I-type photodynamic COF-based liver-targeted therapeutic drug as well as preparation method and application of I-type photodynamic COF-based liver-targeted therapeutic drug

Abstract

The invention discloses a liver-targeted therapeutic drug based on I-type photodynamic COF as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The preparation method comprises the following steps: firstly, synthesizing a novel I-type photosensitizer, modifying imine type COF with the photosensitizer to obtain a series of COF-E with I-type photodynamic capability, then loading a chemotherapeutic drug, and modifying a targeting molecule on the surface of a material through a pi-pi accumulation effect. The biocompatibility and cytotoxicity of the obtained liver tumor targeted nano therapeutic drug are evaluated through the cellular level. The liver tumor targeted therapeutic drug has good stability, can be triggered to release by a tumor microenvironment, and can play a synergistic therapeutic role of type I photodynamic therapy and chemotherapy; in addition, the compound has a good treatment effect in a subcutaneous hypoxia solid tumor model, and is expected to be applied to clinical research.

Description

Technical Field

[0001] This invention relates to a liver-targeted therapeutic drug based on type I photodynamic COF, its preparation method, and its application, belonging to the field of biomedical technology. Background Technology

[0002] Hepatocellular carcinoma (HCC) remains a serious global health challenge with a persistently high mortality rate. Tumor hypoxia, a common feature of solid tumors, plays a crucial role in the development of HCC and its resistance to treatment. Cancer stem cells (CSCs) are increasingly recognized as a core factor driving tumor progression and treatment resistance. Numerous studies have demonstrated the presence of cancer stem cells in HCC, and these cells can survive and proliferate in hypoxic environments, resulting in significant resistance to chemotherapy and radiotherapy, leading to treatment failure and disease recurrence. Therefore, there is an urgent need to develop effective strategies to target and eliminate cancer stem cells enriched in hypoxic regions of HCC.

[0003] Despite significant advancements in nanocarrier technology, tumor hypoxia remains a critical obstacle. Photodynamic therapy (PDT) has emerged as a clinically viable solution due to its non-invasive nature and synergistic effect with chemotherapy. However, conventional type II PDT relies on the presence of oxygen, limiting its efficacy in hypoxic tumors. In contrast, type I PDT generates superoxide anions (O2) through electron transfer. -• ) and hydroxyl radicals (OH) • Reactive oxygen species (ROS) such as photosensitizers are not oxygen-dependent and therefore show effectiveness in hypoxic tumors. However, the highly conjugated structure of photosensitizers leads to poor solubility in aqueous solutions and a tendency to undergo aggregation-induced quenching (ACQ), thereby reducing O2. -• The generation of photosensitizer molecules weakens the therapeutic effect. To address these issues, micellar encapsulation strategies are widely adopted. However, the tight packing of photosensitizer molecules in these systems still inhibits the generation of reactive oxygen species due to the ACQ effect. Notably, existing reports (ACS Nano, 2024, 18(52), 35671-35683) indicate that the structure of covalent organic frameworks (COFs) helps prevent ACQ, thanks to the unique ordered and rigid arrangement of molecular units within their layers and the dislocation stacking between layers (interlayer confinement effect). However, the synthesis conditions of the COFs involved are generally extremely harsh, requiring high temperature and high pressure, which is not conducive to large-scale production. Summary of the Invention

[0004] Technical issues: This invention addresses the problem of aggregation-induced quenching (ACQ) in the aforementioned photosensitizers by providing a novel photosensitizer.

[0005] Furthermore, this invention addresses the aforementioned challenges of insufficient drug targeting, strong side effects, and the need to improve tumor hypoxia associated with traditional chemotherapy drugs. It proposes a type I photodynamic COF drug-loaded nanomaterial that enables precise drug release at the tumor site while simultaneously treating hypoxic tumors. First, the COF material is synthesized via a condensation polymerization method. Then, based on the aforementioned novel photosensitizer, a COF-E material is synthesized through a bond defect functionalization method. Subsequently, a drug is loaded onto the COF-E material using a π-π stacking method to synthesize Drug@COF-E. Finally, the target molecule is linked to the nanomaterial using another π-π stacking method to obtain the nanodrug. This enables the drug to target tumor tissue and be continuously released within it, while simultaneously effectively treating hypoxic tumors.

[0006] Furthermore, this invention utilizes imine COFs with specific structures to prepare nanomedicines with excellent performance. The imine COFs with specific structures can be synthesized in large quantities at room temperature, which is beneficial for clinical use.

[0007] Technical solution: This invention first provides a type I photosensitizer having the following structure: , hereinafter referred to as Enbs-Ar-NH2.

[0008] This invention also provides a method for preparing type I photodynamic covalent organic framework (COF) nanomedicines targeting liver tumors, comprising the following steps: (1) Preparation of COF: TAPB and TF are dispersed in acetonitrile and reacted under stirring. Then, the solid and liquid are separated to collect the precipitate, which is washed to obtain COF. (2) Preparation of photosensitizer-modified COF: The obtained COF and the above type I photosensitizer Enbs-Ar-NH2 are dispersed in ethanol, and the reaction is carried out under heating and stirring. Then the solid and liquid are separated to take the precipitate, washed, and the photosensitizer-modified COF is obtained, denoted as COF-E. (3) Preparation of drug-loaded photosensitizer modified COF: COF-E and drug are dispersed in water and reacted under stirring. Then, solid-liquid separation is performed to collect the precipitate, which is washed to obtain the drug-loaded photosensitizer modified COF, denoted as Drug@COF-E. (4) Preparation of nanomedicine products: COF modified with photosensitizer loaded with drug and reagent containing target structure are dispersed in water and reacted under stirring. Then, solid-liquid separation is performed to collect the precipitate, which is washed to obtain the nanomedicine product.

[0009] In one embodiment of the present invention, the mass ratio of TAPB to TF in step (1) is 2:1.

[0010] In one embodiment of the present invention, the structure of TAPB in step (1) is as follows:

[0011] In one embodiment of the present invention, the structure of TF in step (1) is as follows: .

[0012] In one embodiment of the present invention, the structural unit of COF in step (1) is: .

[0013] In one embodiment of the present invention, the COF mentioned in step (1) is obtained by the following method: TAPB, TF, glacial acetic acid and acetonitrile were mixed and stirred at room temperature. After the reaction was completed, the mixture was centrifuged, the solid was collected, washed and dried to obtain TAPB-Tf-COF.

[0014] In one embodiment of the present invention, during the preparation of COF, the molar ratio of TAPB to TF is 1:1. The volume ratio of glacial acetic acid to acetonitrile is 0.16:1. The concentration of TAPB relative to acetonitrile is 0.008 mmol / mL. The reaction is carried out at room temperature with stirring for 10-15 h.

[0015] In one embodiment of the present invention, the concentration of TAPB relative to acetonitrile in step (1) is 0.5-0.8 mg / mL. Specifically, 0.52 mg / mL may be selected.

[0016] In one embodiment of the present invention, the concentration of TF relative to acetonitrile in step (1) is 0.2-0.5 mg / mL. Specifically, 0.26 mg / mL is optional.

[0017] In one embodiment of the present invention, the reaction time in step (1) is 8-12 hours. Specifically, 10 hours may be selected.

[0018] In one embodiment of the present invention, the reaction temperature in step (1) is 20-30°C, specifically 25°C.

[0019] In one embodiment of the present invention, the concentration of COF relative to ethanol in step (2) is 0.5-1.0 mg / mL. Specifically, 0.52 mg / mL may be selected.

[0020] In one embodiment of the present invention, the concentration of the type I photosensitizer Enbs-Ar-NH2 relative to ethanol in step (2) is 1-2 mg / mL. Specifically, 1.08 mg / mL may be selected.

[0021] In one embodiment of the present invention, the mass ratio of COF to type I photosensitizer in step (2) is 1:(2-3).

[0022] In one embodiment of the present invention, the reaction time in step (2) is 8-12 hours. Specifically, 10 hours may be selected.

[0023] In one embodiment of the present invention, the reaction temperature in step (2) is 50-80°C, specifically 60°C.

[0024] In one embodiment of the present invention, the drug in step (3) is selected from any one or more of the following combinations: varentinib (Len), curcumin (Cur), doxorubicin, sorafenib, 5-fluorouracil, resveratrol, gemcitabine.

[0025] In one embodiment of the present invention, the concentration of COF-E relative to water in step (3) is 8-10 mg / mL. Specifically, 8.58 mg / mL is optional.

[0026] In one embodiment of the present invention, the mass ratio of COF-E to drug in step (3) is 1:(1-2).

[0027] In one embodiment of the present invention, the drugs mentioned in step (3) are varentinib (Len) and curcumin (Cur). The concentration of Len relative to water is 6-8 mg / mL, specifically 7.68 mg / mL; the concentration of Cur relative to water is 3-5 mg / mL, specifically 3.98 mg / mL.

[0028] In one embodiment of the present invention, the reaction time in step (3) is 8-12 hours. Specifically, 10 hours may be selected.

[0029] In one embodiment of the present invention, the reaction temperature in step (3) is 20-30°C, specifically 25°C.

[0030] In one embodiment of the present invention, the reagent containing the target structure in step (4) includes: acetylgalactosamine or galactose, or a monosaccharide or oligosaccharide containing acetylgalactosamine or galactose residues that targets hepatocytes.

[0031] In one embodiment of the present invention, the reagent containing the target structure in step (4) includes: RGD, biotin or folic acid or a tumor-targeting molecule containing RGD, biotin or folic acid molecules.

[0032] In one embodiment of the present invention, the reagent containing the target structure in step (4) is selected from any one or more combinations of the following: RhB-GalNAc and RhB-RGD. Wherein, RhB-GalNAc refers to an N-GalNAc target molecule linked to rhodamine, and RhB-RGD refers to an RGD target molecule linked to rhodamine.

[0033] In one embodiment of the present invention, the concentration of the photosensitizer-modified COF with drug loading in step (4) relative to water is 10-15 mg / mL; specifically, 10.67 mg / mL may be selected.

[0034] In one embodiment of the present invention, when the reagent containing the target structure in step (4) is RhB-GalNAc and RhB-RGD, the concentration of RhB-GalNAc relative to water is 3-5 mg / mL, specifically 3.78 mg / mL; the concentration of RhB-RGD relative to water is 3-5 mg / mL, specifically 3.98 mg / mL.

[0035] In one embodiment of the present invention, in step (4), the mass ratio of the photosensitizer-modified COF to GalNAc (N-acetylgalactosamine) and RGD (tumor-targeting peptide Arg-Gly-Asp) is 1:(0.5-2.5):(0.5-2.5).

[0036] In one embodiment of the present invention, the reaction time in step (4) is 20-40 hours. Specifically, 29 hours may be selected.

[0037] In one embodiment of the present invention, the reaction temperature in step (4) is 20-30°C, specifically 25°C.

[0038] Based on the above method, this invention provides a type I photodynamic covalent organic framework (COF) nanomedicine targeting liver tumors.

[0039] In one embodiment of the present invention, the structure of the type I photodynamic covalent organic framework (COF) nanomedicine targeting liver tumors is shown in Formula 1:

[0040] Formula 1 In the formula, A is selected from one or more of the following same or different chemotherapy or natural drugs: doxorubicin, sorafenib, varentinib, 5-fluorouracil, curcumin, resveratrol, gemcitabine; B represents covalent organic framework materials (COFs) modified with type I photosensitizers; C represents acetylgalactosamine or galactose, or a monosaccharide or oligosaccharide containing acetylgalactosamine or galactose residues that targets hepatocytes. D represents RGD, biotin, or folic acid, or a tumor-targeting molecule containing RGD, biotin, or folic acid.

[0041] This invention also provides the application of the above-mentioned type I photodynamic covalent organic framework (COF) nanomedicine targeting liver tumors in the preparation of anti-liver cancer drugs.

[0042] Beneficial effects: This invention provides a drug with targeting and tracing properties for liver tumor tissue. Its cytotoxicity and biocompatibility have been rigorously evaluated. In vitro studies included the stability of this targeted synergistic therapeutic nanosystem, drug release triggered by the acidic pH and GSH tumor microenvironment, and its synergistic therapeutic effects with photodynamic therapy and chemotherapy in C5WN1, HepG2, Huh7, and SMMC-7721 liver cancer cell lines. Furthermore, in vivo behavioral tracing and therapeutic efficacy evaluation in subcutaneous solid tumor models have demonstrated the drug's excellent tracing and therapeutic effects. Attached Figure Description

[0043] Figure 1 The image shows the 1H NMR spectrum of the compound Enbs-Ar-NHBoc.

[0044] Figure 2 This is the carbon NMR spectrum of the compound Enbs-Ar-NHBoc.

[0045] Figure 3 This is the 1H NMR spectrum of the compound Enbs-Ar-NH2.

[0046] Figure 4 This is the carbon NMR spectrum of the compound Enbs-Ar-NH2.

[0047] Figure 5 The images show the spectral characterization of the photosensitizer Enbs-Ar-NH2. (a) Molecular structures of Enbs-N3 and Enbs-Ar-NH2. (b) UV absorption spectra of Enbs-Ar-NH2 and Enbs-N3. (c) Fluorescence spectra of Enbs-Ar-NH2 and Enbs-N3. (d) Detection of O2 using DHR 123 as a fluorescent probe. -• The fluorescence spectrum. (e) Using Vc as O2 -• The scavenger was tested, and the spectrum of Enbs-Ar-NH2 mixed with 50 μM Vc after irradiation was measured.

[0048] Figure 6TEM image of LC@GR-COF-E. Scale bar, 100 nm. FESEM image of LC@GR-COF-E. Scale bar, 200 nm.

[0049] Figure 7 Cell fluorescence images of the LC@GR-COF-E in vitro co-culture model. Scale bar: 25 μm. Data are expressed as mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, determined by t-test.

[0050] Figure 8 To image intracellular hypoxia in SMMC-7721 cells using the Image-iT™ hypoxia reagent as an anaerobic indicator. Scale bar: 50 μm.

[0051] Figure 9 MTT results for HepG2, C5WN1, Huh7 and SMMC-7721 cells after 48 h of treatment with different concentrations of (a) LC@GR-COF-E and (b) LC@GR-COF-E + Light.

[0052] Figure 10 Live and dead staining experiments for SMMC-7721, scale bar, 50 μm.

[0053] Figure 11 To evaluate the effects of different treatments on mitochondrial membrane potential in C5WN1 cells using the JC-1 assay.

[0054] Figure 12 In vivo fluorescence imaging of mice at 4, 8, 12 and 24 hours after tail vein injection of GR-COF-E, R-COF-E, G-COF-E or COF-E.

[0055] Figure 13(a) Schematic diagram of treatment regimen; (b) Immunofluorescence imaging of tumor sections; Tumor vessels (magenta) stained with anti-CD31 antibody, hypoxia-related proteins stained with anti-HIF-1α antibody (green), and cell nuclei stained with DAPI (blue); Scale bar, 100 μm; (c) Tumor growth curves of each group; (d) Photographs of ex vivo tumor tissue after treatment of C5WN1 subcutaneous tumor model. Saline (1), COF-E (2), Len + Cur (3), L@GR-COF-E (4), LC@COF-E (5), LC@G-COF-E (6), LC@GR-COF-E (7) and LC@GR-COF-E + Light (8); Scale bar, 1 cm; (e) Weight of tumor tissue collected on day 14 (statistical significance was calculated by t-test: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). Detailed Implementation

[0056] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. In the embodiments, all reagents used are commercially available.

[0057] Example 1: Synthesis of photosensitizer Enbs-Ar-NH2 I) Synthesis of compound iii:

[0058] Specific procedures: Compounds i (80 mg, 0.16 mmol), ii (52 mg, 0.24 mmol), copper sulfate pentahydrate (24 mg, 0.10 mmol), and sodium ascorbate (38 mg, 0.19 mmol) were dissolved in a mixed solvent (14 mL, chloroform:ethanol:water = 12:1:1, v / v / v), and then stirred at room temperature for 24 h under nitrogen protection. The solvent was then removed by vacuum distillation, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 20:1, v / v) to give a blue solid iii (108 mg, 99%). 1H NMR(400 MHz, Chloroform-d) δ 11.54 (s, 1H), 9.27 (d, J = 7.9 Hz, 1H), 8.85 (dd,J = 7.8, 1.7 Hz, 1H), 7.90 – 7.82 (m, 2H), 7.78 – 7.66 (m, 4H), 7.44 – 7.33(m, 2H), 7.04 (dd, J = 9.4, 2.6 Hz, 1H), 6.90 (s, 1H), 6.78 (d, J = 2.7 Hz,1H), 6.65 (s, 1H), 4.38 (t, J = 7.0 Hz, 2H), 3.84 – 3.69 (m, 2H), 3.57 (q, J= 7.1 Hz, 4H), 2.01 – 1.82 (m, 4H), 1.58 – 1.48 (m, 11H), 1.47 – 1.38 (m,1H), 1.32 (t, J = 7.1 Hz, 6H). 13C NMR (151 MHz, CDCl3) δ 154.4, 152.7, 150.3,147.3, 140.2, 138.2, 136.8, 135.6, 133.4, 131.9, 131.4, 130.5, 130.0, 126.3,125.9, 125.6, 125.1, 124.9, 119.6, 118.6, 115.8, 104.4, 102.8, 80.6, 50.2,45.6, 44.2, 29.9, 28.6, 28.4, 26.2, 25.9, 12.8. HR-ESI-MS (m / z): Calculated value: C39H46N7O2S+ (M + H+): 676.3428; Detected value: 676.3496.

[0059] II) Synthesis of compound iv:

[0060] Compound iii (108 mg, 0.16 mmol) was dissolved in methanol (5 mL), and 6 M hydrochloric acid aqueous solution (5 mL) was added. The mixture was stirred overnight at room temperature. Subsequently, the solvent was removed under reduced pressure distillation, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 5:1, v / v) to give a blue solid Enbs-Ar-NH2 (86 mg, 93%). 1H NMR (600 MHz, Methanol-d4) δ 8.59 (d, J = 8.0 Hz, 1H), 8.10 (s, 1H), 8.03 (d, J = 8.1 Hz,1H), 7.64 (dd, J = 8.7, 6.8 Hz, 2H), 7.60 – 7.54 (m, 1H), 7.46 – 7.43 (m,2H), 7.17 (dd, J = 9.4, 2.7 Hz, 1H), 6.89 (d, J = 2.7 Hz, 1H), 6.87 (s, 1H), 6.67 – 6.64 (m, 2H), 4.43 (t, J = 6.8 Hz, 2H), 3.58 (q, J = 7.3 Hz, 4H), 3.43(t, J = 7.5 Hz, 2H), 2.03 – 1.95 (m, 2H), 1.79 – 1.71 (m, 2H), 1.54 – 1.46(m, 2H), 1.45 – 1.36 (m, 2H), 1.29 (t, J = 7.2 Hz, 9H). 13C NMR (101 MHz, MeOD) δ 152.8, 151.1, 148.2, 148.0, 139.7, 136.9, 133.5, 133.2, 132.5, 131.7,130.6, 129.2, 126.2, 124.7, 123.9, 122.0, 119.6, 119.3, 117.2, 114.9, 104.6,101.6, 49.8, 48.1, 45.4, 43.8, 29.7, 28.1, 26.0, 25.8, 11.8, 11.8. HR-ESI-MS (m / z): Calculated value: C 34 H 38 N7S + (M + H + ): 576.2904; Detected value: 576.2956.

[0061] The photosensitizer Enbs-Ar-NH2(iv) prepared in Example 1 was analyzed by ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy.

[0062] The experimental results are attached. Figure 5 The UV-Vis spectral characteristics of the photosensitizer Enbs-Ar-NH2(iv) show that the maximum absorption wavelength falls at 660 nm, within the "tissue transparency window (600-1000 nm)". Under 660 nm excitation, Enbs-Ar-NH2 exhibits near-infrared emission, which is beneficial for in vivo tumor imaging. Furthermore, the photosensitizer Enbs-Ar-NH2 follows a Type I photodynamic mechanism.

[0063] Example 2: Preparation of nanomedicine LC@GR-COF-E (1) A mixture of TAPB (1.6 mmol), TF (1.6 mmol), glacial acetic acid (32 mL), and acetonitrile (200 mL) was stirred at 800 rpm for 12 hours at room temperature. The particles were then separated by centrifugation and washed successively with acetonitrile, triethylamine / ethanol (1 / 9, v / v), and ethanol. After freeze-drying for 12 hours, a milky white powder, TAPB-Tf-COF, was obtained. Yield: 0.53 g (63%).

[0064] (2) The products TAPB-Tf-COF and Enbs-Ar-NH2 obtained above (in a weight ratio of 1:1) were mixed with acetic acid solution (3M) and ethanol and heated at 65°C overnight. The resulting particles were collected to produce a blue powder TAPB-Tf-COF-E (COF-E).

[0065] (3) Len, Cur and COF-E were stirred in the dark at room temperature for 12 hours. The resulting particles were collected to produce green powder LC@COF-E.

[0066] (4) LC@COF-E, GalNAc-RhB and RGD-RhB were stirred in the dark at room temperature for 29 hours. The resulting particles were collected to form a dark red powder LC@GR-COF-E.

[0067] The performance of the LC@GR-COF-E nanomaterial prepared in Example 2 was characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

[0068] As attached Figure 6 As shown, the prepared LC@GR-COF-E consists of uniformly distributed spherical nanoparticles with a particle size of 232 nm.

[0069] Example 3: Establishment of an in vitro co-culture model of LC@GR-COF-E To compare the uptake of the nanomaterial LC@GR-COF-E by tumor cells and normal cells, HepG2 liver cancer cells and HEK293 normal cells were co-incubated in the same confocal glass dish. After 12 h of culture, LC@GR-COF-E (1 mL, 32 μg / mL) was added. -1 The cells were incubated for 6 h. The fluorescence values ​​of HepG2 and HEK293 under a 635 nm (Enbs) laser were analyzed by CLSM.

[0070] As attached Figure 7 As shown, the GalNAc and RGD peptides on LC@GR-COF-E enhance the selective uptake of LC@GR-COF-E in HepG2 cells by binding to the ASGPR receptor and αvβ3 on the surface of liver cancer cells.

[0071] Example 4: Establishment of an in vitro hypoxia model The hypoxic conditions were prepared using Anaero Pack-Anaero (1% O2, 5% CO2). Image-iT TM The hypoxia reagent is a cell-permeable compound that enhances fluorescence in a hypoxic environment, serving as a fluorescent indicator of oxygen concentration in vitro. Cells were seeded in confocal glass dishes, and the samples were incubated under normoxic or hypoxic conditions. The cells were then washed three times with PBS and reacted with Image-iT... TM Incubate with hypoxia reagent stock solution (10 μM) for 30 min. After washing the cells three times with PBS, observe the fluorescence images under CLSM at an excitation wavelength of 488 nm.

[0072] As attached Figure 8 As shown, the hypoxia group exhibits strong green fluorescence, confirming the successful construction of the in vitro hypoxia model.

[0073] Example 5: Experiment on the cytotoxicity of drug LC@GR-COF-E C5WN1, HepG2, Huh7, SMMC-7721 and HEK293 cells were respectively loaded at 2 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of 0.5 μg / mL in 96-well plates. After 24 hours of stable growth, the nanomaterial LC@GR-COF-E from Example 2 was added to each well at concentrations of 0, 4, 8, 16, 32, and 64 μg / mL. After co-incubation with the material for 48 hours, the culture medium was removed, and the cells were washed three times with PBS. Then, 100 μL of medium containing 0.5 mg / mL LMT was added to each well, followed by 100 μL of LDMSO. The absorbance (λ = 490 nm) of all wells in the 96-well plate was measured using a microplate reader.

[0074] Appendix Figure 9The graph shows the cytotoxicity results of the nanomaterial LC@GR-COF-E. The results show that the concentration of the nanomaterial in Example 2 is as high as 64 µg / mL. -1 At that time, the activity of cancer cells was greatly reduced.

[0075] Example 6: In vitro therapeutic effects of nanomedicine LC@GR-COF-E The in vitro therapeutic effects of the nanomaterial LC@GR-COF-E were detected using a live-dead staining method.

[0076] SMMC-7721 liver cancer cells were divided into 9×10⁻⁶ cells per well. 4 Cells were seeded at a density of [insert density here] in 96-well plates. After culturing for 24 hours, the cells were incubated for 24 hours in media treated with different groups. Then, the light-treated group was irradiated with a 660 nm light source for 10 min, followed by incubation for another 24 hours, and then Calcein-AM / PI live / dead cell co-staining was performed. The cells were observed under a fluorescence microscope.

[0077] As attached Figure 10 As shown, after LC@GR-COF-E + Light treatment, the entire structure exhibits almost entirely red fluorescence, demonstrating the excellent targeted synergistic therapeutic effect of the dual-target type I covalent organic framework material.

[0078] Example 7: JC-1 detection of changes in mitochondrial membrane potential caused by LC@GR-COF-E in cells Cells were seeded into glass dishes and cultured overnight in a cell culture incubator. After removing the culture medium, the cells were pre-incubated for 6 hours in an incubator with the control group and LC@GR-COF-E, and then washed twice with PBS. Subsequently, the cells in the light-illuminated group were exposed to red light. After another 4 hours of incubation, the cells were mixed with JC-1 (200 μL, 10 μg mL). -1 Incubate together for 10 min and wash twice with PBS. Then, capture fluorescence images. Fluorescence images of monomer were excited by 488 nm light, and fluorescence images of J-aggregate were excited by 532 nm light.

[0079] As attached Figure 11 As shown, targeted chemotherapy alone has a limited effect on Δψ. However, by combining it with photodynamic therapy, the JC-1 monomer (green fluorescence) is directly increased, which exacerbates mitochondrial functional damage and is beneficial for anti-tumor treatment.

[0080] Example 8: In vivo distribution of nanomedicine LC@GR-COF-E The distribution of nanomaterials was studied in mice with subcutaneous hepatocellular carcinoma. The distribution was investigated when the tumor volume reached 200 m³. 3Mice were randomly divided into four groups and intravenously injected with LC@GR-COF-E, LC@G-COF-E, LC@R-COF-E, and LC@COF-E (100 μL, 10 mg / mL), respectively. -1 The in vivo and in vitro RhB fluorescence was monitored at 4, 8, 12 and 24 h after injection using a small animal in vivo imaging system to analyze the in vivo distribution.

[0081] The tumor-targeting ability and biodistribution of GR-COF-E, G-COF-E, R-COF-E, and COF-E in C5WN1 nude mice were investigated. (See attached image) Figure 12 The dual-targeting group showed better targeting ability than the single-targeting group and the non-targeting group, which will help reduce the toxic side effects of free drugs in the body and enhance the drugs to target tumors faster and more effectively.

[0082] Example 9: Nanomedicine LC@GR-COF-E inhibits tumor growth in a mouse model of subcutaneous solid tumors. Subcutaneous tumors in mice exceeding 100 mm in size 3 Mice were randomly divided into 8 groups of 5 mice each, including saline (1), COF-E (2), free drug group Len + Cur (3), L@GR-COF-E (4), LC@COF-E (5), LC@G-COF-E (6), LC@GR-COF-E (7), and LC@GR-COF-E + Light (8). Tumor volume and body weight of mice were monitored and recorded every two days. After 14 days of treatment, five mice in each group were euthanized with carbon dioxide, and tumor tissue was collected, weighed, and photographed. Tumor tissue and major organs (liver, spleen, kidney, heart, and lungs) were fixed with 4% paraformaldehyde, embedded in paraffin, sectioned to a thickness of 4 μm, and stained with H&E.

[0083] As attached Figure 13 As shown, the LC@GR-COF-E+ light irradiation group exhibited a significant inhibitory effect on tumor growth. Based on the tumor volume data obtained on the last experimental day, the tumor inhibition efficiency was calculated (tumor inhibition efficiency = (1 - average tumor volume of the experimental group / average tumor volume of the saline control group) × 100%). The LC@GR-COF-E+ light irradiation group showed the highest inhibition efficiency, at 95.5% ± 1.7%. It can be concluded that the LC@GR-COF-E+ light irradiation group can maximize the inhibition of tumor growth.

[0084] Comparative Example 1: Comparison of different COFs Referring to Example 1, different monomers were substituted to prepare the COF materials shown in Table 1.

[0085] Referring to Example 2, the COF (i.e., TAPB-TF-COF) was replaced with other COFs shown in Table 1, while other steps remained unchanged, to prepare the corresponding nanomedicine containing type I photosensitizer.

[0086] Table 1

[0087] The obtained nanomedicines containing type I photosensitizers were subjected to corresponding performance tests according to Examples 3-9, and the results are shown in Table 2.

[0088] Table 2

[0089] O2 production -• Capability Test: Using dihydrorhodamine 123 (DHR 123) as O2 -• Indicator, in O2 -• In the presence of [a specific substance], it transforms into rhodamine 123, which exhibits fluorescent properties. In an aqueous solution, COF-E (with a uniform photosensitizer concentration of 10 μM) and DHR 123 (10 μM) were added. The cuvette was irradiated with 660 nm red light for different times (0 and 10 minutes), and the fluorescence intensity of rhodamine 123 (I0, I [other values], respectively) was recorded. 10 ). Calculate O2 production -• Ability δI = I 10 - I0.

[0090] Assay for inhibition of hepatocellular carcinoma cell growth rate (%): Hepatocellular carcinoma cells were incubated at 37°C with culture medium or COF-E-containing medium for 4 h, then irradiated with 660 nm red light for 10 min, followed by incubation at 37°C for 44 h. Next, the culture medium was removed, and 20 μL of 0.5 mg / mL MTT solution (dissolved in PBS) was added to each well. After another 4 hours, the medium was removed, and 150 μL of DMSO was added to each well. The plate was shaken for 10 min, and the absorbance at 490 nm was measured using a microplate reader.

[0091] Inhibition of liver cancer cell growth rate (%) = [(Control group - Experimental group) / (Control wells - Blank wells)] × 100% Experimental group (culture medium containing cells, COF-E, MTT, DMSO); control group (culture medium containing cells, MTT, DMSO); blank group (culture medium without cells and test substances, MTT, DMSO).

[0092] Comparative Example 2: Comparison of different photosensitizers Referring to Example 2, Enbs-Ar-NH2 was replaced with other type I photosensitizers shown in Table 3, while other steps remained unchanged, to prepare the corresponding nanomedicine COF-E containing type I photosensitizers.

[0093] Table 3

[0094] COF-E synthesis success rate: Record the initial type I photosensitizer content (W0), thoroughly wash the obtained COF-E, collect the supernatant, and measure the type I photosensitizer content (W0) in the supernatant. t ). Calculate the synthesis success rate = (W0 - W) t )*100%.

[0095] Assay for inhibiting the growth rate (%) of liver cancer cells (in vitro cell model of Type I PDT-COF): Liver cancer cells were incubated at 37°C with culture medium or culture medium containing Type I PDT-COF for 4 h, then irradiated with 660 nm red light for 10 min, followed by incubation at 37°C for 44 h. Next, the culture medium was removed, and 20 μL of 0.5 mg / mL MTT solution (dissolved in PBS) was added to each well. After another 4 hours, the medium was removed, and 150 μL of DMSO was added to each well. The plate was shaken for 10 min, and the absorbance at 490 nm was measured using a microplate reader.

[0096] Inhibition of liver cancer cell growth rate (%) = [(Control group - Experimental group) / (Control wells - Blank wells)] × 100% Experimental group (culture medium containing cells, Type I PDT-COF, MTT, DMSO); control group (culture medium containing cells, MTT, DMSO); blank group (culture medium without cells and test substances, MTT, DMSO).

[0097] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A type I photosensitizer, characterized in that, It has the following structure: 。 2. The application of the type I photosensitizer according to claim 1 in the preparation of type I photodynamic covalent organic framework nanomedicines targeting liver tumors.

3. A method for preparing a type I photodynamic covalent organic framework nanomedicine targeting liver tumors, characterized in that, Includes the following steps: (1) Preparation of COF: TAPB and TF are dispersed in acetonitrile and reacted under stirring. Then, the solid and liquid are separated to collect the precipitate, which is washed to obtain COF. (2) Preparation of photosensitizer-modified COF: The obtained COF and the type I photosensitizer described in claim 1 are dispersed in ethanol, and the reaction is carried out under heating and stirring. Then, the solid and liquid are separated to take the precipitate, washed, and the photosensitizer-modified COF is obtained, denoted as COF-E. (3) Preparation of drug-loaded photosensitizer-modified COF: COF-E and drug are dispersed in water and reacted under stirring. Then, solid-liquid separation is performed to collect the precipitate, which is washed to obtain drug-loaded photosensitizer-modified COF. (4) Preparation of nanomedicine products: COF modified with photosensitizer loaded with drug and reagent containing target structure are dispersed in water and reacted under stirring. Then, solid-liquid separation is performed to collect the precipitate, which is washed to obtain the nanomedicine product.

4. The preparation method according to claim 3, characterized in that, The structure of TAPB described in step (1) is as follows: The structure of TF is .

5. The preparation method according to claim 3, characterized in that, The structural unit of COF mentioned in step (1) is 。 6. The preparation method according to claim 3, characterized in that, In step (2), the concentration of COF relative to ethanol is 0.5-1.0 mg / mL, and the concentration of type I photosensitizer relative to ethanol is 1-2 mg / mL.

7. The preparation method according to claim 3, characterized in that, In step (2), the mass ratio of COF to type I photosensitizer is 1:(2-3).

8. The preparation method according to claim 3, characterized in that, The drug mentioned in step (3) is selected from any one or more of the following combinations: valentinib, curcumin, doxorubicin, sorafenib, 5-fluorouracil, resveratrol, gemcitabine.

9. The preparation method according to claim 3, characterized in that, The concentration of COF-E relative to water in step (3) is 8-10 mg / mL; the mass ratio of COF-E to the drug is 1:(1-2).

10. The preparation method according to claim 3, characterized in that, The reagents containing the target structure mentioned in step (4) include: acetylgalactosamine or galactose, or monosaccharides or oligosaccharides containing acetylgalactosamine or galactose residues that target hepatocytes.

11. The preparation method according to claim 3, characterized in that, The reagents containing target structures mentioned in step (4) include: RGD, biotin or folic acid or tumor-targeting molecules containing RGD, biotin or folic acid molecules.

12. A type I photodynamic covalent organic framework nanomedicine targeting liver tumors prepared by the method according to any one of claims 3-11.

13. A type I photodynamic covalent organic framework nanomedicine targeting liver tumors, characterized in that, Its structure is shown in Equation 1: Formula 1 In the formula, A is selected from one or more of the following same or different chemotherapy or natural drugs: doxorubicin, sorafenib, varentinib, 5-fluorouracil, curcumin, resveratrol, gemcitabine; B is a covalent organic framework material modified with a type I photosensitizer; C represents acetylgalactosamine or galactose, or a monosaccharide or oligosaccharide containing acetylgalactosamine or galactose residues that targets hepatocytes. D represents RGD, biotin, or folic acid, or a tumor-targeting molecule containing RGD, biotin, or folic acid.

14. The application of the type I photodynamic covalent organic framework (COF) nanomedicine targeting liver tumors as described in claim 12 or 13 in the preparation of anti-liver cancer drugs.

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