A near-infrared two-region covalent organic framework nanoprobes and a preparation method and application thereof

Abstract

The application discloses a kind of covalent organic framework nano-probes with photothermal / photodynamic therapy function and near-infrared two area / photothermal imaging function and its preparation method and application.The nano-probe is prepared by Suzuki coupling reaction from tri (4-boronic acid phenol ester phenyl) amine (TPA-3B) and 4,8-di (5-bromo-4- (2-ethylhexyl) thiophene-2-yl) benzo [1,2-C:4,5-C'] bis ([1,2,5] thiazole) (DTBDT).The nano-probe is spherical structure, particle size is uniform, has excellent near-infrared two area (NIR-II) fluorescence imaging and photothermal imaging performance, and has high photothermal conversion efficiency and active oxygen yield under single laser excitation, can realize the precise diagnosis of liver cancer and other deep tumors and photothermal / photodynamic synergistic treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical nanomaterials technology, specifically relating to a multifunctional covalent organic framework (COF) nanoprobe with near-infrared II fluorescence imaging, photothermal imaging, and photothermal / photodynamic synergistic therapy functions, and its preparation method. Background Technology

[0002] Cancer has become one of the most significant public health problems worldwide. Treatment methods for cancer mainly include surgical resection, radiotherapy, and chemotherapy, but these methods still have certain limitations.

[0003] Therapeutic technologies for cancer aim to achieve both precise diagnosis and efficient treatment of tumors simultaneously. Existing therapeutic nanomaterials are primarily limited to near-infrared I (NIR-I, 650-950 nm) imaging, resulting in shallow tissue penetration, low signal-to-noise ratios, and difficulty in accurately locating deep tumors (such as liver cancer). Furthermore, single treatment modalities (such as photothermal or photodynamic therapy alone) often fail to completely eradicate tumors, and traditional photosensitizers are prone to fluorescence quenching in the aggregated state, leading to low ROS yields.

[0004] Covalent organic frameworks (COFs), as porous crystalline materials, have advantages such as tunable structure and high stability. However, existing technologies usually require two different wavelengths of laser to excite them separately, or it is difficult to achieve high-efficiency ROS generation in the NIR-II region at the same time. Developing them into nanoprobes with both NIR-II imaging and efficient PTT / PDT synergistic functions still faces challenges. Summary of the Invention

[0005] This invention aims to solve the problems of insufficient imaging penetration depth and low treatment efficiency in the prior art, and provides a COF nanoprobe based on TPA-3B and DTBDT.

[0006] The technical solution of the present invention is as follows: This invention provides a covalent organic framework nanoprobe (COF), the chemical structural formula of which is as follows: Where n = 100~400, and is an integer; The nanoprobes are polymerized from tris(4-borate pineneol ester phenyl)amine (TPA-3B) as shown in structural formula I and 4,8-di(5-bromo-4-(2-ethylhexyl)thiophen-2-yl)benzo[1,2-C:4,5-C']bis([1,2,5]thiadiazole) (BEH-A COFs) as shown in structural formula II via a Suzuki coupling reaction. The reaction equation is as follows:

[0007] The emission spectrum of the nanoprobe covers the near-infrared II region (1000-1350 nm). The innovation of this invention lies in its specific donor-acceptor structure design, which plays a crucial role in achieving emission in the NIR-II region (1000-1350 nm), overcoming the shortcomings of many existing COFs that only emit light in the visible or NIR-I regions. The prepared covalent organic framework nanoprobe (COF) can be used for both near-infrared II fluorescence imaging and synergistic tumor therapy. This near-infrared II fluorescence imaging effectively improves the problem of insufficient tissue penetration depth in imaging. Simultaneously, COF generates reactive oxygen species (ROS) under excitation light, utilizing photodynamic effects to improve the tumor microenvironment and enhance tumor treatment efficiency. Furthermore, due to the photothermal effect, COF generates a large amount of heat, thereby effectively killing tumor cells. This simultaneous imaging and therapeutic function greatly improves the efficacy and precision of tumor treatment, which is of great significance for tumor therapy. At the same time, the enhanced near-infrared II fluorescence penetration depth helps to achieve real-time monitoring and precise localization of tumor sites. In summary, the covalent organic framework (COF) nanoprobe can effectively realize the diagnosis, imaging, and treatment of liver cancer.

[0008] In some embodiments, the nanoprobes exhibit a monodisperse spherical morphology with a particle size distribution of 80-200 nm. This size range optimizes diagnostic and therapeutic performance through biodistribution regulation, tumor targeting, EPR effect, and enhanced optical properties. Specifically, when the nanomedicine particle size is within this range, it can effectively evade rapid renal clearance, thereby prolonging its circulation time in vivo. Simultaneously, nanoparticles of this size can more effectively penetrate the intercellular spaces of vascular endothelial cells to enter tumor tissue or other target areas, achieving more precise targeted delivery.

[0009] This invention provides a method for preparing the aforementioned covalent organic framework nanoprobe, comprising the following steps: (1) Under an inert atmosphere and in the dark, TPA-3B and DTBDT were dissolved in a mixture of deoxygenated organic solvent and water; (2) Add palladium catalyst and alkaline auxiliaries to the reaction system of step (1); (3) React at 80-100℃ for 48-72 hours; (4) After the reaction is complete, the product is separated by centrifugation, and then washed with organic solvent and water and dried to obtain the solid nanoprobe.

[0010] In some embodiments, in step (1), the molar ratio of TPA-3B to DTBDT is 1:1 to 1:1.5; the mixed solvent is a mixture of tetrahydrofuran (THF) and water, with a volume ratio of 2:1 to 4:1.

[0011] In some embodiments, in step (2), the palladium catalyst is tetrakis(triphenylphosphine)palladium (Pd(PPh3)4); the alkaline auxiliary is an aqueous solution of K2CO3, Cs2CO3 or Na2CO3.

[0012] In some embodiments, in step (4), the organic solvent is selected from one or more of tetrahydrofuran, N,N-dimethylformamide, ethanol or dimethyl sulfoxide.

[0013] Application of the aforementioned covalent organic framework nanoprobe in the preparation of integrated tumor diagnostic and therapeutic formulations.

[0014] In some embodiments, the therapeutic formulation is used for dual-modal diagnosis of solid tumors of liver cancer, breast cancer, or lung cancer using near-infrared II (NIR-II) fluorescence imaging and photothermal imaging.

[0015] In some embodiments, the therapeutic formulation is used for the synergistic treatment of tumors with photothermal therapy (PTT) and photodynamic therapy (PDT).

[0016] In some embodiments, the synergistic therapy refers to the simultaneous generation of thermal energy and reactive oxygen species (ROS) to kill tumor cells under irradiation with 808 nm, 980 nm or 1064 nm laser light.

[0017] The beneficial effects of this invention are: The covalent organic framework nanoprobe (COF) of this invention has excellent NIR-II imaging capabilities: the probe emits wavelengths up to 1000-1350 nm and penetrates biological tissues to a depth of up to 8 mm, which is significantly better than traditional ICG probes (about 2 mm), enabling high signal-to-noise ratio imaging of deep tumors.

[0018] The covalent organic framework nanoprobe (COF) of this invention has a synergistic effect of integrated diagnosis and treatment: under single laser excitation at 808 nm, it simultaneously achieves photothermal heating (up to 74.2 °C at a concentration of 200 μg / mL) and the generation of a large amount of reactive oxygen species (ROS), thereby achieving synergistic enhancement of PTT and PDT, and the tumor inhibition rate can reach 89.8%.

[0019] The covalent organic framework nanoprobe (COF) of this invention has good biocompatibility and targeting: the particle size is controlled within 80-200 nm, and it is effectively enriched at the tumor site by utilizing the EPR effect, reducing toxic side effects on normal tissues. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 (A) Schematic diagram of the synthesis route of COF nanoprobes; (B) Transmission electron microscopy (TEM) image; (C) Powder image.

[0022] Figure 2 (A) Fluorescence emission spectrum; (B) Photothermal heating curve; (C) ESR spectrum (characterization of ROS generation).

[0023] Figure 3 (A) Cytotoxicity test; (B) Intracellular ROS fluorescence imaging; (CD) Cell photothermal imaging of temperature changes.

[0024] Figure 4 (AB) NIR-II fluorescence imaging; (CE) Curves showing the therapeutic effect and tumor volume change in tumor-bearing mice; (F) Comparison of chicken meat penetration depth experiment. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to represent selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1: Preparation of COF nanoprobes like Figure 1As shown in (A): Raw material preparation: Under glove box or nitrogen protection, tris(4-boronic acid pinene ester phenyl)amine (TPA-3B, 0.1 mmol) and 4,8-di(5-bromo-4-(2-ethylhexyl)thiophene-2-yl)benzo[1,2-C:4,5-C']bis([1,2,5]thiadiazole) (DTBDT, 0.15 mmol) were prepared in a molar ratio of 1:1.5. Dissolution: The above raw materials were dissolved in a deoxygenated tetrahydrofuran / water mixed solvent (volume ratio 4:2, 6 mL).

[0027] Catalytic reaction: 10 mg of Pd(PPh3)4 catalyst and 0.4 g of K2CO3 solution (2 M) were added.

[0028] Synthesis: The reaction was carried out at 80°C for 72 hours under nitrogen protection and in the dark.

[0029] Purification: After the reaction is complete, the precipitate is collected by centrifugation and washed several times with ethanol and deionized water to remove unreacted monomers and catalyst.

[0030] Drying: freeze drying yielded dark green powdered COF nanoprobes (BEH-A COFs).

[0031] Example 2: COF Characterization and Performance Testing Morphological characterization: The COF prepared in Example 1 was prepared into a 10 μM aqueous solution and its morphology was observed using a transmission electron microscope (FEIG2 Spirit Biotwin); the crystallinity of the material was characterized using a Panalytical BV (Empyrean) X-ray diffraction instrument, and the X-ray diffraction pattern of COF was obtained by computer simulation.

[0032] like Figure 1 As shown, (B) is a transmission electron microscope image of COF; (C) is a powder image of COFs, and a comparison is made between the material crystallinity measured using a Panalytical BV (Empyrean) X-ray diffractometer and the computer-simulated X-ray diffraction pattern of COF. The results show that the COF prepared in Example 1 has a spherical structure with a particle size distribution of 100–200 nm. Figure 1 B), the powder is green ( Figure 1 C). This COF exhibits good crystallinity, and its X-ray diffraction pattern is highly consistent with the simulated porous COF structure. AA stacking fitting results: Rwp = 6.94%, Rp = 5.24%. This demonstrates its highly crystalline framework characteristics and proves that we have successfully prepared a porous crystalline COF.

[0033] This invention utilizes a Suzuki coupling reaction to precisely crosslink the C3-symmetric node unit TPA-3B with the C2-symmetric connecting unit DTBDT, constructing a covalent organic framework (COF) with long-range ordered topological properties. Unlike traditional amorphous conjugated microporous polymers (CMPs), the nanoprobes prepared in this invention exhibit significant crystallinity. X-ray diffraction (XRD) analysis reveals sharp and high-intensity diffraction peaks in the low-angle region, indicating the formation of a periodically arranged pore structure within the framework. Rietveld refinement fitting of the XRD experimental data shows a high degree of fit between the measured curves and the topological model constructed based on the AA-stacking mode, with a refinement weighted residual (Rwp) of only 6.94%. This extremely low fitting error physically confirms that the monomers achieve a highly ordered geometric arrangement through covalent bonds. Furthermore, high-resolution transmission electron microscopy (HR-TEM) results further confirmed the aforementioned orderliness. Large-scale extended lattice fringes were clearly visible at the edges and interior of the nanospheres, demonstrating the long-range ordered lattice within the material. This highly ordered alternating DA (donor-acceptor) arrangement not only effectively confines the nonradiative transitions of the luminescent centers through a rigid framework but also optimizes the intramolecular charge transfer (ICT) process through π-π stacking effects. This microstructural regularity is the core mechanism underlying the present invention's nanoprobes, enabling them to achieve high-brightness fluorescence emission in the near-infrared II (NIR-II) region, deep tissue penetration (up to 8 mm), and efficient photothermal conversion, significantly outperforming structurally disordered homologous polymer materials.

[0034] Photothermal properties: The emission spectrum of COF will be obtained using an Edinburgh FL980 transient fluorescence spectrometer. Figure 2 A), and simultaneously used an infrared thermal imager (Beijing Jun Chuang Hongye Technology Co., Ltd., Ax5) to record the temperature changes of COF aqueous solutions of different concentrations every 30 seconds. Figure 2 B), using electron spin resonance laser irradiation (ESR, Bruker A300), recorded the ESR signals after irradiation (no irradiation, 808 nm, and 980 nm). Figure 2 C).

[0035] Depend on Figure 2It is known that the emission wavelength range of COF is 1100-1400 nm, with the optimal emission peak at 1300 nm. Meanwhile, COF exhibits good photothermal effect under laser irradiation (808 nm laser, 1 W / cm²), with a maximum temperature of 74.2 °C at 200 μg / mL and 60 °C at 100 μg / mL, and a photothermal conversion efficiency of 13.5%. Electron paramagnetic resonance (EPR) spectroscopy shows that COF has almost no EPR signal under dark conditions, while under laser irradiation (808 / 980 nm laser, 1 W / cm²), a significant singlet oxygen EPR peak is generated, with a ROS generation efficiency of 0.195%, comparable to traditional commercial photosensitizers (ICG literature reports a ROS generation efficiency of 0.2%).

[0036] Example 3: Antitumor effect of COF in cells CCK8 assay for the viability of HepG2 liver cancer cells HepG2 hepatocellular carcinoma cells were seeded into culture plates and cultured in the dark for 24 h. Then, the HepG2 hepatocellular carcinoma cells were incubated with COF at concentrations of 0, 20, 40, 60, 80, and 100 µg / ml for 12 h, and washed three times with PBS. Subsequently, the cells were irradiated with a laser (808 nm, 1 w / cm²) for 5 minutes, while the dark control group was not irradiated. Finally, CCK-8 solution was added, and the absorbance of the CCK-8 solution at 450 nm was measured using a microplate reader to calculate cell viability. The viability of HepG2 hepatocellular carcinoma cells was assessed as follows: Figure 3 As shown in Figure A. Figure 3 As shown in A, cells exhibit dose-dependent cytotoxicity to COF, and the synergistic effect significantly enhances cytotoxicity.

[0037] Photoexcitation of ROS production in COF cells: HepG2 liver cancer cells were seeded in culture plates and cultured in the dark for 12 h. After removing the original culture medium, the cells were incubated in the dark with PBS and COF for 6 h, then irradiated with a laser at 808 nm 1 W / cm² for 5 min, and cultured in the dark for 12 h. DCFH-DA staining was then performed. Intracellular fluorescence images were obtained using confocal laser scanning microscopy (CLSM), and the mean intracellular fluorescence intensity was measured. Figure 3 As shown in B. Figure 3 As shown in B, the relative fluorescence intensity of HepG2 liver cancer cells in the PBS culture group was 0.32. The relative fluorescence intensity of HepG2 liver cancer cells after taking up COF and not being exposed to light was 1.9, while the fluorescence intensity of the light-exposed group was 11.9, which was 6.4 times higher than that of the unexposed group. This proves that COF produced a large amount of ROS in the cells under light conditions.

[0038] COF cells were taken and centrifuged in centrifuge tubes to precipitate them. The COF-cell solution was then irradiated with an 808 nm laser for 5 min, and temperature changes were recorded every 30 s using an infrared thermal imager (Beijing Jun Chuang Hongye Technology Co., Ltd., Ax5). Figure 3 As shown in C and D. From Figure 3 As shown in C and D, the temperature of COF- cells increases with time, and its highest temperature can reach 45.62℃, while the PBS blank control group aqueous solution is only 36.9℃.

[0039] Example 4: NIR-II fluorescence imaging of fluorescence intensity under different wavelengths of excitation light COF nanoprobes were uniformly placed in a 96-well plate, and the samples were excited with excitation light at 808 nm, 980 nm, and 1064 nm, respectively. Near-infrared II imaging images of COF were captured under filters at 1000 nm, 1100 nm, 1200 nm, 1300 nm, and 1400 nm, and the fluorescence intensity was recorded. Figure 4 As shown in A and B. From Figure 4 As shown in A and B, COF emission images can be recorded with excitation light at 808nm, 980nm, and 1064nm and with various filters, indicating that COF has the potential for channeled fluorescence imaging.

[0040] Example 5: In vivo antitumor experiment Model: HepG2 liver cancer-bearing mice.

[0041] Specific steps: PBS, ICG (Shanghai Jizhi Biochemical Technology Co., Ltd.), and COF were injected into tumor-bearing mice via the tail vein, and the mice were divided into PBS, PBS-L, ICG-L, and COF-L groups. After a period of time following injection, the tumor sites of the mice in the PBS-L, ICG-L, COF-L, and COF-LP groups were irradiated with near-infrared light for 5 minutes. To monitor the treatment effect, photographs were taken on days 5, 9, and 13 to observe the size of the tumors.

[0042] like Figure 4 As shown in CE, COF was administered via tail vein injection, once every two days, divided into three doses. Following injection, the patient was irradiated with an 808nm laser for 5 minutes at a power of 1W / cm². 2 . Figure 4 CE images show the treatment of HepG2 liver cancer-bearing mice at different time points, as well as the tumor size at different time points. Among them, the tumors of mice in the COF-L group were significantly inhibited, with a tumor inhibition rate of up to 89.8%.

[0043] like Figure 4As shown in F, chicken meat of different thicknesses was covered in capillaries containing COF aqueous solution for near-infrared II imaging. Currently, the only commercially available near-infrared probe in clinical use—ICG—has a reported penetration depth of only 2 mm in chicken meat, while COF near-infrared II imaging can achieve a penetration depth of 8 mm in chicken meat, demonstrating better penetration depth.

[0044] The covalent organic framework (COF) nanoprobes, their preparation methods, and applications described in this invention have the following significant inventiveness and unexpected technical effects compared to existing technologies: 1. Unique DA framework design and synergistic optimization of NIR-II performance: In the prior art, materials based on benzobisthiadiazole (BBT) derivatives are mostly found in amorphous conjugated polymers (CPs). This invention creatively uses tris(4-boronic acid pinene ester phenyl)amine (TPA-3B) as the donor unit, and constructs a highly crystalline COF structure via a Suzuki coupling reaction with a 4,8-di(5-bromo-4-(2-ethylhexyl)thiophene-2-yl)benzo[1,2-C:4,5-C']bis([1,2,5]thiadiazole) (DTBDT) acceptor unit.

[0045] Experimental data (see Example 2 and XRD data) show that the material exhibits clear lattice fringes and a highly ordered AA stacking pattern (Rwp = 6.94%). This highly ordered molecular arrangement effectively enhances the intramolecular electron transfer (ICT) effect between DA units, successfully stretching the emission spectrum to the near-infrared II region (1000-1350 nm). Compared to amorphous materials, the COF structure described in this invention reduces nonradiative transitions of excited-state energies through a rigid framework, thereby achieving deep tissue penetration (penetration depth up to 8 mm or more) while ensuring excellent imaging resolution.

[0046] 2. Highly efficient integrated diagnosis and treatment capabilities under single laser excitation: The nanoprobe of this invention achieves deep coupling of photothermal (PTT) and photodynamic (PDT) effects under a single 808 nm laser excitation, solving the problem that traditional probes require multiple excitation sources or auxiliary components.

[0047] According to the experimental results of Example 3, after cells took up COF nanoprobes, the intensity of intracellular reactive oxygen species (ROS) production under 808 nm laser irradiation increased significantly by 6.4 times compared to the unirradiated group. Simultaneously, the highest temperature of the cell solution containing the nanoprobes reached 45.62℃, significantly higher than the blank control group (36.9℃). This ability to simultaneously excite efficient electron-hole pair generation and photothermal conversion efficiency at a single wavelength not only simplifies the diagnostic and treatment process but also accelerates blood circulation through the thermal effect of photothermal generation, thereby alleviating the hypoxic state of the tumor microenvironment and further enhancing the photodynamic therapy effect, achieving a synergistic therapeutic goal of "1+1>2".

[0048] 3. Excellent biocompatibility and morphological stability: The COF nanoprobes prepared in this invention exhibit a regular monodisperse spherical morphology with a particle size distribution between 80 and 200 nm. This size range generates an enhanced penetration and retention (EPR) effect, enabling them to accumulate more effectively at tumor sites. Furthermore, due to the strong covalent bond support of COF materials, compared to easily photobleached small-molecule fluorescent dyes, these probes possess extremely high structural stability and resistance to photobleaching in the complex environment of the body, providing a reliable material basis for the precise monitoring and continuous treatment of deep tumors in clinical practice.

[0049] In summary, this invention is not only novel in terms of material structure design, but also demonstrates significantly superior near-infrared II imaging depth and synergistic treatment efficiency compared to existing technologies in biomedical applications, possessing extremely high clinical translational value.

Claims

1. A covalent organic framework nanoprobe, characterized in that, The nanoprobe was prepared via a Suzuki coupling reaction, and its chemical structure is as follows: Where n = 100~400, and is an integer; The emission spectrum of the nanoprobe covers the near-infrared II region (1000-1350 nm).

2. The covalent organic framework nanoprobe according to claim 1, characterized in that, The nanoprobes exhibit a monodisperse spherical morphology with a particle size distribution of 80-200 nm.

3. A method for preparing the covalent organic framework nanoprobe according to claim 1 or 2, characterized in that, Includes the following steps: (1) Under an inert atmosphere and light-protected conditions, TPA-3B and DTBDT are dissolved in a mixed solvent of deoxygenated organic solvent and water, wherein the molar ratio of TPA-3B to DTBDT is 1:1 to 1:1.5; (2) Add palladium catalyst and alkaline auxiliaries to the reaction system of step (1); (3) React at 80-100℃ for 48-72 hours; (4) After the reaction is complete, the product is separated by centrifugation, and then washed with organic solvent and water and dried to obtain the solid nanoprobe.

4. The preparation method according to claim 3, characterized in that: In step (1), the mixed solvent is a mixture of tetrahydrofuran (THF) and water in a volume ratio of 2:1 to 4:

1.

5. The preparation method according to claim 3, characterized in that: In step (2), the palladium catalyst is tetraphenylphosphine palladium (Pd(PPh3)4); the alkaline auxiliary agent is an aqueous solution of K2CO3, Cs2CO3 or Na2CO3.

6. The preparation method according to claim 3, characterized in that: In step (4), the organic solvent is selected from one or more of tetrahydrofuran, N,N-dimethylformamide, ethanol or dimethyl sulfoxide.

7. The application of the covalent organic framework nanoprobe according to claim 1 in the preparation of integrated tumor diagnosis and treatment formulations.

8. The application according to claim 7, characterized in that, The therapeutic formulation is used for dual-modal diagnosis of solid tumors of liver cancer, breast cancer, or lung cancer using near-infrared II (NIR-II) fluorescence imaging and photothermal imaging.

9. The application according to claim 7, characterized in that, The aforementioned integrated diagnostic and therapeutic formulation is used for the synergistic treatment of tumors using photothermal therapy (PTT) and photodynamic therapy (PDT).

10. The application according to claim 9, characterized in that, The synergistic therapy refers to the simultaneous generation of heat energy and reactive oxygen species (ROS) to kill tumor cells under irradiation with 808 nm, 980 nm or 1064 nm lasers.

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