, and the tested size was approximately 1.4 nm, which is in good agreement with the size of TPZ (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Therefore, we envisaged that the DT-COF could effectively encapsulate TPZ. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, TPZ-loaded DT-COF (TPZ@DT-COF) was obtained after 24 h of stirring and several washes with ethanol, which increased the hydrated particle size to approximately 206 nm and changed the zeta potential to 0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 mV. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, TPZ@DT-COF in the DMF solution exhibited the characteristic UV-Vis absorption peaks of DT-COF and TPZ, indicating the successful loading of TPZ. Furthermore, the characteristic absorption peak at 460 nm for gradient concentration of TPZ (7.8\u0026ndash;250 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was used for quantitative loading analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee), and the absorption intensity showed a linear positive correlation with concentration (Fig. S7). Significantly, based on the characteristic absorption peak of TPZ in the supernatant after centrifugation of the TPZ@DT-COF aqueous solution, the encapsulated content (EC) and encapsulated efficiency (EE) of TPZ were calculated from Eq.\u0026nbsp;1 as 43.3% \u0026plusmn; 3.3% and 76.2% \u0026plusmn; 4.4%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). The inset shows the color change from yellow to red in the nanosystem after TPZ loading. In addition, we selected doxorubicin (DOX) as the control drug loading model with the dimensions of length \u0026times; width \u0026times; height as 16.51 \u0026Aring; \u0026times; 12.56 \u0026Aring; \u0026times; 9.78 \u0026Aring;, which was larger than the pore size of DT-COF. As shown in Fig. S8, the EC and EE of DOX were significantly reduced to 23.5% \u0026plusmn; 1.1% and 33.7% \u0026plusmn; 3.7%, respectively. The solution shown in the inset is lighter in color. These results indicated that DT-COF and TPZ possess synergistic sizes and anti-tumor mechanisms to achieve good drug delivery and tumor therapeutic effects. Furthermore, the release behavior of TPZ under different pH conditions was investigated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg, the cumulative release of TPZ was pH-dependent. The TPZ release rate was 16.98\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99% after 96 h of incubation in healthy physiological pH condition (pH\u0026thinsp;=\u0026thinsp;7.4), whereas the release rate was significantly elevated in the slightly acidic environment of tumor tissues, reaching 70.39\u0026thinsp;\u0026plusmn;\u0026thinsp;3.28%, 85.35\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2%, and 89.09\u0026thinsp;\u0026plusmn;\u0026thinsp;4.82% at pH 6.5, 5.0, and 4.0, respectively. To better understand the pH-responsive release behavior of TPZ@DT-COF, the Ritger\u0026ndash;Peppas equation was used to analyze release kinetics. The fitting parameters for the kinetic constant (k) and release exponent (n) are presented in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The n values for TPZ release were all \u0026lt;\u0026thinsp;0.45, suggesting that the release mechanism for TPZ was Fickian diffusion. After incubation at pH 7.4, the morphology of the samples was observed using TEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh), which showed that DT-COF could responsively release the drug in the slightly acidic environment of the tumor with little change in morphology. In addition, the stability of TPZ@DT-COF was characterized by variations in its particle size in different environments. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei, TPZ@DT-COF was co-incubated in water, PBS, and DMEM containing 10% FBS for 7 days to assess its stability for transportation, storage, and physiological applications. The results showed that the particle size remained stable throughout the 7 days.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eDrug delivery and multiphoton performance evaluation of TPZ@DT-COF in vitro\u003c/h3\u003e\n\u003cp\u003eNext, we evaluated the drug delivery, multiphoton activity-mediated fluorescence imaging, and ROS generation effects of DT-COF in vitro. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, the fluorescence intensities of the 1PF, 2PF, and 3PF channels increased gradually with increasing incubation time, with cyan, green, and red fluorescence indicating 1PF, 2PF, and 3PF, respectively. The cell positions of the fluorescence channels and the bright field (BF) channel completely overlapped with each other, suggesting effective cellular internalization and good multiphoton fluorescence imaging performance of the DT-COF. Semi-quantitative data on fluorescence intensity also showed good time dependence and was greatest at 2PF (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Furthermore, the multiphoton fluorescence imaging depth of DT-COF was explored using laser confocal layer-by-layer scanning. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, the 3PF group exhibited deeper fluorescence emission with the strongest fluorescence at a depth of 70 \u0026micro;m, while the strongest fluorescence at a depth of 50 \u0026micro;m in the 2PF group, indicating that the three-photon laser penetrated to a deeper depth to excite DT-COF. These results demonstrate that DT-COF has the potential for multiphoton fluorescence imaging in deep tissues and can be used as an imaging contrast agent. Furthermore, the targeted delivery effects of the TPZ@DT-COF nanosystem were evaluated based on the mitochondrial-targeting ability of thiazoles reported in our previous work [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, the bright violet fluorescence attributed to DT-COF was excited by a two-photon laser and overlapped with the red fluorescence of Mito-tracker. In contrast, the green fluorescence of the DT-COF was distributed in the nuclei of cells localized by DAPI, which may have resulted from the microenvironment-driven release of TPZ in tumor cells. The results of fluorescence co-localization analyses showed a high degree of overlap between DT-COF and mitochondria, as well as between TPZ and the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). This delivery effect is in good agreement with the corresponding therapeutic pathways of TPZ and DT-COF, resulting in precise delivery and effective therapeutic enhancement. To rigorously demonstrate the mitochondria-targeting ability of the DT-COF, we utilized the mitochondria-targeting molecule triphenylphosphine to block the mitochondria. As shown in Fig. S9, the fluorescence of DT-COF incompletely overlapped with that of the mitochondrial green fluorescent probe (MitoTracker). The enrichment of mitochondria was significantly reduced, and a shift in the channel position was clearly detected by co-localization analysis, indicating that DT-COF exhibited a target-dependent property. The cellular uptake of TPZ@DT-COF after different times (1\u0026ndash;24 h) of co-culturing with 4T1 cells was quantified using flow cytometry. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef, the cellular uptake increased gradually with increasing incubation time, reaching 98.8% and 34.7% at 24 h, as determined by fluorescence quantification of DT-COF and TPZ, respectively. Subsequently, the photodynamic ability of DT-COF was evaluated by detecting the generation of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e using DCFH-DA after irradiating 4T1 cells with 200 mW cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e 808 nm laser for 5 min. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg, the control groups without added DT-COF showed no significant green fluorescence under either dark or light conditions in a normoxic environment, and no significant green fluorescence was observed in the DT-COF group alone, whereas the DT-COF group produced obvious green fluorescence after 808 nm laser irradiation. This phenomenon indicates that DT-COF can be activated by the two-photon 808 nm laser to efficiently produce \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e, rather than by DT-COF or the 808 nm laser irradiation itself. We also compared the generation of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e under the same conditions in a hypoxic environment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh), which was consistent with the conclusions drawn in a normoxic environment. However, the intensity and cell volume of the green fluorescence produced by DT-COF after 808 nm laser irradiation under hypoxic conditions were significantly reduced, suggesting that hypoxia limited the photodynamic performance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAntitumor effects of TPZ@DT-COF in vitro\u003c/h2\u003e\u003cp\u003eThe cytotoxicity of DT-COF against tumor cell of 4T1 and normal cells (L929 and MCF-10A) was assessed using the methyl thiazolyl tetrazolium (MTT) assay. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and Fig. S10a, the cell viability after 24 h incubation with DT-COF was still over 95% even at the maximum concentration of 500 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, suggesting good biocompatibility of DT-COF for further biological applications. Moreover, the cell viability was above 90%, even after 48 and 72 h of co-incubation with different concentrations of DT-COF (Fig. S10b). Oxygen deprivation limited the photodynamic performance of DT-COF, whereas TPZ@DT-COF offered excellent delivery and therapeutic potential. The therapeutic effects of the chemotherapy and photodynamic-chemotherapy combinations of TPZ@DT-COF in normoxic and hypoxic environments were evaluated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, the decrease in cell viability was concentration-dependent, and the cell killing efficiency was greatest at a concentration of 50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TPZ@DT-COF (21.6 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for TPZ). Moreover, TPZ@DT-COF exhibited good chemotherapeutic efficacy in rapidly proliferating 4T1 cells and enhanced chemotherapeutic efficacy under hypoxic conditions. The cell viability decreased from about 50% to 40% at a final concentration of 50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which demonstrated the advantages of the hypoxia-activated drug TPZ in hypoxic tumor therapy. Two-photon PDT with 808 nm laser excitation effectively improved the therapeutic efficiency of TPZ@DT-COF in 4T1 cells, and the enhancement effect was more significant in the normoxic environment. These results suggest that the TPZ@DT-COF nanosystem exhibits good synergy between two-photon PDT and hypoxia-activated chemotherapy. The killing effect of TPZ@DT-COF on 4T1 cells was directly observed in vitro by double staining with calcein AM (CA)/propidium iodide (PI). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, live and dead cells were stained with green and red fluorescence, respectively. Almost all 4T1 cells in the untreated control and 808 nm laser or DT-COF treatment groups alone under normoxia and hypoxia were stained green, and the red fluorescence of PI was negligible, indicating that neither the 808 nm laser nor the DT-COF killed 4T1 cells. In contrast, DT-COF plus 808 nm laser irradiation (DT-COF\u0026thinsp;+\u0026thinsp;L) resulted in a significant increase in the number of red fluorescent cells in both normoxic environments, caused by the two-photon photodynamic activity of DT-COF. The TPZ@DT-COF\u0026thinsp;+\u0026thinsp;L group exhibited a significant killing effect in normoxic and hypoxic environments, with a marked increase in red fluorescent cells, indicating a synergistic therapeutic effect. To quantitatively evaluate the apoptotic effect of different treatment groups (1\u0026ndash;9) on tumor cells, the apoptotic reagent annexin-V/PI was used and detected by flow cytometry. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, the percentages of live and dead cells in each group exhibited a trend consistent with the above results. These results demonstrated that DT-COF exhibited good biocompatibility and photodynamic capacity and could effectively synergize with TPZ to enhance the 4T1 treatment efficiency in vitro.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEvaluation of the antitumor efficacy of TPZ@DT-COF in vivo\u003c/h3\u003e\n\u003cp\u003eHaving proven the good delivery effects and efficient photodynamic chemotherapy outcomes of TPZ@DT-COF in vitro, we evaluated its anti-tumor effects in vivo. Primarily, TPZ@DT-COF exhibited good stability and biocompatibility for biological applications because the nanosystem did not cause hemolysis (Fig. S10c). To further investigate the distribution and metabolism in tumor tissues and verify the performance and potential in practical applications, we utilized the 1PF properties of DT-COF in an in vivo imaging system (IVIS) to monitor its distribution and metabolism in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). Within 8 hours of tail vein injection of DT-COF, the fluorescence intensity at the tumor reached a maximum, and then the fluorescence intensity gradually decreased, and even could not be observed due to the limited fluorescence penetration distance. The visual analysis of distribution and metabolism through isolated major organs (Heart, Liver, Spleen, Lung and Kidney) and tumor tissues at the end of the monitoring procedure showed that DT-COF was metabolized mainly by the liver and kidney. Quantitative fluorescence analysis also showed the same distribution and metabolic trends (Fig. S11a and S11b). In addition, when the tumor volumes reached 80\u0026ndash;100 mm\u003csup\u003e3\u003c/sup\u003e, a single intratumoral injection was administered to subcutaneous 4T1 tumor-bearing mice to evaluate the oncological treatment and multiphoton imaging effects of TPZ@DT-COF. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef, neither PBS\u0026thinsp;+\u0026thinsp;L nor DT-COF treatment showed any antitumor efficacy, the tumour volume growth curves showed rapid increases, reaching relative tumor volumes of 10.97\u0026thinsp;\u0026plusmn;\u0026thinsp;2.00 and 10.38\u0026thinsp;\u0026plusmn;\u0026thinsp;2.32 at 15 days, respectively. While DT-COF\u0026thinsp;+\u0026thinsp;L, TPZ@DT-COF and TPZ@DT-COF\u0026thinsp;+\u0026thinsp;L groups led to a rapid and prominent tumor inhibition, distinguished markedly from other treatments. Besides, TPZ@DT-COF\u0026thinsp;+\u0026thinsp;L group-mediated photodynamic-chemotherapy combination strategy effectively enhanced tumor volume growth inhibition compared to the chemotherapy or PDT alone groups (TPZ@DT-COF or DT-COF\u0026thinsp;+\u0026thinsp;L). Tumor weight and morphology of isolated tumors also provided a clear indication of the tumor suppression effects consisted with the above results. The weight monitoring during the treatment period also showed no abnormal changes, indicating that the nano-formulation possessed good biocompatibility (Fig. S12). The survival rate of mice during the 35-day treatment period also indicated that the synergistic treatment effect of the TPZ@DT-COF\u0026thinsp;+\u0026thinsp;L group significantly prolonged the survival period (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). The penetration depth of DT-COF fluorescence-induced by 1PF, 2PF and 3PF laser exposure was assessed through tumor tissues. The mice were executed once the tumor volume exceeded 1000 mm\u003csup\u003e3\u003c/sup\u003e. At the same depth of 80 \u0026micro;m, almost no fluorescence emission from group 1PF was noted, the 2PF and 3PF groups had significant fluorescence emission, and the 3PF group possessed the largest fluorescence emission area (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg), indicating that DT-COF-mediated multiphoton fluorescence exhibited deeper tissue imaging capability. In addition, the penetration depths of 1PF, 2PF and 3PF of DT-COF were also examined by layer-by-layer scanning. As shown in Fig. S13, the strongest intensity depth of 1PF was at 30 \u0026micro;m, the fluorescence intensity of 2PF was higher at 40 and 60 \u0026micro;m, and 3PF reached 100 and 120 \u0026micro;m, which suggested that the development of MPPSs exhibited the potential to break the limitation of the light penetration depth. Then, the therapeutic effects were studied by the histopathological analysis of tumor tissues. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eh, Hematoxylin and eosin (H\u0026amp;E) staining showed intact tumor tissue without significant abnormalities and lesions in the PBS and DT-COF groups, whereas the other three groups showed extensive tissue necrosis. It was clear from the tightness of the tumor tissues in the DT-COF\u0026thinsp;+\u0026thinsp;L, TPZ@DT-COF and TPZ@DT-COF\u0026thinsp;+\u0026thinsp;L groups became loose and necrosis. Notably, H\u0026amp;E of major organs in all groups of mice showed no abnormalities, and the biomarkers such as ALT, AST, CRE and BUN were detected and all were within the normal range and showed no abnormalities (Fig. S14 and Fig. S15a-S15d), indicating the long-term safety of the treatment in mice. Meanwhile, the immunofluorescent staining of TUNEL revealed results consistent with those of H\u0026amp;E staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ei). The apoptotic and necrotic cells in the TPZ@DT-COF\u0026thinsp;+\u0026thinsp;L group were stained with the greatest intensity and area of green fluorescence, reflecting the superiority of combined chemo-photodynamic therapy for tumor treatment. These results fully demonstrated that the nanosystem exhibited good drug delivery and theranostic performance and showed great potential for anti-tumor applications. In addition, we also explored the \u003cem\u003ein vivo\u003c/em\u003e distribution of DT-COF using its single-photon fluorescence properties. Firstly, all indicators in the whole blood analysis were within the normal range compared with the control group (Fig. S15e). These results indicated that BT-nHOFs exhibited good blood safety and did not destroy blood cells and cause obvious blood abnormalities. Acute toxicity experiments at high doses (25 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) also showed that DT-COF and TPZ@DT-COF did not cause significant damage to major organs in mice (Fig. S16), suggesting that DT-COF exhibited potential for clinical application.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we efficiently constructed DT-COF with strong multiphoton activity, utilizing thiazolothiazole as the nonlinear optical unit, whose framework structure extended the π-electron system, attenuated π-π stacking, and enhanced charge transfer. The DT-COF not only exhibited multiphoton fluorescence imaging and PDT but also specifically and efficiently encapsulated TPZ, a hypoxia-activated chemotherapeutic drug. The pore size of DT-COF matched the size of TPZ, as well as the cascade of anti-tumor mechanisms between DT-COF and TPZ. In addition, the nanosystem exhibited excellent subcellular organelle-targeted delivery performance with precise anti-tumor effects. The anti-tumor results in vitro and in vivo showed deep penetration depth (80 µm) for multiphoton fluorescence imaging and enhanced photodynamic-chemotherapeutic therapy. This work presents a promising design guideline for drug delivery and combined therapy and sheds new light on the theranostics of deep-seated tumors.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eHistopathological analysis\u003c/h2\u003e\u003cp\u003eHematoxylin and eosin (H\u0026amp;E) and immunofluorescent staining of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) were performed following the manufacturer's instructions to evaluate histological therapy of the tumor, livers, kidneys, spleens, lungs, and hearts of mice. The prepared slices of tumor, livers, kidneys, spleens, lungs, and hearts were visualized under an inverted fluorescence microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eStatistical analysis and reproducibility\u003c/h2\u003e\u003cp\u003eThe experimental data are presented as means ± standard deviation (means ± SD). The statistical analyses were conducted by two-tailed Student's t-test or one-way analysis of variance. Survival curves were analyzed by using the log-rank (Mantel-Cox) test. Statistical significance was defined as *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, no significance (ns). Each experiment in this study was designed to use the minimum number of animals required to obtain informative results. Tumor-bearing mice were randomized before treatment. No data were excluded from the analysis.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Experimental Section","content":"\u003ch2\u003eMaterials\u003c/h2\u003e\u003cp\u003e1,3,5-triformylbenzene (TFB) and dithioacetamide (DTO) were obtained from Tengqian Biotechnology Co., Ltd. (Shanghai, China). 1,3-diphenylisobenzofuran (DPBF), 2,2,6,6-tetramethylpiperidine (TEMP) and N,N-dimethylformamide (DMF) were purchased from Energy Chemicals (Shanghai, China) without other purification. Other chemicals were obtained from Sinopharm Chemical Reagent (SCR) and used as received. tirapazamine (TPZ) was synthesized according to previous literature\u003csup\u003e39\u003c/sup\u003e. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 2′,7′-Dichlorofluorescein diacetate (DCFH-DA), 4′,6-diamidino-2-phenyl-indole (DAPI), calcein-AM, doxorubicin hydrochloride and propidium iodide (PI) cell apoptosis kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Gibco Life Technologies (USA). All cell lines used in this study were obtained from the Chinese Type Culture Collection (Wuhan University). The animal experiments were completed at Huazhong Agricultural University. All animal experiments conformed to the guidelines of the Chinese Regulations for the Administration of Affairs Concerning Experimental Animals and were approved by the Institutional Animal Care and Use Committee of Huazhong Agricultural University, China (HZAUMO-2023–0311).\u003c/p\u003e\u003ch2\u003eSynthesis of DT-COF\u003c/h2\u003e\u003cp\u003eTo synthesize DT-COF nanowires, TFB (0.4 mmol, 1.0 equiv.) and DTO (0.6 mmol, 1.5 equiv.) were added into 10 mL of DMF after 1 h of nitrogen blowdown. The reaction mixture was stirred vigorously at 157°C for 6 h. After cooling to room temperature, the reaction mixture was then filtered. The yellow precipitate was collected and washed 3 times with DMF, then dispersed in a phosphate buffer solution (PBS) for further use.\u003c/p\u003e\u003ch2\u003ePreparation of TPZ@DT-COF\u003c/h2\u003e\u003cp\u003eTo encapsulate the TPZ into the DT-COF, 5 mg of DT-COF dispersed in PBS was co-mixed with 5 mg of TPZ in PBS solution co-solubilized with DMSO and then stirred for 24 h at room temperature. The mixed solution was washed by centrifugation until the supernatant was free of TPZ. The loading of doxorubicin hydrochloride was done by stirring in PBS solution and in the same steps as described above.\u003c/p\u003e\u003ch2\u003eCharacterization of DT-COF and TPZ@DT-COF\u003c/h2\u003e\u003cp\u003eMorphology and elemental distribution of DT-COF were assessed by scanning electron microscope (SEM; Nano Surface Division, Bruker, USA) and transmission electron microscopy (TEM; Hitachi HT7700, Japan) at 200 kV. Particle size and zeta potential of DT-COF and TPZ@DT-COF were analyzed with Zetasizer Nano-S90 (Malvern Instruments, UK). The photophysical and chemical properties of DT-COF were detected by UV-Vis and UV-Vis-NIF spectrometers (UV-2600 and 3700 DUV, Shimadzu). The loading of TPZ and Doxorubicin hydrochloride was visualized by applying a UV-vis detector (UV-2600, Shimadzu). To verify the macropores and mesopores in DT-COF, Brunauer-Emmett-Teller (BET) surface area analysis was measured with a Micromeritics ASAP 2420 analyzer. Powder X-ray diffraction (PXRD) was performed using a Rigaku MiniFlex600 Focus Powder Diffractometer with Cu Kα line focused radiation. Confocal laser scanning microscope images were acquired with a Nikon AX and an Olympus FVMPE-RS equipped with the femtosecond laser.\u003c/p\u003e\u003ch2\u003eDensity functional theory calculations\u003c/h2\u003e\u003cp\u003eGeometric optimization of the position of the unit of DT-COF and the closed DT-COF, as well as calculation of HOMO and LUMO were using the Dmol\u003csup\u003e3\u003c/sup\u003e module in the Materials Studio software. All calculations were performed using the generalized gradient approximation (GGA) with the Perdew–Burke–Ernzerh (PBE) of functional in conjunction with the double number plus polarization function (DNP). In addition, the dipole moment was calculated by Chem3D (PerkinElmer, version 19.0.0.22).\u003c/p\u003e\u003ch2\u003eEncapsulated content and encapsulated efficiency\u003c/h2\u003e\u003cp\u003eTo determine the encapsulated content (EC) and encapsulated efficiency (EE) of TPZ and doxorubicin hydrochloride, the standard curves of TPZ and doxorubicin hydrochloride were constructed based on their UV-vis absorption. EC and EE were respectively calculated as follows: EC% = The weight of TPZ or doxorubicin hydrochloride in DT-COF∕The total weight of nanosystem, EE% = The weight of loaded TPZ or doxorubicin hydrochloride in DT-COF∕The feed weight of TPZ or doxorubicin hydrochloride, Eq.\u0026nbsp;1.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2PF spectroscopy and two-photon absorption (2PA) cross-section\u003c/b\u003e\u003c/p\u003e\u003cp\u003e2PF spectra were obtained by a femtosecond laser pulse and a Ti: sapphire system (650–820 nm, 80 MHz, 140 fs) as the light source. The concentration of DT-COF was 100 µg mL\u003csup\u003e− 1\u003c/sup\u003e. The reference sample is Fluorescein with a concentration of 1.0 × 10\u003csup\u003e− 3\u003c/sup\u003e M. 2PA cross-section was calculated by using the following equation:\u003c/p\u003e\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\sigma\\:={\\sigma\\:}_{\\text{r}\\text{e}\\text{f}}\\frac{{{\\varnothing}}_{\\text{r}\\text{e}\\text{f}}{\\text{c}}_{ref}{\\text{n}}_{ref}F}{{\\varnothing}\\text{c}\\text{n}{F}_{\\text{r}\\text{e}\\text{f}}}$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHere, ref stands for reference sample, \u003cem\u003eδ\u003c/em\u003e is the two-photon absorption cross-section, Ф is fluorescence quantum yield, c is the concentration of the sample, n is the refractive index, and F is two-photon fluorescence integral area. The value of the two-photon absorption cross-section of the reference sample is derived from the literature [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003e3PF spectroscopy and 3PA cross-section\u003c/b\u003e\u003c/p\u003e\u003cp\u003e3PF spectra were obtained by Coherent Astrella + TOPAS Prime (1050–1300 nm, 1 kHz, 120 fs) as the light source. The reference sample is rhodamine 6G (1.0 × 10\u003csup\u003e− 3\u003c/sup\u003e M). The concentration of DT-COF was 100 µg mL\u003csup\u003e− 1\u003c/sup\u003e. The three-photon absorption cross-section was calculated by using the following equation:\u003c/p\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\sigma\\:=\\frac{{\\gamma\\:}}{{N}_{A}{d}_{0}{10}^{-3}}\\times\\:{\\left(\\frac{hc}{\\lambda\\:}\\right)}^{2}$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHere, γ is the three-photon absorption coefficient, λ is the wavelength of the incident light, N\u003csub\u003eA\u003c/sub\u003e is the Avogadro constant, and d\u003csub\u003e0\u003c/sub\u003e is the concentration of the sample (0.1 mg mL\u003csup\u003e− 1\u003c/sup\u003e) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003eEvaluation of singlet oxygen generation\u003c/h2\u003e\u003cp\u003eDPBF was used as an indicator to study the singlet oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e) generation measuring the attenuation of DPBF absorbance at 425 nm by UV-vis spectrophotometer. 8 µL DMSO solution of DPBF (2.7 mg mL-1) was mixed with an aqueous solution of DT-COF, (1 mL, 100 µg mL\u003csup\u003e− 1\u003c/sup\u003e). Under 808 nm laser irradiation, the absorbance changes of DPBF at 280 nm were recorded at regular time points.\u003c/p\u003e\u003cp\u003eNext, to detect the generation of intracellular \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e, DCFH-DA was used as the indicator with inverted fluorescence microscope imaging. Briefly, 4T1 cells were seeded in 6-well plates for 24 h and then incubated with the medium containing PBS, PBS + Light, DT-COF and DT-COF + Light (in hypoxia or normoxia conditions) for another 4 h. For hypoxia conditions, the Oxygen content was regulated at 1% with N\u003csub\u003e2\u003c/sub\u003e. Subsequently, the cells were treated with 10 µM DCFH-DA for 30 min before observation.\u003c/p\u003e\u003ch2\u003eCell culture and cellular uptake\u003c/h2\u003e\u003cp\u003eThe mouse fibroblast cells (L929) and mouse breast cancer cells (4T1) were cultured in DMEM medium with the addition of 10% FBS, and 1% penicillin/streptomycin in an atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e at 37°C to evaluate the biocompatibility and the therapeutic effects of DT-COF and TPZ@DT-COF. To evaluate the cellular uptake, 4T1 cells were incubated with DT-COF or TPZ@DT-COF at 37°C at different times. After washing with PBS, cells were collected and analyzed by CLSM and flow cytometry. For multiphoton fluorescence imaging, 100 µg mL\u003csup\u003e− 1\u003c/sup\u003e DT-COF was co-cultured with 4T1 cells for different times and the imaging effects of 1PF, 2PF and 3PF in the 500–600 nm range were monitored using 405 nm, 808 nm and 1250 nm excitation light at 0.2 W cm\u003csup\u003e− 2\u003c/sup\u003e, respectively. In addition, the mitochondrial targeting and nuclear delivery capabilities of TPZ@DT-COF were detected by fluorescent probe co-localization analysis.\u003c/p\u003e\u003ch2\u003eIn vitro cellular cytotoxicity\u003c/h2\u003e\u003cp\u003e4T1 cell line was cultured at a density of 10\u003csup\u003e4\u003c/sup\u003e cells per well in 96-well plates under normoxic or hypoxic conditions, respectively, for 24 h. A fresh culture medium containing serial concentrations of TPZ@DT-COF was added to each well to incubate with cells for another 24 h. The cells in the PDT group were exposed to an 808 nm laser at the power density of 200 mW cm\u003csup\u003e− 2\u003c/sup\u003e for 5 min. The cells in the chemotherapy group without light exposure remained in the dark. After being further incubated for another 24 h, and then the fresh culture medium containing 5 mg mL\u003csup\u003e− 1\u003c/sup\u003e MTT was added to incubate for another 4 h to generate formazan crystals. The purple formazan crystals in each well were dissolved by 150 µL DMSO and the absorbance was detected on a microplate reader at a wavelength of 490 nm.\u003c/p\u003e\u003cp\u003eIn addition, cell apoptosis was further visualized by an inverted fluorescence microscope with Calcein-AM/PI double staining. Generally, the cells were seeded in a 24-well plate and treated in the same way mentioned before, the concentration of DT-COF and TPZ@DT-COF was 15 µg mL\u003csup\u003e− 1\u003c/sup\u003e and 25 µg mL\u003csup\u003e− 1\u003c/sup\u003e (TPZ content was 10.8 µg mL\u003csup\u003e− 1\u003c/sup\u003e). After being washed with PBS twice, the cells were stained with Calcein-AM (green fluorescence, live cells) and PI (red fluorescence, dead cells) for 15 min. Finally, the culture medium was replaced with fresh PBS before microscope observation.\u003c/p\u003e\u003ch2\u003eHemolysis assay\u003c/h2\u003e\u003cp\u003eAs for the hemolysis assay, whole blood of mice was collected and washed using PBS to obtain red blood cells (RBCs), which were further mixed with water, PBS, 125, 250, and 500 µg mL\u003csup\u003e− 1\u003c/sup\u003e DT-COF. All samples were incubated at 37°C for 6 hours, and the supernatants were centrifuged to test for UV absorption at 450 nm to assess the rate of hemolysis. All samples were incubated at 37°C for 6 h and centrifuged to obtain supernatant for hemolysis detection.\u003c/p\u003e\u003ch2\u003eTumor model\u003c/h2\u003e\u003cp\u003eThe right lower limb of mice was subcutaneously inoculated with 1×10\u003csup\u003e6\u003c/sup\u003e 4T1 cells to establish a hypoxic tumor model. The tumor volume was calculated as V = d\u003csup\u003e2\u003c/sup\u003e × D∕2 (d and D: the shortest point and longest point of the tumor, respectively).\u003c/p\u003e\u003ch2\u003eIn vivo multiphoton fluorescence imaging study after intratumoral injection\u003c/h2\u003e\u003cp\u003eWhen the tumors reached about 80–100 mm\u003csup\u003e3\u003c/sup\u003e, DT-COF was administrated intratumorally at an equivalent dose (10 mg kg\u003csup\u003e− 1\u003c/sup\u003e). Then, solid tumors were then dissected and subjected to multiphoton fluorescence imaging of deep tissues.\u003c/p\u003e\u003ch2\u003eIn vivo antitumor effect after intratumoral injection\u003c/h2\u003e\u003cp\u003eMice were randomly divided into 4 groups (n = 5) with treatments of PBS + L, DT-COF, DT-COF + L, TPZ@DT-COF, and TPZ@DT-COF + L (10 mg kg\u003csup\u003e− 1\u003c/sup\u003e based on DT-COF). Two hours after intratumoral administration, the treatment groups used for laser activation were irradiated continuously for 5 minutes using a 200 mW cm\u003csup\u003e− 2\u003c/sup\u003e 808 nm laser, and the tumor volumes and body weights were recorded every two days. At the end day of the treatment, mice were euthanized, and the tumors in different groups were collected for final observation. The histological study was performed with hematoxylin/eosin (H\u0026amp;E) staining and immunofluorescent staining of TUNEL on tumors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eSupporting Information\u003c/h2\u003e\u003cp\u003eThe online version contains supplementary material available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/******\u003c/span\u003e\u003cspan address=\"10.1186/******\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003eAll animal experiments conformed to the guidelines of the Chinese Regulations for the Administration of Affairs Concerning Experimental Animals and were approved by the Institutional Animal Care and Use Committee of Huazhong Agricultural University, China (HZAUMO-2023-0311).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.L. and L.H. conceived the project idea, analyzed the data, and wrote the paper. S.L., Y.L. and G.D. prepared and characterized the nanosystem. L.H. and Y.W. performed the cell and animal experiments. All authors read and edited the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eThis work was financially supported by the following funding: Hubei Provincial Natural Science Foundation of China (Grant No. 2025AFB070), State Key Laboratory of New Textile Materials and Advanced Processing (FZ20230024).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data are available in the main text, supporting information, and are also on request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHopper C. Photodynamic therapy: a clinical reality in the treatment of cancer. Lancet Oncol. 2000;1:12\u0026ndash;219.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKarges J. Clinical development of metal complexes as photosensitizers for photodynamic therapy of cancer. Angew Chem Int Edit. 2022;61:e202112236.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLv L, Fan B, Ji X, Liu Y, Chen T, Li Y, Gao X, Chen P, Tang B, Chen G. From the clinical perspective of photodynamic therapy and photothermal therapy: structure-activity-practice. Coordin Chem Rev. 2024;507:215733.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePham TC, Nguyen VN, Choi Y, Lee S, Yoon J. Recent strategies to develop innovative photosensitizers for enhanced photodynamic therapy. Chem Rev. 2021;121:13454\u0026ndash;619.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi M, Xu Y, Peng X, Kim JS. From low to no O\u003csub\u003e2\u003c/sub\u003e-dependent hypoxia photodynamic therapy (hPDT): a new perspective. Acc Chem Res. 2022;55:3253\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Y, Du L, Li F, Deng Z, Zeng S. Intelligent nanotransducer for deep-tumor hypoxia modulation and enhanced dual-photosensitizer photodynamic therapy. ACS Appl Mater Interfaces. 2022;14:14944\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao X, Liu J, Fan J, Chao H, Peng X. Recent progress in photosensitizers for overcoming the challenges of photodynamic therapy: from molecular design to application. Chem Soc Rev. 2021;50:4185\u0026ndash;219.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Z, Lu J, Li X. Recent progress in thermally activated delayed fluorescence photosensitizers for photodynamic therapy. Chem Eur J. 2024;30:e202401001.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen K, He P, Wang Z, Tang BZ. A feasible strategy of fabricating type I photosensitizer for photodynamic therapy in cancer cells and pathogens. ACS Nano. 2021;15:7735\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang J, Gong Q, Jiao L, Hao E. Research advances in BODIPY-assembled supramolecular photosensitizers for photodynamic therapy. Coordin Chem Rev. 2023;496:215367.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTavakkoli Yaraki M, Liu B, Tan YN. Emerging strategies in enhancing singlet oxygen generation of nano-photosensitizers toward advanced phototherapy. Nano-micro lett. 2022;14:123.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen J, Zhou Q, Cao W. Multifunctional porphyrin-based sonosensitizers for sonodynamic therapy. Adv Funct Mater. 2024;34:2405844.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJuvekar V, Lee DJ, Park TG, Samanta R, Kasar P, Kim C, Rotermund F, Kim HM. Two-photon excitation photosensitizers for photodynamic therapy: From small-molecules to nano-complex systems. Coordin Chem Rev. 2024;506:215711.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCao H, Fang B, Liu J, Shen Y, Shen J, Xiang P, Zhou Q, De Souza SC, Li D, Tian Y, Luo L, Zhang Z, Tian X. Photodynamic therapy: photodynamic therapy directed by three-photon active rigid plane organic photosensitizer. Adv Healthc Mater. 2021;10:2001489.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu Z, Jiang Y, Shen Y, Tang L, Hu Z, Lin G, Law WC, Ma M, Dong B, Yong KT, Xu G. A biocompatible photosensitizer with a high intersystem crossing efficiency for precise two-photon photodynamic therapy. Mater Horiz. 2022;9:1283\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTian S, He J, Lyu D, Li S, Xu QH. Aggregation enhanced photoactivity of photosensitizer conjugated metal nanoparticles for multimodal imaging and synergistic phototherapy below skin tolerance threshold. Nano Today. 2022;45:101534.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDeng X, Zhao R, Tang Y, Yi M, Wang D, Lin W, Wang G. FeS\u003csub\u003e2\u003c/sub\u003e@COF based nanocarrier for photothermal-enhanced chemodynamic/thermodynamic tumor therapy and immunotherapy via reprograming tumor-associated macrophages. J Nanobiotechnol. 2024;22:711.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMa J, Shu T, Sun Y, Zhou X, Ren C, Su L, Zhang X. Luminescent covalent organic frameworks for biosensing and bioimaging applications. Small. 2022;18:2103516.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi S, Zhang K, Ma Z, Zhang W, Song Z, Wang W, Han H. Biomimetic nanoplatelets to target delivery hirudin for site-specific photothermal/photodynamic thrombolysis and preventing venous thrombus formation. Small. 2022;18:2203184.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSeki S, Paitandi RP, Choi W, Ghosh S, Tanaka T. Electron transport over 2D molecular materials and assemblies. Acc Chem Res. 2024;57:2665\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhou Y, Jing S, Liu S, Shen X, Cai L, Zhu C, Zhao Y, Pang M. Double-activation of mitochondrial permeability transition pore opening via calcium overload and reactive oxygen species for cancer therapy. J Nanobiotechnol. 2022;20:188.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang L, Wang S, Zhou Y, Wang C, Zhang XZ, Deng H. Covalent organic frameworks as favorable constructs for photodynamic therapy. Angew Chem Int Edit. 2019;58:14213\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZeng JY, Wang XS, Xie BR, Li MJ, Zhang XZ. Covalent organic framework for improving near-infrared light induced fluorescence imaging through two‐photon induction. Angew Chem Int Edit. 2020;59:10087\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchmidt K, Leclercq A, Zojer E, Lawson PV, Jang SH, Barlow S, Jen A, K-Y, Marder SR, Br\u0026eacute;das JL. Order of magnitude effects of thiazole regioisomerism on the near-IR two‐photon cross‐sections of dipolar chromophores. Adv Funct Mater. 2008;18:794\u0026ndash;801.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBeverina L, Fu J, Leclercq A, Zojer E, Pacher P, Barlow S, Van Stryland EW, Hagan DJ, Br\u0026eacute;das J-L, Marder SR. Two-photon absorption at telecommunications wavelengths in a dipolar chromophore with a pyrrole auxiliary donor and thiazole auxiliary acceptor. J Am Chem Soc. 2005;127:7282\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKang G, Nasiri Avanaki K, Mosquera MA, Burdick RK, Villabona-Monsalve JP, Goodson T III, Schatz GC. Efficient modeling of organic chromophores for entangled two-photon absorption. J Am Chem Soc. 2020;142:10446\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBramley SE, Dupplin V, Goberdhan DG, Meakins GD. The hantzsch thiazole synthesis under acidic conditions: change of regioselectivity. J Chem Soc. 1987; 639\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu CJ, Li XY, Li TR, Shao MZ, Niu LJ, Lu XF, Kan JL, Gen Y, Dong YB. Natural sunlight photocatalytic synthesis of benzoxazole-bridged covalent organic framework for photocatalysis. J Am Chem Soc. 2022;144:18750\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEl-Kaderi HM, Hunt JR, Mendoza-Cort\u0026eacute;s JL, C\u0026ocirc;t\u0026eacute; AP, Taylor RE, O'Keeffe M, Yaghi OM. Designed synthesis of 3D covalent organic frameworks. Science. 2007;316:268\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi B, Lu X, Tian Y, Li D. Embedding multiphoton active units within metal-organic frameworks for turning on high-order multiphoton excited fluorescence for bioimaging. Angew Chem Int Edit. 2022;61:e202206755.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMatito E, Sol\u0026agrave; M. The role of electronic delocalization in transition metal complexes from the electron localization function and the quantum theory of atoms in molecules viewpoints. Coordin Chem Rev. 2009;253:647\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZou Q, Abbas M, Zhao L, Li S, Shen G, Yan X. Biological photothermal nanodots based on self-assembly of peptide-porphyrin conjugates for antitumor therapy. J Am Chem Soc. 2017;139:1921\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHu L, Xu Y, Zhao Y, Mei Z, Xiong C, Xiao J, Zhang J, Tian J. Supramolecular nanovesicles with in-situ switchable photothermal/photodynamic effects for precisely controllable cancer phototherapy. Chem Eng J. 2023;476:146829.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnbazhagan R, Krishnamoorthi R, Kumaresan S, Thankachan D, Van DTT, Wang JS, Tsai HC. Benzobisthiazole-bridged white fluorescent emitting covalent organic framework for simultaneous mercury detection and removal. React Funct Polym. 2021;169:105083.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhu X, Tian C, Jin T, Wang J, Mahurin SM, Mei W, Xiong Y, Hu J, Feng X, Liu H, Dai S. Thiazolothiazole-linked porous organic polymers. Chem Commun. 2014;50:15055\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHou Y, Zhou P, Liu F, Tong K, Lu Y, Li Z, Liang J, Tong M. Rigid covalent organic frameworks with thiazole linkage to boost oxygen activation for photocatalytic water purification. Nat Commun. 2024;15:7350.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFeng Z, Zhu T, Wang L, Yuan T, Jiang Y, Tian X, Tian Y, Zhang Q. Coordination-regulated terpyridine-Mn(II) complexes for photodynamic therapy guided by multiphoton fluorescence/magnetic resonance imaging. Inorg Chem. 2022;61:12652\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHu L, Cao J, Lv Y. A mitochondria-targeted biocompatible photothermal nanoscale hydrogen-bonded organic framework for effective multimodal-imaging guided breast cancer therapy. Chem Eng J. 2024;492:152270.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShi X, Mandel SM, Platz MS. On the mechanism of reaction of radicals with tirapazamine. J Am Chem Soc. 2007;129:4542\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang Q, Lu X, Wang H, Tian X, Wang A, Zhou H, Wu J, Tian Y. A benzoic acid terpyridine-based cyclometalated iridium(III) complex as a two-photon fluorescence probe for imaging nuclear histidine. Chem Commun. 2018;54:3771\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFeng Z, Li D, Zhang M, Shao T, Shen Y, Tian X, Zhang Q, Li S, Wu J, Tian Y. Enhanced three-photon activity triggered by the AIE behaviour of a novel terpyridine-based Zn(II) complex bearing a thiophene bridge. Chem Sci. 2019;10:7228\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Multiphoton, Covalent-organic framework, Drug delivery, Photodynamic-chemotherapeutic therapy, Deep-seated tumor","lastPublishedDoi":"10.21203/rs.3.rs-7635511/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7635511/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMultiphoton photosensitizers activated by near-infrared light have attracted considerable attention in the fields of homogeneous photocatalysis, optical imaging, and phototherapy because of their negligible interference, low dilapidation, and deep penetration. Nonetheless, most multiphoton photosensitizers still face issues such as poor long-range ordering, strong π-π stacking hindrances, and potential metal toxicity. In this study, a rationally designed thiazole-based covalent organic framework (DT-COF) with multiphoton activity was efficiently harvested via one-step preparation from inactive small molecules\u0026mdash;namely, dithiooxamide and 1,3,5-triformylbenzene. Theoretical calculation and characterization tests revealed that the good performances of deeper multiphoton fluorescence and photodynamic therapy benefited from the donor-π-acceptor configuration, highly ordered long-range structure, weakened π-π stacking interaction between the layers, and good nonlinear optical properties. Furthermore, tirapazamine (TPZ), a hypoxia-activated chemotherapeutic drug, was encapsulated into DT-COF pores with anastomosing dimensions (encapsulation efficiency\u0026thinsp;=\u0026thinsp;75%). The DT-COF targeted the mitochondria and delivered TPZ to the nucleus. Additionally, the DT-COF consumed oxygen to generate \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e, promoting TPZ activation and realizing a combination of photodynamic and chemotherapeutic anti-tumor therapy. This study provides a new strategy for the development of multiphoton photosensitizers and demonstrates their potential in cancer theranostics.\u003c/p\u003e","manuscriptTitle":"Rationally Designed Thiazole-Based COF with Multiphoton Activity for Drug Delivery and Synergistic Therapy of Deep-Seated Tumors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-06 16:52:02","doi":"10.21203/rs.3.rs-7635511/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-02T15:57:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-26T08:31:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"327459914242141975731595039292369450993","date":"2025-11-25T09:21:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"277341542780414852154296202142700917660","date":"2025-11-23T03:50:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-04T02:47:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"266421958522966629675889371848481716786","date":"2025-10-29T23:06:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-27T17:36:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-19T13:05:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-17T09:39:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2025-09-17T04:08:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3bcb3bea-a35a-4506-a2f6-aa4cb171a1c6","owner":[],"postedDate":"November 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T16:02:33+00:00","versionOfRecord":{"articleIdentity":"rs-7635511","link":"https://doi.org/10.1186/s12951-026-04188-6","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2026-02-21 15:57:52","publishedOnDateReadable":"February 21st, 2026"},"versionCreatedAt":"2025-11-06 16:52:02","video":"","vorDoi":"10.1186/s12951-026-04188-6","vorDoiUrl":"https://doi.org/10.1186/s12951-026-04188-6","workflowStages":[]},"version":"v1","identity":"rs-7635511","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7635511","identity":"rs-7635511","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}