Fluorescent molecular probe coupled with indocyanine green and hepatocellular carcinoma specific targeting protein as well as preparation method and application of fluorescent molecular probe

Abstract

The invention discloses a fluorescent molecular probe coupled with indocyanine green and hepatocellular carcinoma specific targeting protein as well as a preparation method and application of the fluorescent molecular probe. According to the invention, a targeting ligand of hepatocellular carcinoma specific protein GPC3 is coupled with IRDye 800CW to generate a fluorescence signal, and a tumor is displayed through a clinically used fluorescence laparoscope system; and animal models of two human-derived tumors are constructed for evaluating the difference of the targeting performance among various different targeting molecules. The fluorescent molecular probe disclosed by the invention is high in liver cancer targeting specificity and low in background signal; the detention time in hepatocellular carcinoma in-situ tumor and subcutaneous tumor tissues is long, tumor boundaries can be stably displayed without diffusion, and toxic and side effects are avoided; therefore, the fluorescent molecular probe disclosed by the invention has a good application prospect.

Description

Technical Field

[0001] The present invention relates to a fluorescent molecular probe in which indocyanine green is conjugated with a hepatocellular carcinoma-specific targeting protein, and a preparation method and application thereof, belonging to the technical field of tumor imaging and localization. Background Art

[0002] At present, a variety of protein molecules have been proven to have tumor-specific targeting ability, but the linked fluorescent groups (such as FITC, etc.) are not compatible with clinical fluorescence imaging systems, resulting in these targeting proteins not being widely used clinically.

[0003] Glypican-3 (GPC3) protein is a hepatocellular carcinoma-specific antigen protein, which is usually expressed in the tumor cells of embryos and patients with hepatocellular carcinoma (HCC). GPC3, also known as DGSX, GTR2-2, MXR7, OCI-5, contains a core protein of 580 amino acids and two HS chains at the C-terminus. The GPC3 core protein is anchored to the cell membrane surface through glycosylphosphatidylinositol (GPI), and can be cleaved into an N-terminal soluble protein (sGPC3) of about 40KDa and a 30KDa C-terminal membrane protein. The cleaved sGPC3 can be released from cancer cells into the circulation.

[0004] A variety of amino acid sequences have been identified to have the ability to bind to GPC3 protein, such as GPC3-2 (DHLASLWWGTEL, SEQ ID NO: 1), GPC3-3 (DYEMHLWWGTEL, SEQ ID NO: 2) and GPC3-4 (SNDRPPNILQKR, SEQ ID NO: 3). However, there has been no application of conjugating these targeting polypeptide sequences with a near-infrared fluorescent group (such as ICG) and being compatible with clinical fluorescence devices for intraoperative tumor localization. Summary of the Invention

[0005] The object of the present invention is: aiming at the deficiencies of the prior art, the present invention provides a fluorescent molecular probe in which indocyanine green is conjugated with a hepatocellular carcinoma-specific targeting protein, and a preparation method and application thereof. The present invention conjugates a targeting ligand of the hepatocellular carcinoma-specific protein GPC3 with IRDye 800CW to generate a fluorescent signal, and uses a clinically used fluorescence laparoscope system to display tumors; and constructs two animal models of human tumors to evaluate the differences in targeting performance among various different targeting molecules.

[0006] To achieve the above object, the present invention provides a fluorescent molecular probe, and the fluorescent molecular probe is selected from any one of the following formulas I - III:

[0007]

[0008] The present invention also provides a method for preparing the above-mentioned fluorescent molecular probe, which includes the following steps:

[0009] Step 1: Prepare a polypeptide compound with a coupling group, NH2-PEG6-CH2CH2CO-Asp-His-Leu-Ala-Ser-Leu-Trp-Trp-Gly-Thr-Glu-Leu, NH2-PEG6-CH2CH2CO-Asp-Tyr-Glu-Met-His-Leu-Trp-Trp-Gly-Thr-Glu-Leu, or NH2-PEG6-CH2CH2CO-Ser-Asn-Asp-Arg-Pro-Pro-Asn-Ile-Leu-Gln-Lys-Arg, by solid-phase synthesis method. The polypeptide compound is a GPC3-targeting polypeptide modified with a coupling group NH2-PEG6-CH2CH2CO-, and the amino acid sequence of the GPC3-targeting polypeptide is any one of those shown in SEQ ID NO: 1-3;

[0010] Step 2: Couple the polypeptide compound synthesized in Step 1 with the fluorescent dye activated ester IR Dye 800CW NHS respectively to prepare the fluorescent probe.

[0011] In some embodiments of the present invention, the synthesis of the polypeptide in Step 1 adopts the standard Fmoc polypeptide synthesis method. The specific steps include: sequentially coupling with Fmoc-protected amino acid molecules on the solid-phase synthesis resin, coupling the corresponding amino acids, and then carrying out a coupling reaction with Fmoc-NH-PEG6-CH2CH2COOH to couple NH2-PEG6-CH2CH2CO-.

[0012] In some embodiments of the present invention, the specific steps for synthesizing NH2-PEG6-CH2CH2CO-Asp-His-Leu-Ala-Ser-Leu-Trp-Trp-Gly-Thr-Glu-Leu in Step 1 include: sequentially coupling with Fmoc-protected amino acid molecules Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Trp(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-His(Boc), Fmoc-Asp(tBu)-OH on the solid-phase synthesis resin, coupling the corresponding amino acids, and then carrying out a coupling reaction with Fmoc-NH-PEG6-CH2CH2COOH to couple NH2-PEG6-CH2CH2CO-.

[0013] In some embodiments of the present invention, the specific steps for synthesizing NH2-PEG6-CH2CH2CO-Asp-Tyr-Glu-Met-His-Leu-Trp-Trp-Gly-Thr-Glu-Leu in step 1 include: sequentially coupling with Fmoc-protected amino acid molecules Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Trp(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Leu-OH, Fmoc-His(Boc)-OH, Fmoc-Met-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH on a solid-phase synthesis resin to couple the corresponding amino acids. Then, a coupling reaction is carried out with Fmoc-NH-PEG6-CH2CH2COOH to couple NH2-PEG6-CH2CH2CO-.

[0014] In some embodiments of the present invention, the specific steps for synthesizing NH2-PEG6-CH2CH2CO-Ser-Asn-Asp-Arg-Pro-Pro-Asn-Ile-Leu-Gln-Lys-Arg in step 1 include: sequentially coupling with Fmoc-protected amino acid molecules Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH on a solid-phase synthesis resin to couple the corresponding amino acids. Then, a coupling reaction is carried out with Fmoc-NH-PEG6-CH2CH2COOH to couple NH2-PEG6-CH2CH2CO-.

[0015] Preferably, in the coupling reaction, the coupling reaction of the solid-phase synthesis resin with the first amino acid is carried out in an organic base N,N-diisopropylethylamine (DIEA) and DMF solution; the remaining coupling reactions are carried out in a coupling reagent HOBt / DIC (1-hydroxybenzotriazole / N,N'-diisopropylcarbodiimide) and DMF solution.

[0016] Preferably, the solid-phase synthesis resin is 2-chlorotrityl chloride resin.

[0017] Preferably, in the coupling reaction, a deprotection reaction step is further included before coupling the next molecule, and the deprotection reaction is carried out in a piperidine / DMF solution under an inert atmosphere condition.

[0018] The present invention also provides the application of the above-mentioned fluorescent molecular probe in the preparation of an imaging agent targeting hepatocellular carcinoma.

[0019] The present invention also provides the application of the above-mentioned fluorescent molecular probe in the preparation of a product for diagnosing or tracing hepatocellular carcinoma.

[0020] The present invention also provides a system for diagnosing or tracing hepatocellular carcinoma, which includes the above-mentioned fluorescent molecular probe and a fluorescence laparoscope.

[0021] Preferably, the hepatocellular carcinoma includes subcutaneous hepatocellular carcinoma and orthotopic tumor.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The fluorescent molecular probe of the present invention has high targeting specificity for liver cancer and low background signal; it has a long retention time in the orthotopic tumor and subcutaneous tumor tissues of hepatocellular carcinoma, can stably display the tumor boundary without spreading, and has no toxic and side effects; therefore, the fluorescent molecular probe of the present invention has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the chemical structural formula of the fluorescent molecular probe GPC3-02;

[0025] Figure 2 is the chemical structural formula of the fluorescent molecular probe GPC3-03;

[0026] Figure 3 is the chemical structural formula of the fluorescent molecular probe GPC3-04;

[0027] Figure 4 is the imaging result of three fluorescent molecular probes in the orthotopic tumor of hepatocellular carcinoma in nude mice;

[0028] Figure 5 is the imaging result of three fluorescent molecular probes in the orthotopic tumor of hepatocellular carcinoma in nude mice. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] To make the present invention more obvious and understandable, preferred embodiments are described in detail below in conjunction with the accompanying drawings.

[0030] For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product instructions; the materials, reagents, etc. used, unless otherwise specified, are all conventional commercially available products.

[0031] Example 1: Synthesis of Fluorescent Molecular Probe GPC3-02

[0032] 1. Synthesize the polypeptide using the standard Fmoc solid-phase peptide synthesis method:

[0033] 1) Resin preparation: In a solid-phase peptide synthesis tube, 2-chlorotrityl chloride resin (sub: 1.09 mmol / g, 0.16 mmol, 150 mg) was soaked in DMF for 0.5 hours to swell, and the solution was removed by vacuum filtration.

[0034] 2) Coupling of the first amino acid monomer: A solution of DIEA (3.0 eq) and Fmoc-Leu-OH (3.0 eq) in DMF (1.1 mL, 7V) was added to the resin, and the mixture was stirred with nitrogen at 15 - 20 °C for 3 hours. The solution was removed by vacuum filtration. The resin was washed with DMF (1.1 mL * 3). A solution of DIEA (3.0 eq) / MeOH (20.0 eq) / DMF (1.1 mL, 7V) was added to the resin, and the mixture was stirred with nitrogen at 15 - 20 °C for 1 hour. The resin was washed with DMF (1.1 mL * 6). The solution was removed by vacuum filtration.

[0035] 3) Deprotection: A 20% piperidine / DMF (1.1 mL) solution was added, and the resin was stirred with N2 at 15 - 20 °C for 20 minutes. The resin was washed with DMF (1.1 mL * 6) and filtered to obtain the resin.

[0036] 4) Coupling: A DMF (1.1 mL) solution containing HOBt (3.0 eq), DIC (3.8 eq), and Fmoc-Glu(tBu)-OH (3.0 eq) was added to the resin, and the mixture was stirred with N2 at 15 - 20 °C for 30 minutes. The resin was washed with DMF (1.1 mL * 6).

[0037] 5) Repeat the above steps 3 to 4 to couple the following amino acids:

[0038] # Raw material Coupling reagent 3 Fmoc-Thr(tBu)-OH / 3.0eq HOBt(3.00eq), DIC(3.80eq) 4 Fmoc-Gly-OH / 3.0eq HOBt(3.00eq), DIC(3.80eq) 5 Fmoc-Trp(Boc)-OH / 3.0eq HOBt(3.00eq), DIC(3.80eq) 6 Fmoc-Trp(Boc)-OH / 3.0eq HOBt(3.00eq), DIC(3.80eq) 7 Fmoc-Leu-OH / 3.0eq HOBt(3.00eq), DIC(3.80eq) 8 Fmoc-Ser(tBu)-OH / 3.0eq HOBt(3.00eq), DIC(3.80eq) 9 Fmoc-Ala-OH / 3.0eq HOBt(3.00eq), DIC(3.80eq) 10 Fmoc-Leu-OH / 3.0eq HOBt(3.00eq), DIC(3.80eq) 11 Fmoc-His(Boc)-OH / 3.0eq HOBt(3.00eq), DIC(3.80eq) 12 Fmoc-Asp(tBu)-OH / 3.0eq HOBt(3.00eq), DIC(3.80eq) 13 <![CDATA[Fmoc-NH-PEG6-CH2CH2COOH / 3.0eq]]> HOBt(3.00eq), DIC(3.80eq)

[0039] 6) A DMF (2.1 mL) solution containing 20% piperidine was added, and the resin was stirred with nitrogen at 15 - 20 °C for 20 minutes. The resin was washed with DMF (2.1 mL * 6) and filtered to obtain the resin.

[0040] 7) Cleavage, isolation and purification of the polypeptide: After synthesis, the polypeptide was washed with MeOH (2.1 mL * 3), and then dried overnight under nitrogen to obtain 300 mg of resin. It was transferred to a flask, and the cleavage solution (4.4 mL TFA / 122 mg phenol / 122 μL H2O / 122 μL TIPS / 122 μL phenyl sulfide) was added to the flask containing the resin at 15 - 20 °C, and stirred for 2.5 hours. The solution was precipitated with cold MTBE (methyl tert-butyl ether, 49 mL) and centrifuged (3000 rpm, 3 minutes). The crude peptide was treated with the solution (70 mL TFA, 30 mL H2O) at 15 - 20 °C for 24 h, and purified by Prep-HPLC (column: UniSil-C18, 30 mm * 250 mm, 10 μm; mobile phase: ACN-H2O (0.1% TFA-H2O)) to obtain 9 mg of polypeptide (Mass: 1784.36 (M+Na + ), HPLC purity: 97.06%).

[0041] 2. Fluorescent dye coupling: A PBS solution (0.5 mL, pH = 8.4) of IR Dye 800CW NHS (5.8 mg, 1 eq) was added to a PBS solution (0.5 mL, pH = 8.4) of the polypeptide (9 mg, 1 eq), and stirred in the dark at room temperature for 2 hours. The product was purified by reverse-phase HPLC to obtain 4 mg of freeze-dried GPC3-02 (Mass: 1374.0 (M+2H + ), MW: 2747.9, HPLC purity 98.9%), and its chemical structural formula is as Figure 1 shown.

[0042] Example 2: Synthesis of the fluorescent molecular probe GPC3-03

[0043] 1. Synthesis of the polypeptide using the standard Fmoc polypeptide synthesis method:

[0044] 1) Resin preparation: In a polypeptide solid-phase synthesis tube, 2-chlorotrityl chloride resin (sub: 1.09 mmol / g, 0.16 mmol, 150 mg) was soaked in DMF for swelling for 0.5 hours, and the solution was removed by vacuum filtration.

[0045] 2) Coupling of the first amino acid monomer: A solution of DIEA (3.0 eq) and Fmoc-Leu-OH (3.0 eq) in DMF (1.1 mL, 7V) was added to the resin, and the mixture was stirred with nitrogen at 15 - 20 °C for 3 hours. The solution was removed by vacuum filtration. The resin was washed with DMF (1.1 mL * 3). A solution of DIEA (3.0 eq) / MeOH (20.0 eq) / DMF (1.1 mL, 7V) was added to the resin, and the mixture was stirred with nitrogen at 15 - 20 °C for 1 hour. The resin was washed with DMF (1.1 mL * 6). The solution was removed by vacuum filtration.

[0046] 3) Deprotection: A 20% piperidine / DMF (1.1 mL) solution was added, and the resin was stirred with N2 at 15 - 20 °C for 20 minutes. The resin was washed with DMF (1.1 mL * 6) and filtered to obtain the resin.

[0047] 4) Coupling: A DMF (1.1 mL) solution containing HOBt (3.0 eq), DIC (3.8 eq), and Fmoc-Glu(OtBu)-OH (3.0 eq) was added to the resin, and the mixture was stirred with N2 at 15 - 20 °C for 30 minutes. The resin was washed with DMF (1.1 mL * 6).

[0048] 5) Repeat the above steps 3 to 4 to couple the following amino acids:

[0049] # Raw material Coupling reagent 3 Fmoc-Thr(tBu)-OH / 3.0eq HOBt(3.00eq), DIC(3.80eq) 4 Fmoc-Gly-OH / 3.0eq HOBt(3.00eq), DIC(3.80eq) 5 Fmoc-Trp(Boc)-OH / 3.0eq HOBt(3.00eq), DIC(3.80eq) 6 Fmoc-Trp(Boc)-OH / 3.0eq HOBt(3.00eq), DIC(3.80eq) 7 Fmoc-Leu-OH / 3.0eq HOBt(3.00eq), DIC(3.80eq) 8 Fmoc-His(Boc)-OH / 3.0eq HOBt(3.00eq), DIC(3.80eq) 9 Fmoc-Met-OH / 3.0eq HOBt(3.00eq), DIC(3.80eq) 10 Fmoc-Glu(OtBu)-OH / 3.0eq HOBt(3.00eq), DIC(3.80eq) 11 Fmoc-Tyr(tBu)-OH / 3.0eq HOBt(3.00eq), DIC(3.80eq) 12 Fmoc-Asp(OtBu)-OH / 3.0eq HOBt(3.00eq), DIC(3.80eq) 13 <![CDATA[Fmoc-NH-PEG6-CH2CH2COOH / 3.0eq]]> HOBt(3.00eq), DIC(3.80eq)

[0050] 6) A DMF (2.1 mL) solution containing 20% piperidine was added, and the resin was stirred with nitrogen at 15 - 20 °C for 20 minutes. The resin was washed with DMF (2.1 mL * 6) and filtered to obtain the resin.

[0051] 7) Cleavage, separation, and purification of the polypeptide: After synthesis, the polypeptide was washed with MeOH (2.1 mL * 3), then dried overnight under nitrogen to obtain 300 mg of resin. Transferred to a flask, a cleavage solution (4.4 mL TFA / 122 mg phenol / 122 μL H2O / 122 μL TIPS / 122 μL phenylsulfide) was added to the flask containing the resin at 15 - 20 °C, and stirred for 2.5 hours. The solution was precipitated with cold MTBE (49 mL) and centrifuged (3000 rpm, 3 minutes). The crude peptide was treated with a solution (70 mL TFA, 30 mL H2O) at 15 - 20 °C for 24 h, and purified by Prep-HPLC (column: UniSil-C18, 30 mm * 250 mm, 10 μm; mobile phase: ACN-H2O (0.1% TFA-H2O)) to obtain 10 mg of polypeptide (Mass: 1936.39 (M + Na + )), HPLC purity: 96.56%).

[0052] 2. Fluorescent dye coupling:

[0053] Add the PBS solution (0.5 mL, pH = 8.4) of IR Dye 800CW NHS (5.75 mg, 1 eq) to the PBS solution (0.5 mL, pH = 8.4) of the polypeptide (10 mg, 1 eq), stir in the dark at room temperature for 2 hours, purify the product by reverse-phase HPLC, and obtain 5 mg of freeze-dried GPC3-03 (Mass: 1451.2 (M+2H + ), MW: 2900.3, HPLC purity 98.9%), and its chemical structural formula is as Figure 2 shown.

[0054] Example 3: Synthesis of fluorescent molecular probe GPC3-04

[0055] 1. Synthesize the polypeptide using the standard Fmoc polypeptide synthesis method:

[0056] 1) Resin preparation: In the polypeptide solid-phase synthesis tube, 2-chlorotrityl chloride resin (sub: 1.09 mmol / g, 0.16 mmol, 150 mg) is soaked in DMF for swelling for 0.5 hour, and the solution is removed by vacuum filtration.

[0057] 2) Coupling of the first amino acid monomer: Add the solution of DIEA (3.0 eq) and Fmoc-Arg(Pbf)-OH (3.0 eq) dissolved in DMF (1.1 mL, 7V) to the resin, and stir the mixture with nitrogen at 15 - 20 °C for 3 hours. Remove the solution by vacuum filtration. Wash the resin with DMF (1.1 mL * 3). Add the DIEA (3.0 eq) / MeOH (20.0 eq) / DMF (1.1 mL, 7V) solution to the resin, and stir the mixture with nitrogen at 15 - 20 °C for 1 hour. Wash the resin with DMF (1.1 mL * 6). Remove the solution by vacuum filtration.

[0058] 3) Deprotection: Add 20% piperidine / DMF (1.1 mL) solution, and stir the resin with N2 at 15 - 20 °C for 20 minutes. Wash the resin with DMF (1.1 mL * 6) and filter to obtain the resin.

[0059] 4) Coupling: Add the DMF (1.1 mL) solution containing HOBt (3.0 eq), DIC (3.8 eq) and Fmoc-Lys(Boc)-OH (3.0 eq) to the resin, and stir the mixture with N2 at 15 - 20 °C for 30 minutes. Wash the resin with DMF (1.1 mL * 6).

[0060] 5) Repeat the above steps 3 to 4 to couple the following amino acids:

[0061] # Raw material Coupling reagent 3 Fmoc-Gln(Trt)-OH / 3.0eq HOBt (3.00 eq), DIC (3.80 eq) 4 Fmoc-Leu-OH / 3.0 eq HOBt (3.00 eq), DIC (3.80 eq) 5 Fmoc-Ile-OH / 3.0 eq HOBt (3.00 eq), DIC (3.80 eq) 6 Fmoc-Asn(Trt)-OH / 3.0 eq HOBt (3.00 eq), DIC (3.80 eq) 7 Fmoc-Pro-OH / 3.0 eq HOBt (3.00 eq), DIC (3.80 eq) 8 Fmoc-Pro-OH / 3.0 eq HOBt (3.00 eq), DIC (3.80 eq) 9 Fmoc-Arg(Pbf)-OH / 3.0 eq HOBt (3.00 eq), DIC (3.80 eq) 10 Fmoc-Asp(OtBu)-OH / 3.0 eq HOBt (3.00 eq), DIC (3.80 eq) 11 Fmoc-Asn(Trt)-OH / 3.0 eq HOBt (3.00 eq), DIC (3.80 eq) 12 Fmoc-Ser(tBu)-OH / 3.0 eq HOBt (3.00 eq), DIC (3.80 eq) 1 <![CDATA[Fmoc-NH-PEG6-CH2CH2COOH / 3.0eq]]> HOBt (3.00 eq), DIC (3.80 eq)

[0062] 6) Add a solution of DMF (2.1 mL) containing 20% piperidine, and stir the resin with nitrogen at 15 - 20 °C for 20 minutes. Wash the resin with DMF (2.1 mL * 6), and filter to obtain the resin.

[0063] 7) Cleavage, separation, and purification of the polypeptide: After synthesis, wash the polypeptide with MeOH (2.1 mL * 3), and then dry it overnight under nitrogen to obtain 300 mg of resin. Transfer it to a flask, and add the cleavage solution (4.4 mL TFA / 122 mg phenol / 122 μL H2O / 122 μL TIPS / 122 μL phenyl sulfide) to the flask containing the resin at 15 - 20 °C, and stir for 2.5 hours. Precipitate the solution with cold MTBE (49 mL), and centrifuge (3000 rpm, 3 minutes). Treat the crude peptide with a solution (70 mL TFA, 30 mL H2O) at 15 - 20 °C for 24 h, and purify it by Prep-HPLC (column: UniSil-C18, 30 mm * 250 mm, 10 μm; mobile phase: ACN-H2O (0.1% TFA-H2O)) to obtain 9 mg of polypeptide (Mass: 1794.56 (M+Na + )), HPLC purity: 96.30%).

[0064] 2. Fluorescent dye coupling:

[0065] Add a PBS solution (0.5 mL, pH = 8.4) of IR Dye 800CW NHS (5.75 mg, 1 eq) to a PBS solution (0.5 mL, pH = 8.4) of the polypeptide (10 mg, 1 eq), stir in the dark at room temperature for 2 hours, and purify the product by reverse-phase HPLC to obtain 5 mg of freeze-dried GPC3-04 (Mass: 1379.3 (M+2H + ), MW: 2758.5, HPLC purity 98.7%), and its chemical structural formula is as Figure 3 shown.

[0066] Example 4: Evaluation of probe targeting performance

[0067] (I) Verification of the small molecule polypeptide fluorescent probe in the subcutaneous tumor of nude mice with hepatocellular carcinoma

[0068] In the above Examples 1-3, three small molecule polypeptide fluorescent probes, namely GPC3-02, GPC3-03, and GPC3-04, were synthesized. Each was injected into the tail vein of nude mice with subcutaneous hepatocellular carcinoma xenografts at a dose of 3 mg / kg. The fluorescence of the subcutaneous tumors was observed using a fluorescence laparoscope, and the fluorescence contrast between the tumor and the surrounding tissues and the retention time of the fluorescent probe in the tumor were recorded. It was found that the retention times of the three fluorescent molecular probes in the subcutaneous tumors were all as long as 132 hours, and almost no background fluorescence was generated in other organs. There was good contrast and a long retention time in the subcutaneous tumors. The results are as Figure 4 shown.

[0069] (II) Verification of small molecule polypeptide fluorescent probes in orthotopic hepatocellular carcinoma xenografts in nude mice

[0070] The three small molecule polypeptide fluorescent probes synthesized in the above Examples 1-3, namely GPC3-02, GPC3-03, and GPC3-04, were each injected into the tail vein of nude mice with orthotopic hepatocellular carcinoma xenografts at a dose of 3 mg / kg. After laparotomy, the fluorescence of the orthotopic tumors was observed using a fluorescence laparoscope, and the fluorescence contrast between the tumor and the surrounding tissues and the retention time of the fluorescent probe in the tumor were recorded. It was found that the retention times of the three fluorescent molecular probes in the orthotopic tumors were 96H, 96H, and 107H respectively, and almost no background fluorescence was generated in other organs. There was good contrast and a long retention time in the orthotopic tumors. The results are as Figure 5 shown.

[0071] As mentioned above, the above are only the preferred embodiments of the present invention, and there are no limitations in any form or substance to the present invention. It should be pointed out that for those of ordinary skill in the art in this technical field, without departing from the premise of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A fluorescent molecular probe, characterized in that, The fluorescent molecular probe is selected from any one of the following formulas I - III:

2. The preparation method of the fluorescent molecular probe according to claim 1, characterized in that, Comprising the following steps: Step 1: Prepare a GPC3-targeting polypeptide modified with a coupling group NH2-PEG6-CH2CH2CO by solid-phase synthesis method, and the amino acid sequence of the GPC3-targeting polypeptide is any one of those shown in SEQ ID NO: 1 - 3; Step 2: Perform a coupling reaction on the polypeptide compound synthesized in Step 1 with the fluorescent dye active ester IR Dye 800CW NHS respectively to prepare the fluorescent probe.

3. Use of the fluorescent molecular probe according to claim 1 in the preparation of an imaging agent for targeting hepatocellular carcinoma.

4. Use of the fluorescent molecular probe according to claim 1 in the preparation of a product for diagnosing or tracing hepatocellular carcinoma.

5. A system for diagnosing or tracing hepatocellular carcinoma, characterized in that, Comprising the fluorescent molecular probe according to claim 1 and a fluorescence laparoscope system.

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