Octadecanedioic acid modified hepatocellular carcinoma specific targeting fluorescent probe

Abstract

The invention discloses an octadecanedioic acid modified specific targeting fluorescent probe for hepatocellular carcinoma. The fluorescent probe disclosed by the invention is obtained by connecting an SP94 liver cancer specific probe with octadecanedioic acid molecules and IRDye 800CW fluorescent molecules. In the invention, the SP94 peptide is responsible for targeting glucose regulatory protein 78 on the surface of a liver cancer cell, and the protein is expressed in a normal cell endoplasmic reticulum and is abnormally expressed on the surface of the cell in the liver cancer; oDDA is responsible for being combined with albumin, so that the probe is stabilized not to be degraded in vivo, the targeting property is further improved, and the residence time of the probe in tumors is further prolonged; the IRDye 800CW molecule is responsible for generating a fluorescence signal and displaying a tumor. The probe disclosed by the invention is high in liver cancer targeting specificity and low in background signal; the detention time in tumor tissues is long, and tumor boundaries can be stably displayed without diffusion. No toxic or side effect exists. Therefore, the fluorescent probe disclosed by the invention has a good application prospect.

Description

Technical Field

[0001] The present invention relates to an octadecanedioic acid-modified hepatocellular carcinoma-specific targeting fluorescent probe, belonging to the technical field of tumor imaging and localization. Background Art

[0002] One method for tumor localization during surgery is to use indocyanine green (ICG) fluorescent dye. This dye can be absorbed and retained by hepatocellular carcinoma tissues, facilitating real-time observation of liver cancer lesions during surgery. However, ICG lacks specificity for liver cancer because it can also accumulate in cirrhotic nodules, degenerative nodules, bile duct hyperplasia, and necrotic areas, resulting in a false positive rate as high as 40%.

[0003] Octadecanedioic acid (ODDA) has been reported to bind to human serum albumin (HSA), thereby enhancing the therapeutic effect of anti-tumor drugs such as paclitaxel. However, there is currently no report on the use of ODDA to enhance the tumor display ability of fluorescent probes. Summary of the Invention

[0004] The object of the present invention is to provide an octadecanedioic acid-modified hepatocellular carcinoma-specific targeting fluorescent probe. The present invention uses the liver cancer-specific targeting molecule SP94 peptide (SFSIIHTPILPL, SEQ ID NO: 1) to connect the octadecanedioic acid molecule and the IRDye 800CW fluorescent molecule to form this probe. The present invention introduces ODDA onto the probe molecule. ODDA can effectively bind to albumin, enabling the probe to effectively resist degradation by various enzymes during in vivo circulation, and due to the enhanced permeability and retention effect (EPR), it can achieve higher accumulation inside tumor tissues.

[0005] To achieve the above object, the present invention provides a fluorescent probe, and the chemical structural formula of the fluorescent probe is shown as follows:

[0006]

[0007] Wherein, m is an integer of 1 or greater than 1, and n is an integer of 0 or greater than 0. Wherein, m represents the number of coupled NH 2 -PEG6-CH 2 CH 2 CO-groups, and n represents the number of repeatedly coupled SP94 peptides. When m = 1 and n = 0, 1 NH 2 -PEG6-CH 2 CH 2 CO-group and 1 SP94 peptide are coupled.

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

[0009] Step 1: Prepare SP94 peptide by solid-phase synthesis method, and sequentially couple NH 2 -PEG6-CH 2 CH 2 COOH, lysine (Lys), and octadecanedioic acid to obtain a polypeptide compound HOOC(CH 2 ) 16 -CO-NH-Lys-CO-NH-PEG6-CH 2 CH 2 CO-NH-Ser-Phe-Ser-Ile-Ile-His-Thr-Pro-Ile-Leu-Pro-Leu;

[0010] Step 2: Couple the polypeptide compound synthesized in Step 1 with the fluorescent dye activated ester IR Dye 800CW NHS to prepare the fluorescent probe (m = 1, n = 0); when m and n are other values, repeat the corresponding coupling steps according to the values of m and n to couple multiple corresponding groups or SP94 peptides.

[0011] In some embodiments of the present invention, the polypeptide compound synthesized in Step 1 is prepared by Fmoc solid-phase peptide synthesis method.

[0012] In some embodiments of the present invention, the specific steps for synthesizing the polypeptide compound in Step 1 include: sequentially performing coupling reactions with Fmoc-protected amino acid molecules Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH on a solid-phase synthesis resin to couple the corresponding amino acids, and then sequentially performing coupling reactions with Fmoc-NH-PEG6-CH 2 CH 2 COOH, Fmoc-Lys(Boc)-OH, and monoter-butyl octadecanedioate (18-(tert-Butoxy)-18-oxooctadecanoic acid) to couple NH 2 -PEG6-CH 2 CH 2 COOH, lysine (Lys), and octadecanedioic acid.

[0013] Preferably, in the coupling reaction, the coupling reaction of the solid-phase synthesis resin with Fmoc-Leu-OH (coupling the first amino acid Leu) 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.

[0014] Preferably, the solid-phase synthesis resin is 2-chlorotrityl chloride resin.

[0015] Preferably, in the coupling reaction, before coupling the next molecule, it further includes a step of deprotection reaction, and the deprotection reaction is carried out in piperidine / DMF solution and under an inert atmosphere condition.

[0016] The present invention also provides the application of the above fluorescent probe in the preparation of an imaging agent for targeting hepatocellular carcinoma.

[0017] The present invention also provides the application of the above fluorescent probe in the preparation of a product for diagnosing or tracing hepatocellular carcinoma.

[0018] In the present invention, the SP94 peptide is responsible for targeting the glucose-regulated protein 78 (GRP78) on the surface of hepatoma cells. This protein is expressed in the endoplasmic reticulum of normal cells, while it is abnormally expressed on the cell surface in hepatocellular carcinoma; ODDA is responsible for binding to albumin, thereby stabilizing the probe from degrading in vivo and further increasing the targeting property and the retention time in tumors; the IRDye 800CW molecule is responsible for generating a fluorescent signal to show the tumor.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The probe of the present invention has high targeting specificity for hepatocellular carcinoma and low background signal; it has a long retention time in tumor tissues, can stably show the tumor boundary without spreading, and has no toxic and side effects; therefore, the fluorescent probe of the present invention has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the chemical structural formula of the fluorescent probe of the present invention.

[0022] Figure 2 It is the imaging diagram of subcutaneous tumor of hepatocellular carcinoma in nude mice.

[0023] Figure 3 It is the imaging diagram of orthotopic tumor of hepatocellular carcinoma in nude mice. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] To make the present invention more obvious and understandable, the preferred embodiments are described in detail below in conjunction with the accompanying drawings.

[0025] For the experimental methods without specific conditions noted 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 are all conventional commercially available products without special instructions.

[0026] Example 1

[0027] (I) Synthesis of Fluorescent Probe

[0028] 1. Synthesis of polypeptide:

[0029] The polypeptide HOOC(CH 2 ) 16 -CO-NH-Lys-CO-NH-PEG6-CH 2 CH 2 CO-NH-Ser-Phe-Ser-Ile-Ile-His-Thr-Pro-Ile-Leu-Pro-Leu was synthesized by the standard Fmoc chemical method. The specific steps are as follows:

[0030] 1) Resin preparation: In the polypeptide solid-phase synthesis tube, 2-chlorotrityl chloride resin (sub: 1.09 mmol / g, 0.16 mmol, 150 mg), and the solution was removed by vacuum filtration.

[0031] 2) Coupling of the first amino acid monomer: A solution of DIEA (3.0 eq) and Fmoc-Leu-OH (3.0 eq) in DMF (2.1 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 (2.1 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 (2.1 mL * 6). The solution was removed by vacuum filtration.

[0032] 3) Deprotection: 20% piperidine / DMF (2.1 mL) solution was added, and the resin was stirred with N 2 at 15 - 20 °C for 20 minutes. The resin was washed with DMF (2.1 mL * 6) and filtered to obtain the resin.

[0033] 4) Coupling: A DMF (1.1 mL) solution containing HOBt (3.0 eq), DIC (3.8 eq), and Fmoc-Pro-OH (3.0 eq) was added to the resin, and the mixture was stirred with N 2 at 15 - 20 °C for 30 minutes. The resin was washed with DMF (2.1 mL * 6).

[0034] 5) Repeat the above steps 3 to 4 to couple the following amino acids:

[0035]

[0036]

[0037] 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.

[0038] 7) Cleavage, separation, and purification of the polypeptide: After synthesis, wash the polypeptide with MeOH (2.1 mL * 3), 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 H 2 O / 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 H 2 O) 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-H 2 O (0.1% TFA-H 2 O)), to obtain 10 mg of white polypeptide powder (Mass: 2099.48 (M + H + ), HPLC purity: 98.62%).

[0039] 2. Fluorescent dye coupling:

[0040] Add a PBS solution (0.5 mL, pH = 8.4) of IR Dye 800CW NHS (CAS No.: 956579-01-4, 5.8 mg, 1 eq) to a PBS solution (0.5 mL, pH = 8.4) of the polypeptide (10 mg, 1 eq), stir at room temperature in the dark for 2 hours, and purify the product by reverse-phase HPLC to obtain 5 mg of freeze-dried fluorescent probe (Mass: 1028.2 (M + 3H + ), MW: 3082.8, HPLC purity 98.2%). The chemical structural formula of the probe is as Figure 1 shown.

[0041] (II) Subcutaneous tumor imaging experiment of hepatocellular carcinoma in nude mice

[0042] The above-prepared fluorescent probe was intravenously injected into the caudal vein of nude mice with subcutaneous hepatocellular carcinoma xenografts at a dose of 3 mg / kg, and the fluorescence of the subcutaneous tumors was observed using a fluorescence laparoscope. It was found that the fluorescence retention time in the tumors was up to more than 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. As Figure 2 shown.

[0043] (III) Imaging experiment of orthotopic hepatocellular carcinoma xenografts in nude mice

[0044] The above-prepared fluorescent probe was intravenously injected into the caudal 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. It was found that the fluorescence retention time in the tumors was up to more than 83 hours, and almost no background fluorescence was generated in other organs. There was good contrast and a long retention time in the orthotopic tumors. As Figure 3 shown.

[0045] As described above, the above are only the preferred embodiments of the present invention, and there are no limitations in any form and substance to the present invention. It should be noted that for those of ordinary skill in the art of this technology, several improvements and supplements can still be made without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A fluorescent probe, characterized in that The chemical structural formula of the fluorescent probe is as follows: Wherein, m is any integer of 1 or greater than 1, and n is any integer of 0 or greater than 0.

2. The method for preparing the fluorescent probe according to claim 1, characterized in that: The following steps are involved: Step 1: Prepare SP94 peptide by solid phase synthesis, and sequentially couple NH2-PEG6-CH2CH2COOH, lysine (Lys), and octadecane dioic acid to obtain the peptide compound HOOC(CH2) 16 -CO-NH-Lys-CO-NH-PEG6-CH2CH2CO-NH-Ser-Phe-Ser-Ile-Ile-His-Thr-Pro-Ile-Leu-Pro-Leu; Step 2: The polypeptide compound synthesized in step 1 is coupled with the fluorescent dye activated ester IR Dye 800CW NHS to prepare the fluorescent probe.

3. The preparation method according to claim 2, characterized in that: The specific steps of synthesizing the polypeptide compound in step 1 include: coupling the corresponding amino acids with Fmoc-protected amino acid molecules Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Ser(tBu)-OH in sequence on a solid phase synthesis resin, and then coupling with Fmoc-NH-PEG6-CH2CH2COOH, Fmoc-Lys(Boc)-OH, and octadecanedioic acid mono-tert-butyl ester in sequence to couple NH2-PEG6-CH2CH2COOH, lysine (Lys), and octadecanedioic acid.

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

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

Images