Preparation method and application of liver cancer highlight activated diagnosis and treatment probe IR-FTDH4P-Gal-SSSFc

Abstract

The application belongs to the technical field of anti-hepatocellular carcinoma phototherapy agents, and particularly relates to a preparation method and application of a liver cancer high-brightness activation type diagnosis and treatment probe IR-FTDH4P-Gal-SSSFc. The IR-FTDH4P-Gal-SSSFc compound provided by the application has a structure shown in formula 1, can be preferentially accumulated in tumor tissues, has target specificity, and can realize fluorescence quenching through a photo-induced electron transfer mechanism of coupled ferrocene and trisulfide. The IR-FTDH4P-Gal-SSSFc compound can activate site-specific near-infrared II fluorescence in liver tumors, and significantly improves the diagnosis accuracy of liver cancer and the anti-tumor effect. In formula 1, n is 10-16; the R6 is selected from the molar ratio of 1:1.

Description

Technical Field

[0001] This invention belongs to the field of phototherapy technology for hepatocellular carcinoma, specifically relating to the preparation method and application of the high-brightness activation-type diagnostic probe IR-FTDH4P-Gal-SSSFc for liver cancer. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a major global health problem, with its high mortality rate primarily stemming from the difficulty of early diagnosis and the inherent limitations of existing treatment strategies. Current challenges in HCC management can be categorized as follows: (i) Inadequate diagnosis: Traditional imaging modalities (CT, MRI) and serum biomarkers lack the sensitivity and specificity required for reliable detection of early HCC. This deficiency hinders the precise localization and real-time monitoring of small or heterogeneous tumors, delaying timely intervention; (ii) Inherent limitations of traditional treatment strategies: While surgical resection is the primary method, it is associated with risks of tissue trauma, incomplete tumor resection, or iatrogenic metastasis. Furthermore, due to poor selectivity between malignant and healthy tissues, the clinical application of chemotherapy and radiotherapy is often limited by acquired treatment resistance and significant systemic toxicity. Currently, traditional chemotherapeutic drugs and radiosensitizers exhibit suboptimal pharmacokinetic characteristics, characterized by extensive nonspecific biodistribution (leading to high background signal and off-target effects) and rapid clearance, limiting preferential tumor accumulation, reducing the target-to-background ratio, narrowing the treatment window, and ultimately affecting treatment outcomes. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing and applying a high-brightness activation-type diagnostic and therapeutic probe for hepatocellular carcinoma, IR-FTDH4P-Gal-SSSFc. The IR-FTDH4P-Gal-SSSFc compound provided by this invention exhibits targeting specificity and preferential accumulation in tumor tissue, and can activate site-specific near-infrared II fluorescence in hepatocellular carcinoma, significantly improving the diagnostic accuracy and anti-tumor efficacy of hepatocellular carcinoma.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] An IR-FTDH4P-Gal-SSSFc compound having the structure shown in Formula 1:

[0006]

[0007] In Equation 1, n is 10 to 16; R6 is selected from... The The molar ratio is 1:1.

[0008] This invention provides a method for preparing the IR-FTDH4P-Gal-SSSFc compound described above, comprising the following steps:

[0009] In a protective gas atmosphere, IR-FTDH4P-Gal compound, triphenylphosphine and organic solvent were mixed and reduced to obtain IR-FTDH4P-Gal-NH2 compound.

[0010] The IR-FTDH4P-Gal-NH2 compound, FcSSS-COOH compound, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and organic solvent were mixed and coupled to obtain the IR-FTDH4P-Gal-SSSFc compound with the structure shown in Formula 1.

[0011] The IR-FTDH4P-Gal compound has the structure shown in Formula 2, where R4 is selected from... And N3, the The molar ratio of R4 to N3 is 1:1; the IR-FTDH4P-Gal-NH2 compound has the structure shown in Formula 3, wherein R4 in Formula 3 is selected from... and NH2, the The molar ratio of NH2 to NH2 is 1:1; the FcSSS-COOH compound has the structure shown in Formula 4;

[0012]

[0013] Preferably, the preparation method of the IR-FTDH4P-Gal compound includes the following steps:

[0014] The IR-FTDH compound, sodium azide, and organic solvent were mixed and subjected to an azide reaction to obtain the IR-FTDH4P compound.

[0015] The IR-FTDH4P compound, copper(I)-thiophene-2-carboxylate, acetylaceton-polyethylene glycol-galactose, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine and organic solvent were mixed and subjected to a superimposed-alkyl click cyclization reaction to obtain the IR-FTDH4P-Gal compound.

[0016] The IR-FTDH compound has the structure shown in Formula 5, and the IR-FTDH4P compound has the structure shown in Formula 6; in Formula 6, R3 is selected from... And N3, the The molar ratio of N to N3 is 1:1;

[0017]

[0018] Preferably, the mass ratio of the IR-FTDH4P-Gal compound to triphenylphosphine is 2:1.

[0019] Preferably, the mass ratio of the IR-FTDH4P-Gal-NH2 compound to the FcSSS-COOH compound is 25-26:6.5; and the mass ratio of the IR-FTDH4P-Gal-NH2 compound to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 25-26:10.

[0020] Preferably, the molar ratio of the IR-FTDH compound to sodium azide is 0.069:0.72; the mass ratio of the IR-FTDH4P compound, acetylacetone-polyethylene glycol-galactose, copper(I)-thiophene-2-carboxylate, and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine is 92-94:280:10:5.

[0021] This invention provides the application of the IR-FTDH4P-Gal-SSSFc compound described in the above technical solution or the IR-FTDH4P-Gal-SSSFc compound obtained by the above preparation method in the preparation of targeted drugs for hepatocellular carcinoma.

[0022] This invention provides a targeted drug for treating hepatocellular carcinoma, comprising the IR-FTDH4P-Gal-SSSFc compound described in the above technical solution or the IR-FTDH4P-Gal-SSSFc compound obtained by the preparation method described above.

[0023] This invention provides the application of the IR-FTDH4P-Gal-SSSFc compound described in the above technical solution or the IR-FTDH4P-Gal-SSSFc compound obtained by the above preparation method in the preparation of near-infrared II phototherapy reagents.

[0024] This invention provides a near-infrared II phototherapy agent, comprising the IR-FTDH4P-Gal-SSSFc compound described in the above technical solution or the IR-FTDH4P-Gal-SSSFc compound obtained by the preparation method described above.

[0025] This invention provides an IR-FTDH4P-Gal-SSSFc compound. The IR-FTDH4P-Gal-SSSFc compound possesses a highly conjugated and hydrophobic alkylthiophene moiety, which enhances the planarity of the core chromophore and induces a significant dark shift when the excitation and emission wavelengths further enter the near-infrared II window (>1000 nm). Simultaneously, the alkylthiophene increases hydrophobicity and rigidity, effectively mitigating aggregation-induced quenching (ACQ) in aquatic physiological environments, thereby significantly improving in vivo fluorescence brightness and quantum yield. A ketene-polyethylene glycol-galactose ligand serves as the Gal-PEG ligand, which is a targeting carrier for hepatocellular carcinoma, facilitating preferential accumulation of the IR-FTDH4P-Gal-SSSFc compound in tumor tissue. Furthermore, a ferrocene quencher is introduced into the IR-FTDH4P-Gal-SSSFc compound via a trisulfide linker, utilizing a photoinduced electron transfer (PET) mechanism; the proximal ferrocene effectively quenches NIR-II fluorescence. Based on the expected specific cleavage of trisulfide bonds in the tumor microenvironment (TME), ferrocene partially dissociates, selectively restoring fluorescence emission at the tumor site. This significantly reduces background signal interference from non-target organs, ultimately greatly improving the signal-to-noise ratio (SNR) for contrast diagnosis and precise treatment guidance. Furthermore, the core structure of the IR-FTDH4P-Gal-SSSFc compound employs a donor-acceptor-donor (DAD) structure with SO conformational locking, giving the conjugated backbone high planarity and rigidity, thereby promoting efficient intramolecular charge transfer (ICT) processes. The IR-FTDH4P-Gal-SSSFc compound achieves a tumor-specific activation mechanism through bifunctionalization, activating site-specific NIR-II fluorescence in liver tumors.

[0026] Furthermore, the IR-FTDH4P-Gal-SSSFc compound shows great potential for developing advanced NIR-II agents. NIR-II agents containing the IR-FTDH4P-Gal-SSSFc compound exhibit superior brightness, targeting specificity, activatable high-contrast imaging, and powerful photothermal therapy capabilities, significantly improving the molecular photothermal conversion efficiency (PCE) and enhancing its potential for effective photothermal therapy (PTT). In addition, the IR-FTDH4P-Gal-SSSFc compound combines PTT with chemical kinetics (CDT) and gas therapy (GT), establishing a synergistic trimodal treatment modality that significantly improves the diagnostic accuracy and antitumor efficacy of liver cancer. Attached Figure Description

[0027] Figure 1 The flowchart for preparing the IR-FTDH4P-Gal-SSSFc compound is shown in the example.

[0028] Figure 2The 1H NMR spectrum of the IR-FTDH compound prepared for the example ( 1 H NMR spectrum;

[0029] Figure 3 Carbon NMR spectra of IR-FTDH compounds prepared for examples 13 C NMR spectrum;

[0030] Figure 4 High-resolution mass spectra (HRMS) of IR-FTDH compounds were prepared for this example.

[0031] Figure 5 The proton nuclear magnetic resonance spectrum of the IR-FTDH4P compound prepared for the example ( 1 H NMR spectrum;

[0032] Figure 6 Carbon NMR spectra of IR-FTDH4P compounds prepared for examples ( 13 C NMR spectrum;

[0033] Figure 7 The proton nuclear magnetic resonance spectrum of the IR-FTDH4P-Gal compound prepared for the example ( 1 H NMR spectrum;

[0034] Figure 8 Carbon NMR spectrum of the IR-FTDH4P-Gal compound prepared for the example ( 13 C NMR spectrum;

[0035] Figure 9 The proton nuclear magnetic resonance spectrum of the IR-FTDH4P-Gal-Fc compound prepared for the example ( 1 H NMR spectrum;

[0036] Figure 10 Carbon NMR spectra of the IR-FTDH4P-Gal-Fc compound prepared for the example ( 13 C NMR spectrum;

[0037] Figure 11 The proton nuclear magnetic resonance spectrum of the IR-FTDH4P-Gal-SSSFc compound prepared for the example ( 1 H NMR spectrum;

[0038] Figure 12 Carbon NMR spectra of the IR-FTDH4P-Gal-SSSFc compound prepared for the example ( 13 CNMR diagram;

[0039] Figure 13 Graphs showing the targeting specificity and cellular uptake of the compounds prepared for the examples and comparative examples;

[0040] Figure 14 In vivo near-infrared 2nd region fluorescence imaging of the compounds prepared for the examples and comparative examples;

[0041] Figure 15 The images show the effects of the compounds prepared in situ on liver cancer models in the examples and comparative examples. Detailed Implementation

[0042] This invention provides an IR-FTDH4P-Gal-SSSFc compound having the structure shown in Formula 1:

[0043]

[0044] In Equation 1, n can be 10 to 16; in this specific embodiment of the invention, n is 12. R6 is selected from... The The molar ratio is 1:1. In a specific embodiment of the present invention, R6 consists of 4 units. and 4

[0045] This invention provides a method for preparing the IR-FTDH4P-Gal-SSSFc compound described above, comprising the following steps:

[0046] In a protective gas atmosphere, IR-FTDH4P-Gal compound, triphenylphosphine and organic solvent were mixed and reduced to obtain IR-FTDH4P-Gal-NH2 compound.

[0047] The IR-FTDH4P-Gal-NH2 compound, FcSSS-COOH compound, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and organic solvent were mixed and coupled to obtain the IR-FTDH4P-Gal-SSSFc compound with the structure shown in Formula 1.

[0048] The IR-FTDH4P-Gal compound has the structure shown in Formula 2, where R4 is selected from... And N3, the The molar ratio of R4 to N3 is 1:1; in a specific embodiment of the present invention, R4 consists of 4 molecules. And 4 N3; the IR-FTDH4P-Gal-NH2 compound has the structure shown in Formula 3, wherein R4 in Formula 3 is selected from and NH2, the The molar ratio of R4 to NH2 is 1:1. In a specific embodiment of the present invention, R4 consists of four molecules. And 4 NH2; the FcSSS-COOH compound has the structure shown in Formula 4;

[0049]

[0050] In this invention, unless otherwise specified, all raw materials / components used in the preparation are commercially available products well known to those skilled in the art.

[0051] This invention involves a reduction reaction of an IR-FTDH4P-Gal compound, triphenylphosphine, and an organic solvent under a protective gas atmosphere to obtain the IR-FTDH4P-Gal-NH2 compound. In one embodiment, the mass ratio of the IR-FTDH4P-Gal compound to triphenylphosphine can be 2:1, and the organic solvent can be tetrahydrofuran (THF). The amount of organic solvent used is not particularly important, as long as the reduction reaction proceeds smoothly. Preferably, the organic solvent is degassed before use. The protective gas can be nitrogen.

[0052] In one embodiment of the present invention, the reduction reaction can be carried out in a Schlenk flask. Before the reduction reaction, the present invention preferably uses a protective gas to purge the gas in the Schlenk flask three times. The temperature of the reduction reaction can be 15-30°C, and the time can be 10-16 hours. The reduction reaction is carried out under stirring. After the reduction reaction, a reduction reaction solution is obtained. The present invention can perform post-treatment on the reduction reaction solution to obtain the IR-FTDH4P-Gal-NH2 compound. In the present invention, the post-treatment can include: cooling the reduction reaction solution under ice-water bath conditions, adding cold water dropwise to the cooled reduction reaction solution, continuing to maintain it under ice-water bath conditions for 2 hours, washing and drying the resulting processed solution to obtain the IR-FTDH4P-Gal-NH2 compound. The washing reagent can be methyl tert-butyl ether (MTBE), and the number of washings can be 3. The drying can be freeze-drying. The IR-FTDH4P-Gal-NH2 compound does not need to be purified and can be directly subjected to subsequent coupling reactions.

[0053] After obtaining the IR-FTDH4P-Gal-NH2 compound, the present invention mixes the IR-FTDH4P-Gal-NH2 compound, the FcSSS-COOH compound, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and an organic solvent to carry out a coupling reaction to obtain the IR-FTDH4P-Gal-SSSFc compound with the structure shown in Formula 1. As one embodiment of the present invention, the mass ratio of the IR-FTDH4P-Gal-NH2 compound to the FcSSSS-COOH compound can be 25-26:6.5, specifically 25.7:6.5; the mass ratio of the IR-FTDH4P-Gal-NH2 compound to the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide can be 25-26:10, specifically 25.7:10; the organic solvent can be THF. The present invention does not have special requirements on the amount of the organic solvent used, as long as the coupling reaction proceeds smoothly.

[0054] In one embodiment of the present invention, the coupling reaction temperature can be 45–55°C, specifically 50°C, and the time can be 9–11 hours, specifically 10 hours; the coupling reaction is carried out under stirring. After the coupling reaction, a coupling reaction solution is obtained. Preferably, the coupling reaction solution is post-treated to obtain a pure IR-FTDH4P-Gal-SSSFc compound; the post-treatment preferably includes: washing the coupling reaction solution with methyl tert-butyl ether (MTBE) to remove lipid-soluble organic impurities, and then... The compound is washed with water in a centrifugal filter to remove water-soluble impurities, and then dried to obtain the IR-FTDH4P-Gal-SSSFc compound. The MTBE washing can be performed 3 times, and the water washing can be performed 10 times. The drying process can be freeze-drying.

[0055] As one embodiment of the present invention, the preparation method of the IR-FTDH4P-Gal compound includes the following steps:

[0056] The IR-FTDH compound, sodium azide, and organic solvent were mixed and subjected to an azide reaction to obtain the IR-FTDH4P compound.

[0057] The IR-FTDH4P compound, copper(I)-thiophene-2-carboxylate, acetylaceton-polyethylene glycol-galactose, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine and organic solvent were mixed and subjected to a superimposed-alkyl click cyclization reaction to obtain the IR-FTDH4P-Gal compound.

[0058] The IR-FTDH compound has the structure shown in Formula 5, and the IR-FTDH4P compound has the structure shown in Formula 6; in Formula 6, R3 is selected from... And N3, the The molar ratio of R3 to N3 is 1:1. In a specific embodiment of the present invention, R3 consists of four molecules. And 4 N3;

[0059]

[0060] This invention involves mixing an IR-FTDH compound, sodium azide, and an organic solvent to perform an azide reaction, yielding an IR-FTDH4P compound. In one embodiment of this invention, the molar ratio of the IR-FTDH compound to sodium azide is 0.069:0.72; the organic solvent can be DMF. This invention does not have specific requirements regarding the amount of organic solvent used, as long as the azide reaction proceeds smoothly.

[0061] In one embodiment of the present invention, the temperature of the azide reaction can be 65-75°C, specifically 70°C, and the holding time can be 4-6 hours, specifically 5 hours. After the azide reaction, an azide reaction solution is obtained. Preferably, the azide reaction solution is post-treated to obtain the IR-FTDH4P compound. The post-treatment preferably includes: diluting the azide reaction solution with water, extracting the diluted reaction solution with ethyl acetate to obtain an organic phase; drying the organic phase and removing the solvent to obtain a crude product; purifying the crude product by column chromatography to obtain a pure IR-FTDH4P compound. In this invention, the extraction can be performed twice, and the organic phases from each extraction are combined and dried; the drying reagent can be magnesium sulfate; the solvent removal method can be vacuum evaporation; the column chromatography purification can be flash column chromatography, and the eluent can be petroleum ether (PE) and ethyl acetate (EA), with a volume ratio of PE to EA of 2:1.

[0062] After obtaining the IR-FTDH4P compound, the present invention mixes the IR-FTDH4P compound, copper(I)-thiophene-2-carboxylate, acetylacetonate-polyethylene glycol-galactose, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) and an organic solvent, and performs a superimposed-alkyl click cyclization reaction to obtain the IR-FTDH4P-Gal compound. In one embodiment of the present invention, the acetylacetone-polyethylene glycol-galactose can be acetylacetone-polyethylene glycol 600-galactose (Alkyne-PEG600-Gal), and the copper(I)-thiophene-2-carboxylate can be cuprous(I) thiophene-2-carboxylate (CuTc); the mass ratio of the IR-FTDH4P compound, acetylacetone-polyethylene glycol-galactose, copper(I)-thiophene-2-carboxylate, and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine is 92-94:280:10:5; the IR-FTDH4P compound and the acetylacetone-polyethylene glycol... The mass ratio of alcohol to galactose can be 92-94:280, specifically 93:280; the mass ratio of the IR-FTDH4P compound to copper(I)-thiophene-2-carboxylate can be 92-94:10, specifically 93:10; the mass ratio of the IR-FTDH4P compound to tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine can be 93:5, specifically 93:5; the organic solvent can be THF. This invention does not have special requirements for the amount of organic solvent used, as long as the superimposed-alkyl click cyclization reaction proceeds smoothly.

[0063] In one embodiment of the present invention, the temperature of the superimposed-alkyl click cyclization reaction can be 15-30°C, and the time can be 0.5 h. The superimposed-alkyl click cyclization reaction is carried out under stirring. In the present invention, after the superimposed-alkyl click cyclization reaction, a cyclization reaction solution is obtained. Preferably, the cyclization reaction solution is post-treated to obtain the IR-FTDH4P-Gal compound. The post-treatment preferably includes: filtering the cyclization reaction solution with diatomaceous earth to obtain a liquid product; removing the solvent from the liquid product to obtain a crude product; purifying the crude product by column chromatography to obtain a first-purified product; and recrystallizing the first-purified product with methyl tert-butyl ether to obtain a pure IR-FTDH4P-Gal compound. In the present invention, the solvent removal method can be vacuum evaporation. The elution solvent used in the column chromatography can be petroleum ether (PE) and ethyl acetate (EA), and the volume ratio of PE to EA can be 2:1. The present invention does not have special requirements for the specific implementation of the recrystallization.

[0064] This invention provides the application of the IR-FTDH4P-Gal-SSSFc compound described in the above technical solution or the IR-FTDH4P-Gal-SSSFc compound obtained by the above preparation method in the preparation of targeted drugs for hepatocellular carcinoma.

[0065] This invention provides a targeted drug for treating hepatocellular carcinoma, comprising the IR-FTDH4P-Gal-SSSFc compound described in the above technical solution or the IR-FTDH4P-Gal-SSSFc compound obtained by the preparation method described above.

[0066] This invention provides the application of the IR-FTDH4P-Gal-SSSFc compound described in the above technical solution or the IR-FTDH4P-Gal-SSSFc compound obtained by the above preparation method in the preparation of near-infrared II phototherapy reagents.

[0067] This invention provides a near-infrared II phototherapy agent, comprising the IR-FTDH4P-Gal-SSSFc compound described in the above technical solution or the IR-FTDH4P-Gal-SSSFc compound obtained by the preparation method described above.

[0068] Figure 1 This is a flowchart illustrating the preparation of the IR-FTDH4P-Gal-SSSFc compound according to an embodiment of the present invention. To further illustrate the present invention, the technical solutions provided by the present invention are described in detail below with reference to embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0069] Example 1

[0070] (1) Preparation of compound 16: 2-bromo-9,9-bis(6-bromohexyl)-9H-fluorene (compound 2) (3 g, 5.28 mmol) and tributyl(thiophene-2-yl)tin (compound 15) (2.96 g, 7.92 mmol) were added to a 100 mL round-bottom flask. Under nitrogen protection, Pd(PPh3)4 (200 mg) and anhydrous toluene (40 mL) were added to the reaction system. The mixture was stirred at 130 °C for 3 h. The resulting product was then... The system was extracted twice with ethyl acetate and water. The organic phase was collected, dried over MgSO4, filtered, and the filtrate was vacuum evaporated to remove the solvent. The crude product was subjected to silica gel column chromatography with petroleum ether (PE) and dichloromethane (DCM) as the eluents, with a volume ratio of PE to DCM of 4:1, to obtain 2-(9,9-bis(6-bromohexyl)-9H-fluorene-2-yl)thiophene (compound 16) (2.46 g, 81.6%), which was a light yellow oil.

[0071] 1H NMR (500MHz, CDCl3) δ7.69 (dd, J=7.6, 3.2Hz, 2H), 7.60 (dd, J=8.0, 1.6Hz, 1H), 7.55(dJ=1.6Hz,1H),7.38(dJ=3.6Hz,1H),7.32(ddd,J=15.8,7.0,3.5Hz,4H), 7.11(dd,J=5.1,3.6Hz,1H),3.26(t,J=6.8Hz,4H),2.03-1.93(m,4H),1.67-1. 61(m,4H),1.22-1.15(m,4H),1.12-1.04(m,4H),0.65(dd,J=15.8,7.8Hz,4H).

[0072] 13 C NMR (126MHz, CDCl3) δ151.15,150.50,145.11,140.72,140.67,133.32,128.08,127.23,127.00,125.02,124.55, 122.93,122.80,120.14,120.08,119.79,77.27,77.02,76.77,55.05,40.24,33.90,32.62,29.03,27.74,23.52.

[0073] (2) Synthesis of compound 17: Compound 16 (2.00 g, 3.50 mmol) was dissolved in 30 mL of DMF at -10 °C under nitrogen protection. 10 mL of DMF containing NBS (0.75 g, 4.20 mmol) was added dropwise. The reaction mixture was heated to room temperature and stirred for 5 h. The resulting product system was extracted twice with ethyl acetate. The organic phase was collected, dried over MgSO4, and filtered. The filtrate was evaporated under vacuum to remove the solvent. The crude product was subjected to silica gel column chromatography. The elution solvents were petroleum ether (PE) and dichloromethane (DCM) in a volume ratio of 8:1, yielding 2-(9,9-bis(6-bromohexyl)-9H-fluorene-2-yl)-5-bromothiophene (compound 17) (1.53 g, 67.3%), which was a pale yellow oil.

[0074] (3) Synthesis of compound 20: Under nitrogen protection, 3,4-bis(6-bromohexoxy)-2-thienyltributyltinane (compound 8) (1.29 g, 1.77 mmol) and compound 17 (1.72 g, 2.65 mmol) were placed in a 100 mL round-bottom flask, and Pd(PPh3)4 (150 mg) and 40 mL of anhydrous toluene were added. The mixture was stirred at 130 °C for 3 h. The resulting product system was extracted twice with ethyl acetate, and the organic phase was collected. After drying with MgSO4 and filtering, the filtrate was vacuum evaporated to remove the solvent. The crude product was then subjected to silica gel column chromatography using petroleum ether (PE) and dichloromethane (DCM) as the eluents, with a volume ratio of PE to DCM of 10:1. This yielded 5'-(9,9-bis(6-bromohexyl)-9H-fluorene-2-yl)-3,4-bis((6-bromohexyl)oxy)-2,2'-bithiophene (compound 20) (1.46 g, 81.6%) as a pale yellow solid.

[0075] 1 H NMR(500MHz, CDCl3)δ7.69(d,J=7.8Hz,2H),7.60(d,J=7.9Hz,1H),7.54(s,1H),7.31(dd,J=13.4,3.4Hz,4H), 7.23(d,J=3.8Hz,1H),6.05(s,1H),4.15(t,J=6.5Hz,2H),4.00(t,J=6.3Hz,2H),3.43(dt,J=13.9,6.8Hz,4H) ,3.27(t,J=6.8Hz,4H),2.00(t,J=8.2Hz,4H),1.88(ddd,J=20.5,12.4,5.5Hz,8H),1.64(dd,J=14.5,7.1Hz,4 H),1.33-1.28(m,4H),1.22-1.17(m,4H),1.09(dd,J=15.0,7.5Hz,4H),0.97-0.76(m,4H),0.73-0.57(m,4H).

[0076] 13C NMR(500MHz,CDCl3)δ151.16,150.48,150.29,143.60,143.22,142.14,140.67,134.20,133.22,127.23,127.01,124.68,124.23,122.80,120.19,120.14,119.79,119.60,114.05,77.30,77.04,76.79,72.61,69.81,55.05,40.29,33.97,33.86,33.74,32.82,32.69,32.65,31.95,31.46,30.21,30.09,29.72,29.34,29.07,29.04,28.03,27.88,27.77,25.38,25.30,23.55,22.71,14.14.

[0077] HRMS(ESI)calcd for C 45 H 59 Br4O2S2,([M+H + ])1015.06436,Found1015.06666。

[0078] (4) IR-FTDH: Compound 20 (2.06 g, 2.03 mmol) was dissolved in 30 mL of THF at -78 °C under nitrogen protection, and n-BuLi solution (solvent: hexane, concentration: 1.6 M, volume: 2.55 mL, 3.20 mmol) was added dropwise; the mixture was stirred at -78 °C for 2 h, and tributyltin chloride (1.06 g, 3.30 mmol) was added. The reaction system was heated to room temperature and stirred for another 1 h. The resulting product system was extracted twice with ethyl acetate, the organic phase was collected, dried over MgSO4, filtered, and the filtrate was vacuum evaporated to remove the solvent, yielding 5'-[9,9-bis(6-bromohexyl)-9H-fluorene-2-yl]-3,4-bis[(6-bromohexyl)oxy]-[2,2'-bithiophene]-5-yltributyltinane (compound 21). Compound 21 (2.65 mg, 2.04 mmol) and BBTD (237.95 mg, 0.68 mmol) were transferred together to a 100 mL round-bottom flask. Under a nitrogen atmosphere, Pd(PPh3)2Cl2 (120 mg) and 30 mL of anhydrous toluene were added. The mixture was stirred at 130 °C for 3 h. The resulting product system was extracted twice with ethyl acetate. The organic phase was collected, dried over MgSO4, filtered, and the filtrate was evaporated under vacuum to remove the solvent. The crude product was subjected to silica gel column chromatography using petroleum ether (PE) and dichloromethane (DCM) as the eluents, with a PE to DCM volume ratio of 2:1. The green solid obtained was the IR-FTDH compound (841.4 mg, 56%). The 1H NMR spectrum of the IR-FTDH compound was obtained. 1 HNMR (HNMR) image as follows Figure 2 As shown; Carbon NMR spectrum ( 13 (C NMR) image as follows Figure 3 As shown, the high-resolution mass spectrometry (HRMS) image is as follows. Figure 4 As shown.

[0079] 1 HNMR(500MHz,CDCl3)δ7.72(t,J=6.8Hz,4H),7.66-7.55(m,4H),7.35(ddd,J=17.3,14.3,9.1Hz,10H),4.36-4.22(m,4H),4.11(t,J=6.4Hz,4H),3 .46-3.26(m,16H),1.98(ddd,J=30.5,15.6,7.7Hz,16H),1.71-1.61(m,1 6H), 1.49-1.34 (m, 8H), 1.24-1.08 (m, 24H), 0.67 (dq, J = 13.7, 7.1Hz, 8H).

[0080] 13C NMR (126MHz, CDCl3) δ152.69,151.26,150.51,149.97,145.47,140.97,140.81,140.6 0,139.14,133.37,133.02,127.35,127.06,125.19,124.75,123.09,122.83,120.28,1 19.86,119.87,77.29,77.04,76.79,73.47,72.84,55.09,40.31,33.97,33.80,33.69,32.81,32.65,32.52,31.45,30.23,29.86,29.09,28.15,27.79,25.43,25.05,23.57.

[0081] HRMS(ESI) for C 96 H 114 Br8N4O4S6,([M+H + ])2220.0523,Found2220.0593.

[0082] (5) Preparation of IR-FTDH4P-Gal compound:

[0083] The IR-FTDH compound (100 mg, 0.069 mmol) and sodium azide (47 mg, 0.72 mmol) were dissolved in 10 mL of DMF and heated at 70 °C for 5 h. Water was added to the reaction system and the mixture was stirred until the solid dissolved. The mixture was extracted twice with ethyl acetate. The organic phase was collected, dried over MgSO4, and filtered. The filtrate was evaporated under vacuum to remove the solvent. The crude product was subjected to silica gel column chromatography on silica gel. The elution solvents were petroleum ether (PE) and ethyl acetate (EA), with a volume ratio of PE to EA of 2:1. The resulting dark green solid was the IR-FTDH4P compound (93 mg, 0.071 mmol). The IR-FTDH4P compound was dissolved in 5 mL of THF, and 10 mg of copper thiophene-2-carboxylate (I) (CuTc), 280 mg of acetylacetone-PEG600-galactose, and 5 mg of tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) were added. The mixture was stirred at room temperature for 0.5 h. The resulting reaction system was filtered through diatomaceous earth, and the filtrate was evaporated under vacuum to remove the solvent. The crude product was subjected to silica gel column chromatography, using petroleum ether (PE) and ethyl acetate (EA) as the eluents, with a PE to EA volume ratio of 2:1. The substance obtained from column chromatography was recrystallized from methyl tert-butyl ether to obtain the IR-FTDH4P-Gal compound as a dark green solid (130.2 mg, 57.20%). The 1H NMR spectrum of the IR-FTDH4P compound was... 1 H NMR (image) Figure 5 As shown; Carbon NMR spectrum ( 13 CNMR) diagram as follows Figure 6 As shown. The 1H NMR spectrum of the IR-FTDH4P-Gal compound (…). 1 H NMR (image) Figure 7 As shown; Carbon NMR spectrum ( 13 (C NMR) image as follows Figure 8 As shown.

[0084] IR-FTDH4P-Gal compound: 1 H NMR (500MHz, CDCl3) δ7.73 (dd, J=9.4, 6.8Hz, 8H), 7.37 (dd, J=13.5, 3.7Hz, 10H), 3.65 (s, 176H), 1.70-1.64 (m, 8H), 1.39-1.29 (m, 40H), 1.03 (d, J = 5.1Hz, 16H), 0.86-0.81 (m, 8H).

[0085] IR-FTDH4P-Gal compound: 13C NMR (126MHz, CDCl3) δ151.06,150.84,140.64,137.25,126.91,124.38,12 3.49,122.90,119.93,77.33,77.07,76.82,70.54,55.32,55.27,40.52,3 4.99,34.45,34.03,32.65,31.87,31.52,30.14,29.72,29.62,29.56,29. 35,29.28,29.11,28.08,27.82,26.82,23.88,22.65,17.43,14.10,13.63.

[0086] (6) Preparation of IR-FTDH4P-Gal-SSSFc compound:

[0087] Add 40 mg of IR-FTDH4P-Gal compound and 20 mg of triphenylphosphine to a Schlenk flask, add a magnetic stir bar, seal the flask, and purge with N2 three times; add 2 mL of degassed tetrahydrofuran to the Schlenk flask and stir at room temperature for 12 h; place the Schlenk flask in an ice-water bath for 5 min, add 0.1 mL of cold water to the reaction system, and then maintain the reaction in an ice-water bath for 2 h; wash the resulting reaction system three times with 30 mL of tert-butyl methyl ether (MTBE) to obtain 25.7 mg of IR-FTDH-GAl-NH2 compound, which is a foul-smelling green solid; freeze-dry to remove residual water, and use the IR-FTDH-GAl-NH2 compound directly in the next step without further purification. The FcSSS-COOH compound (6.5 mg) was dissolved in THF (5 mL), added to a THF solution containing the IR-FTDH-GA1-NH2 compound (2 mL), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) (10 mg) was added. The mixture was stirred for 10 h. The resulting reaction system was washed three times with tert-butyl methyl ether to remove lipid-soluble organic impurities. The solution was then purified using 15 mL of [unspecified solution]. The centrifuged filter was washed 10 times with water to remove water-soluble impurities; the purified solid was freeze-dried to obtain the IR-FTDH4P-Gal-SSSFc compound as a green solid (18.6 mg). The 1H NMR spectrum of the IR-FTDH4P-Gal-SSSFc compound is shown below. 1 H NMR (image) Figure 11 As shown; Carbon NMR spectrum ( 13 (C NMR) image as follows Figure 12 As shown.

[0088] 1H NMR (500MHz, CDCl3) δ7.76-7.72(m,10H),7.39-7.36(m,8H),4.45(s,6H),4.22-4.15(m,30H),3. 65(s,180H),1.65(d,J=8.1Hz,8H),1.30(td,J=15.9,8.7Hz,46H),0.90(dd,J=13.2,7.8Hz,18H).

[0089] 13 C NMR (126MHz, CDCl3) δ151.05,131.28,119.93,106.60,77.33,77.08,76.82,73.56,71.75,70.50,70.05,68.72,67.92,5 1.98,40.53,36.15,34.03,31.88,31.52,30.13,29.57,29.29,28.05,26.84,23.87,22.64,18.26,17.43,14.10,13.63.

[0090] Comparative Example 1

[0091] The preparation was essentially the same as in Example 1, except that the FcSSS-COOH compound in step (6) was replaced with the Fc-COOH compound to obtain the IR-FTDH4P-Gal-Fc compound. The 1H NMR spectrum of the IR-FTDH4P-Gal-Fc compound (…) 1 HNMR (HNMR) image as follows Figure 9 As shown; Carbon NMR spectrum ( 13 (C NMR) image as follows Figure 10 As shown.

[0092] 1 H NMR(500MHz, CDCl3) δ7.75(dd,J=17.7,9.5Hz,10H),7.36(dd,J=9.4,7.3Hz,8H),4.33-3.98(m,14H),3.65( s,18H),3.58-3.36(m,18H),3.32-3.23(m,4H),1.69-1.59(m,8H),1.50-1.25(m,40H),0.93-0.75(m,24H).

[0093] 13C NMR (126MHz, CDCl3) δ151.18,143.51,141.47,141.38,140.61,136.14,122.89,121.46,119.91,116 .54,77.34,77.08,76.83,75.15,71.93,71.37,70.55,70.20,68.53,67.91,67.44,59.04,55.40,55. 25,54.03,67.91,67.64,34.02,33.02,32.63,32.47,31.93,31.59,31.54,29.37,29.32,29.27,28.36,27.80,25.13,24.88,24.50,23.82,22.70,22.64,17.73,14.71,14.32,14.15,14.10,9.53,0.01.

[0094] Test Example 1: Assessment of Target Specificity and Cellular Uptake

[0095] The IR-FTDH4P-Gal-SSSFc compound can be abbreviated as FTDH4P-Gal-SSSFc, the IR-FTDH4P-Gal-Fc compound can be abbreviated as FTDH4P-Gal-Fc, the IR-FTDH4P-Gal compound can be abbreviated as FTDH4P, and the IR-FTDH compound can be abbreviated as FTDH. FTDH4P-Gal-Fc, FTDH4P-Gal, FTDH4P, and PBS served as control groups.

[0096] Figure 13 Figures a and b show the preparation and nano-self-assembly of FTDH4P-Gal-SSSFc. Figure 13 Figure c shows the targeting specificity and cellular uptake effect of the compounds prepared in the examples and comparative examples; Figure 13 In the middle, d represents a schematic diagram of the fluorescence signals of FTDH4P-Gal-SSSFc in LX-2 cells, HepG-2 cells without pre-blocking, HepG-2 cells pre-blocked with NEM, and HepG-2 cells pre-blocked with Gal. Figure 13 In the image, 'e' represents the ·OH staining images of FTDH4P-Gal-SSSFc and the control group under laser irradiation. Figure 13 f in the figure represents the relationship between the fluorescence signals of FTDH4P-Gal-SSSFc and FTDH4P-Gal and time. Figure 13 The bar chart in section g shows the ·OH and H2S content of cells treated with FTDH4P-Gal-SSSFc and the control group. Figure 13In the middle, h represents the H2S staining images of FTDH4P-Gal-SSSFc and the control group under laser irradiation. Figure 13 In the image, i represents the GPX staining images of FTDH4P-Gal-SSSFc and the control group under laser irradiation. Figure 13 The figure in the middle (k) shows the COX I and GPX results for FTDH4P-Gal-SSSFc and control group cells. Figure 13 In the middle, j and l are fluorescence images and bar graphs showing the survival and mortality rates of HepG-2 cells in FTDH4P-Gal-SSSFc and the control group. Figure 13 In the diagram, I represents PBS+L, II represents FTDH4P+L, III represents FTDH4P-Gal+L, IV represents FTDH4P-Gal-Fc+L, and V represents FTDH4P-Gal-SSSFc+L.

[0097] Figure 13 Figure 'a' illustrates the process of obtaining FTDH4P-Gal-SSSFcNPs from IR-FTDH self-assembly. Figure 13 Figure b indicates that the diameter range of the obtained FTDH4P-Gal-SSSFc is 108.46±0.71 nm. The targeting ability of FTDH4P-Gal-SSSFc to hepatocellular carcinoma cells was detected. FTDH4P (non-targeted group), FTDH4P-Gal (targeted non-quenched group), and FTDH4P-Gal-SSSFc (targeted quenched activation group) were incubated with HepG-2 cells, and the targeting and fluorescence activation of tumor cells were detected. Figure 13 As shown in Figure f, compared with the non-targeted control group (FTDH4P), the mean fluorescence intensity (MFI) of HepG-2 cells treated with lactose-modified nanoparticles (FTDH4P-Gal and FTDH4P-Gal-SSSFc, after activation) was approximately 3 times higher. Figure 13 As shown in Figure c, the fluorescence growth rate of FTDH4P-Gal-SSSFc is slower than that of FTDH4P-Gal. This kinetic difference is attributed to the gradual breakdown of the trisulfide linker by intracellular glutathione (GSH), thereby disrupting the PET quenching mechanism and restoring fluorescence emission. Conversely, the fluorescence signal generated by incubating FTDH4P-Gal-SSSFc with normal human hepatocytes (LX-2) is negligible and remains constant during incubation. These results indicate that galactose modification significantly increases the uptake of nanoparticles by tumor cells.

[0098] NIR-II fluorescence imaging further validated the results of the confocal experiments. Figure 13As shown in Figure d, FTDH4P-Gal-SSSFc activated fluorescence in HepG-2 cells after 1 hour of treatment, while LX-2 cells with lower GSH levels did not show significant fluorescence activation. Furthermore, pretreatment of HepG-2 cells with the GSH inhibitor n-ethylmaleimide (NEM, 1.0 mM) significantly reduced the fluorescence signal of FTDH4P-Gal-SSSFc. Comparing FTDH4P-Gal-SSSFc with a non-targeted but quenched control (FTDH4P-SSSFc, assuming a similar structure without Gal), the galactose-targeted group showed a significantly higher fluorescence signal after 60 minutes of incubation. These results indicate that FTDH4P-Gal-SSSFc achieves selectivity for tumor cells through active targeting by the galactose ligand and activation of fluorescence via GSH-mediated trisulfide bonds, thereby specifically dequenching the cells in the high-GSH tumor microenvironment.

[0099] The generation of intracellular hydroxyl radicals (·OH) after nanoparticle treatment and laser irradiation was detected using the fluorescent probe aminophenyl fluorescein (APF). CLSM imaging showed that, compared with non-cleavable control cells treated with laser (assuming stable ligation) (FTDH4P-Gal-Fc+L), cells treated with FTDH4P-Gal-SSSFc+L exhibited significantly enhanced green fluorescence. Figure 13 (e and g). The MFI in the FTDH4P-Gal-SSSFc+L group was 3.97 times that of the control group ( Figure 13 The presence of g indicates effective ·OH generation, which may be related to photothermal effects or related processes.

[0100] In addition, this test case also investigated the effect of treatment on glutathione peroxidase 4 (GPX4), a key enzyme regulating ferroptosis, using immunofluorescence staining. Figure 13 (i and k). The control group (PBS treatment) maintained high GPX4 levels. The FTDH4P-Gal-SSSFc+L group showed the most significant decrease in GPX4 fluorescence intensity and relative expression level, indicating that the combination therapy significantly downregulated GPX4 expression, which may be one of the reasons for its overall therapeutic effect.

[0101] Intracellular hydrogen sulfide (H2S) release was monitored using the hydrogen sulfide (H2S)-specific probe WSP-1, which is likely caused by the breaking of trisulfide bonds. Cells incubated with FTDH4P-Gal-SSSFc (30 μM) showed resolvable fluorescence in the presence of H2S, while the control group showed a negligible signal. Figure 10(h and g). Enhanced fluorescence after 808 nm laser irradiation indicated an increase in intracellular H2S concentration. The results showed that internalized FTDH4P-Gal-SSSFc releases H2S via GSH-mediated trisulfide cleavage, and laser irradiation accelerated this reaction. Studies have found that H2S induces acute toxicity by inhibiting mitochondrial cytochrome c oxidase IV (COX IV), thereby reducing ATP production. Simultaneously, reduced ATP production decreases HSP70 expression, thus counteracting tumor heat resistance. Therefore, this test case investigated the effect of FTDH4P-Gal-SSSFc on COX IV activity.

[0102] Quantitative analysis of the cytotoxic effects of FTDH nanoparticles on HepG-2 cells using live / dead cell staining. Figure 13 (j and l). At a concentration of 30 μM, after irradiation with 808 nm laser, the cell viability rates of the PBS group, FTDH4P group, FTDH4P-Gal group, FTDH4P-Gal-Fc (non-lysis control) group, and FTDH4P-Gal-SSSFc group were 96.76% (3.24% cell killing), 76.1% (23.9% cell killing), 53.17% (46.83% cell killing), 22.48% (77.52% cell killing), and 4.85% (95.15% cell killing), respectively. FTDH4P-Gal-SSSFc exhibits good anti-hepatocellular carcinoma activity, and its mechanism may be related to the combination of targeting, activation, and photothermal / photodynamic effects, further confirming that FTDH4P-Gal-SSSFc has a highly efficient tumor cell killing ability under laser irradiation. Among the molecules tested, FTDH4P-Gal-SSSFc elicited the most significant therapeutic response.

[0103] Test Example 2: In vivo near-infrared II fluorescence imaging

[0104] Figure 14 In vivo near-infrared 2nd region fluorescence imaging of the compounds prepared for the examples and comparative examples; Figure 14 Figure a shows the relationship between the fluorescence signal of FTDH4P-Gal-SSSFc, FTDH4P-Gal, and FTDH4P at the tumor site and time. Figure 14 Figure b shows a schematic diagram illustrating the aggregation capabilities of FTDH4P-Gal-SSSFc, FTDH4P-Gal, and FTDH4P at the tumor site. Figure 14 In the middle, c represents the signal fitting curves of FTDH4P-Gal-SSSFc for experimental groups with different lipid layer thicknesses. Figure 14 In the middle d, the graph shows the relationship between the temperature of the tumor site and the control group over time. Figure 14Figure e shows the changes in FTDH4P-Gal-SSSFc levels in blood and fecal samples over 48 hours. Figure 14 f in the figure represents the signal ratio between tumor and normal tissue at different time points for FTDH4P-Gal-SSSFc, FTDH4P-Gal, and FTDH4P. Figure 14 In the middle, g represents the signal ratio between tumor and normal tissue for FTDH4P-Gal-SSSFc, FTDH4P-Gal, and FTDH4P. Figure 14 The graph in Figure h represents the change in the concentration of FTDH4P-Gal-SSSFc in blood over time. Figure 14 The figure in Figure i represents the change in the content of FTDH4P-Gal-SSSFc in feces over time.

[0105] Based on the high in vitro selectivity of FTDH4P-Gal-SSSFc for tumor cells, this test case used an in situ tumor-bearing mouse model of HepG-2 cells to evaluate its in vivo fluorescence imaging effect. Figure 14 In Figure a, it is explained that intravenous administration of FTDH4P, FTDH4P-Gal, and FTDH4P-Gal-SSSFc resulted in a time-dependent enhancement of tumor fluorescence signal, reaching a plateau phase approximately 12 hours post-injection. Subsequent euthanasia and dissection allowed for in vitro assessment of biodistribution via fluorescence imaging of major organs. Figure 14 Figure b shows that, compared with the FTDH4P control group, FTDH4P-Gal and FTDH4P-Gal-SSSFc showed significantly higher tumor accumulation, thus confirming the effectiveness of galactose modification in in vivo tumor targeting. Notably, in vivo lateral imaging and ex vivo organ analysis showed that the fluorescence background signal in the liver and spleen of the FTDH4P-Gal-SSSFc group was lower than that of the FTDH4P and FTDH4P-Gal groups. This observation suggests that although FTDH4P-Gal-SSSFc accumulates in the liver, its fluorescence may be partially quenched in the "off" state through photoinduced electron transfer (PET) mechanism, thereby reducing the background signal. Furthermore, quantitative region of interest (ROI) analysis was performed to assess the tumor-to-normal tissue (T / N) signal ratio. Figure 14 (f) According to Figure 14As shown in the figure, the T / N ratio of FTDH4P-Gal-SSSFc at a dose of 30 μM (100 μL) was 12.7 12 h post-injection. The imaging penetration depth of FTDH4P-Gal-SSSFc was further evaluated by utilizing its excellent excitation and emission characteristics within the second near-infrared (NIR-II) window. Capillaries containing FTDH4P-Gal-SSSFc were activated with glutathione (GSH) and then covered with 1% fat emulsions of varying thicknesses to simulate tissue scattering. Imaging was performed within the NIR-II window. Analysis of the fluorescence signal attenuation in the lipid layer indicated that FTDH4P-Gal-SSSFc possessed good imaging penetration, with an estimated optical penetration depth of 7–9 mm. Figure 14 (c). These findings highlight the potential of FTDH4P-Gal-SSSFc for deep tissue bioimaging applications. These results demonstrate that FTDH4P-Gal-SSSFc possesses robust NIR-II fluorescence imaging performance, exhibiting significant tissue penetration, high contrast, and accurate localization of liver tumor regions.

[0106] Subsequently, this test case investigated the efficacy of in vivo hypothermic photothermal therapy (HPTT) guided by fluorescence imaging. Twelve hours after injection of FTDH4P-Gal-SSSFc, the tumor area was irradiated with an 808nm laser (0.33W / cm²). 2 (10 min). In mice treated with FTDH4P-Gal-SSSFc, the tumor surface temperature rapidly increased to approximately 44°C. In contrast, under the same irradiation conditions, the tumor temperatures in the PBS group, FTDH4P group, and FTDH4P-Gal group only reached approximately 34.6°C, 41.8°C, and 42.1°C, respectively. Figure 14 (d). These results demonstrate the effectiveness of FTDH4P-Gal-SSSFc in achieving thermotherapy in vivo, potentially enabling effective HPTT.

[0107] The pharmacokinetic characteristics of FTDH4P-Gal-SSSFc were evaluated in healthy mice. Blood and fecal samples were collected at predetermined time points after intravenous administration. In this test case, plasma concentration-time curves and cumulative fecal excretion were monitored over 48 hours. Figure 14 (e). Pharmacokinetic analysis showed that the circulating half-life of FTDH4P-Gal-SSSFc was approximately 2.37 h. Figure 14 (h). Furthermore, fecal excretion analysis showed that approximately 49.63% (of the administered dose) Figure 14 (i). It was excreted in feces within 48 hours after injection, suggesting that hepatobiliary clearance is an important excretory route. Figure 14In the diagram, I stands for FTDH4P, II for FTDH4P-Gal, and III for FTDH4P-Gal-SSSFc.

[0108] Test Example 3

[0109] Figure 15 Graphs showing the effects of in situ liver cancer models of the compounds prepared in the examples and comparative examples; Figure 15 Image a shows the bioluminescence signal observed in mice using luciferase-labeled tumor sites via in vivo imaging. Figure 15 Figure b shows the changes in tumor luminescence signal over time in mice after injection of FTDH4P-Gal-SSSFc and in the control group, respectively. Figure 15 In the figure, 'c' represents the body weight curves of mice injected with FTDH4P-Gal-SSSFc and the control group over 14 days. Figure 15 In the middle, d represents the time-varying curves of relevant tumor signals in FTDH4P-Gal-SSSFc and the control group. Figure 15 The diagram in image e shows the liver tumor regions of mice injected with FTDH4P-Gal-SSSFc and the control group, respectively. Figure 15 The graph in figure f shows the cell survival rates after injection of FTDH4P-Gal-SSSFc and the control group, respectively. Figure 15 The graph in section g shows the tumor volume of FTDH4P-Gal-SSSFc and the control group within 14 days after initial tumor elimination. Figure 15 The middle image shows the tumor apoptosis detection results of FTDH4P-Gal-SSSFc and the control group using KI-67, HE, GPX4, and HSP70 staining, respectively.

[0110] After establishing an orthotopic liver cancer model, this test case detected and treated liver malignancy during a 14-day observation period. Figure 15 (a). To non-invasively monitor tumor progression in vivo, the in vivo experiment first used HepG-2 cells pre-transfected with the luciferase reporter gene. During a 14-day observation period, bioluminescent signals from xenografts expressing luciferase were longitudinally monitored using an IVIS imaging system. Figure 15 (b) Quantitative assessment of tumor volume by measuring bioluminescence intensity; such as... Figure 15 As shown in Figure d, tumor growth was significantly accelerated in the PBS group. Conversely, the FTDH4P, FTDH4P-Gal, FTDH4P-Gal-Fc, and FTDH4P-Gal-SSSFc treatment groups showed significant tumor growth inhibition. Figure 15As can be seen from Figure g, the relative anti-tumor effects are as follows: FTDH4P (HPTT: 56.5%) < FTDH4P-Gal (Target-HPTT: 87.9%) < FTDH4P-Gal-Fc (Target-HPTT+CDT: 93.2%) < FTDH4P-Gal-SSSFc (target-activated HPTT+CDT+GT: 97.36%). It is worth noting that in the FTDH4P-Gal-SSSFc group, the tumors completely regressed after the initial treatment, and there was no evidence of tumor recurrence during the entire study period. This result is consistent with the stable 100% survival rate on day 14 ( Figure 15 Figure f), indicating that FTDH4P-Gal-SSSFc has a strong ability to inhibit liver tumor proliferation in vivo.

[0111] In this test example, in vitro analysis was performed on the livers of mice with different treatment regimens (FTDH4P, FTDH4P-Gal, FTDH4P-Gal-Fc, and FTDH4P-Gal-SSSFc). Gross morphological examination showed that the livers of the FTDH4P-Gal-SSSFc cohort had a healthy red appearance and the smallest residual tumor area ( Figure 15 Figure e), which is consistent with the in vivo bioluminescence imaging data ( Figure 15 Figure b).

[0112] Meanwhile, a significant downregulation of the proliferation marker KI-67 was observed in this test example ( Figure 15 Figure h), indicating that the proliferation ability of tumor cells was significantly inhibited, accompanied by an increase in the level of apoptosis. Further mechanism studies involved histopathological evaluation by hematoxylin-eosin (HE) staining. These analyses confirmed a significant increase in apoptosis in the tumor sections of the effective treatment groups, especially in FTDH4P-Gal-SSSFc.

[0113] In addition, after laser irradiation, the expression of GPX4 protein was downregulated, showing efficient GSH consumption and activation of ferroptosis by ROS generation. The heat resistance of tumors is due to the high expression of HSPS as Figure 15 shown in Figure h. Compared with the PBS+L group, the expression of HSP70 increased in the FTDH4P+L, FTDH4P-Gal+L, and FTDH4P-Gal-Fc+L groups due to the increase in temperature. However, the expression of HSP70 was absent in the FTDH4P-Gal-SSSFc+L group, which may be due to H2S-induced mitochondrial dysfunction leading to limited ATP generation induced by COX IV. Based on the HE staining images of major organs and blood biochemical analysis, FTDH4P-Gal-SSSFc NPs showed good biocompatibility, which can also be reflected by the Figure 15 unchanged body weight of the mice in Figure c. Figure 15In the diagram, I represents PBS+L, II represents FTDH4P+L, III represents FTDH4P-Gal+L, IV represents FTDH4P-Gal-Fc+L, and V represents FTDH4P-Gal-SSSFc+L.

[0114] In summary, the results indicate that FTDH4P-Gal-SSSFc is a precise and efficient integrated treatment platform combining chemodynamic therapy (CDT), thermophotothermal therapy (HPTT), and gas therapy (GT).

[0115] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An IR-FTDH4P-Gal-SSSFc compound having the structure shown in Formula 1: Formula 1; R6 is selected from and The and The molar ratio is 1:1; in Equation 1, n is 10~16.

2. The method for preparing the IR-FTDH4P-Gal-SSSFc compound according to claim 1, comprising the following steps: In a protective gas atmosphere, IR-FTDH4P-Gal compound, triphenylphosphine and organic solvent were mixed and reduced to obtain IR-FTDH4P-Gal-NH2 compound. The IR-FTDH4P-Gal-NH2 compound, the FcSSS-COOH compound, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and an organic solvent were mixed and coupled to obtain the IR-FTDH4P-Gal-SSSFc compound with the structure shown in Formula 1. The IR-FTDH4P-Gal compound has the structure shown in Formula 2, where R4 is selected from... And N3, the The molar ratio of R4 to N3 is 1:1; the IR-FTDH4P-Gal-NH2 compound has the structure shown in Formula 3, wherein R4 in Formula 3 is selected from... and NH2, the The molar ratio of NH2 to NH2 is 1:1; the FcSSS-COOH compound has the structure shown in Formula 4; Formula 2; Formula 3; Formula 4.

3. The preparation method according to claim 2, characterized in that, The preparation method of the IR-FTDH4P-Gal compound includes the following steps: The IR-FTDH compound, sodium azide, and organic solvent were mixed and subjected to an azide reaction to obtain the IR-FTDH4P compound. The IR-FTDH4P compound, copper(I)-thiophene-2-carboxylate, acetylaceton-polyethylene glycol-galactose, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine and organic solvent were mixed and subjected to a superimposed-alkyl click cyclization reaction to obtain the IR-FTDH4P-Gal compound. The IR-FTDH compound has the structure shown in Formula 5, and the IR-FTDH4P compound has the structure shown in Formula 6; in Formula 6, R3 is selected from... And N3, the The molar ratio of N to N3 is 1:1; Equation 5, Formula 6.

4. The preparation method according to claim 2, characterized in that, The mass ratio of the IR-FTDH4P-Gal compound to triphenylphosphine is 2:

1.

5. The preparation method according to claim 2, characterized in that, The mass ratio of the IR-FTDH4P-Gal-NH2 compound to the FcSSS-COOH compound is 25~26:6.5; the mass ratio of the IR-FTDH4P-Gal-NH2 compound to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 25~26:

10.

6. The preparation method according to claim 3, characterized in that, The molar ratio of the IR-FTDH compound to sodium azide is 0.069:0.72; the mass ratio of the IR-FTDH4P compound, acetylacetone-polyethylene glycol-galactose, copper(I)-thiophene-2-carboxylate, and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine is 92~94:280:10:

5.

7. The use of the IR-FTDH4P-Gal-SSSFc compound of claim 1 or the IR-FTDH4P-Gal-SSSFc compound obtained by any one of claims 2 to 6 in the preparation of a targeted drug for hepatocellular carcinoma.

8. An anti-hepatocellular carcinoma targeted drug, comprising the IR-FTDH4P-Gal-SSSFc compound of claim 1 or the IR-FTDH4P-Gal-SSSFc compound obtained by the preparation method of any one of claims 2 to 6.

9. The use of the IR-FTDH4P-Gal-SSSFc compound of claim 1 or the IR-FTDH4P-Gal-SSSFc compound obtained by the preparation method of any one of claims 2 to 6 in the preparation of near-infrared II phototherapy reagents.

10. A near-infrared II phototherapy agent, comprising the IR-FTDH4P-Gal-SSSFc compound of claim 1 or the IR-FTDH4P-Gal-SSSFc compound obtained by the preparation method of any one of claims 2 to 6.

Images