An ir820-gpc3-gd compound and nanomaterial thereof for liver cancer photodiagnosis and magnetic resonance imaging, a preparation method and application thereof

Abstract

This invention provides an IR820-GPC3-Gd compound and its nanomaterials (IGD NPs) for phototherapy and magnetic resonance imaging of liver cancer, along with their preparation method and applications, belonging to the field of medicinal chemistry. The IR820-GPC3-Gd compound exhibits strong targeting, good phototherapy performance, and stable imaging. The IGD NPs demonstrate excellent active targeting ability for hepatocellular carcinoma (HCC); under 808nm laser irradiation, they exhibit outstanding photothermal and photodynamic properties, making them suitable for treating unresectable HCC. The combined fluorescence imaging and magnetic resonance imaging capabilities of the IGD NPs can precisely locate residual tumor boundaries and provide real-time surgical navigation, improving the conversion therapy efficiency of HCC and ultimately significantly reducing postoperative recurrence rates, providing an effective strategy for tumor downstaging and surgical navigation.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to an IR820-GPC3-Gd compound for phototherapy and magnetic resonance imaging of liver cancer, its nanomaterials, preparation method and application. Background Technology

[0002] Hepatocellular carcinoma (HCC), a major malignant liver tumor, is often diagnosed at an intermediate or advanced stage, making surgical resection difficult. Conversion therapy refers to converting unresectable tumors into resectable ones, followed by surgical removal to maximize patient survival. Combined conversion therapies, including molecular targeted therapy, chemotherapy, immunotherapy, and local treatment, have been applied clinically, achieving high objective response rates and median survival. However, repeated interventions with these synergistic therapies can gradually impair liver function. Therefore, it is necessary to continue exploring new treatment methods and integrating multiple therapies to ensure a good prognosis.

[0003] Near-infrared (NIR) phototherapy offers advantages such as efficient phototherapy downstaging and multimodal deep tissue surgical navigation. Traditional near-infrared fluorescence imaging (FLI) boasts high sensitivity, rapid response, and non-invasiveness, but is limited by low resolution and tissue penetration. Existing indocyanine green (ICG) and neo-indocyanine green (IR820) are clinically used NIR imaging contrast agents with good biocompatibility, used for liver reserve function assessment and surgical guidance. However, existing NIR imaging contrast agents suffer from insufficient photostability, low photothermal conversion efficiency (PCE), and poor tumor targeting, limiting their application in deep and high-precision HCC surgical navigation. Summary of the Invention

[0004] In view of this, the present invention provides an IR820-GPC3-Gd compound, which has the characteristics of strong targeting of liver cancer cells, good photothermal and photodynamic properties, and stable imaging.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides an IR820-GPC3-Gd compound with the structural formula shown in Formula I:

[0007]

[0008] GPC3 is a GPC3 targeting peptide, and its amino acid sequence is shown in SEQ ID NO:1.

[0009] This invention provides a method for preparing the IR820-GPC3-Gd compound, comprising the following steps:

[0010] Under alkaline conditions, phloroglucinol and 1,3-dibromopropane undergo a substitution reaction to give compound 1;

[0011] Compound 1 was subjected to an azide reaction with sodium azide, and the product was coupled with IR820 to obtain compound 2.

[0012] Under copper (I) catalysis, compound 2 and PEG-modified GPC3 targeting peptide were subjected to a click chemical reaction to obtain compound 3;

[0013] The compound 3 and the gadolinium-macrocyclic ligand were subjected to a click chemical reaction under copper (I) catalysis to obtain the IR820-GPC3-Gd compound.

[0014] Preferably, the temperature of the substitution reaction is 20–30°C, and the time of the substitution reaction is 10–14 h;

[0015] The temperature of the azide reaction is 65–75°C, and the time of the azide reaction is 10–14 h;

[0016] The coupling reaction is carried out at a temperature of 0–5°C for 2.5–3.5 h.

[0017] The temperature of the click chemical reaction is 65–75°C, and the time of the click chemical reaction is 2.5–3.5 h.

[0018] Preferably, in the substitution reaction, potassium carbonate is used to adjust the alkaline environment; the molar ratio of phloroglucinol, 1,3-dibromopropane and potassium carbonate is 1:(1.9-2.5):(1.9-2.5).

[0019] The molar ratio of compound 1, sodium azide, and IR820 is 1:(2-3):(0.08-0.12);

[0020] In the coupling reaction, sodium hydride is used to adjust the alkaline environment; the molar ratio of IR820 to sodium hydride is 1:(6-7);

[0021] The reagents used in the copper (I) catalysis include sodium ascorbate and copper acetate; the molar ratio of compound 2, PEG-modified GPC3 targeting peptide, sodium ascorbate and copper acetate is 1:(1.5-2.5):(0.1-0.3):(0.05-0.1);

[0022] The molar ratio of compound 3 to the gadolinium-macrocyclic ligand is 1:(0.5-1).

[0023] This invention provides an IR820-GPC3-Gd self-assembled nanomaterial, which is obtained by self-assembling the IR820-GPC3-Gd compound in water.

[0024] This invention provides a method for preparing the IR820-GPC3-Gd self-assembled nanomaterial, comprising the following steps:

[0025] The IR820-GPC3-Gd compound was dissolved in water to obtain IR820-GPC3-Gd self-assembled nanomaterials.

[0026] This invention provides the application of the IR820-GPC3-Gd compound, the IR820-GPC3-Gd compound prepared by the preparation method, the IR820-GPC3-Gd self-assembled nanomaterial, or the IR820-GPC3-Gd self-assembled nanomaterial prepared by the preparation method in the preparation of phototherapy materials and / or contrast agents.

[0027] This invention provides the application of the IR820-GPC3-Gd compound, the IR820-GPC3-Gd compound prepared by the preparation method, the IR820-GPC3-Gd self-assembled nanomaterial, or the IR820-GPC3-Gd self-assembled nanomaterial prepared by the preparation method in the preparation of drugs for treating liver cancer.

[0028] This invention provides a drug for treating cancer, wherein the active ingredient is the IR820-GPC3-Gd self-assembled nanomaterial or the IR820-GPC3-Gd self-assembled nanomaterial prepared by the preparation method.

[0029] This invention provides an aggregation-induced emission molecule, including the IR820-GPC3-Gd self-assembled nanomaterial or the IR820-GPC3-Gd self-assembled nanomaterial prepared by the preparation method.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] This invention provides an IR820-GPC3-Gd compound. Conjugation of the GPC3 targeting peptide with an IR820 derivative improves the precision of photoimaging and phototherapy. Further conjugation with a gadolinium-macrocyclic ligand enhances imaging clarity and stability. The IR820-GPC3-Gd compound, through the action of the GPC3 peptide, promotes active targeting of HCC, while also possessing strong phototherapy properties (photothermal and photodynamic properties) and imaging capabilities. It can be used to treat unresectable HCC, precisely locate residual tumor boundaries, and provide real-time surgical navigation, ultimately significantly reducing postoperative recurrence rates.

[0032] This invention provides a method for preparing the IR820-GPC3-Gd compound, comprising the following steps: under alkaline conditions, phloroglucinol and 1,3-dibromopropane undergo a substitution reaction to obtain compound 1; compound 1 is azidated with sodium azide, and the resulting product is coupled with IR820 to obtain compound 2; under copper (I) catalysis, compound 2 and a PEG-modified GPC3 targeting peptide undergo a click chemistry reaction to obtain compound 3; under copper (I) catalysis, compound 3 and a gadolinium-macrocyclic ligand (DOTA-Gd) undergo a click chemistry reaction to obtain the IR820-GPC3-Gd compound. This invention, through the rational design of IR820 derivatives, after azide modification, utilizes a click chemistry reaction to couple with a PEG-modified GPC3 targeting peptide and DOTA-Gd to obtain the IR820-GPC3-Gd compound. The IR820-GPC3-Gd compound prepared by the method of this invention has different parts stably linked and is not affected by the modifier, exhibiting good fluorescence performance. It inherits the dual emission characteristics of IR820 and shows higher photothermal conversion ability than IR820. The coupling with GPC3 targeting peptide improves targeting and the coupling with DOTA-Gd exhibits good magnetic resonance paramagnetism.

[0033] This invention provides an IR820-GPC3-Gd self-assembled nanomaterial (IGD NPs), obtained by the self-assembly of an IR820-GPC3-Gd compound in water. Experimental results show that IGD NPs exhibit good active targeting ability for HCC, which is beneficial for nanoparticle endocytosis. Under non-invasive 808nm laser irradiation, IGD NPs exhibit excellent photothermal and photodynamic properties, which can be used to treat unresectable HCC. Subsequently, the combined NIR FLI (fluorescence imaging) and MRI (magnetic resonance imaging) capabilities of IGD NPs can accurately locate residual tumor boundaries and provide real-time surgical navigation, improving the conversion therapy efficiency of HCC and ultimately significantly reducing the postoperative recurrence rate, providing an effective strategy for tumor downstaging and surgical navigation. The photothermal and photodynamic properties of IGD NPs show that IGD NPs exhibit higher photothermal conversion capacity than IR820, while also promoting ROS production in cells and possessing strong photodynamic properties. Fluorescence and magnetic resonance imaging (MRI) results of IGD NPs showed that they maintain high fluorescence stability and exhibit good paramagnetism, demonstrating significant potential for HCC imaging and localization. Furthermore, this invention evaluated the in vivo antitumor effect of IGD NPs using an animal tumor model. Results showed that IGD NPs highly accumulated in liver tissue, and the fluorescence signal in the tumor region remained at a high level for an extended period, up to 24 hours, with optimal contrast. In contrast, the fluorescence completely disappeared 4 hours after ICG injection, with no accumulation at the tumor site. Fluorescence imaging of major organs and tumors in mice showed that the fluorescence intensity in the IGD NPs group was significantly higher than that in normal liver tissue, with clear boundaries. T1-weighted MRI results in tumor-bearing mice and mice with orthotopic tumors demonstrated the excellent MRI capabilities of IGD NPs. Dual-mode FLI / MARI, with its extended in vivo cycle time, allows for multimodal imaging analysis of HCC, providing a longer imaging window for precise surgical localization and guided resection. In vivo antitumor therapy results showed that the tumors in the IGD NPs group heated up rapidly and to a high temperature, resulting in a significant reduction in tumor volume and weight, while the tumors in the ICG group heated up slowly and to a low temperature, with only a slight inhibitory effect on the tumor. Therefore, utilizing the phototherapy advantages of IGD NPs can promote tumor downstaging, creating favorable conditions for subsequent surgical resection. Furthermore, the IGD NPs described in this invention have good biocompatibility, providing safe and effective phototherapy for liver tumors, and can completely remove tumor tissue under FLI guidance, reducing postoperative recurrence rates. Attached Figure Description

[0034] Figure 1 A schematic diagram illustrating the principle of using IR820-GPC3-Gd compound self-assembled nanomaterials for phototherapy;

[0035] Figure 2 The reaction diagram for the preparation of the IR820-GPC3-Gd compound;

[0036] Figure 3 For compound 1 1 H NMR spectrum;

[0037] Figure 4 For compound 1 13 C NMR spectrum;

[0038] Figure 5 Here is the HRMS plot of compound 1;

[0039] Figure 6 For compound 2 1 H NMR spectrum;

[0040] Figure 7 For compound 2 13 C NMR spectrum;

[0041] Figure 8 Here is the HRMS plot of compound 2;

[0042] Figure 9 For compound 3 1 H NMR spectrum;

[0043] Figure 10 For compound 3 13 C NMR spectrum;

[0044] Figure 11 For IR820-GPC3-Gd compounds 1 H NMR spectrum;

[0045] Figure 12 For IR820-GPC3-Gd compounds 13 C NMR spectrum;

[0046] Figure 13 A graph showing the results of SEC analysis of the differences in molecular weight of different compounds;

[0047] Figure 14The figures show the physicochemical characteristics of IGD NPs; where A is a TEM image of IGD NPs; B is a hydration particle size distribution of IGD NPs, with a scale bar of 50 nm; C is the absorption spectrum of IR820 and IGD NPs in aqueous phase; D is the fluorescence spectrum of IR820 and IGD NPs under excitation at 488 nm and 808 nm, respectively; E is the photothermal heating curve of IGD NPs at different concentrations; F is the photothermal heating curve of IGD NPs at different concentrations; G is the statistical graph of DCF fluorescence values ​​in aqueous solutions of PBS, IR820, and IGD NPs (488 nm) after laser irradiation for different times; H is the linear fitting curve of fluorescence signals in aqueous solutions of IGD NPs at different concentrations; and I is the linear fitting curve of magnetic resonance signals in aqueous solutions of IGD NPs at different concentrations.

[0048] Figure 15 Zeta potential plots of IGD NPs (n=3);

[0049] Figure 16 The graphs show the photothermal performance, where a is the photothermal temperature rise curve of IR820 and IGD NPs, and b is the photothermal temperature rise curve of IGD NPs at different powers.

[0050] Figure 17 A statistical graph showing the DCF fluorescence values ​​(488 nm) in PBS, IR820, and IGD NPs aqueous solutions after 5 min of laser irradiation;

[0051] Figure 18 Figure 1 shows the results of FLI and fluorescence values ​​in mice with subcutaneous tumors at different time points after tail vein injection of free ICG and IGD NPs (n=4); where a is the fluorescence graph and b is the fluorescence value statistical graph.

[0052] Figure 19 The images show the FLI and MRI results of IGD NPs in tumor-bearing mice. A and C are statistical graphs of FLI and signal values ​​in orthotopic tumor-bearing mice measured at different time points after tail vein injection of free ICG and IGD NP probes (n=4). * indicates p<0.05, and *** indicates p<0.001. B and D are statistical graphs of FLI and signal values ​​of tumors and major ex vivo organs 24 hours after intravenous injection of free ICG and IGD NP probes (n=4). E is a T1-weighted MRI image of mice at different time points after tail vein injection of IGD NP probes. ST represents subcutaneous tumors, and PIS represents orthotopic tumors.

[0053] Figure 20 The graph shows the signal values ​​of t1-weighted magnetic resonance imaging in mice at different time points, where ST represents subcutaneous tumors and PIS represents in situ tumors (n=4).

[0054] Figure 21 The following figures evaluate the effects of IGD NPs phototherapy on subcutaneous tumors in mice. Figures A and B show thermal imaging and temperature change curves at different time intervals during 808nm laser irradiation after tail vein injection of PBS, free ICG, and IGD NPs. Figure C shows photographs of subcutaneous tumor tissue changes in different groups 15 days after phototherapy. Figures D and E show curves of tumor volume and weight changes in different groups 15 days after phototherapy. Figures F and G show photographs and weight differences of tumors in different groups 15 days after phototherapy. (n=4) ** indicates p<0.01, *** indicates p<0.001.

[0055] Figure 22 Figure showing the evaluation results of IGD NPs phototherapy in mice with orthotopic tumors;

[0056] Figure 23 Differences in routine hematological and biochemical tests in mice from different treatment groups (n=4);

[0057] Figure 24 H&E staining results of various organs in mice from different treatment groups. Detailed Implementation

[0058] This invention provides an IR820-GPC3-Gd compound having the structure shown in Formula I:

[0059]

[0060] GPC3 is a GPC3 targeting peptide, and its amino acid sequence is shown in SEQ ID NO:1 (ALLANHEELFET).

[0061] In this invention, IR820 is a near-infrared imaging contrast agent; compared with normal liver tissue, HCC shows increased GPC3 peptide expression, and the GPC3 targeting peptide can bind to the GPC3 peptide, possessing the ability to actively target HCC. The coupling of the PEG-modified GPC3 targeting peptide with the IR820 derivative can enhance the targeting of IR820. The PEG-modified GPC3 targeting peptide has the structure shown in Formula 4:

[0062]

[0063] Gadolinium-macrocyclic ligand (DOTA-Gd), as a contrast agent in magnetic resonance imaging (MRI), can improve image resolution and contrast, and possesses good water solubility, low toxicity, and good stability. This invention involves the rational design of modifying IR820 with an azide group to obtain an IR820 derivative, which is then coupled with a GPC3-targeting peptide and DOTA-Gd using click chemistry. The resulting IR820-GPC3-Gd compound exhibits the ability to actively target HCC, while also possessing strong phototherapy properties (photothermal and photodynamic properties) and imaging capabilities. It can be used to treat unresectable HCC, precisely locate residual tumor boundaries, and provide real-time surgical navigation, ultimately significantly reducing postoperative recurrence rates.

[0064] This invention provides a method for preparing the IR820-GPC3-Gd compound, comprising the following steps:

[0065] Under alkaline conditions, phloroglucinol and 1,3-dibromopropane undergo a substitution reaction to give compound 1;

[0066] Compound 1 was subjected to an azide reaction with sodium azide, and the product was coupled with IR820 to obtain compound 2.

[0067] Under copper (I) catalysis, compound 2 and PEG-modified GPC3 targeting peptide were subjected to a click chemical reaction to obtain compound 3;

[0068] The compound 3 and the gadolinium-macrocyclic ligand were subjected to a click chemical reaction under copper (I) catalysis to obtain the IR820-GPC3-Gd compound.

[0069] In this invention, under alkaline conditions, phloroglucinol and 1,3-dibromopropane undergo a substitution reaction to yield compound 1.

[0070] In this invention, during the substitution reaction, potassium carbonate is preferably used to adjust the alkaline environment. Before the substitution reaction, N,N-dimethylformamide (DMF) is preferably used to dissolve phloroglucinol and 1,3-dibromopropane. The molar ratio of phloroglucinol, 1,3-dibromopropane, and potassium carbonate is preferably 1:(1.9–2.5):(1.9–2.5), more preferably 1:(1.95–2):(1.95–2), and most preferably 1:1.98:1.99. The ratio of the organic solvent to phloroglucinol is preferably (8–12) mL:0.016 mol, more preferably 10 mL:0.016 mol. The temperature of the substitution reaction is preferably 20–30°C, more preferably 22–28°C, and most preferably 25°C. The time of the substitution reaction is preferably 10–14 h, more preferably 11–13 h, and most preferably 12 h. After the substitution reaction, it is preferable to further remove the organic solvent and alkaline residue. The removal of organic solvents and alkaline residues is preferably achieved by extracting the product with a mixture of water and ethyl acetate (EA) (water to EA volume ratio of 1:1), followed by rotary evaporation to remove EA from the product, yielding a crude product. After obtaining the crude product, it is preferably purified by silica gel column chromatography and rotary evaporation to obtain a white solid, which is compound 1. The eluent used for silica gel column chromatography purification is preferably a mixture of petroleum ether (PE) and ethyl acetate (EA). The volume ratio of PE to EA is preferably 8:1. Compound 1 has the structure shown in Formula 1:

[0071]

[0072] After obtaining compound 1, the present invention performs an azidization reaction on compound 1 and sodium azide to obtain a clear, pale yellow liquid product. After obtaining the clear, pale yellow liquid product, the present invention performs a coupling reaction on the pale yellow liquid product and IR820 to obtain compound 2. In the present invention, the molar ratio of compound 1, sodium azide and IR820 is preferably 1:(2-3):(0.08-0.12), more preferably 1:(2.5-2.9):(0.09-0.11), and most preferably 1:2.8:0.1. Before the azidization reaction, compound 1 and sodium azide are preferably dissolved in an organic solvent. The organic solvent is preferably N,N-dimethylformamide (DMF). The molar ratio of the organic solvent to compound 1 is preferably (8-12) mL:0.0022 mol, more preferably 10 mL:0.0022 mol. In this invention, the temperature of the azidation reaction is preferably 65–75°C, more preferably 68–72°C, and most preferably 70°C; the time of the azidation reaction is preferably 10–14 h, more preferably 11–13 h, and most preferably 12 h. The azidation reaction is preferably accompanied by stirring, and the stirring speed is preferably 250 rpm. The crude product obtained after the azidation reaction is preferably purified by silica gel column chromatography, followed by rotary evaporation to obtain a clear, light yellow liquid product; the eluent used for silica gel column chromatography purification is preferably a mixture of petroleum ether (PE) and ethyl acetate (EA). The volume ratio of PE to EA is preferably 4:1. Sodium hydride is preferably used to adjust the alkaline environment in the coupling reaction. Before the coupling reaction, the light yellow liquid product and IR820 are preferably dissolved in an organic solvent. The organic solvent is preferably N,N-dimethylformamide (DMF). The molar ratio of IR820 to sodium hydride is 1:(6-7), more preferably 1:(6.2-6.8), and most preferably 1:6.47; the molar ratio of organic solvent to IR820 is preferably (8-12) mL:0.000235 mol, more preferably 10 mL:0.000235 mol. The coupling reaction temperature is 0-5℃, preferably 1-4℃, and most preferably 3℃; the coupling reaction time is preferably 2.5-3.5 h, more preferably 2.8-3.2 h, and most preferably 3 h. After the coupling reaction, the product is preferably filtered using a hydrophobic filter, then washed with a mixture of PE and EA at a volume ratio of 1, centrifuged, and rotary evaporated to obtain a dark green solid, which is compound 2. The filter pore size is preferably 0.45 mm. The centrifugation rate is 500 rpm, and the centrifugation time is preferably 20 min. Compound 2 has the structure shown in Formula 2:

[0073]

[0074] After obtaining compound 2, the present invention performs a click chemical reaction between compound 2 and PEG-modified GPC3 targeting peptide under copper (I) catalysis to obtain compound 3.

[0075] In this invention, the copper(I)-catalyzed reagents include sodium ascorbate and copper acetate. The solvent for the click chemistry reaction is a 50% (v / v) isopropanol aqueous solution. The molar ratio of compound 2, the PEG-modified GPC3 targeting peptide, sodium ascorbate, and copper acetate is preferably 1:(1.5–2.5):(0.1–0.3):(0.05–0.1), more preferably 1:(1.6–2):(0.11–0.2):(0.06–0.08), and most preferably 1:1.73:0.12:0.07. The type of PEG is preferably PEG1000. The ratio of the reaction solvent to the PEG-modified GPC3 targeting peptide is preferably (1.8–2.2) mL:0.032 mol, more preferably 2 mL:0.032 mol. The temperature of the click chemistry reaction is preferably 65–75°C, more preferably 68–72°C, and most preferably 70°C. The click chemical reaction time is 2.5–3.5 h, more preferably 2.8–3.2 h, and most preferably 3 h. After the click chemical reaction, the obtained product is preferably dried, then dialyzed through a 2 kD dialysis bag for 24 hours and freeze-dried to obtain a black solid, which is compound 3. Compound 3 has the structure shown in Formula 3:

[0076]

[0077] After obtaining compound 3, the present invention performs a click chemical reaction between compound 3 and a gadolinium-macrocyclic ligand under copper (I) catalysis to obtain IR820-GPC3-Gd compound.

[0078] In this invention, the copper(I) catalyzed reagent, the solvent for the click reaction, the reaction time, and the temperature are all the same as those used in the click chemistry reaction of compound 2 and the PEG-modified GPC3 targeting peptide, and will not be repeated here. The molar ratio of compound 3 to the gadolinium-macrocyclic ligand is 1:(0.5-1), more preferably 1:(0.6-0.8), and most preferably 1:0.65. After the click reaction, the crude product is preferably purified. The purification method is the same as that for the crude product of compound 3, and will not be repeated here. The black solid obtained after purification is IGD.

[0079] In this invention, IR820 is modified with an azide group by a rational design to obtain an IR820 derivative, which is then coupled with a GPC3 targeting peptide and DOTA-Gd using click chemistry to obtain the IR820-GPC3-Gd compound. Coupling the GPC3 targeting peptide with the IR820 derivative improves the accuracy of photoimaging and phototherapy, and further coupling with a gadolinium-macrocyclic ligand improves the clarity and stability of the imaging. The IR820-GPC3-Gd prepared by the method of this invention exhibits stable linkage between different parts, unaffected by modifiers, and displays excellent fluorescence performance. It inherits the dual-emission characteristics of IR820 and shows higher photothermal conversion capability than IR820. Coupling with the GPC3 targeting peptide improves targeting, and coupling with DOTA-Gd exhibits good magnetic resonance paramagnetism.

[0080] This invention provides a self-assembled IR820-GPC3-Gd nanomaterial (IGD NPs) obtained by self-assembling the IR820-GPC3-Gd compound in water.

[0081] In this invention, the IGD NPs exhibit a near-spherical structure with an average diameter of 70.4 ± 1.1 nm and a Zeta potential of -16.1 ± 0.49 mV. IGD NPs demonstrate excellent active targeting capability for HCC, facilitating cellular endocytosis of nanoparticles. Under non-invasive 808 nm laser irradiation, IGD NPs exhibit outstanding photothermal and photodynamic properties, making them suitable for treating unresectable HCC. Subsequently, the combined NIR, FLI, and MRI capabilities of IGD NPs can precisely locate residual tumor boundaries and provide real-time surgical navigation, improving the efficiency of HCC conversion therapy and ultimately significantly reducing postoperative recurrence rates, providing an effective strategy for tumor downstaging and surgical navigation.

[0082] In one embodiment of the present invention, the photothermal and photodynamic properties of IGD NPs were tested. The results showed that IGD NPs exhibited higher photothermal conversion capacity than IR820, with a temperature rise of 21°C after 10 minutes of laser irradiation. The temperature increased with increasing laser power and concentration, and IGD NPs promoted ROS production in cells, demonstrating strong photodynamic properties. In another embodiment of the present invention, the fluorescence and magnetic resonance imaging signals of IGD NPs were detected. The results showed that IGD NPs were unaffected by the modifier and maintained high fluorescence stability. Simultaneously, due to the chelation effect of DOTA-Gd, they exhibited good magnetic resonance paramagnetism. In another embodiment of the present invention, the in vivo antitumor effect of IGD NPs was evaluated using an animal tumor model. The results showed that IGD NPs could highly accumulate in liver tissue, and the fluorescence signal in the tumor area remained at a high level for a long time, up to 24 hours, with optimal contrast. ICG completely disappeared 4 hours after injection, with no accumulation at the tumor site. Fluorescence imaging results of major organs and tumors in mice showed that the fluorescence intensity of the IGD NPs group in the tumor was significantly higher than that in normal liver tissue, with clear boundaries. T1-weighted MRI results in tumor-bearing mice demonstrated that IGD NPs possess excellent MRI capabilities. In vivo antitumor therapy results showed that the tumors in the IGD NPs group rapidly and at high temperatures, resulting in a significant reduction in tumor volume and weight, while the ICG group experienced slow and low temperature increases, with only a slight inhibitory effect on tumors. Therefore, utilizing the phototherapy advantages of IGD NPs can promote tumor downstaging, creating favorable conditions for subsequent surgical resection. Furthermore, the IGD NPs described in this invention exhibit good biocompatibility, providing safe and effective phototherapy for liver tumors, and enabling complete tumor resection under FLI guidance, reducing postoperative recurrence rates.

[0083] This invention provides a method for preparing the IR820-GPC3-Gd self-assembled nanomaterial, comprising the following steps:

[0084] The IR820-GPC3-Gd compound was dissolved in water to obtain IR820-GPC3-Gd self-assembled nanomaterials.

[0085] In this invention, the mass ratio of IR820 to water is preferably 1:(500-700), more preferably 1:(550-650), and most preferably 1:600. Dissolution is preferably performed at room temperature, with a preferred temperature of 20-30°C, more preferably 22-28°C, and most preferably 25°C. Dissolution is preferably accompanied by stirring, with a preferred stirring rate of 400-600 rpm, more preferably 450-550 rpm, and most preferably 500 rpm. The stirring time is preferably 8-12 min, more preferably 9-11 min, and most preferably 10 min. The method described in this invention produces IGD NPs with excellent photothermal and photodynamic properties, and stable imaging.

[0086] This invention provides the application of the IR820-GPC3-Gd compound, the IR820-GPC3-Gd compound prepared by the preparation method, the IR820-GPC3-Gd self-assembled nanomaterial, or the IR820-GPC3-Gd self-assembled nanomaterial prepared by the preparation method in the preparation of phototherapy materials and / or contrast agents.

[0087] This invention provides the application of the IR820-GPC3-Gd compound, the IR820-GPC3-Gd compound prepared by the preparation method, the IR820-GPC3-Gd self-assembled nanomaterial, or the IR820-GPC3-Gd self-assembled nanomaterial prepared by the preparation method in the preparation of drugs for treating liver cancer.

[0088] This invention provides a drug for treating cancer, wherein the active ingredient is the IR820-GPC3-Gd self-assembled nanomaterial or the IR820-GPC3-Gd self-assembled nanomaterial prepared by the preparation method.

[0089] In this invention, the active ingredient in the drug is preferably IR820-GPC3-Gd self-assembled nanomaterials, and the concentration of the active ingredient is preferably 50-200 μM, more preferably 100-180 μM, and most preferably 150 μM. The dosage form of the drug is preferably an aqueous solution. The method of administration of the drug is preferably intravenous injection. The method of treating liver cancer with the drug is preferably phototherapy following intravenous injection.

[0090] This invention provides an aggregation-induced emission molecule comprising the IR820-GPC3-Gd self-assembled nanomaterial or the IR820-GPC3-Gd self-assembled nanomaterial prepared by the preparation method.

[0091] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes an IR820-GPC3-Gd compound for phototherapy and magnetic resonance imaging of liver cancer, its nanomaterials, preparation method, and applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0092] 1. Source of materials

[0093] GPC3-targeting peptide-PEG and DOTA-Gd were purchased from Chinapeptides Co., Ltd.

[0094] Cell counting kit (CCK8), AM / PI staining kit, and 2',7'-dichlorofluorescein diacetate (DCFH-DA) were purchased from Abbkine Scientific.

[0095] D-fluorescein potassium salt was purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.

[0096] Organic synthetic chemicals were purchased from Shanghai Titan Technology Co., Ltd.

[0097] The fluorescently labeled human hepatocellular carcinoma cells were HepG2-luc cells (Human Hepatocellular Carcinoma Cell Line-Luciferase labeled) obtained from the U.S. Type Culture Collection.

[0098] DMEM culture medium, trypsin, and fetal bovine serum were purchased from Dalian Meilun Biotechnology Co., Ltd.

[0099] All cells were cultured in a humidified incubator at 37°C with 5% CO2.

[0100] The experimental mice were 4-5 week old male BALB / c nude mice, purchased from Hunan Shengjia Experimental Animal Co., Ltd., and housed in the SPF Animal Room of the Department of Animal Science, Nanhua University. All animal experiments were approved by Nanhua University and followed the ethical review of animal experiments and the health guidelines for the care and use of experimental animals of national research institutes.

[0101] Example 1

[0102] The specific reaction formula for preparing the IR820-GPC3-Gd compound is as follows: Figure 2 .

[0103] Synthesis of Compound 1:

[0104] Phloroglucinol (2.0 g, 0.016 mol) and 1,3-dibromopropane (2.0 g, 0.016 mol) were added to a 100 mL dry round-bottom flask, followed by the sequential addition of K₂CO₃ (4.4 g, 0.0318 mol) and N,N-dimethylformamide (10 mL, DMF). The mixture was reacted at room temperature for 12 h to obtain a crude product. The crude product was extracted with an equal volume of water and ethyl acetate (EA) to remove excess K₂CO₃ and DMF. EA was then removed by rotary evaporation. The crude product was then separated using a column packed with silica gel powder. The eluent used was a mixture of petroleum ether (PE) and EA at a volume ratio of 8:1. After separation, the product was rotary evaporated to obtain a white solid compound 1 (3.8 g), with a yield of 65.0%.

[0105] Figure 3 For compound 1 1 H-NMR spectrum, Figure 4 For compound 1 13 C NMR spectrum, Figure 5 The HRMS plot for compound 1 is shown below.

[0106] 1 H NMR (500MHz, CDCl3) δ6.01 (t, J=2.1Hz, 1H), 5.96 (d, J=2.1Hz, 2H), 3.98 (dd, J=7.1, 4.5Hz, 6H), 3.52 (t, J=6.4Hz, 6H). 13 C12H17O3Br2+ NMR (126MHz, CDCl3) δ 160.75, 157.38, 95.23, 95.05, 94.50, 94.30, 77.27, 77.01, 76.76, 65.44, 32.29, 29.90. HRMS (ESI) calculated value of C12H17O3Br2+, ([M+H+]) 368.9512, measured value 368.9518.

[0107] Synthesis of compound 2 (IR820-N3):

[0108] Compound 1 (0.8 g, 0.0022 mol) and sodium azide (0.4 g, 0.00615 mol) were dissolved in N,N-dimethylformamide (DMF, 10 mL). The mixture was stirred overnight at 250 rpm under anhydrous and oxygen-free conditions at 70 °C to obtain a crude product. The crude product was purified by silica gel column chromatography (using a mixture of PE and EA as eluent, with a PE to EA volume ratio of 4:1), followed by rotary evaporation and drying to obtain a clear, pale yellow liquid product (0.6 g), with a yield of 94.5%. IR820 (0.2 g, 0.000235 mol), NaH (35 mg, 0.00152 mol), and DMF (10 mL) were added to the pale yellow liquid product (0.6 g), and the reaction was carried out at low temperature (0 °C) for 3 h. The crude product was filtered through a hydrophobic filter, then washed with a mixture of PE and EA at a volume ratio of 1, centrifuged twice, and dried to obtain a dark green solid compound 2 (0.2 g), with a yield of 77.0%.

[0109] Figure 6 For compound 1 1 H-NMR spectrum, Figure 7 For compound 1 13 C NMR spectrum, Figure 8 The HRMS plot for compound 1 is shown below.

[0110] 1 HNMR(500MHz,CD3OD_SPE)δ8.09(s,3H),7.94(d,J=2.8Hz,4H),7.57(d,J=3.7Hz,4H),7.43(s,2H),7.33(s,1H),7.14(s,1H),6.21(d,J=14.3 Hz,4H),4.25(d,J=4.5Hz,2H),3.45(d,J=5.4Hz,2H),3.31(dd,J=10.5,8.9Hz,16H),2.89(s,4H),2.79(d,J=5.8Hz,4H),2.03–1.94(m,18H). 13 C NMR(500MHz,CD3OD_SPE)δ160.75,157.38,95.23,95.05,94.50,94.30,77.27,77.01,76.76,65.44,32.29,29.90.HRMS(ESI)calcd forC 58 H 65 O9N8S2 + ,([M+H + ])1081.4321,Found 1081.4373.

[0111] Synthesis of compound 3 (IR820-GPC3):

[0112] Compound 2 (20 mg, 0.0185 mol) and GPC3-targeting peptide-PEG (65 mg, 0.032 mol) were added to a 25 mL dry flask. Sodium ascorbate (0.46 mg, 0.0023 mol) and copper acetate (0.24 mg, 0.0013 mol) were added to centrifuge tubes containing 1 mL of water and 1 mL of isopropanol, respectively. The centrifuge tubes were ultrasonically cleaned and shaken for 5 min, then added to the reaction flask. The mixture was stirred at 250 rpm for 3 h under anhydrous anaerobic conditions at 70 °C to obtain the crude product. The crude product was dried, dialyzed through a 2 kD dialysis bag for 24 h, and then lyophilized to obtain the final black solid compound 3 (65 mg), with a yield of 78.1%.

[0113] Figure 9 For compound 3 1 H-NMR spectrum, Figure 10 For compound 3 13 C NMR spectrum, specific data are as follows:

[0114] 1 H NMR (500MHz, CD3OD_SPE) δ 8.26 (d, J = 8.5Hz, 3H), 7.99 (d, J = 10.7Hz, 4H), 7.62 (s, 4H), 7.51 (s, 2H), 7.22 (s, 1H), 7.10 (s, 1H), 6.21 (d, J = 14.0Hz, 4H), 4.22 (d, J = 51.9Hz, 18H), 3.87 (s, 188H), 2.73 (d, J = 50.8Hz, 18H). 13 C NMR (500MHz, CD3OD_SPE) δ160.75, 157.38, 95.23, 95.05, 94.50, 94.30, 77.27, 77.01, 76.76, 65.44, 32.29, 29.90.

[0115] Synthesis of IGD (IR820-GPC3-Gd):

[0116] Compound 3 (65 mg, 0.0217 mol) and DOTA-Gd (10 mg, 0.0142 mol) were added to a 25 mL dry flask. Sodium ascorbate (0.46 mg, 0.0023 mol) and copper acetate (0.24 mg, 0.0013 mol) were added to centrifuge tubes containing 1 mL of water and 1 mL of isopropanol, respectively. The centrifuge tubes were ultrasonically cleaned and shaken for 5 min, then added to the reaction flask. The mixture was stirred at 250 rpm for 3 h under anhydrous anaerobic conditions at 70 °C to obtain the crude product. The crude product was dried, dialyzed through a 2 kD dialysis bag for 24 h, and then lyophilized to obtain a black solid IGD (68 mg), with a yield of 92.3%.

[0117] Figure 11 For IR820-GPC3-Gd 1 H-NMR spectrum, Figure 12 For IR820-GPC3-Gd 13 C NMR spectrum, specific data are as follows:

[0118] 1 H NMR (500MHz, CD3OD_SPE) δ8.26(s,3H),7.98(s,4H),7.62(s,8H),6.21(s,4H),4.22–3.33(m,287H),2.91–2.65(m,21H). 13 C NMR (500MHz, CD3OD_SPE) δ160.75, 157.38, 95.23, 95.05, 94.50, 94.30, 77.27, 77.01, 76.76, 65.44, 32.29, 29.90.

[0119] Example 2

[0120] The steps for preparing IR820-GPC3-Gd self-assembled nanoparticles are as follows:

[0121] The IGD (5 mg) prepared in Example 1 was added to 3 mL of water and stirred at room temperature for 10 min at a stirring rate of 500 rpm to obtain IR820-GPC3-Gd self-assembled nanoparticles (IGD NPs).

[0122] Test Example 1

[0123] Physicochemical characteristics of IGD NPs

[0124] 1. The structures of GPC3-PEG, IR820-GPC3, and IR820-GPC3-Gd nanoparticles and their precursors were analyzed using size exclusion chromatography (SEC). SEC analysis results showed that the retention time of IR820 was shortened after coupling with different fractions. Figure 13 The observed molecular weight differences among the different molecules, particularly in GPC3-PEG (2900), IR820-GPC3 (4100), and IR820-GPC3-Gd (4700), indicate that different parts are stably linked. The statistical results of the molecular weight differences among the different compounds are shown in Table 1.

[0125] Table 1. Molecular weight differences of GPC3-PEG, IR820-GPC3, and IR820-GPC3-Gd

[0126]

[0127] 2. The morphology and size distribution of IGD NPs were examined using transmission electron microscopy (TEM) and dynamic light scattering (DLS). The results showed that the IGD NPs exhibited a spherical structure with an average diameter of 70.4 ± 1.1 nm. Figure 14 (A and B), the Zeta potential is -16.1 ± 0.49 mV ( Figure 15 ).

[0128] The UV-Vis-NIR absorption spectra of IR820 (50 μM) and IGD NPs (50 μM) in aqueous phase were measured using a spectrometer. The results show that, compared to IR820, IGD NPs exhibit a redshift absorption, with the maximum peak located at 810 nm. Figure 14 (C)

[0129] The fluorescence spectra of IR820 and IGD NPs under excitation at 488 nm and 808 nm were measured by fluorescence spectroscopy. The results showed that IGD NPs inherited the dual emission characteristics of IR820, with peak values ​​at 930 nm (λex: 808 nm) and 560 nm (λex: 488 nm). Figure 14 (D).

[0130] Test Example 2

[0131] Evaluation of the photothermal and photodynamic properties of IGD NPs

[0132] 1. Evaluation of photothermal performance

[0133] Add 1.0 mL of IR820 aqueous solution (50 μM) or 1.0 mL of IGD NPs aqueous solution (50 μM) to a laser at an intensity of 1.0 W / cm². 2The solution was irradiated with an 808 nm laser for 10 min under the specified conditions, and the temperature was recorded using an infrared thermal imager. The results show that IGDNPs exhibit higher photothermal conversion capabilities than IR820, with a temperature rise of 21 °C after 10 min of laser irradiation. Figure 16 a)

[0134] Furthermore, 1.0 mL of IGD NPs aqueous solution (50 μM) was subjected to different laser intensities (0.25 W / cm²). 2 0.5W / cm 2 0.75W / cm 2 and 1W / cm 2 Under certain conditions, the solution was irradiated with an 808 nm near-infrared laser for 10 min, and the temperature evolution curve was recorded using an infrared thermal imager. The results show that the temperature of IGD NPs increases with increasing laser power. Figure 16 (b)

[0135] Furthermore, 1.0 mL of IGD NPs aqueous solutions of different concentrations (0 μM, 10 mL, 25 μM, 50 μM, 75 μM, and 100 μM) were subjected to laser intensities (1 W / cm²). 2 Under certain conditions, the solution was irradiated with an 808 nm laser for 10 min, and the temperature was recorded using an infrared thermal imager. The photothermal heating curves and thermograms showed that IGD NPs exhibited concentration-dependent photothermal properties, rapidly heating to 45 °C at a low concentration of 75 μM, which was sufficient to effectively kill tumor cells. Figure 14 (E and F in the middle).

[0136] 2. Evaluation of photodynamic performance

[0137] Reactive oxygen species (ROS) levels were measured using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). Specifically, Hep-G2 cells (1×10⁻⁶) were seeded in 12-well plates. 5 Cells were incubated using different treatment methods: PBS, IR820 (50 μM), and IGD NPs (50 μM). After 4 hours of incubation, the cells were washed three times with PBS to remove residual sample. ROS levels were then detected using a DCFH (Sigma-Aldrich Co.) analyzer.

[0138] The DCF fluorescence results showed that after irradiation with an 808nm laser for 5 minutes, the DCF fluorescence of IR820 and IGD NPs reached its maximum intensity at 525nm. Figure 17 This indicates that ROS is released from IGD NPs; and the amount of ROS released increases with the extension of laser irradiation time. Figure 14(G). Therefore, IGD NPs can promote the production of ROS in cells and have strong photodynamic properties.

[0139] The results in summary indicate that IGD NPs have good potential for photothermal and photodynamic therapy.

[0140] Test Example 3

[0141] Evaluation of FLI and MRI function

[0142] The fluorescence imaging (FLI) and MRI functions of different concentrations of IGD NPs (25 μM, 50 μM, 75 μM, and 100 μM) were measured using a near-infrared II spectrometer. Linear fitting curves of fluorescence signals in aqueous solutions of different concentrations of IGD NPs showed that the FLI imaging signal gradually increased linearly with increasing IGD NP concentration (R0). 2 =0.98568)( Figure 14 In the H+ group, IGD NPs are unaffected by the modifier and maintain high fluorescence stability. Simultaneously, due to the chelating effect of DOTA-Gd, IGD NPs exhibit good magnetic resonance paramagnetism. Linear fitting curves of magnetic resonance signals in aqueous solutions of IGD NPs at different concentrations show that the T1-weighted signal gradually increases with increasing IGD NP concentration (R+). 2 =0.98078), the relaxation rate of r1 is 252 mM-1S-1 ( Figure 14 Middle I).

[0143] The results in summary demonstrate that IGD NPs possess sensitive fluorescence and magnetic resonance imaging signals, and that IGD NPs have great potential for HCC imaging and localization.

[0144] Test Example 4

[0145] Animal tumor model experiments

[0146] The pre-phototherapy phase of IGD NPs in mice with subcutaneous tumors showed excellent photothermal and photodynamic properties against tumor cells, which further promoted research on their in vivo anti-tumor effects.

[0147] In vivo FLI and MRI subcutaneous tumor models and in situ tumor models were established for subsequent animal imaging experiments. The experimental mice were 4-5 week old male BALB / c nude mice purchased from Hunan Shengjia Experimental Animal Co., Ltd., and housed in the SPF animal laboratory of the Department of Animal Science, Nanhua University. All animal experiments were approved by Nanhua University and followed animal experiment ethics review and the national research institute's guidelines for the care and use of laboratory animals.

[0148] Subcutaneous tumor model: Hep-G2 cells (1.0 × 10⁻⁶) were subcutaneously injected into the thigh of BALB / c mice. 7A subcutaneous tumor model was established using cells / mL. When the tumor volume reached 100 mm², a subcutaneous tumor model was established. 3 At that time, internal treatment is performed.

[0149] In situ tumor model: BALB / c mice were anesthetized, and the skin and peritoneum of the upper abdomen were cut open to expose the right lobe of the liver. Hep-G2 cells (2.0 × 10⁻⁶) were slowly injected under the capsule of the right lobe of the liver. 7 Cells / mL, 50μL), apply sterile cotton ball to the puncture site to stop bleeding and prevent leakage; suture the incision continuously with prilin sutures, disinfect the incision and bandage the abdominal incision with sterile gauze; after surgery, inject an appropriate amount of ceftriaxone sodium subcutaneously into the buttocks of nude mice to prevent infection, and after anesthesia recovery, return them to their cages for normal feeding; subsequently, disinfect the wound daily, feed and observe, and detect the growth of the tumor in the mice by CT imaging. When the tumor volume reaches a suitable size, in vivo treatment is performed.

[0150] 1. FLI capability assessment experiment of ICG and IGD NPs

[0151] Subcutaneous tumor mice and orthotopic tumor mice were injected via tail vein with PBS solution containing free ICG (200 μM, 100 μL) and IGDNPs (200 μM, 100 μL), respectively. The FLI and signal values ​​in the subcutaneous tumor mice were observed at different time points after injection. The results are as follows: Figure 18 .according to Figure 18 It was found that after tail vein injection in mice, IGD NPs were rapidly distributed throughout the body via blood vessels and highly accumulated in liver tissue, resulting in low contrast between tumor and normal liver tissue. Figure 19 (A and C). Over time, the fluorescence signal in normal tissues weakened, while the fluorescence signal in the tumor area remained at a high level until 24 hours, when the contrast was optimal. The metabolic and degradation patterns of ICG showed significant differences; after ICG injection, the fluorescence signal rapidly concentrated in the liver tissue, then weakened in the abdominal region, and disappeared completely after 4 hours, with no accumulation at the tumor site.

[0152] Twenty-four hours after tail vein injection into mice, fluorescence imaging (FLI) was performed on the major organs and tumors of mice treated with ICG and IGD NPs. Figure 19 As shown in Figures B and D, the fluorescence signals were mainly located in the liver, kidneys, and tumors. The fluorescence intensity of the IGD NPs group in the tumor was significantly higher than that in normal liver tissue, with clear boundaries. This is because the active GPC3-targeting peptide was successfully introduced into the IGD NPs, resulting in long-term enrichment and retention of the IGD NPs in HCC tissues, with stronger fluorescence signals, while there was no significant difference in fluorescence between the liver and tumor tissues in the ICG group.

[0153] 2. MRI capability assessment experiment of IGD NPs

[0154] T1-weighted MRI was examined in tumor-bearing mice and mice with orthotopic tumors 0 h, 24 h, and 48 h after tail vein injection. The results showed that mice treated with IGD NPs exhibited high T1-weighted MRI signal peaks that persisted for more than 48 hours. Figure 19 China E and Figure 20 This indicates that IGD NPs have excellent MRI capabilities.

[0155] In summary, IGD NPs possess dual-mode FLI / MARI functionality. Dual-mode FLI / MARI extends the in vivo cycle time, enabling multi-mode imaging analysis of HCC, thus providing a longer imaging window for precise surgical localization and guided resection.

[0156] 3. In vivo anti-tumor therapy experiments

[0157] Nude mice with subcutaneous tumors were divided into three groups and intravenously injected with PBS, ICG (200 μM 100 μL), and IGD NPs (200 μM 100 μL), respectively.

[0158] Mice with orthotopic tumors were divided into two groups and intravenously injected with PBS (200 μM 100 μL) and IGD NPs (200 μM 100 μL), respectively.

[0159] Each group was irradiated with an 808nm laser for 10 minutes, and thermal imaging and temperature change curves were recorded at different time intervals during the laser irradiation.

[0160] Subcutaneous tumor thermography and temperature change curves showed that the tumor temperature in the IGD NPs group rose rapidly and significantly, exceeding 51°C within 10 minutes. At this temperature, the tumor could be effectively ablated while minimizing damage to surrounding healthy tissue. In contrast, the ICG group heated up slowly, reaching 44.0°C after 10 minutes. The PBS group showed slow tumor temperature rise, averaging only 38.8°C after 10 minutes, far below the threshold required for effective tumor ablation. Figure 21 (A and B in the middle).

[0161] Measuring the therapeutic effect of subcutaneous tumors after phototherapy is a key step in evaluating the in vivo efficacy of IGD NPs. The tumor volume and weight of each tumor were examined every 3 days, and subcutaneous tumors were harvested for morphological observation after 15 days.

[0162] Tumor volume and weight measurements showed a significant reduction in tumor volume and weight in the IGD NPs group, indicating that laser irradiation significantly inhibited tumor growth. Morphological observation of subcutaneous tumors 15 days later further validated the tumor-suppressing effect of the IGD NPs group. Figure 21(F and G). In contrast, the ICG group showed a smaller ablation effect, accompanied by a slight inhibition of tumor growth (F and G). Figure 21 (C to E).

[0163] Changes in in situ tumors were detected during phototherapy, and the results showed that IGD NPs had a good therapeutic effect on in situ tumors. Figure 22 ).

[0164] IGD NPs possess excellent photothermal and photodynamic properties, and can utilize the effective synergistic effect of PTT and PDT to reduce tumor volume and decrease tumor activity. The phototherapy advantages of IGD NPs can promote tumor downstaging and create favorable conditions for subsequent surgical resection.

[0165] 3. Biosafety assessment experiment

[0166] Fifteen days after phototherapy, blood biochemical parameters and H&E staining were performed on mice in each group. Routine hematological and biochemical tests showed that routine hematological and biochemical tests were normal in both the PBS and IGD NPs treatment groups. Figure 23 H&E staining results showed that no substantial damage or obvious abnormalities were found in the vital organs of each group. Figure 24 Therefore, IGD NPs have good biocompatibility.

[0167] The results in summary indicate that IGD NPs have a safe and effective phototherapy effect on liver tumors, and can completely remove tumor tissue under FLI guidance, reducing the postoperative recurrence rate.

[0168] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An IR820-GPC3-Gd compound, with the structural formula shown in Formula I: Equation I; GPC3 is a GPC3 targeting peptide, and its amino acid sequence is shown in SEQ ID NO:

1.

2. The method for preparing the IR820-GPC3-Gd compound according to claim 1, comprising the following steps: Under alkaline conditions, phloroglucinol and 1,3-dibromopropane undergo a substitution reaction to give compound 1; Compound 1 has the structure shown in Formula 1: Formula 1; Compound 1 was subjected to an azide reaction with sodium azide, and the product was coupled with IR820 to obtain compound 2. Compound 2 has the structure shown in Formula 2: Formula 2; Under copper (I) catalysis, compound 2 and PEG-modified GPC3 targeting peptide were subjected to a click chemical reaction to obtain compound 3; Compound 3 has the structure shown in Formula 3: Formula 3; The compound 3 and the gadolinium-macrocyclic ligand were subjected to a click chemical reaction under copper (I) catalysis to obtain the IR820-GPC3-Gd compound.

3. The preparation method according to claim 2, characterized in that, The temperature of the substitution reaction is 20~30℃, and the time of the substitution reaction is 10~14h; The temperature of the azidation reaction is 65~75℃, and the time of the azidation reaction is 10~14h; The coupling reaction is carried out at a temperature of 0~5℃ for 2.5~3.5h. The temperature of the click chemical reaction is 65~75℃, and the time of the click chemical reaction is 2.5~3.5h.

4. The preparation method according to claim 2 or 3, characterized in that, In the substitution reaction, potassium carbonate is used to adjust the alkaline environment; the molar ratio of phloroglucinol, 1,3-dibromopropane, and potassium carbonate is 1:(1.9~2.5):(1.9~2.5). The molar ratio of compound 1, sodium azide, and IR820 is 1:(2~3):(0.08~0.12). In the coupling reaction, sodium hydride is used to adjust the alkaline environment; the molar ratio of IR820 to sodium hydride is 1:(6~7). The reagents used in the copper (I) catalysis include sodium ascorbate and copper acetate; the molar ratio of compound 2, PEG-modified GPC3-targeting peptide, sodium ascorbate, and copper acetate is 1:(1.5~2.5):(0.1~0.3):(0.05~0.1). The molar ratio of compound 3 to the gadolinium-macrocyclic ligand is 1:(0.5~1).

5. An IR820-GPC3-Gd self-assembled nanomaterial, obtained by self-assembling the IR820-GPC3-Gd compound of claim 1 in water.

6. The preparation method of the IR820-GPC3-Gd self-assembled nanomaterial according to claim 5, comprising the following steps: The IR820-GPC3-Gd compound was dissolved in water to obtain IR820-GPC3-Gd self-assembled nanomaterials.

7. The application of the IR820-GPC3-Gd self-assembled nanomaterial of claim 5 or the IR820-GPC3-Gd self-assembled nanomaterial prepared by the preparation method of claim 6 in the preparation of phototherapy materials and / or contrast agents.

8. The use of the IR820-GPC3-Gd self-assembled nanomaterial according to claim 5 or the IR820-GPC3-Gd self-assembled nanomaterial prepared by the preparation method according to claim 6 in the preparation of drugs for treating liver cancer.

9. A drug for treating cancer, characterized in that, The active ingredient is the IR820-GPC3-Gd self-assembled nanomaterial as described in claim 5 or the IR820-GPC3-Gd self-assembled nanomaterial prepared by the preparation method described in claim 6.

10. An aggregation-induced emission molecule, characterized in that, This includes the IR820-GPC3-Gd self-assembled nanomaterial as described in claim 5 or the IR820-GPC3-Gd self-assembled nanomaterial prepared by the preparation method described in claim 6.

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