Preparation method and application of dual-targeting hepatocellular carcinoma nano-drug

Abstract

The invention discloses a preparation method and application of a dual-targeting hepatocellular carcinoma nano-drug, and belongs to the field of biological medicines. According to the invention, a double-targeting target molecule with myeloperoxidase-asialoglycoprotein receptor double-targeting property is synthesized, and the double-targeting target molecule is modified on a covalent organic nano molecule COF-366 through pi-pi stacking, wherein the covalent organic nano molecule COF-366 is coated with chemotherapeutic drugs sorafenib and chloroquine and has photodynamic characteristics. The obtained nano-drug can be gradually delivered to hepatocytes under the guidance of double-targeting molecules, drug release is started in acidity of a tumor acidic microenvironment, the synergistic treatment effect of photodynamic therapy and chemotherapy can be played, and the synergistic effect causes tumor injury, so that the inflammation targeting of the nano-drug is further enhanced. The nano-drug has good stability and biological safety, in-vivo behavior tracing and treatment efficacy evaluation are carried out in subcutaneous solid tumor and in-situ tumor mouse models, and the nano-drug is expected to play a huge role in clinical application.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a method for preparing and applying a dual-targeting hepatocellular carcinoma nanomedicine. Background Technology

[0002] Hepatocellular carcinoma (HCC), the most common primary malignant tumor of the liver, has become a major global public health problem. It ranks fourth among cancer-related deaths worldwide, with a continuously rising incidence and limited treatment options. Nat. Rev. Dis. Primers (July 6, 2021). Current conventional treatments include surgical resection, liver transplantation, image-guided ablation, radiotherapy, and systemic therapy, but these generally face clinical challenges such as high recurrence rates, treatment resistance, and poor patient tolerance. Nat Med , 2014, 20, 1138-1146). Given these limitations, the development of more effective treatment strategies is urgently needed. In recent years, targeted combination therapies based on nanomaterials, through multimodal synergistic mechanisms, have provided new avenues for overcoming tumor drug resistance, hypoxic solid tumors, and metastasis. Chem Rev (2017, 22, 13566-13638). Various nanomedicine delivery systems (such as liposomes, supramolecular structures, micelles, and exosomes) have been developed. Traditional non-selective or single-target delivery systems are prone to non-specific cytotoxicity, causing systemic damage to normal tissues; however, recent studies have shown that dual-targeting strategies, which can simultaneously target tumor cells and regulate the tumor microenvironment (TME), have become an important direction for the combination therapy of hepatocellular carcinoma (HCC).

[0003] Sorafenib (Sor) was the first targeted drug approved for HCC, but studies have shown that sorafenib can induce pro-survival autophagy by inhibiting mTORC1 and activating HIF-1α-dependent autophagy, thereby weakening its therapeutic effect.

[0004] Simultaneously, photodynamic therapy (PDT) generates singlet oxygen through a photosensitizer-mediated type II reaction. 1 O2 causes oxidative damage and apoptosis in tumors. However, this process consumes a lot of oxygen, exacerbating tumor hypoxia and thus activating pro-survival autophagy. Summary of the Invention

[0005] [Technical Issues] (1) The problem of tumor cell drug resistance: Sorafenib (Sor) can induce cancer cells to start "protective autophagy", that is, cancer cells provide energy and remove damage through "self-digestion" to resist drug action, leading to treatment failure. The present application can prevent the fusion and degradation of autophagosomes and acidic lysosomes by co-delivery of Sor and chloroquine (CQ), paralyzing the "self-rescue" process of cancer cells, thereby effectively reversing Sor resistance and significantly enhancing the killing effect of Sor.

[0006] (2) The problem of tumor metastasis and immunosuppression: Neutrophils in the tumor microenvironment can form neutrophil extracellular traps (NETs). NETs not only can capture and protect cancer cells, promote their metastasis, but also can inhibit the function of immune cells, forming an immunosuppressive environment. The present application uses the dual function of chloroquine (CQ) to block the TLR9 / PAD4 signaling pathway, thereby inhibiting the formation of NETs from the source and relieving immunosuppression and inhibiting cancer cell metastasis.

[0007] (3) The problem of insufficient targeting accuracy: Traditional nanomedicines (such as those relying only on ASGPR targeting) have "off-target" effects and are not high enough in enrichment efficiency, resulting in insufficient drug concentration in the tumor site and increasing systemic toxicity. The present application realizes a cascade double-targeting delivery mechanism by designing a double-targeting molecule. First level: using the high activity of myeloperoxidase (MPO) in the tumor inflammatory microenvironment to activate the substrate peptide D5HT modified nanoparticles, so that they can be anchored and enriched in the tumor area. Second level: specific binding of galactose (Gal) to the asialoglycoprotein receptor (ASGPR) highly expressed on the surface of hepatoma cells to achieve efficient uptake by cells.

[0008] (4) The problem of static targeting strategy that cannot be sustained: Once the drug is released, the targeting process is over and cannot cope with the dynamically changing tumor microenvironment. The present application introduces a light-driven self-enhancing positive feedback loop by using a covalent organic nanoframe COF-366 with photodynamic properties. The COF-366 carrier generates a large amount of active oxygen (O2) under 660 nm laser irradiation. 1 O2). 1 O2kills tumor cells while triggering acute inflammation, recruiting more neutrophils to the tumor site. These cells release more MPO, further enhancing the efficiency of the first level MPO targeting.

[0009] [Technical solutions] This invention provides a method for preparing a dual-targeting hepatocellular carcinoma nanomedicine, comprising synthesizing covalent organic nanomaterials with photodynamic properties, encapsulating the chemotherapy drugs sorafenib (Sor) and chloroquine (CQ) by electrostatic adsorption, simultaneously synthesizing the dual-targeting molecule Gal-D5HT, and completing the surface modification of the nanoparticles by utilizing π-π stacking interaction. The nanoparticles are labeled with Rhodamine B (RhB) fluorescence, and finally SC@GRT-COF-366 nanoparticles are obtained.

[0010] A method for preparing a dual-targeting hepatocellular carcinoma nanomedicine, the method comprising the following steps: (1) Preparation of covalent organic nanomaterials: 5,10,15,20-tetra(4-aminophenyl)porphyrin, terephthalaldehyde and polyvinylpyrrolidone were dissolved in a mixed solvent of DMF and methanol, trifluoroacetic acid was added, and the mixture was heated to obtain covalent organic nanomaterials. (2) Preparation of dual-target molecules: Azide-treated galactose raw material A and raw material B containing alkyne and 5-hydroxytryptamine structure are dissolved in a mixed solvent of water and DMF; and ascorbic acid and sulfuric acid pentahydrate are added to react and obtain dual-target molecules; (3) First, the covalent organic nanomaterials and chemotherapy drugs are mixed and stirred to obtain drug-encapsulated nanomaterials; then the obtained drug-encapsulated nanomaterials are mixed with dual-target molecules and tracer molecules to obtain the final nanomedicine product. The structure of the azidated galactose raw material A is as follows: ; The structure of raw material B, which contains alkyne and 5-hydroxytryptamine, is as follows: .

[0011] In one embodiment of the present invention, in step (1), the heating reaction is carried out under reflux and stirring at 120°C for 30 minutes.

[0012] In one embodiment of the present invention, after the reaction is completed in step (1), the following post-processing is further included: The reaction mixture was poured into a mixture of methanol and triethylamine, centrifuged at 12,000 rpm for 15 min, and the resulting solid precipitate was collected. The solid was washed five times with methanol and then freeze-dried for 48 h to obtain the final product COF-366.

[0013] In one embodiment of the present invention, the molar ratio of 5,10,15,20-tetra(4-aminophenyl)porphyrin to terephthalaldehyde in step (1) is 1:2.

[0014] In one embodiment of the present invention, the mass ratio of polyvinylpyrrolidone to 5,10,15,20-tetrakis(4-aminophenyl)porphyrin in step (1) is 1:(0.05-0.10); specifically, 1:0.07 may be selected.

[0015] In one embodiment of the present invention, the volume ratio of DMF to methanol in step (1) is 1:(2-5); specifically, 1:4 can be selected.

[0016] In one embodiment of the present invention, the ratio of trifluoroacetic acid to 5,10,15,20-tetra(4-aminophenyl)porphyrin in step (1) is 10-15 mL / mmol. Specifically, 12.5 mL / mmol is optional.

[0017] In one embodiment of the present invention, in step (1), the concentration of 5,10,15,20-tetra(4-aminophenyl)porphyrin relative to the mixed solvent is 0.002-0.008; specifically, 0.005 mmol / mL.

[0018] In one embodiment of the present invention, in step (2), the azide-treated galactose raw material A is prepared by the following method:

[0019] 2.1) Synthesis of compound 1 (Gal-Ac-Br): Boron trifluoride diethyl ether was added to a dichloromethane solution containing 1,2,3,4,6-penta-O-acetyl-d-galactopyranose and 2-bromoethanol (Gal-Ac), and stirred overnight at room temperature under nitrogen protection to obtain compound 1. 2.2) Synthesis of compound 2 (Gal-Ac-N3): Compound 1 was dissolved with sodium azide in DMF solution and reacted under argon protection to obtain compound 2; 2.3) Synthesis of compound 3 (Gal-N3): Compound 2 and sodium methoxide were dissolved in methanol under nitrogen protection and reacted at room temperature to obtain compound 3, which is the azidated galactose raw material A.

[0020] In one embodiment of the present invention, the molar ratio of compound 1 to sodium azide in step 2.2) is 1:2.

[0021] In one embodiment of the present invention, the molar ratio of compound 2 and sodium methoxide in step 2.3) is 2:1.

[0022] In one embodiment of the present invention, in step (2), the raw material B containing alkyne and 5-hydroxytryptamine structure is prepared by the following method:

[0023] 2.4) Synthesis of compound 4 (D-5HT-Boc): N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) were dissolved in DMF and added to a DMF solution of Boc-DL-Glu. The mixture was stirred at room temperature for activation. Subsequently, a DMF solution containing 5-hydroxytryptamine and triethylamine was added, and the mixture was stirred to obtain compound 4. 2.5) Synthesis of Compound 6 (D-5HT): Compound 4 was dissolved in dichloromethane, and trifluoroacetic acid (TFA) was added and stirred overnight at room temperature. After removing dichloromethane and excess TFA under reduced pressure, the residue was dissolved in DMF, and excess triethylamine was added. Subsequently, a solution of 4-pentenoic acid activated in DMF for 30 minutes with NHS / EDC or HOBt / HATU was added to the above mixture, and the reaction yielded compound 6, which is the starting material B containing alkyne and 5-hydroxytryptamine structure.

[0024] In one embodiment of the present invention, in step (2), after the reaction is completed, the mixture is filtered and then purified by reversed-phase chromatography (acetonitrile / water) to obtain dual-target molecules.

[0025] In one embodiment of the present invention, in steps 2.4) to 2.5), the molar ratio of the carboxyl activator to the amino ligand is 1:2, wherein NHS and EDC are twice the amount of the carboxyl activator and the molar ratio is 1:1.

[0026] In one embodiment of the present invention, in step 2.5), the volume ratio of dichloromethane to trifluoroacetic acid is 5:1.

[0027] In one embodiment of the present invention, the molar ratio of compound 3 and compound 6 or 7 in step (3) is 2:1, and the final ratio of solvent water to DMF is 1:1, wherein sodium ascorbate and copper sulfate are added after being dissolved, and can only be dissolved in water.

[0028] In one embodiment of the present invention, step (3) includes the following process: (3.1) Encapsulation of chemotherapy drugs: The obtained covalent organic nanomaterials and chemotherapy drugs were dissolved in DMSO solution, stirred and mixed at room temperature, then centrifuged, washed with ultrapure water, and freeze-dried to obtain nanomaterials encapsulated with drugs; (3.2) Modification of dual-target molecules: The drug-loaded nanomedicine and the dual-target molecules Gal-D5HT and tracer molecules were dissolved in water, stirred and mixed at room temperature, then centrifuged, washed with ultrapure water, and freeze-dried to obtain dual-target hepatocellular carcinoma nanomedicine.

[0029] In one embodiment of the present invention, in step (3.1), the chemotherapy drugs are Sor and CQ.

[0030] In one embodiment of the present invention, the mass ratio of the covalent organic nanomaterial, Sor and CQ is (1-2):1:(1-2); specifically, 1:1:1, 1:1:2, 2:1:1 or 2:1:2 can be selected.

[0031] In one embodiment of the present invention, in step (3.2), the tracer molecule is rhodamine B.

[0032] In one embodiment of the present invention, in step (3.2), the mass ratio of the drug-loaded nanomedicine to the dual-target molecules is 1:1.

[0033] In one embodiment of the present invention, in step (3.2), the mass ratio of the drug-loaded nanomedicine to the tracer molecule is 1:1.

[0034] In one embodiment of the present invention, in step (3.2), the concentration of the drug-loaded nanomedicine in water is 5 mg / mL.

[0035] Based on the above preparation method, this invention provides a dual-targeting hepatocellular carcinoma nanomedicine with myeloperoxidase-desialylglycoprotein receptor.

[0036] This invention also provides the application of the above-mentioned dual-target hepatocellular carcinoma nanomedicine in the preparation of anti-liver cancer drugs.

[0037] The beneficial effects achieved by this invention are as follows: The nanomedicine SC@GRT-COF-366 constructed in this invention is uniformly spherical with a particle size of approximately 100 nm and an electrical potential of approximately [missing value]. At 28 mV, the drug loadings of Sor and CQ were 8.5 ± 0.35% and 7.8 ± 0.24%, respectively. The drug release exhibited excellent pH-responsive characteristics, achieving release rates of 94.3 ± 2.0% and 72.4 ± 3.4% for CQ and Sor, respectively, even in the slightly acidic tumor microenvironment. Furthermore, this nanomaterial possessed good stability and excellent photodynamic properties, generating a large amount of singlet oxygen (SQ) under 660 nm laser irradiation. 1 O2) is beneficial for further killing of tumor cells.

[0038] The platform’s targeting capability was validated through a dual in vitro mechanism. Compared with no targeting and single targeting, the dual-targeting SC@GRT-COF-366 responded to the specific aggregation effect triggered by MPO / H2O2 in the tumor microenvironment and completed the uptake of ASGPR-positive HCC cells under the mediation of ASGPR receptor. The cytotoxicity results also showed excellent ASGPR targeting specificity and photodynamic properties.

[0039] In summary, we have successfully constructed nanomedicines with uniform particle size, stable properties, and dual targeting of myeloperoxidase-desialylglycoprotein receptor to inhibit hepatocellular carcinoma proliferation and metastasis, laying the foundation for subsequent biological evaluation. Attached Figure Description

[0040] Figure 1 Transmission electron microscopy characterization of nanomaterial COF-366 (1); Transmission electron microscopy characterization of nanodrug SC@GRT-COF-366 (2); Representative images and particle size characterization of COF-366 and SC@GRT-COF-366 (3); Schematic diagram of the composition of SC@GRT-COF-366.

[0041] Figure 2 This is a synthetic route for the dual-target molecule Gal-D5HT.

[0042] Figure 3 The image shows the 1H NMR spectrum of compound 1 (Gal-Ac-Br).

[0043] Figure 4 The image shows the 1H NMR spectrum of compound 2 (Gal-Ac-N3).

[0044] Figure 5 The image shows the 1H NMR spectrum of compound 3 (Gal-N3).

[0045] Figure 6 The image shows the 1H NMR spectrum of compound 4 (D-5HT-Boc).

[0046] Figure 7 This is the 1H NMR spectrum of compound 5 (D-TA).

[0047] Figure 8 The image shows the 1H NMR spectrum of compound 6 (D-5HT).

[0048] Figure 9 The image shows the 1H NMR spectrum of compound 7 (D-TA).

[0049] Figure 10 The image shows the 1H NMR spectrum of compound 8 (Gal-D5HT).

[0050] Figure 11 The image shows the 1H NMR spectrum of compound 9 (Gal-DTA). Detailed Implementation

[0051] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0052] Example 1: Synthesis of COF-366 5,10,15,20-tetratetra(4-aminophenyl)porphyrin (TAPP, 14 mg, 0.02 mmol), terephthalaldehyde (TPAL, 5.36 mg, 0.04 mmol), and polyvinylpyrrolidone (PVP, 200 mg) were dissolved in a mixed solvent of anhydrous DMF and methanol (4 mL, v / v, 1:4). Trifluoroacetic acid (TFA, 0.25 mL) was then added, and the mixture was stirred at 120°C for 30 minutes. After the reaction was complete, the reaction mixture was poured into a mixture of methanol and triethylamine, centrifuged at 12000 rpm for 10 minutes, and the resulting solid precipitate was collected. The solid was washed five times with methanol and then freeze-dried for 48 hours to obtain the final product COF-366.

[0053] Example 2: Synthesis of dual-target Gal-D5HT Synthetic routes such as Figure 2 As shown.

[0054] 2.1) Synthesis of Compound 1 (Gal-Ac-Br) Boron trifluoride diethyl ether (BF3·Et2O, 2.6 mL, 0.021 mmol) was added to a 30 mL solution of dichloromethane containing 1,2,3,4,6-penta-O-acetyl-d-galactopyranose (5.0 g, 0.013 mmol) and 2-bromoethanol (2.80 mL, 0.02 mmol). The mixture was stirred overnight at room temperature under nitrogen protection, then washed with 4% sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by column chromatography (ethyl acetate / n-hexane = 25%) to give compound 1 (4.78 g, 75% yield).

[0055] 1H NMR (400 MHz, CDCl3) δ 5.38 (d, J = 3.4 Hz, 1H), 5.22–5.15 (m, 1H), 5.02 (ddd, J = 10.5, 3.4, 0.9 Hz, 1H), 4.47 (dd, J = 7.9, 1.0 Hz, 1H), 4.21–4.08 (m, 2H), 4.02–3.96 (m, 1H), 3.91 (t, J = 6.7 Hz, 1H), 3.68 (td, J = 9.1,4.2 Hz, 1H), 3.51–3.44 (m, 2H), 2.14 (s, 3H), 2.07 (s, 3H), 2.04 (s, 3H), 1.98 (s, 3H).

[0056] 2.2) Synthesis of Compound 2 (Gal-Ac-N3) Compound 1 (4.79 g, 0.01 mmol) was reacted with sodium azide (NaN3, 1.22 g, 0.02 mmol) in DMF (13.5 mL) and stirred at 70°C under argon protection for 2 hours. Ethyl acetate was added after the reaction, the mixture was washed with water, dried over anhydrous sodium sulfate, and concentrated. Purification by column chromatography (ethyl acetate / n-hexane = 50%) gave compound 2 (4.31 g, 95% yield).

[0057] 1 H NMR (400 MHz, CDCl3) δ 5.37 (dd, J = 3.5, 1.2 Hz, 1H), 5.18 (dd, J= 10.5, 7.9 Hz, 1H), 5.00 (dd, J = 10.5, 3.4 Hz, 1H), 4.45 (d, J = 7.9 Hz,1H), 4.14 (qd, J = 11.1, 6.6 Hz, 2H), 3.98–3.87 (m, 2H), 3.78 (t, J = 5.9 Hz,1H), 3.62–3.49 (m, 2H), 3.39–3.29 (m, 2H), 2.13 (s, 3H), 2.04 (d, J = 6.8 Hz, 6H), 1.97 (s, 3H).

[0058] 2.3) Synthesis of Compound 3 (Gal-N3) Compound 2 (4.31 g, 0.0095 mmol) and sodium methoxide (MeONa, 171 mg, 0.0048 mmol) were dissolved in methanol (100 mL) under nitrogen protection and stirred at room temperature for 2 hours. After neutralization with Amberlite IR-12 resin, the mixture was filtered, the filtrate was concentrated, and purified by column chromatography (methanol / chloroform = 10%) to give compound 3 (2.66 g, 98% yield).

[0059] 1 H NMR (400 MHz, CDCl3) δ 4.41 (d, J = 7.9 Hz, 1H), 4.02 (dt, J =10.4, 6.2 Hz, 1H), 3.94 (d, J = 3.4 Hz, 1H), 3.82–3.74 (m, 3H), 3.68 (ddd, J= 18.2, 8.9, 4.0 Hz, 2H), 3.55–3.46 (m, 3H), 1.93 (p, J = 6.5 Hz, 2H).

[0060] 2.4) Synthesis of Compound 4 (D-5HT-Boc) N-hydroxysuccinimide (NHS, 230 mg, 2 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 382 mg, 2 equiv) were added to 4 mL of DMF solution containing Boc-DL-Glu (247 mg, 1 mmol) and stirred at room temperature for 30 minutes. Then, a DMF solution (2 mL) containing 5-hydroxytryptamine (5-HT, 440 mg, 2 mmol) and triethylamine (Et3N, 200 µL, 2 equiv) was added. After stirring the mixture for 2 hours, it was filtered and purified by reversed-phase chromatography (acetonitrile / water) to give compound 4 (383 mg, 68% yield).

[0061] 1H NMR (400 MHz, deuterated methanol) δ 7.14 (dd, J = 8.7, 4.7 Hz, 2H), 7.01 (d,J = 3.7 Hz, 2H), 6.93 (dd, J = 4.0, 2.4 Hz, 2H), 6.66 (dt, J = 8.6, 2.1 Hz,2H), 3.51–3.39 (m, 4H), 3.20 (q, J = 7.3 Hz, 2H), 2.99 (s, 2H), 2.86 (d, J =3.4 Hz, 4H), 2.17 (t, J = 7.6 Hz, 2H), 1.95 (s, 1H), 1.80 (dt, J = 14.4, 7.9Hz, 1H), 1.42 (d, J = 6.4 Hz, 9H).

[0062] 2.5) Synthesis of Compound 5 (D-TA) The synthesis steps of compound 5 are the same as those of compound 4, except that tryptamine (TA) is used instead of 5-hydroxytryptamine.

[0063] 1 ¹H NMR (400 MHz, deuterated methanol) δ 7.53–7.49 (m, 2H), 7.28 (dd, J = 8.1, 5.1 Hz, 2H), 7.06–7.00 (m, 4H), 6.95 (tdd, J = 7.1, 2.4, 1.1 Hz, 2H), 3.44 (dt, J = 19.1, 7.7 Hz, 4H), 3.27 (p, J = 1.7 Hz, 2H), 2.94–2.85 (m, 4H), 2.12 (t, J = 7.5 Hz, 2H), 1.91 (d, J = 7.6 Hz, 1H), 1.83–1.65 (m, 1H), 1.38 (s, 9H).

[0064] 2.6) Synthesis of Compound 6 (D-5HT) Compound 4 (383 mg, 0.68 mmol) was dissolved in 2 mL of dichloromethane, and 0.4 mL of trifluoroacetic acid (TFA) was added. The mixture was stirred overnight at room temperature. After removing dichloromethane and excess TFA under reduced pressure, the residue was dissolved in DMF, and excess triethylamine was added. Subsequently, a solution of 4-pentenoic acid (133 mg, 2 equiv) activated in DMF (2 mL) with NHS (2 equiv) and EDC (2 equiv) for 30 min was added to the above mixture. The reaction was carried out for 2 h, and the mixture was purified by reversed-phase chromatography (acetonitrile / water) after filtration to give compound 6 (162 mg, yield 54%).

[0065] 1 ¹H NMR (400 MHz, deuterated methanol) δ 7.11 (dd, J = 8.6, 4.3 Hz, 2H), 6.97 (s, 2H), 6.89 (d, J = 2.3 Hz, 2H), 6.62 (dt, J = 8.7, 2.1 Hz, 2H), 4.22 (dd, J = 8.9, 5.3 Hz, 1H), 3.41 (dt, J = 11.3, 7.1 Hz, 4H), 3.27 (p, J = 1.7 Hz, 4H), 3.15 (q, J = 7.3 Hz, 1H), 2.95 (s, 1H), 2.83–2.79 (m, 4H), 2.43–2.34 (m, 4H), 2.16–2.12 (m, 2H), 2.03–1.93 (m, 1H), 1.86–1.73 (m, 1H), 1.25 (d, J = 7.3 Hz, 2H).

[0066] In step 2.6), during the 4-pentenoic acid linkage step, HOBt / HATU can be used instead of NHS / EDC as the condensation reagent system. Results: The yield is comparable to or slightly improved compared to the original method (e.g., from 65% to 75%), and byproducts may be reduced. Analysis: HOBt (or HOAt) effectively inhibits racemization, and HATU is a highly efficient condensation reagent. For amidation reactions containing chiral amino acid substrates, the HOBt / HATU system typically provides purer products and higher yields.

[0067] 2.7) Synthesis of Compound 7 (D-TA) The synthesis of compound 7 was performed in the same manner as that of compound 6, using compound 5 as the starting material (146 mg, yield 58%).

[0068] 1¹H NMR (400 MHz, deuterated methanol) δ 7.55 (d, J = 7.8 Hz, 2H), 7.31 (dd, J = 8.1, 4.5 Hz, 2H), 7.11–7.03 (m, 4H), 7.03–6.96 (m, 2H), 4.26 (dd, J = 8.9, 5.3 Hz, 1H), 3.51–3.42 (m, 4H), 2.93 (td, J = 7.2, 3.4 Hz, 4H), 2.46–2.38 (m, 4H), 2.21–2.12 (m, 3H), 2.00 (dtd, J = 15.6, 8.0, 7.4, 5.4 Hz, 1H). 1.84 (ddd, J = 13.7, 8.6, 6.8 Hz, 1H).

[0069] 2.8) Synthesis of Compound 8 (Gal-D5HT) Compound 3 (171 mg, 0.6 mmol) and compound 6 (162 mg, 0.3 mmol) were dissolved in water (2 mL) and DMF (4 mL). Ascorbic acid (115 mg, 0.6 mmol) and copper sulfate pentahydrate (CuSO4·5H2O, 7.2 mg, 0.03 mmol) were dissolved in water (1 mL) and added to the reaction system in sequence. The mixture was stirred overnight, filtered, and purified by reversed-phase chromatography (acetonitrile / water) to obtain compound 8 (215 mg, yield 89%).

[0070] 1H NMR (400 MHz, D2O) δ 7.04 (d, J = 6.0 Hz, 1H), 6.78 (dd, J = 14.6,8.3 Hz, 1H), 4.34 (t, J = 6.8 Hz, 2H), 4.19 (d, J = 7.6 Hz, 1H), 4.00 (d, J =7.4 Hz, 1H), 3.86 (s, 1H), 3.80–3.74 (m, 2H), 3.72 (s, 2H), 3.66 (s, 2H), 3.60 (d, J = 8.3 Hz, 1H), 3.55–3.50 (m, 2H), 3.43 (dt, J = 12.1, 6.4 Hz, 3H),3.36 (s, 2H), 3.22 (d, J = 7.4 Hz, 1H), 2.88 (s, 3H), 2.61 (t, J = 6.4 Hz,1H), 2.50 (t, J = 6.9 Hz, 1H), 2.09–2.01 (m, 2H), 1.83 (d, J = 7.6 Hz, 1H), 1.36 (d, J = 6.3 Hz, 4H), 1.29 (s, 4H), 0.89 (s, 4H).

[0071] 2.9) Synthesis of Compound 9 (Gal-DTA) The synthesis of compound 9 was performed in the same manner as that of compound 8, using compound 7 as the starting material (206 mg, 94% yield).

[0072] 1¹H NMR (600 MHz, deuterated methanol) δ 7.56 (dd, J = 9.0, 7.8 Hz, 2H), 7.31 (t, J = 8.0 Hz, 2H), 7.14–7.04 (m, 4H), 7.02–6.95 (m, 2H), 4.59 (s, 6H), 4.45 (t, J = 6.8 Hz, 2H), 4.26 (dd, J = 9.1, 4.9 Hz, 1H), 4.20–4.14 (m, 1H), 3.82 (s, 2H), 3.74 (t, J = 6.6 Hz, 2H), 3.64 (s, 1H), 3.52 (dd, J = 7.1, 2.9 Hz, 2H). 3H), 3.46 (d, J = 3.2 Hz, 4H), 3.02–2.90 (m, 5H), 2.57 (d, J = 7.1 Hz, 1H), 2.11 (dd, J = 14.6, 7.4 Hz, 3H).

[0073] ESI-MS m / z: [M + H] + 775.38 (100.0%), 776.38 (43.1%), 777.388 (11.8%); Measured value 775.25.

[0074] Example 3: Synthesis of the nanomedicine SC@GRT-COF-366 3.1) COF-366, Sor, and CQ were dissolved separately in 1 mL of DMSO solution and stirred at room temperature for 12 hours at different ratios (1:1:1, 1:1:2, 2:1:1, and 2:1:2, c / c / c). Then, the mixture was centrifuged at 12000 rpm for 20 minutes and washed three times with ultrapure water. The resulting material was freeze-dried for 48 hours to obtain SC@COF-366.

[0075] 3.2) Next, SC@COF-366 (1 mL, 5 mg / mL H2O solution) was mixed with Gal-D5HT or Gal-DTA (1 mL, 5 mg / mL H2O solution) and Rhodamine B (RhB, 1 mL, 5 mg / mL H2O solution) and stirred at room temperature for 12 hours. Then, it was centrifuged at 12000 rpm for 20 minutes and washed three times with ultrapure water. The resulting material was freeze-dried for 48 hours to obtain SC@GRT-COF-366 or SC@GRD-COF-366.

[0076] Example 4: Characterization of COF-366 and SC@GRT-COF-366 4.1) Ultraviolet-Vis Spectrophotometer (UV-Vis): The COF-366 prepared in Example 1 and its raw material TAPP were dissolved in methanol solution, and the synthesis of COF-366 was detected by UV-Vis spectrophotometry. Methanol solutions of different concentrations of Sor (265 nm), CQ (332 nm), Gal-D5HT or Gal-DTA (309 nm), and RhB (555 nm) were prepared and scanned with UV-Vis, and standard curves were plotted at appropriate absorbance positions. Furthermore, the supernatant from the synthesis of SC@COF-366 and SC@GRT-COF-366 was collected and diluted with methanol solution, and the corresponding drug loading efficiency (DLE) and encapsulation efficiency (DEE) were calculated by UV-Vis.

[0077] DLE (%) = (Amount of drug loaded into nanomaterials / Total mass of drug-loaded nanomaterials) × 100% DEE (%) = Amount of drug loaded into nanomaterials / Total mass of drug added × 100%.

[0078] The results showed that the synthesis of COF-366 exhibited an absorption band at 425 nm, a blue shift of 12 nm compared to the monomeric precursor TAPP. This blue shift indicates the formation of H-aggregates of porphyrin S30 units in the stacked structure, confirming successful synthesis. Simultaneously, absorption peaks for Sor (265 nm), Gal-D5HT (309 nm), CQ (335 nm), and RhB (550 nm) were also present in SC@GRT-COF-366, confirming successful loading. Based on these measurements, an optimal drug loading ratio (COF-366:Sor:CQ=1:1:2, c / c / c) was established, with DLC values ​​of 8.5±0.35% for Sor and 7.8±0.24% for CQ in SC@GRT-COF-366.

[0079] 4.2) Infrared Spectrometer The COF-366 prepared in Example 1, along with its raw materials TAPP and TPAL, and the SC@COF-366 and SC@GRT-COF-366 prepared in Example 3, were ground and compressed into tablets with a certain amount of potassium bromide. The characteristic peaks of each component were analyzed using Fourier transform infrared spectroscopy (FTIR) to characterize the successful synthesis of SC@GRT-COF-366.

[0080] The results showed that in the Fourier transform infrared spectrum of COF-366, 1696 cm⁻¹ -1 and 3370 cm-1 The stretching vibrations are attributed to the C=O bonds in TPAL and the NH bonds in TAPP, respectively. At 1620 cm⁻¹ -1 The appearance of a vibrational band at 1706 cm⁻¹ corresponds to a C=N bond, indicating a successful condensation reaction between the aldehyde and amine groups. In the FTIR spectra of SC@COF-366 and SC@GRT-COF-366, compared to COF-366, the band at 1706 cm⁻¹ is [missing information]. -1 (C=O) and 1556 cm -1 The stretching vibrations at (NH) show a redshift, which is attributed to the bonds in Sor and CQ. The significant enhancement of the hydroxyl peak in SC@GRT-COF-366 indicates that Gal-D5HT has been encapsulated.

[0081] 4.3) Transmission electron microscopy (TEM) imaging The COF-366 prepared in Example 1, the SC@GRT-COF-366 prepared in Example 3, and the GRT-COF366 and GRD-COF366 treated with MPO and H2O2 were respectively prepared into suspensions of 50 μg / ml and ultrasonically dispersed in ddH2O. A portion of the suspension was taken by the inoculation loop and placed on a copper grid. After drying at room temperature overnight, the particle size and morphology were observed.

[0082] The results showed that COF-366 was a uniform spherical state with a particle size of about 100 nm. After being encapsulated by the drug and the targeting molecule, the overall morphology of SC@GRT-COF-366 did not change much compared to COF366, indicating that the drug was encapsulated inside the pores.

[0083] 4.4) Dynamic Light Scattering Analyzer (DLS) The nanomaterial COF-366 prepared in Example 1, SC@COF-366 and SC@GRT-COF-366 prepared in Example 3 were dispersed in PBS buffer (pH=7.4), and the particle size, uniformity and zeta potential of the nanomaterials were analyzed by DLS.

[0084] The results showed that dynamic light scattering (DLS) analysis revealed that COF-366 and SC@GRT-COF-366 were uniformly sized spheres with a particle size of approximately 100 nm, and the size of SC@GRT-COF-366 increased slightly after encapsulation. Simultaneously with encapsulation, the sites of COF-366 gradually shifted from negative charge to positive charge, resulting in a gradual increase in the site size of both SC@COF-366 and SC@GRT-COF-366.

[0085] Example 5: Performance evaluation of covalent organic nanoframework SC@GRT-COF-366 5.1) Stability of SC@GRT-COF-366 To investigate the stability of SC@GRT-COF-366 nanomaterials, they were dispersed in PBS buffer (pH=7.4) and DMEM serum-free medium, and the changes in hydrated particle size of the nanomaterials were monitored using DLS over 7 days.

[0086] Stability testing showed that the particle size of SC@GRT-COF-366 did not change significantly in buffer and DMEM within seven days, indicating that the nanoparticles have a certain degree of stability.

[0087] 5.2) Drug release capability of SC@GRT-COF-366 Five mg of SC@GRT-COF-366 nanoframeworks were suspended in 20 mL of PBS buffer containing different pH values ​​(pH 7.4 or pH 5.0) and stirred at 37°C. At different time points (h 1, 2, 4, 8, 12, 24, 36, and 48), 2 mL of supernatant was aspirated and replenished with an equal volume of fresh buffer. The amount of drug released at each time point was measured using UV-Vis, and the total amount of drug released was calculated using a release-cumulative method.

[0088] The results are shown in Table 1.

[0089] Table 1

[0090] 5.3) Production of SC@GRT-COF-366 1 O2 capacity DPBF was used as a probe. A final concentration of 50 μM DPBF was added to a DMSO solution with a concentration of 30 μg / mL SC@GRT-COF-366. The probe was then analyzed using a wavelength of 660 nm and a power density of 0.1 W / cm². 2 The mixture was irradiated with a laser for different durations (0, 3, 6, 9, 12 and 15 min), and the absorbance of the mixture after irradiation was measured using UV-Vis.

[0091] The results showed that 1,3-diphenylisobenzofuran (DPBF) was used as the molecular probe for quantitative detection. The results indicated that... 1 The generation of O2 requires the presence of 660 nm light (SC@GRT-COF-366). When using a density of 20 mW / cm²... -2 When irradiated with 660 nm light for 15 minutes, the absorbance of DPBF decreased to ~0.1, confirming... 1 The efficient production of O2. It is noteworthy that we observed... 1The concentration- and time-dependent increase in O2 generation is evidenced by the accelerated degradation of DPBF with increasing concentration. These results confirm the efficiency of SC@GRT-COF-366 in PDT under 660 nm illumination.

[0092] Example 6: Cytotoxicity assay of covalent organic nanoframework SC@GRT-COF-366 To investigate ASGPR-targeted synergistic therapy, HepG2, Huh7, HEK293, and C5WN1 cells were cultured at 5 × 10⁶ cells per well. 3 Individual samples were seeded at a density of [number] cells / well in 96-well plates and incubated for 24 hours. Subsequently, each system was supplemented with a solution containing sorafenib (2 μg / mL). -1 ), chloroquine (3 μg·mL) -1 COF-366 (20 μg·mL) -1 ), SC@COF-366 (25 μg·mL) -1 ) or SC@GRT-COF-366 (30 μg·mL -1 The cells were cultured in DMEM medium for 24 hours. Afterward, they were irradiated with a 660 nm laser for 15 minutes and cultured for another 24 hours. Cell viability was assessed using the MTT assay.

[0093] The results showed that, to further explore the synergistic therapeutic efficacy of SC@GRT-COF-366, the cytotoxic effects of its components were evaluated using the MTT assay. The concentration of the encapsulated drug corresponded to 30 μg / mL. -1 The SC@GRT-COF-366 assay showed negligible cytotoxicity under light-free conditions; however, under 660 nm irradiation, cell viability decreased sharply (C5WN1 decreased to 51%, Huh7 to 56%, HepG2 to 62%, and SMMC-7721 to 54%), indicating a significant photodynamic therapy (PDT) effect. Sorafenib (Sor) and chloroquine (CQ) treatments induced only a small number of apoptosis, with cell viability maintained at around 80%. In contrast, SC@COF-366, lacking targeting ability, reduced liver cancer cell viability to 50% under light-free conditions and further to 30% under 660 nm irradiation, demonstrating a synergistic effect of PDT / chemotherapy (CT). Notably, the targeted synergistic therapy of SC@GRT-COF-366 combined with light irradiation significantly reduced cell viability to approximately 10%.

[0094] Example 7: In vitro ASGPR targeting detection of covalent organic nanoframework SC@GRT-COF-366 To validate ASGPR targeting in vitro, a galactose competition assay was used to detect the cell targeting of SC@GRT-COF-366. Specifically, 1×10 4 Or 5×10 4 Cells were seeded into 48-well or 6-well plates and cultured at 37°C and 5% CO2 for 24 hours. Then, SC@GRT-COF-366 (30 μg / mL) was added. -1 Before culturing, cells were pretreated with 1 mM galactose for 12 hours, followed by culturing at 37°C and 5% CO2 for 5 hours. Cells were washed three times with PBS buffer (pH 7.4) before fluorescence observation (Zeiss, Germany) and flow cytometry analysis (BD, USA).

[0095] Results showed that, to investigate the in vitro liver-targeting ability of SC@GRT-COF-366, its cellular uptake behavior in ASGPR-positive cell lines (ASGPR+, including C5WN1, Huh7, HepG2, and SMMC-7721) and ASGPR-negative cell lines (ASGPR-, HEK293) was studied using fluorescence microscopy and flow cytometry. Fluorescence results showed that, compared to HEK293 cells (ASGPR-), the fluorescence intensity of rhodamine B (RhB) and COF-366-derived TAPP was significantly enhanced in ASGPR-positive cells, indicating that SC@GRT-COF-366 mainly enters tumor cells through the ASGPR receptor. When cells were pretreated with galactose (Gal+, 1 mM) for 12 hours to competitively block ASGPR, the fluorescence intensity in ASGPR-positive cells decreased sharply due to receptor occupancy, while only a weak RhB signal was observed in HEK293 cells. These results demonstrate that galactose recognition of ASGPR-mediated cellular uptake can be competitively inhibited, further confirming that the ASGPR pathway is the primary route for nanoparticle internalization. Flow cytometry analysis further validated these results: the mean RhB fluorescence intensity of galactose-pretreated (Gal+) HepG2 cells was three-fold lower than that of untreated (Gal-) cells. These results collectively demonstrate that ASGPR-mediated endocytosis is crucial for the hepatocyte targeting ability of SC@GRT-COF-366.

[0096] Example 8: Detection of in vitro aggregation of covalent organic nanoframework SC@GRT-COF-366 under the action of MPO and H2O2 To verify in vitro that SC@GRT-COF-366 can aggregate under the action of MPO and H2O2, the surface of nanoparticles were modified with Gal-D5HT and its negative control Gal-DTA, and the following experiments were conducted: In the in vitro experiment, 1×10 4Cells were seeded in 48-well plates and cultured at 37°C and 5% CO2 for 24 hours. The experimental groups were treated with GRT-COF-366 (1 mg / mL). -1 ), or GRT-COF-366 (1 mg·mL) -1 ) and MPO (50 μg·mL -1 Cells were treated with MPO and H2O2 (0.5 mmol) and cultured at 37°C for 12 hours; the control group received no MPO or H2O2. Cells were washed three times with PBS buffer (pH 7.4) before fluorescence observation.

[0097] The results showed that, to further investigate the MPO / H2O2 response behavior, the changes in GRT-COF-366 and GRD-COF-366 nanoparticles modified with Gal-D5HT and Gal-DTA after 24 hours of incubation with MPO / H2O2 were evaluated. Transmission electron microscopy (TEM) analysis revealed significant morphological differences: GRD-COF-366, serving as a negative control, maintained its spherical morphology (diameter 100-200 nm); in stark contrast, GRT-COF-366 nanoparticles significantly aggregated under MPO / H2O2 induction, forming large cluster structures. Quantitative dynamic light scattering (DLS) detection further confirmed this aggregation phenomenon: after MPO / H2O2 treatment, the hydrodynamic diameter increased sharply from 180±26 nm to 603±45 nm. More importantly, when studying the MPO / H2O2-mediated intracellular aggregation of GRT-COF-366 in Huh7 cells, fluorescence imaging showed that Gal-D5HT-modified GRT-COF-366 exhibited numerous strong fluorescent spots in the perinuclear region, indicating significant enrichment of RhB signal. These results clearly demonstrate that Gal-D5HT can be synergistically activated by MPO / H2O2, leading to the aggregation of modified nanoparticles in the PBS environment and within cells. This mechanism endows SC@GRT-COF-366 with MPO-dependent targeting and aggregation capabilities.

[0098] Example 9: In vivo targeting detection of covalent organic nanoframework SC@GRT-COF-366 In a mouse model of subcutaneous HCC, the tumor volume reached 100 mm. 3 Animals were randomly divided into four groups and injected with different nano-formulations (100 μL, 10 mg·kg⁻¹) via the tail vein. -1The formulations included SC@RHB-COF-366 (untargeted), SC@GRD-COF-366 (ASGPR-targeted), SC@RT-COF-366 (MPO-targeted), and SC@GRT-COF-366 (ASGPR / MPO dual-targeted). In vivo distribution of RhB-labeled nanoparticles was monitored at 4, 8, 12, and 24 hours post-injection using a Bruker In Vivo Xtreme II imaging system (Bruker, USA).

[0099] The results showed that, based on the above findings, the tumor-targeting ability and biodistribution behavior of SC@GRD-COF-366 (ASGPR single-target), SC@RT-COF-366 (MPO single-target), and SC@GRT-COF-366 (ASGPR-MPO dual-target) in C5WN1 tumor-bearing nude mice were further investigated, with SC@RhB-COF-366 (without targeting function) serving as a negative control. All nanoparticles were additionally loaded with RhB for convenient real-time tracking and in vivo imaging. RhB fluorescence was monitored at 4, 8, 12, and 24 hours after injection via tail vein injection. In the non-targeted group, a small amount of SC@RhB-COF-366 accumulated at the tumor site 8 hours after injection through enhanced permeation-retention (EPR) effect. In contrast, the targeted group showed significantly enhanced and time-dependent tumor site fluorescence signals from 4 to 24 hours after injection. Notably, 4 hours after injection, the tumor fluorescence intensity of SC@RT-COF-366 (MPO-targeted) and SC@GRT-COF-366 (dual-targeted) was higher than that of SC@GRD-COF-366 (ASGPR-targeted), indicating that MPO-mediated targeting achieves tumor enrichment faster than ASGPR targeting. Particularly noteworthy is that SC@GRT-COF-366 maintained a strong tumor fluorescence signal for up to 24 hours after injection, demonstrating superior retention compared to other formulations. This enhanced tumor accumulation can be attributed to the aggregation effect of the MPO-targeted nanomaterials, thereby prolonging their retention time in the tumor microenvironment. Ex vivo fluorescence imaging results validated the above in vivo observations. At all time points, the fluorescence intensity of SC@GRT-COF-366 in tumor tissue was significantly higher than that in other organs and the single-targeted group. Most notably, even 24 hours after injection, SC@GRT-COF-366 still maintained a large amount of fluorescence accumulation in tumor tissue, exhibiting excellent tumor retention performance.

[0100] To investigate the effect of light irradiation on the distribution of nanoparticles in tumor tissue, the following experimental protocol was designed: Eight hours after the initial injection of nanoparticles (determined as the optimal time for initial tumor accumulation), the tumor site was irradiated with a 660 nm laser for 15 minutes. A second dose was then injected 24 hours after irradiation, and the accumulated amount of nanoparticles within the tumor was measured 4 hours later. In vitro and in vivo fluorescence imaging results showed that the fluorescence intensity of all targeted groups (SC@GRD-COF-366, SC@RT-COF-366, and SC@GRT-COF-366) was significantly enhanced 4 hours after laser irradiation compared to before irradiation, indicating that laser stimulation can significantly enhance the enrichment of nanomaterials in the tumor region. To evaluate the correlation between fluorescence imaging results and neutrophil infiltration and MPO expression in tumor tissue, immunohistochemical analysis was performed on mouse tumor tissues treated with different methods. The results showed that compared with the unirradiated control group, the number of neutrophils and the level of MPO protein were significantly increased in the irradiated group. These results demonstrate that laser irradiation can induce an acute inflammatory response in the tumor microenvironment, characterized by increased neutrophil infiltration and upregulation of myeloperoxidase (MPO) expression. These two factors synergistically enhance the tumor accumulation of SC@RT-COF-366 and SC@GRT-COF-366 through an MPO-dependent targeting mechanism. Furthermore, acute inflammation in the tumor microenvironment enhances the EPR effect, thereby promoting the accumulation of ASGPR-targeted SC@GRD-COF-366 nanoparticles.

[0101] Example 10: Synergistic antitumor effect of covalent organic nanoframework SC@GRT-COF-366 in subcutaneous tumor mice In vivo anti-tumor efficacy evaluation: In a subcutaneous tumor model, the tumor volume reached 50 mm. 3 Mice were randomly divided into nine groups (n = 5) and intravenously injected with 100 μL of different formulations, including physiological saline, sorafenib (2 mg / kg), etc. -1 COF-366 (12 mg·kg) -1 Sor@COF-366 (12 mg·kg) -1 ), SC@COF-366 (12 mg·kg) -1 ), SC@GR-COF-366 (12 mg·kg -1 ), SC@RT-COF-366 (12 mg·kg -1 ), SC@GRT-COF-366 (12 mg·kg -1 ) and SC@GRT-COF-366+ light therapy group (12 mg·kg -1Tumor volume and body weight in mice were monitored and recorded every two days. After 14 days of treatment, mice were euthanized with CO2, and tumor tissue was removed, weighed, and photographed. Tumor tissue and major organs (liver, spleen, kidney, heart, and lungs) were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and used for immunofluorescence analysis.

[0102] The results showed that, based on the aforementioned excellent tumor-targeting properties, the anti-hepatocellular carcinoma efficacy of SC@GRT-COF-366 in a C5WN1 subcutaneous xenograft model was systematically evaluated. When the tumor volume reached approximately 50 mm... 3 Treatment began at a specific time, with nine experimental groups (n=5): saline group, COF-366 group, sorafenib (Sor) group, Sor@COF-366 group, SC@COF-366 group, SC@GRD-COF-366 group, SC@RT-COF-366 group, SC@GRT-COF-366 group, and SC@GRT-COF-366 + light irradiation group. All drugs were administered via tail vein injection every three days for two weeks. The light irradiation group received 660 nm laser irradiation 8 hours after injection. Tumor growth was monitored every two days, and final tumor weight was analyzed after sacrifice. The tumor growth inhibition rate (TGI) was calculated for each treatment group, with the saline group serving as a control. Comparative TGI analysis revealed significant differences in efficacy among different treatment regimens: the COF-366 group experienced rapid tumor progression with a TGI of only 8%, confirming that the blank nanomaterials had no significant anti-tumor activity; the TGIs of sorafenib monotherapy (Sor) and its complex (Sor@COF-366) were 24.2% and 36.5%, respectively; notably, the combination of sorafenib and chloroquine (CQ) (SC@COF-366) significantly increased the TGI to 53.8% (P<0.001), a 1.5-2.2-fold increase compared to sorafenib monotherapy, clearly demonstrating that CQ-mediated autophagy inhibition can enhance the efficacy of sorafenib. Targeted modification further improved treatment outcomes: the TGI of SC@GRD-COF-366 (ASGPR-targeted) reached 62.0%, SC@RT-COF-366 (MPO-targeted) rose to 73.2%, and the dual-targeted SC@GRT-COF-366 reached 81.1%. Most notably, SC@GRT-COF-366 combined with phototherapy exhibited remarkable anti-tumor efficacy, with a TGI of up to 93%, showing a statistically significant improvement compared to monotherapy (p<0.01), highlighting the synergistic effect of PDT and nanomedicine. This evidence fully demonstrates the synergistic effect of chemotherapy (CT) and photodynamic therapy (PDT) in this delivery system, and that targeted modification and combination therapy can gradually enhance anti-tumor efficacy.

[0103] During treatment, there were no significant changes in the body weight of mice in any group. Histopathological examination (H&E staining) showed that the major organ structures were intact, with no obvious pathological changes, indicating that SC@GRT-COF-366 has good biocompatibility and low toxicity. Liver and kidney function indicators (ALT, AST, ALP, CREA, BUN) were all within the normal range, confirming no liver or kidney toxicity; blood routine analysis (WBC, RBC, HGB, PLT) showed that hematopoietic function was not affected and there were no signs of systemic infection. Based on the good biocompatibility and excellent tumor suppression effect, we further evaluated the efficacy of this synergistic therapy through staining experiments.

[0104] Example 11: Detection of synergistic inhibition of tumor lung metastasis in mice by covalent organic nanoframework SC@GRT-COF-366 orthotopic tumor To evaluate the inhibitory effect of nanomedicines on metastasis in an in situ HCC model, mice bearing tumors for 10 days were randomly divided into three groups (n = 5), receiving intravenous injections of saline, SC@GRT-COF-366, and SC@GRT-COF-366 plus light treatment, respectively. The frequency, duration, and dosage of drug administration were consistent with those in subcutaneous tumor model mice. Intraoperative photodynamic therapy (iPDT) was performed using a custom-designed 660 nm laser system. Following the method described by Hu et al., an optical fiber (0.6 mm in diameter) was integrated with a 19G percutaneous peritoneal puncture needle (Nanjing Zhandian Technology Co., Ltd.) for light-guided intervention (Acta Biomater, 2022, 20, 206-217).

[0105] The results showed that C5WN1 cells, obtained by transforming rat liver progenitor cells and transfecting them with green fluorescent protein (GFP), exhibited stable lung metastasis characteristics. To further investigate the anti-metastatic effect of SC@GRT-COF-366, we established an orthotopic hepatocellular carcinoma model by directly injecting C5WN1 cells into the left lobe of the mouse liver, thus constructing a C5WN1 tumor-bearing mouse model with spontaneous lung metastasis characteristics. On day 10 after tumor inoculation, SC@GRT-COF-366 was injected via the tail vein, and 8 hours later, the tumor was irradiated with a 660 nm laser for PDT treatment. Lung metastasis was monitored by in vivo micro-computed tomography (Micro-CT; Bruker, USA) on days 0, 7, and 14 post-treatment.

[0106] Three-dimensional micro-CT reconstruction and lung density analysis confirmed that all groups were tumor-free and had smooth lung surfaces on day 0. By days 7 and 14, all control groups showed varying degrees of lung parenchymal loss, indicating metastatic invasion. In stark contrast, tumor progression was significantly slowed in the SC@GRT-COF-366 treatment group. Notably, the SC@GRT-COF-366 + phototherapy combination therapy group maintained the best lung volume stability throughout the study. These findings strongly demonstrate the remarkable efficacy of this synergistic therapy in inhibiting metastatic lesion growth.

[0107] In summary, this invention discloses a nanomedicine with dual targeting of myeloperoxidase-desialylglycoprotein receptor to inhibit hepatocellular carcinoma proliferation and metastasis. In vivo experiments show that the SC@GRT-COF-366 combined therapy platform achieves multiple synergistic therapeutic strategies: (i) the co-loaded sorafenib / chloroquine (Sor / CQ) exhibits stronger cytotoxicity against HCC cells, and the targeting function and PDT further enhance the therapeutic effect of the dual drugs; (ii) sorafenib-induced protective autophagy is reversed through chloroquine-mediated autophagy inhibition, thereby overcoming drug resistance; (iii) the reactive oxygen species (ROS) generated by PDT amplify the oxidative stress effect, promoting apoptosis while disrupting autophagic flux. In vivo evaluation shows that compared with single-targeting systems, this dual-targeting system has a faster tumor aggregation rate and a longer retention time. In terms of therapeutic efficacy, this platform significantly delays tumor growth in subcutaneous xenograft models and effectively inhibits primary liver tumors and lung metastases in orthotopic HCC models, while maintaining excellent biocompatibility. These findings collectively validate that the MPO-ASGPR dual-targeting strategy is an effective approach for HCC treatment, demonstrating that the synergistic combination of PDT and autophagy inhibition can significantly enhance the efficacy of sorafenib chemotherapy while overcoming drug resistance mechanisms. The SC@GRT-COF-366 platform offers a promising multimodal treatment strategy for advanced HCC.

[0108] Comparative Example 1 Referring to Example 1, the corresponding COF-366 product was obtained by simply not adding PVP and keeping other reaction parameters unchanged.

[0109] Results: The COF-366 product collected by centrifugation was severely agglomerated and difficult to redisperse. TEM images showed that the product was an amorphous blocky structure with uneven size and severe aggregation, rather than regular nanospheres. Specific surface area: BET tests showed a significantly reduced specific surface area (e.g., ≤ 200 m²). 2 This is because, lacking the template effect of PVP, the monomers rapidly and disorderly cross-link, forming dense, poorly crystallized aggregates. This product easily precipitates in water, exhibits extremely poor dispersibility, and cannot form stable nano-suspensions, making it unsuitable for subsequent cell experiments or drug delivery.

[0110] Comparative Example 2 Referring to Example 3, the dual-targeting molecule Gal-D5-HT in step 3.2) was omitted to obtain the corresponding nanomedicine SC@RhB-COF-366 (untargeted).

[0111] Referring to Example 3, the dual-targeting molecule Gal-D5-HT in step 3.2) was replaced with the ASGPR single-targeting molecule Gal-DTA to obtain the corresponding nanomedicine SC@GRD-COF-366 (ASGPR single-targeting).

[0112] Referring to Example 3, the dual-targeting molecule Gal-D5-HT in step 3.2) was replaced with the MPO single-targeting molecule D5HT to obtain the corresponding nanomedicine SC@RT-COF-366 (MPO single-targeting).

[0113] The obtained nanomedicines were characterized according to the test methods in Examples 9-10. The results showed that the non-targeted group had very low tumor aggregation at all time points, indicating poor targeting. The targeted group, however, showed significantly enhanced, time-dependent tumor site fluorescence signals from 4 to 24 hours after injection. Notably, 4 hours after injection, the tumor fluorescence intensity of SC@RT-COF-366 (MPO-targeted) and SC@GRT-COF-366 (dual-targeted) was higher than that of SC@GRD-COF-366 (ASGPR-targeted), indicating that MPO-mediated targeting achieves tumor enrichment faster than ASGPR targeting. Particularly noteworthy is that the dual-targeted SC@GRT-COF-366 maintained a strong tumor fluorescence signal for 24 hours after injection, demonstrating superior retention capacity compared to other formulations.

[0114] Subcutaneous tumor treatment results showed that targeted modification further improved treatment efficacy: the TGI of SC@GRD-COF-366 (ASGPR-targeted) reached 62.0%, SC@RT-COF-366 (MPO-targeted) rose to 73.2%, and the dual-targeted SC@GRT-COF-366 reached 81.1%. Most significantly, SC@GRT-COF-366 combined with phototherapy exhibited excellent anti-tumor effects, with a TGI as high as 93%, which was statistically significantly improved compared with monotherapy (p<0.01), highlighting the synergistic effect of PDT and nanomedicine.

[0115] Comparative Example 3 Solvent and temperature optimization for the nucleophilic substitution reaction in the synthesis of compound 2 (Gal-Ac-N3): Original step 2.2): Compound 1 reacts with sodium azide in DMF at 70°C for 2 hours to obtain compound 2.

[0116] Comparative Experiment 1: With other conditions unchanged, the solvent was changed from DMF to acetone, and the reaction was carried out at 56°C (acetone reflux temperature) for 2 hours. Result: The yield was as low as about 30%.

[0117] Comparative Experiment 2: In DMF, the reaction temperature was lowered to room temperature (25°C) and the reaction was carried out for 2 hours. Result: The reaction was incomplete, with a large amount of raw material remaining, and the product yield was less than 20%.

[0118] Comparative Example 4 Solvent screening for click chemistry reactions in the synthesis of compound 8 (Gal-D5HT): Original step 2.8): Compound 3 (terminal azide) and compound 6 (terminal alkyne) were reacted overnight at room temperature in a water / DMF mixed solvent using a sodium ascorbate / copper sulfate pentahydrate catalytic system to obtain compound 8.

[0119] Comparative experiment: keeping other conditions unchanged, but using a water / tert-butanol (v / v=1:1) mixed solvent and reacting overnight at room temperature.

[0120] Results: The reaction could still proceed, but the reaction rate was significantly reduced. TLC monitoring showed that a large amount of starting material remained, indicating incomplete reaction. After the same reaction time as under the original conditions, the separation yield was only about 50%. Furthermore, the product purity also decreased, requiring further purification by column chromatography.

[0121] Comparative Example 5 Selection of purification method for compound 8 / 9 (Gal-D5HT / Gal-DTA): Original steps 2.8) / 2.9): After the reaction, filter and purify the crude product directly by reversed-phase chromatography (acetonitrile / water).

[0122] Comparative experiment: Before performing reversed-phase chromatography, we first tried purification using preparative thin-layer chromatography.

[0123] Results: Preparative thin-layer chromatography (TLC) is feasible for small samples at the milligram level, but when the reaction scale is scaled up to hundreds of milligrams or more, the separation effect is poor, the product recovery rate is low, and it is difficult to separate structurally very similar byproducts. Analysis: Reversed-phase chromatography (especially preparative HPLC or MPLC) has the advantages of high resolution and high sample loading for purifying amphiphilic molecules like the target compound (containing both the polar part of the sugar and the hydrophobic part of the indole and alkyl chain). Preparative TLC is more suitable for analysis or micro-preparation and is difficult to achieve efficient separation and purification of large quantities of products. This comparison highlights the importance of the choice of purification method for experimental efficiency and success rate.

[0124] 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 preparation method of a dual-targeted hepatocellular carcinoma nanodrug, characterized in that, The method comprises the following steps: (1) Preparation of covalent organic nanomaterial: 5,10,15,20-tetra(4-aminophenyl) porphyrin, p-phenylenedimethylene and polyvinylpyrrolidone are dissolved in a mixed solvent of DMF and methanol, trifluoroacetic acid is added, and the reaction is heated to obtain a covalent organic nanomaterial; (2) Preparation of a dual-target targeting molecule: azidated galactose raw material A and an alkyne and 5-hydroxytryptamine structure-containing raw material B are dissolved in a mixed solvent of water and DMF; and ascorbic acid and sulfuric acid pentahydrate are added, and a dual-target targeting molecule is obtained by reaction; (3) The covalent organic nanomaterial and the chemotherapeutic drug are mixed and stirred to obtain a drug-loaded nanomaterial; then the obtained drug-loaded nanomaterial is mixed with the dual-target targeting molecule and the tracer molecule to obtain a final nanodrug product; In the formula, the structure of the azidated galactose raw material A is ; The structure of the starting material B containing an alkyne and a serotonin structure is .

2. The method of claim 1, wherein, In step (1), the molar ratio of 5,10,15,20-tetra(4-aminophenyl) porphyrin to p-phenylenedimethylene is 1:2; and the mass ratio of polyvinylpyrrolidone to 5,10,15,20-tetra(4-aminophenyl) porphyrin is 1:(0.05-0.10).

3. The method of claim 1, wherein, In step (1), the volume ratio of DMF to methanol is 1:(2-5).

4. The method of claim 1, wherein, In step (2), the azidated galactose raw material A is prepared by the following method: 2.1) Synthesis of compound 1 (Gal-Ac-Br): trifluoroboron ether is added to a dichloromethane solution containing 1,2,3,4,6-penta-O-acetyl-d-galactopyranose and 2-bromoethanol (Gal-Ac), stirred at room temperature under nitrogen protection overnight to obtain compound 1; 2.2) Synthesis of compound 2 (Gal-Ac-N3): compound 1 is dissolved in a DMF solution with sodium azide, and reacted under argon protection to obtain compound 2; 2.3) Synthesis of compound 3 (Gal-N3): compound 2 and sodium methoxide are dissolved in methanol under nitrogen protection, and reacted at room temperature to obtain compound 3, which is the azidated galactose raw material A.

5. The method of claim 1, wherein, In step (2), the alkyne and 5-hydroxytryptamine structure-containing raw material B is prepared by the following method: 2.4) Synthesis of compound 4 (D-5HT-Boc): N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide are dissolved in DMF and added to a DMF solution of Boc-D-L-Glu, and stirred at room temperature for activation; then a DMF solution containing 5-hydroxytryptamine and triethylamine is added, and the mixture is stirred to react to obtain compound 4; 2.5) Synthesis of compound 6 (D-5HT): compound 4 is dissolved in dichloromethane, trifluoroacetic acid is added, and stirred at room temperature overnight; after removing dichloromethane and excess TFA under reduced pressure, the residue is dissolved in DMF, and excess triethylamine is added; then a solution of 4-pentenoic acid activated in DMF by NHS / EDC or HOBt / HATU is added to the above mixture, and reacted to obtain compound 6, which is the alkyne and 5-hydroxytryptamine structure-containing raw material B.

6. The method of claim 1, wherein, In step (3), the chemotherapeutic drug is sorafenib and chloroquine; and the mass ratio of the covalent organic nanomaterial, sorafenib and chloroquine is (1-2):1:(1-2).

7. The method of claim 6, wherein, In step (3), the tracer molecule is rhodamine B.

8. The method according to any one of claims 1 to 7, characterized in that, In step (3), the mass ratio of the drug-loaded nanodrug, the double-targeting targeting molecule and the tracer molecule is 1:1:

1.

9. A double-targeting hepatocellular carcinoma nanodrug prepared by the method of any one of claims 1-8.

10. The double-targeting hepatocellular carcinoma nanodrug of claim 9 for use in the preparation of an anti-liver cancer drug.

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