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CN114796528B - Tumor-specific targeting polypeptides and uses thereof - Google Patents

Tumor-specific targeting polypeptides and uses thereof Download PDF

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CN114796528B
CN114796528B CN202210359385.5A CN202210359385A CN114796528B CN 114796528 B CN114796528 B CN 114796528B CN 202210359385 A CN202210359385 A CN 202210359385A CN 114796528 B CN114796528 B CN 114796528B
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yqga
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CN114796528A (en
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顾月清
涂远彪
刘培飞
王芳
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China Pharmaceutical University
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    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
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    • A61K49/0017—Fluorescence in vivo
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

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Abstract

The invention discloses a tumor-specific targeting polypeptide and application thereof. The series of high affinity polypeptides can be specifically combined with various tumor cells, and the high affinity characteristics can be used for optical imaging and nuclear medicine imaging of malignant tumors. The high-affinity polypeptide coupled fluorescent dye can be used as a tumor specific targeting molecular probe, can achieve the effect of accurately positioning tumor boundaries, can bring real-time performance to preoperative and intra-operative image navigation, and has the advantage of improving operation accuracy. The series of polypeptides can be coupled with radionuclides to detect malignant tumors in real time in vivo so as to achieve the purpose of disease diagnosis or treatment.

Description

Tumor-specific targeting polypeptides and uses thereof
Description of the division
The application is a divisional application of the application of which the application date is 2020, 05, 11 and 202010392677X and the name is tumor targeting polypeptide and application thereof.
Technical Field
The invention belongs to the technical field of bioengineering pharmacy and the fields of protein polypeptide medicaments and biomedical engineering, and particularly relates to tumor targeting polypeptides and application thereof.
Background
Tumor has become a major source of health and life threat, and early diagnosis of tumor and effective treatment of tumor are therefore important and urgent. For tumors, the conventional image diagnosis technologies mainly comprise B-ultrasonic, CT and MRI, the image diagnosis technologies achieve diagnosis results by displaying the functional changes of tissues, and the image diagnosis technologies have good application value, but have certain defects in differential diagnosis, whole body stage and early curative effect evaluation. It is undeniable that the screening and optimizing of polypeptides targeting tumors is a new approach, and can develop novel molecular imaging drugs for tumor diagnosis, stage and operation guidance, and can find more tiny lesions, thereby achieving the purpose of early diagnosis.
The cyanine dye has the advantages of small molecular weight, low toxicity, wide wavelength adjustable range, large molar extinction coefficient and the like, so that the cyanine dye is widely applied to the field of fluorescent marking. The cyanine dye is modified in structure, is connected with reactive groups, then reacts with amino or carboxyl of specific target molecules such as antibodies, proteins, short peptides, small molecules and the like to form stable covalent bonds, and forms a specific targeting molecular probe for fluorescent molecular living imaging, so that the fluorescent dye is an important application of near infrared fluorescent dyes. Single photon emission computed tomography (SPECT-CT) is a novel nuclear medicine imaging technology developed in recent 20 years and popularized clinically, mainly uses short half-life radionuclides to label specific targeted ligands for tracer imaging, can display in-vivo information such as substance metabolism, cell proliferation, receptor distribution and the like, and is used for diagnosis of diseases and research of human vital activities. Therefore, the specific targeted ligand is critical for fluorescence imaging and radionuclide imaging.
Based on the above consideration, the applicant designs a novel tumor targeting polypeptide, the polypeptide can specifically target tumors, and the coupled fluorescent dye can perform optical imaging to assist doctors in accurately positioning tumor boundaries in surgery when using molecular image surgery navigation equipment, so that the aim of accurately cutting tumors is fulfilled, thereby reducing trauma to patients and reducing the risk of postoperative recurrence. In addition, the targeting polypeptide can be coupled with radionuclides for nuclide imaging so as to achieve the purposes of early diagnosis and treatment of tumors.
Disclosure of Invention
The primary aim of the invention is to provide several polypeptides and sequences thereof with novel structure and tumor specific targeting;
another object of the present invention is to provide a method for preparing several tumor-specific targeted fluorescent probes;
another object of the invention is to provide a method for preparing several tumor-specific targeted radioactive probes;
it is a further object of the present invention to provide the use of several of the probes described in optical and SPECT imaging.
A tumor-specific targeting polypeptide selected from any one of the following:
YQGA-2：D-Asp-Arg-Val-Tyr-Ile-His-Pro-D-Phe
YQGA-3：D-Asp-homoArg-Nva-(3-I-Tyr)-Nle-His-Hyp-(4-F-Phe)
YQGA-4：[Sar]-homoArg-Nva-(3-Cl-Tyr)-Nle-His-Hyp-Nle
YQGA-5：D-Asp-Arg-Val-Tyr-NH 2
YQGA-6：D-Asp-homoArg-Nva-Tyr-NH 2
YQGA-7：D-Asp-homoArg-Nva-(4-OCH 3 -Phe)
YQGA-8：Asp-homoArg-Nva-Tyr-NH 2
YQGA-9: mpa-D-Asp-Arg-Val-Tyr-Lys-Cys, wherein Mpa-Cys disulfide bonds form a ring;
YQGA-10: mpa-D-Asp-Arg-Val-Tyr-Cys-Lys, wherein Mpa-Cys disulfide bonds form a ring;
YQGA-11: beta-Ala-D-Asp-Arg-Val-Tyr-Lys-Asp (beta-Ala amino and Asp backbone carboxyl form a ring)
YQGA-12: beta-Ala-D-Asp-Arg-Val-Tyr-Asp-Lys (beta-Ala amino and Asp backbone carboxyl form a ring)
Wherein: D-Asp: d-aspartic acid; homoArg: homoarginine; nva: norvaline; nle: norleucine; hyp: hydroxyproline; 4-F-Phe: 4-fluoro-phenylalanine; 4-OCH 3 Phe: 4-fluoro-phenylalanine; [ Sar ]]: n-methylglycine; 3-Cl-Try: 3-chloro-tyrosine; 3-I-Try: 3-iodo-tyrosine; mpa: 3-mercaptopropionic acid.
The application of the tumor specific targeted polypeptide in preparing a tumor diagnostic reagent is preferably the application in preparing a tumor diagnostic imaging agent; further preferred is the use in the preparation of an agent for the precise localization of tumor boundaries and intra-operative image navigation imaging or in the preparation of radionuclide imaging; the tumor specific targeting polypeptide is selected from any one of the following polypeptides:
YQGA-1：Asp-Arg-Val-Tyr-Ile-His-Pro-Phe
YQGA-2：D-Asp-Arg-Val-Tyr-Ile-His-Pro-D-Phe
YQGA-3：D-Asp-homoArg-Nva-(3-I-Tyr)-Nle-His-Hyp-(4-F-Phe)
YQGA-4：[Sar]-homoArg-Nva-(3-Cl-Tyr)-Nle-His-Hyp-Nle
YQGA-5：D-Asp-Arg-Val-Tyr-NH 2
YQGA-6：D-Asp-homoArg-Nva-Tyr-NH 2
YQGA-7：D-Asp-homoArg-Nva-(4-OCH 3 -Phe)
YQGA-8：Asp-homoArg-Nva-Tyr-NH 2
YQGA-9: mpa-D-Asp-Arg-Val-Tyr-Lys-Cys, wherein Mpa-Cys disulfide bonds form a ring;
YQGA-10: mpa-D-Asp-Arg-Val-Tyr-Cys-Lys, wherein Mpa-Cys disulfide bonds form a ring;
YQGA-11: beta-Ala-D-Asp-Arg-Val-Tyr-Lys-Asp (beta-Ala amino and Asp backbone carboxyl form a ring)
YQGA-12: beta-Ala-D-Asp-Arg-Val-Tyr-Asp-Lys (beta-Ala amino and Asp backbone carboxyl form a ring)
Wherein: D-Asp: d-aspartic acid; homoArg: homoarginine; nva: norvaline; nle: norleucine; hyp: hydroxyproline; 4-F-Phe: 4-fluoro-phenylalanine; 4-OCH 3 Phe: 4-fluoro-phenylalanine; [ Sar ]]: n-methylglycine; 3-Cl-Try: 3-chloro-tyrosine; 3-I-Try: 3-iodo-tyrosine; mpa: 3-mercaptopropionic acid.
A polypeptide compound with tumor targeting fluorescent imaging function comprises a polypeptide for targeting tumor and an infrared fluorescent dye structure for optical imaging, wherein the structure of the polypeptide compound is shown in the following formula (I):
the structure of the polypeptide contains a polypeptide R for targeting tumors and a near infrared fluorescent dye structure MPA for optical imaging, and a connecting agent L for increasing the distance between the targeting polypeptide and the near infrared fluorescent dye and regulating in vivo pharmacokinetics.
The polypeptide R is selected from any one of the tumor specific targeting polypeptides YQGA-X (X=1-12) provided by the invention.
The drug linker L is selected from L1, L2, L3 and L4 shown in a structural formula (II);
L 1
L 2
L3
L 4
the invention also provides a method for preparing the polypeptide fluorescent probe, which comprises the following steps:
1) Synthesis of near infrared fluorescent dye MPA
Mixing glacial acetic acid, p-hydrazinobenzenesulfonic acid, methyl isopropyl ketone and sodium acetate for reaction, and purifying to obtain a product 2, 3-trimethyl [3H ] -indole-5-sulfonic acid; adding o-dichlorobenzene into the mixture of 2, 3-trimethyl [3H ] -indole-5-sulfonic acid and 1, 3-propane sulfonic acid lactone to prepare 2, 3-trimethyl-5-sulfonic acid-1- (3-sulfonic acid-propyl) - [3H ] -indole. And then reacting the product with N- [ (3- (anilinometer) -2-chloro-1-cyclopen-1-yl) methyl ] -aniline monohydrochloride to obtain green carbocyanine dye, and finally reacting the carbocyanine dye with mercaptopropionic acid and triethylamine to prepare the liquid phase separation and purification to obtain the water-soluble near infrared dye MPA.
2) Synthesis of MPA-L-YQGA-X (X=1-12)
Dissolving near infrared dye MPA obtained by separation and purification and L-YQGA-X (X=1-12) polypeptide synthesized by solid phase into dimethyl sulfoxide, adding a proper amount of N, N-Diisopropylethylamine (DIPEA), reacting at room temperature overnight, and preparing a liquid phase after the reaction is completed, purifying and separating to obtain the target fluorescent compound.
The invention relates to application of a polypeptide compound with tumor targeting fluorescence imaging function in preparation of a tumor diagnosis reagent; preferably in the preparation of tumor diagnostic imaging agents; further preferred is the use in the preparation of an agent for the precise localization of tumor boundaries and intra-operative image navigation imaging or in the preparation of a radionuclide imaging agent.
On the basis, the invention further provides a radionuclide probe, which is a radionuclide technetium-labeled monomer polypeptide complex and a radionuclide dimer polypeptide complex, and the structural formulas are shown as (III), (IV) and (V):
the targeting complex in the form of a monomer is simpler to prepare than a dimer, the dimer structure of the targeting complex comprises a polypeptide YQGA-X for targeting tumors and a bifunctional chelating agent 6-hydrazinopyridine-3-carboxylic acid (HYNIC) for radiolabelling, a scaffold (3L-E) with glutamic acid connected with a three-molecule linker L, and a linker L which can increase the distance between the targeting polypeptide and a radionuclide ligand N-tris (hydroxymethyl) methylglycine (Tricine) and triphenylphosphine trimetaphosphate salt (TPPTS) and regulate in vivo pharmacokinetic properties, wherein L is selected from L1, L2, L3 and L4. Wherein the bifunctional chelator is altered, e.g. replaced by the bifunctional chelator DOTA, NOTA, MAG 3 Or DTPA, radionuclide optionally removed 99m Other radionuclides than Tc, e.g. 68 Ga， 64 Cu， 67 Ga， 90 Y， 111 In or In 177 Lu is used for diagnosis or treatment of diseases.
The invention also provides a method for preparing the monomeric and dimeric radionuclide probes, comprising:
1) Synthesis of bifunctional chelating agent HYNIC-L-NHS
Adding 6-chloronicotinic acid and 80% hydrazine hydrate into ethanol, heating and refluxing for reaction, decompressing and steaming the solvent after the reaction is completed, adding the obtained sticky substance into distilled water, adjusting the PH to about 5.5, separating out solid, filtering and drying to obtain yellow solid, and determining the product to be 6-dihydrazide nicotinic acid through ESI-MS mass spectrum and nuclear magnetic hydrogen spectrum. Adding the obtained 6-dihydrazide nicotinic acid and parA-Aminobenzaldehyde into dimethyl sulfoxide (DMSO), heating for reaction for 5-6 hours, adding into water for precipitation after the reaction is completed, filtering to obtain a solid, drying the solid, adding the solid, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) into the DMSO for reaction at room temperature, adding into water for precipitation after the reaction is completed, purifying the solid through a silica gel column, determining the solid as an intermediate HYNIC-NHS through ESI-MS mass spectrum and nuclear magnetic hydrogen spectrum, then reacting the intermediate with a connector L under alkaline condition, activating with an activator EDCI and NHS, and purifying to obtain the HYNIC-L-NHS solid for later use.
2) Synthesis of scaffold (2L-E)
Taking appropriate amount of Boc-cerealDissolving ammonia acid in DMSO, adding EDCI and NHS with 2 times molar quantity, heating at 60 ℃ for 30min, HPLC analyzing that glutamic acid dual-activated ester is generated, adding linker L with 2 times molar quantity and DIPEA with 3 times molar quantity, heating at 60 ℃ for 30min, and HPLC analyzing 2 PEG 4 The molecule is connected with glutamic acid, then equal volume of TFA is added for reaction at room temperature overnight to remove Boc protection, and finally the crude product is prepared for liquid phase separation and freeze drying for standby.
3) Synthesis of intermediate 3L-E-HYNIC-NHS
Dissolving the prepared stent 2L-E in DMSO, adding the same molar amount of HYNIC-L-NHS, adding 3 times of DIPEA, reacting for 2 hours at room temperature, separating and purifying by a preparation liquid phase after the reaction is finished, confirming a target compound by mass spectrometry, activating the purified product by EDCI and NHS, and purifying to obtain 3L-E-HYNIC-NHS for later use.
4)(YQGA-X) 2 Synthesis of-3L-E-HYNIC
Purified intermediate 3L-E-HYNIC-NHS was dissolved in DMSO, added with 0.5 molar amount of targeting peptide YQG-X, then added with 2 molar amount of DIPEA, reacted at room temperature for 1 hour, and after completion of the reaction, separated and purified by preparation of liquid phase and confirmed by mass spectrometry.
5) Radioactive probe 99m Tc-HYNIC-3L-E-(YQGA-X) 2 Is synthesized by (a)
Preparing 100.0mg/mL TPPTS (triphenylphosphine sodium tri-m-sulfonate) solution, 130.0mg/mL Tricine (trimethylglycine) solution, 102.4mg/mL succinic acid-sodium succinate buffer (77.0 mg succinic acid and 25.4mg succinic acid), respectively taking 10.0uL TPPTS solution, 10.0uL Tricine solution, 10.0uL succinic acid-sodium succinate buffer and 10.0uL (1.0 mg/mL) of HYNIC-3L-E- (YQGA-X) solution 2 Mix in a penicillin bottle and then add 10mCi Na 99m TcO 4 Heating in metal bath at 100deg.C for 20 min, cooling to room temperature after the reaction is completed, and respectively preparing polypeptide radiopharmaceuticals, and analyzing and identifying the product by Agilent ZORBAX SB-Aq analysis column.
The radionuclide probe is applied to the preparation of tumor diagnosis reagents; preferably in the preparation of tumor diagnostic imaging agents; further preferred is the use in the preparation of an agent for the precise localization of tumor boundaries and intra-operative image navigation imaging or in the preparation of a radionuclide imaging agent.
The polypeptide compound can specifically target to a tumor part, has good uptake and retention capacity at the tumor part, has higher target/non-target ratio, is suitable for being used as a fluorescent tumor imaging agent, a radionuclide imaging agent and a therapeutic agent, and can be used for preparing optical imaging medicaments for image navigation in tumor operation and accurate positioning of tumor boundaries.
Compared with the prior art, the novel polypeptide and the fluorescence and radionuclide probe constructed by the series of polypeptides have the beneficial effects that:
1. the YQGA-X series polypeptide discovered by the invention is a low molecular weight polypeptide, and a plurality of or more amino acids of the series polypeptide are modified unnatural amino acids, and the introduction of the unnatural amino acids can greatly improve the stability of the series polypeptide in vivo.
2. The YQGA-X series polypeptide has excellent imaging effect on various tumors, including liver cancer, lung cancer, breast cancer, pancreatic cancer, colorectal cancer, cervical cancer and the like, through in vivo optical and radionuclide imaging results. The probe constructed by the series of polypeptides can specifically target the tumor part, so that the nuclear medicine diagnosis and treatment of malignant tumors can be possibly realized, and an optical imaging guides a surgeon to conduct operation navigation, so that the accurate excision of focus is achieved.
3. The near infrared fluorescent dye MPA with more ideal stability and water solubility is used as an optical imaging group in the invention, so that the pharmacokinetics of the medicine in vivo is improved.
4. In the present invention, a plurality of water-soluble PEG are introduced 4 Or PEG (polyethylene glycol) 6 Molecules to further improve pharmacokinetic properties, particularly clearance kinetics from non-tumor tissues.
5. In the present invention, HYNIC is used as a bifunctional chelating agent, and Tricine and TPPTS are simultaneously used as synergistic ligands, so that' 99m Tc-HYNIC core has better in-vivo and in-vitro stability.
The invention is further described below with reference to the drawings and examples.
Drawings
FIG. 1 shows the fluorescent compound MPA-PEG prepared in example 1 4 Fluorescence imaging of YQGA-1 in hepatoma HepG2 tumor-bearing mice.
FIG. 2 is a photograph of the sample of example 2 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-1 in liver cancer HepG2 tumor-bearing mice (A) and brain glioma U87MG tumor-bearing mice (B).
FIG. 3 shows the radioactive compound prepared in example 4 99m SPECT-CT imaging of Tc-HYNIC-YQGA-2 in tumor-bearing mice: a is SPECT-CT imaging in a liver cancer HepG2 tumor-bearing mouse, B is SPECT-CT imaging in a cervical cancer HeLa tumor-bearing mouse, C is SPECT-CT imaging in a breast cancer MCF-7 tumor-bearing mouse, and D is SPECT-CT imaging in a liver cancer MHCC97-H tumor-bearing mouse.
FIG. 4 shows the radioactive compound prepared in example 8 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-3 in liver cancer HepG2 tumor-bearing mice.
FIG. 5 shows the radioactive compound prepared in example 9 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-4 in liver cancer HepG2 tumor-bearing mice.
FIG. 6 shows the radioactive compound prepared in example 10 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-5 in tumor-bearing mice: a SPECT-CT imaging is carried out in a liver cancer HepG2 tumor-bearing mouse body; SPECT-CT imaging of B in liver cancer MHCC97-H tumor-bearing mice; SPECT-CT imaging of cervical cancer HeLa tumor-bearing mice in vivo; d SPECT-CT imaging in breast cancer MCF-7 tumor-bearing mice.
FIG. 7 shows the radioactive compound prepared in example 14 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-6 in tumor-bearing mice: a is HepG2 of liver cancer; b is SPECT-CT imaging in cervical cancer HeLa tumor-bearing mice; c is SPECT-CT imaging in lung cancer A549 tumor-bearing mice; d is SPECT-CT imaging in breast cancer MCF-7 tumor-bearing mice; SPECT-CT imaging of E in pancreatic carcinoma SW1990 tumor-bearing mice; f is SPECT-CT imaging in colorectal cancer HT29 tumor-bearing mice; g is BON-1 in neuroendocrine tumorSPECT-CT imaging in tumor-bearing mice.
FIG. 8 shows the radioactive compound prepared in example 14 99m Tc-HYNIC-YQGA-6 in situ colorectal cancer tumor-bearing mice 1h18min SPECT-CT imaging results
FIG. 9 shows the radioactive compound prepared in example 22 99m Tc-HYNIC-3PEG 4 -E-(YQGA-6) 2 SPECT-CT imaging in pancreatic cancer CFPAC-1 tumor-bearing mice.
FIG. 10 is a fluorescence image of the fluorescent compound MPA-Aca-YQGA-6 prepared in example 23 in tumor-bearing mice: a is fluorescence imaging in a liver cancer HepG2 tumor-bearing mouse; b is fluorescence imaging in breast cancer MCF-7 tumor-bearing mice.
FIG. 11 shows the radioactive compound prepared in example 25 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-7 in cervical cancer HeLa tumor-bearing mice.
FIG. 12 shows the radioactive compound prepared in example 26 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-8 in tumor-bearing mice: a is SPECT-CT imaging in a liver cancer HepG2 tumor-bearing mouse; b is SPECT-CT imaging in the pancreas cancer CFPAC-1 tumor-bearing mice.
FIG. 13 shows the radioactive compound prepared in example 28 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-9 in breast cancer MCF-7 tumor-bearing mice.
FIG. 14 shows the radioactive compound prepared in example 29 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-10 in pancreatic cancer SW1190 tumor-bearing mice.
FIG. 15 shows the radioactive compound prepared in example 30 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-11 in liver cancer HepG2 tumor-bearing mice.
FIG. 16 shows the radioactive compound prepared in example 31 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-12 in breast cancer MCF-7 tumor-bearing mice.
Detailed Description
The invention is further illustrated by the following specific examples and application examples: wherein the chemical substances used in the synthesis step are all existing substances or commercial products. The polypeptides involved in each example were synthesized by Hangzhou solid-topology biotechnology limited.
Fluorescent compound MPA-PEG prepared in example 1 4 Fluorescence imaging of YQGA-1 in liver cancer HepG2 tumor-bearing mice
Weighing and entrusting PEG of solid-phase synthesis of Hangzhou solid-state biological technology Co., ltd 4 10mg of YQGA-1 compound, 12.38mg of prepared dye MPA pure product is added into 200 mu L of dimethyl sulfoxide (DMSO), then 2.3mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) coupling agent and 3.82mg of N-hydroxysuccinimide (NHS) are added, 4.1mg of N, N-Diisopropylethylamine (DIPEA) are added after being uniformly mixed, the reaction is carried out at room temperature overnight, and separation and purification are carried out by using a preparation liquid phase after the reaction is completed, wherein the preparation liquid phase conditions are as follows: an Agilent 1220 Infinicity II series HPLC system was used equipped with a Agilent ZORBAX SB-C18 semi-preparative column (9.4X105 mm,5 um) with a gradient elution for 60 minutes at a flow rate of 2mL/min, wherein mobile phase A was ultrapure water (0.01% TFA) and B was acetonitrile (0.01% TFA). The elution gradient was set as: 95% A and 5% B at 0-5 min, 80% A and 20% B at 15 min, 50% A and 50% B at 45 min, 5%A and 95% B at 60 min. The finally prepared green product is confirmed to be the expected product MPA-PEG by analytical HPLC and ESI-MS mass spectrometry 4 -YQGA-1，ESI-MS：[M-3H] 3- = 728.42 and [ M-4H] 4- = 546.21. In the above preparation process, the PEG used in the step is replaced with a YQGA-X or L-YQGA-X polypeptide synthesized in a solid phase 4 -YQGA-1 polypeptide, thus obtaining other various fluorescent polypeptide compounds of the invention. The prepared compound MPA-PEG 4 YQGA-1 was formulated into a physiological saline solution (100 nmol/mL), and 0.1mL (about 10 nmol) was injected into tail veins of 3 liver cancer HepG2 tumor-bearing nude mice (body weight of about 22 g), respectively, and optical signal collection was performed at 1h, 2h, 4h, 8h, 10h, and 12h after administration. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. Compound MPA-PEG 4 The imaging results of YQGA-1 in 3 tumor-bearing nude mice are basically consistent, and the probe is obviously ingested in tumors from the imaging chart of 2h, and the probe is deduced to be mainly metabolized through kidneys.
Example 2 radioactivity preparedCompounds of formula (I) 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-1 in liver cancer HepG2 tumor-bearing mice
1) Bifunctional chelating agent HYNIC-PEG 4 Synthesis of NHS
1g of 6-chloronicotinic acid and 2.0mL of 80% hydrazine hydrate are added into 10mL of ethanol, the mixture is heated and refluxed for 4 hours, the solvent is distilled under reduced pressure after the reaction is completed, the obtained sticky substance is added into distilled water, the pH value is regulated to about 5.5, solids are separated out, 0.86g of yellow solid is obtained through suction filtration and drying, and the product is determined to be 6-dihydrazide nicotinic acid through ESI-MS mass spectrum and nuclear magnetic hydrogen spectrum. The obtained 0.86g of 6-dihydrazide nicotinic acid and 0.61g of p-aminobenzaldehyde are added into 3.0mL of dimethyl sulfoxide (DMSO), the mixture is heated and reacted for 5 to 6 hours, and the mixture is added into water to be separated out after the reaction is finished, filtered by suction, and dried to obtain 1.2g of solid. The dried 1.2g of solid was then reacted with 2.5g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) and 1.5g N-hydroxysuccinimide (NHS) together in DMSO at room temperature, after the reaction was completed, water was added to precipitate a solid, which was purified by a silica gel column, dried, weighed 1.3g, and identified as the target product by ESI-MS mass spectrum and nuclear magnetic resonance spectrum, ESI-MS: [ M+H ]]= 382.1508. After purification of the product, 1 molar amount of PEG was added 4 After the reaction is completed, EDCI and NHS with 2 times of molar quantity are added for activation, and the target product is obtained after purification, freeze-drying and mass spectrum verification, and ESI-MS: [ M+H ]]= 630.3 and [ m+na ]]＝652.3。
2) Purified 5mg of intermediate HYNIC-PEG 4 NHS is dissolved in 0.3mL DMSO, then 5mg YQGA-1 is added, then 5.6mg DIPEA is added, the reaction is carried out for 3 hours at room temperature, after the reaction is finished, the product is separated and purified through a preparation liquid phase, finally 2.8mg of yellow solid is obtained, the product is confirmed to be a target product through mass spectrum, and ESI-MS: [ M+2H ]] 2+ = 780.1 and [ m+3h] 3+ ＝520.5。
3) Radioactive compounds 99m Tc-HYNIC-PEG 4 Synthesis of YQGA-1
Preparing 100.0mg/mL TPPTS (triphenylphosphine sodium tri-m-sulfonate) solution, 130.0mg/mL Tricine (trimethylglycine) solution, 102.4mg/mL succinic acid-sodium succinate buffer solution (77.0 mg succinic acid and 25.4mg sodium succinate), and respectively dissolving 10.0uL TPPTS solution10.0uL Tricine solution, 10.0uL succinic acid-sodium succinate buffer and 10.0uL (1.0 mg/mL) of the HYNIC-PEG, respectively 4 -YQGA-1 was mixed in penicillin bottles, then 10mCi Na was added 99m TcO 4 Heating in metal bath at 100deg.C for 20 min, cooling to room temperature after reaction, and making into polypeptide radiopharmaceuticals 99m Tc-HYNIC-PEG 4 The product is identified by analysis of YQGA-1 through Agilent ZORBAX SB-Aq analytical column. The HPLC method used was an Agilent 1220 Infinicity II series HPLC system equipped with a radioactive online detector (Flow-RAM) and a Agilent ZORBAX SB-Aq analytical column (4.6X105 mm,5 um). Gradient elution was carried out for 45 minutes at a flow rate of 1mL/min, wherein mobile phase A was ultrapure water (0.01% TFA) and B was acetonitrile (0.01% TFA). The elution gradient was set as: 95% A and 5% B at 0-5 min, 70% A and 30% B at 15 min, 65% A and 35% B at 20 min, 45% A and 55% B at 25 min, 5%A and 95% B at 45 min.
4) Radioactive compounds 99m Tc-HYNIC-PEG 4 YQGA-1 was formulated into physiological saline solution (3 mCi/mL), 0.1mL (about 300 μCi) was injected into the tail vein of 3 liver cancer HepG2 tumor-bearing nude mice, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, 3h, and 4h after administration, respectively. The distribution of radionuclide probes in mice was observed and enrichment in tumor areas was observed. The 1.5h imaging results are shown in FIG. 2A, from which the probe can be seen 99m Tc-HYNIC-PEG 4 The YQGA-1 has obvious aggregation at the tumor part, which shows that the probe can target liver cancer HepG2 tumor and is metabolized out of the body mainly through kidney.
Example 3 radioactive compound prepared 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-1 in brain glioma U87MG tumor-bearing mice
Radioactive compound prepared in example 2 99m Tc-HYNIC-PEG 4 YQGA-1 was formulated into physiological saline solution (3 mCi/mL), 0.1mL (about 300. Mu. Ci) was injected into 3 brain glioma U87MG tumor-bearing nude mice, respectively, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The 1h SPECT-CT imaging result is shown in FIG. 2B, and the probe can be used for target recognition of brain glioma U87MG。
Radioactive compound prepared in example 4 99m SPECT-CT imaging of Tc-HYNIC-YQGA-2 in liver cancer HepG2 tumor-bearing mice
Preparation of radioactive Compounds by the method of reference example 2 99m Tc-HYNIC-YQGA-2 (HYNIC-YQGA-2 confirmed by mass spectrum, ESI-MS: [ M+2H)] 2+ = 657.0 and [ m+3h] 3+ =438.5). Will be described in the same manner as in example 3 99m Tc-HYNIC-YQGA-2 is respectively injected into 3 liver cancer HepG2 tumor-bearing nude mice, and SPECT-CT signal acquisition is carried out at 0.5h, 1h, 2h and 4h after administration. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The 1h SPECT-CT imaging result is shown in FIG. 3A, and the probe can be used for target recognition of liver cancer HepG2 tumor.
Radioactive compound prepared in example 5 99m SPECT-CT imaging of Tc-HYNIC-YQGA-2 in cervical cancer HeLa tumor-bearing mice
Will be described in the same manner as in example 3 99m Tc-HYNIC-YQGA-2 is respectively injected into 3 cervical cancer HeLa tumor-bearing nude mice, and SPECT-CT signal acquisition is carried out at 0.5h, 1h, 2h and 4h after administration. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The 1h SPECT-CT imaging result is shown in FIG. 3B, and the probe can be used for targeted identification of cervical cancer HeLa tumor.
Radioactive compound prepared in example 6 99m SPECT-CT imaging of Tc-HYNIC-YQGA-2 in breast cancer MCF-7 tumor-bearing mice
Will be described in the same manner as in example 3 99m Tc-HYNIC-YQGA-2 is injected into 3 liver cancer MCF-7 tumor-bearing nude mice respectively, and SPECT-CT signal acquisition is carried out at 0.5h, 1h, 2h and 4h after administration. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The results of 1h SPECT-CT imaging are shown in FIG. 3C, and the probe can be seen from the figure to target and identify breast cancer MCF-7 tumor.
Radioactive compound prepared in example 7 99m SPECT-CT imaging of Tc-HYNIC-YQGA-2 in liver cancer MHCC97-H tumor mice
Will be described in the same manner as in example 3 99m Tc-HYNIC-YQGA-2 is injected into 3 liver cancer MHCC97-H tumor-bearing nude mice respectively after administrationSPECT-CT signal acquisition is carried out for 0.5h, 1h, 2h and 4 h. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The 1H SPECT-CT imaging result is shown in figure 3D, and the probe can be used for targeting and identifying liver cancer MHCC97-H tumor.
Radioactive compound prepared in example 8 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-3 in liver cancer HepG2 tumor-bearing mice
Preparation of radioactive Compound by reference example 2 99m Tc-HYNIC-PEG 4 -YQGA-3(HYNIC-PEG 4 The YQGA-3 is confirmed by mass spectrum, ESI-MS: [ M+2H ]] 2+ = 866.5 and [ m+3h] 3+ = 577.6). Will be described in the same manner as in example 3 99m Tc-HYNIC-PEG 4 YQGA-3 was injected into 3 liver cancer HepG2 tumor-bearing nude mice, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration, respectively. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The 1h SPECT-CT imaging result is shown in figure 4, and the probe can be used for target recognition of liver cancer HepG2 tumor.
Radioactive compound prepared in example 9 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-4 in liver cancer HepG2 tumor-bearing mice
Preparation of radioactive Compound by reference example 2 99m Tc-HYNIC-PEG 4 -YQGA-4(HYNIC-PEG 4 The YQGA-4 is confirmed by mass spectrum, ESI-MS: [ M+2H ]] 2+ =772.8 and [ m+3h] 3+ =515.2). Will be described in the same manner as in example 3 99m Tc-HYNIC-PEG 4 YQGA-4 was injected into 3 liver cancer HepG2 tumor-bearing nude mice, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration, respectively. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The 1h SPECT-CT imaging result is shown in figure 5, and the probe can be used for target recognition of liver cancer HepG2 tumor.
Radioactive compound prepared in example 10 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-5 in liver cancer HepG2 tumor-bearing mice
Preparation of radioactive Compound by reference example 2 99m Tc-HYNIC-PEG 4 -YQGA-5(HYNIC-PEG 4 The YQGA-5 is confirmed by mass spectrum, ESI-MS: [ M+2H ]] 2+ = 509.6 and [ m+3h] 3+ = 339.7). Will be described in the same manner as in example 3 99m Tc-HYNIC-PEG 4 YQGA-5 was injected into 3 liver cancer HepG2 tumor-bearing nude mice, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration, respectively. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The 1.5h SPECT-CT imaging result is shown in FIG. 6A, and the probe can be used for target recognition of liver cancer HepG2 tumor.
Radioactive compound prepared in example 11 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-5 in liver cancer MHCC97-H tumor-bearing mice
Will be described in the same manner as in example 3 99m Tc-HYNIC-PEG 4 YQGA-5 was injected into 3 liver cancer MHCC97-H tumor-bearing nude mice, respectively, and SPECT-CT signal acquisition was performed at 0.5H, 1H, 2H, and 4H after administration. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The 1H SPECT-CT imaging result is shown in FIG. 6B, and the probe can be used for targeting and identifying liver cancer MHCC97-H tumor.
Radioactive compound prepared in example 12 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-5 in cervical cancer HeLa tumor-bearing mice
Will be described in the same manner as in example 3 99m Tc-HYNIC-PEG 4 YQGA-5 was injected into 3 cervical cancer HeLa tumor-bearing nude mice, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration, respectively. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The 1h SPECT-CT imaging result is shown in FIG. 6C, and the probe can be used for targeted recognition of cervical cancer HeLa tumor.
Radioactive compound prepared in example 13 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-5 in breast cancer MCF-7 tumor-bearing mice
Will be described in the same manner as in example 3 99m Tc-HYNIC-PEG 4 YQGA-5 was injected into 3 breast cancer MCF-7 tumor-bearing nude mice, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The 1h SPECT-CT imaging result is shown in the figure6D, it can be seen from the figure that the probe can target and recognize breast cancer MCF-7 tumor.
Radioactive compound prepared in example 14 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-6 in liver cancer HepG2 tumor-bearing mice
Preparation of radioactive Compounds by the method of reference example 2 99m Tc-HYNIC-PEG 4 -YQGA-6(HYNIC-PEG 4 The YQGA-6 is confirmed by mass spectrum, ESI-MS is [ M+2H ]] 2+ = 539.9 and [ m+3h] 3+ = 360.3). Will be described in the same manner as in example 3 99m Tc-HYNIC-PEG 4 YQGA-6 was injected into 3 liver cancer HepG2 tumor-bearing nude mice, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration, respectively. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The 1.5h SPECT-CT imaging result is shown in FIG. 7A, and the probe can be used for target recognition of liver cancer HepG2 tumor.
Radioactive compound prepared in example 15 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-6 in cervical cancer HeLa tumor-bearing mice
Will be described in the same manner as in example 3 99m Tc-HYNIC-PEG 4 YQGA-6 was injected into 3 cervical cancer HeLa tumor-bearing nude mice, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration, respectively. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The 1h SPECT-CT imaging result is shown in FIG. 7B, and the probe can be used for targeted recognition of cervical cancer HeLa tumor.
Radioactive compound prepared in example 16 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-6 in lung cancer A549 tumor-bearing mice
Will be described in the same manner as in example 3 99m Tc-HYNIC-PEG 4 YQGA-6 was injected into 3 lung cancer A549 tumor-bearing nude mice, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The 1h SPECT-CT imaging result is shown in FIG. 7C, and the probe can be used for targeted identification of lung cancer A549 tumor.
Radioactive compound prepared in example 17 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-6 in breast cancer MCF-7 tumor-bearing mice
Will be described in the same manner as in example 3 99m Tc-HYNIC-PEG 4 YQGA-6 was injected into 3 lung cancer A549 tumor-bearing nude mice, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The results of 1h SPECT-CT imaging are shown in FIG. 7D, from which it can be seen that the probe can target and recognize breast cancer MCF-7 tumor.
Radioactive compound prepared in example 18 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-6 in pancreatic cancer SW1990 tumor-bearing mice
Will be described in the same manner as in example 3 99m Tc-HYNIC-PEG 4 YQGA-6 was injected into 3 tumor-bearing nude mice with pancreatic carcinoma SW1990, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration, respectively. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The results of 1h SPECT-CT imaging are shown in FIG. 7E, from which it can be seen that the probe can target and recognize pancreatic cancer SW1990 tumor.
Radioactive compound prepared in example 19 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-6 in colorectal cancer HT29 tumor-bearing mice
Will be described in the same manner as in example 3 99m Tc-HYNIC-PEG 4 YQGA-6 was injected into 3 colorectal cancer HT29 tumor-bearing nude mice, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration, respectively. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The results of 1h SPECT-CT imaging are shown in FIG. 7F, from which it can be seen that the probe can target and recognize colorectal cancer HT29 tumor.
The radioactive compound prepared in example 20 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-6 in neuroendocrine tumor BON-1 tumor-bearing mice
Will be described in the same manner as in example 3 99m Tc-HYNIC-PEG 4 YQGA-6 was injected into 3 neuroendocrine tumor BON-1 tumor-bearing nude mice, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. 1hSPECT-CT imaging results are shown in FIG. 7G, from which it can be seen that the probe can target and recognize neuroendocrine tumor BON-1.
Radioactive compound prepared in example 21 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-6 in situ colorectal cancer tumor-bearing mice
Will be described in the same manner as in example 3 99m Tc-HYNIC-PEG 4 YQGA-6 was injected into 3 tumor-bearing nude mice with colorectal cancer in situ, and SPECT-CT signal acquisition was performed at 0.5h, 1h18min, 2h, and 4h after administration, respectively. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The SPECT-CT imaging result of 1h18min is shown in FIG. 8, and the probe can be used for targeted identification of colorectal cancer tumor in situ.
Radioactive compound prepared in example 22 99m Tc-HYNIC-3PEG 4 -E-(YQGA-6) 2 SPECT-CT imaging in pancreatic cancer CFPAC-1 tumor-bearing mice
Prepared radiopharmaceuticals 99m Tc-HYNIC-3PEG 4 -E-(YQGA-6) 2 The synthesis steps of (2) are as follows:
1) Bifunctional chelating agent HYNIC-PEG 4 Synthesis of NHS
1g of 6-chloronicotinic acid and 2.0mL of 80% hydrazine hydrate are added into 10mL of ethanol, the mixture is heated and refluxed for 4 hours, the solvent is distilled under reduced pressure after the reaction is completed, the obtained sticky substance is added into distilled water, the pH value is regulated to about 5.5, solids are separated out, 0.86g of yellow solid is obtained through suction filtration and drying, and the product is determined to be 6-dihydrazide nicotinic acid through ESI-MS mass spectrum and nuclear magnetic hydrogen spectrum. The obtained 0.86g of 6-dihydrazide nicotinic acid and 0.61g of p-aminobenzaldehyde are added into 3.0mL of dimethyl sulfoxide (DMSO), the mixture is heated and reacted for 5 to 6 hours, and the mixture is added into water to be separated out after the reaction is finished, filtered by suction, and dried to obtain 1.2g of solid. 1.2g of the dried solid was reacted with 2.5g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) and 1.5-g N-hydroxysuccinimide (NHS) together in DMSO at room temperature, after the reaction was completed, water was added to precipitate a solid, the solid was purified by a silica gel column, dried, weighed 1.3g, and determined as the target product by ESI-MS mass spectrometry and nuclear magnetic hydrogen spectrometry, and after the purification of the product, 1 molar amount of PEG was added 4 After the reaction is completed, 2 times mole of the catalyst is addedThe EDCI and NHS are activated in molar quantity, and the purified EDCI and NHS are freeze-dried for later use.
2) Support (PEG) 4 ) 3 Synthesis of E
5.0g of t-butyloxycarbonyl (t-Butyloxy carbony) protected glutamic acid, 8.3g of Dicyclohexylcarbodiimide (DCC) and 4.6. 4.6g N-hydroxysuccinimide (NHS) were added to 100mL of an organic solvent Tetrahydrofuran (THF), the mixture was stirred overnight at room temperature to activate the dicarboxyl group, the reaction was then filtered with suction, the filtrate was washed with THF, and after the washing was completed, it was directly dissolved in 50mL of dimethyl sulfoxide (DMSO) without further purification, and 10g of PEG was then added 4 Finally, 14.6g of DIPEA is added for reaction for 2 hours at room temperature, 3.0mL of trifluoroacetic acid (TEA) is added to the reaction for removing Boc protecting group after the reaction is finished, separation and purification are carried out through a preparation liquid phase, finally 7.8g of thick solid is obtained after drying, and the solid is verified to be an expected target (PEG) through mass spectrum 4 ) 2 -E。
3) Intermediate (PEG) 4 ) 3 Synthesis of-E-HYNIC-2 NHS
The resulting 0.5g scaffold (PEG 4 ) 2 E was dissolved in DMSO and then 0.31g HYNIC-PEG was added 4 -NHS, 0.32g DIPEA, and then EDCI and NHS were added for 2 hours at room temperature for activation, and after completion of the reaction, 0.34g of yellow solid was obtained by separation and purification by preparative liquid phase and freeze-drying, which was verified by mass spectrometry to be the expected target compound (PEG 4 ) 3 -E-HYNIC-2NHS，ESI-MS:[M+2H] 2+ = 675.5 and [ m+3h] 3+ ＝450.6。
4)HYNIC-3PEG 4 -E-(YQGA-6) 2 Is synthesized by (a)
Purified 5mg of intermediate 3PEG 4 E-HYNIC-2NHS is dissolved in 0.3mL DMSO, 7.8mg YQGA-6 is added after the reaction is completed, then 5.6mg DIPEA is added, the reaction is carried out for 3 hours at room temperature, separation and purification are carried out through a preparation liquid phase after the reaction is completed, 3.5mg yellow solid is finally obtained, the yellow solid is confirmed to be a target product through mass spectrum, and ESI-MS is shown as [ M+3H ]] 3+ = 825.7 and [ m+4h] 4+ ＝619.5。
5) 99m Tc-HYNIC-3PEG 4 -E-(YQGA-6) 2 Is prepared from
Preparing 100.0mg/mL TPPTS (triphenylphosphine sodium tri-m-sulfonate) solution, 130.0mg/mL Tricine (trimethylglycine) solution, 102.4mg/mL succinic acid-sodium succinate buffer solution (77.0 mg succinic acid and 25.4mg succinic acid), respectively taking 10.0uL TPPTS solution, 10.0uL Tricine solution, 10.0uL succinic acid-sodium succinate buffer solution and 10.0uL (1.0 mg/mL) of HYNIC-3PEG 4 -E-(YQGA-6) 2 Mix in a penicillin bottle and then add 10mCi Na 99m TcO 4 Heating in metal bath at 100deg.C for 20 min, cooling to room temperature after reaction, and making into polypeptide radiopharmaceuticals 99m Tc-HYNIC-3PEG 4 -E-(YQGA-6) 2 The product is analyzed and identified by a Agilent ZORBAX SB-Aq analysis column. The HPLC method used was an Agilent 1220 Infinicity II series HPLC system equipped with a radioactive online detector (Flow-RAM) and a Agilent ZORBAX SB-Aq analytical column (4.6X105 mm,5 um). Gradient elution was carried out for 45 minutes at a flow rate of 1mL/min, wherein mobile phase A was ultrapure water (0.01% TFA) and B was acetonitrile (0.01% TFA). The elution gradient was set as: 95% A and 5% B at 0-5 min, 70% A and 30% B at 15 min, 65% A and 35% B at 20 min, 45% A and 55% B at 25 min, 5%A and 95% B at 45 min.
6) Will be described in the same manner as in example 3 99m Tc-HYNIC-3PEG 4 -E-(YQGA-6) 2 Injected into 3 pancreatic cancer CFPAC-1 tumor-bearing nude mice respectively, and SPECT-CT signal acquisition is carried out at 0.5h, 1h, 2h and 4h after administration. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The results of the SPECT-CT imaging for 1h18min are shown in FIG. 9, and the probe can be used for targeting and identifying pancreatic cancer CFPAC-1 tumor.
Fluorescent imaging of fluorescent Compound MPA-Aca-YQGA-6 prepared in example 23 in liver cancer HepG2 tumor-bearing mice
Fluorescent compound MPA-Aca-YQGA-6 was prepared and formulated as physiological saline solution (100 nmol/mL) as in example 1, 0.1mL (about 10 nmol) was injected into tail veins of 3 liver cancer HepG2 tumor-bearing nude mice (weighing about 22 g) respectively, and optical signal collection was performed at 1h, 2h, 4h, 8h, 10h and 12h after administration. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The imaging results of the compound MPA-Aca-YQGA-6 in 3 tumor-bearing nude mice were substantially identical, and from the 2h imaging plot it can be seen that the probe had been significantly taken up in the tumor, and it can be deduced that the probe was mainly metabolized by the kidney (fig. 10A).
Fluorescent imaging of the fluorescent Compound MPA-Aca-YQGA-6 prepared in example 24 in breast cancer MCF-7 tumor-bearing mice
Fluorescent compound MPA-Aca-YQGA-6 was prepared and formulated in physiological saline solution (100 nmol/mL) as in example 1, 0.1mL (about 10 nmol) was injected into tail veins of 3 breast cancer MCF-7 tumor-bearing nude mice (weighing about 22 g) respectively, and optical signal collection was performed at 1h, 2h, 4h, 8h, 10h and 12h after administration. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The imaging result of the compound MPA-Aca-YQGA-6 in 3 tumor-bearing nude mice is basically consistent, and the probe is obviously ingested in tumors from an imaging chart of 2 hours (figure 10B), and the probe is deduced to be mainly metabolized by kidneys.
Radioactive compound prepared in example 25 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-7 in cervical cancer HeLa tumor-bearing mice
Preparation of radioactive Compounds by the method of reference example 2 99m Tc-HYNIC-PEG 4 -YQGA-7(HYNIC-PEG 4 The YQGA-7 is confirmed by mass spectrum, ESI-MS: [ M+2H ]] 2+ = 546.8 and [ m+3h] 3+ = 364.5). Will be described in the same manner as in example 3 99m Tc-HYNIC-PEG 4 YQGA-7 was injected into 3 cervical cancer HeLa tumor-bearing nude mice, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration, respectively. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The results of 1hSPECT-CT imaging are shown in FIG. 11, and from the graph, the probe can be used for targeting and identifying cervical cancer HeLa tumor.
Radioactive compound prepared in example 26 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-8 in liver cancer HepG2 tumor-bearing mice
Preparation of radioactive Compound by the same procedure as in example 2 99m Tc-HYNIC-PEG 4 -YQGA-8(HYNIC-PEG 4 The YQGA-8 is confirmed by mass spectrum, ESI-MS is [ M+2H ]] 2+ = 539.8 and [ m+3h] 3+ =360.2). Will be described in the same manner as in example 3 99m Tc-HYNIC-PEG 4 YQGA-8 was injected into 3 liver cancer HepG2 tumor-bearing nude mice, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration, respectively. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The 1h SPECT-CT imaging result is shown in FIG. 12A, and the probe can be used for target recognition of liver cancer HepG2 tumor.
Radioactive compound prepared in example 27 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-8 in pancreatic cancer CFPAC-1 tumor-bearing mice
Will be described in the same manner as in example 3 99m Tc-HYNIC-PEG 4 YQGA-8 was injected into 3 pancreatic cancer CFPAC-1 tumor-bearing nude mice, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration, respectively. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The results of 1h SPECT-CT imaging are shown in FIG. 12B, from which it can be seen that the probe can target and recognize pancreatic cancer CFPAC-1 tumor.
Radioactive compound prepared in example 28 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-9 in breast cancer MCF-7 tumor-bearing mice
Preparation of radioactive Compounds by the method of reference example 2 99m Tc-HYNIC-PEG 4 -YQGA-9(HYNIC-PEG 4 The YQGA-9 is confirmed by mass spectrum, ESI-MS: [ M+2H ]] 2+ = 691.1 and [ m+3h] 3+ = 460.6). Will be described in the same manner as in example 3 99m Tc-HYNIC-PEG 4 YQGA-9 was injected into 3 breast cancer MCF-7 tumor-bearing nude mice, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The results of 1h SPECT-CT imaging are shown in FIG. 13, from which it can be seen that the probe can target and recognize breast cancer MCF-7 tumor.
Radioactive compound prepared in example 29 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-10 in pancreatic cancer SW1190 tumor-bearing mice
Preparation of radioactive Compounds by the method of reference example 2 99m Tc-HYNIC-PEG 4 -YQGA-10(HYNIC-PEG 4 The YQGA-10 is confirmed by mass spectrum, ESI-MS: [ M+2H ]] 2+ = 691.1 and [ m+3h] 3+ = 460.7). Will be described in the same manner as in example 3 99m Tc-HYNIC-PEG 4 YQGA-10 was injected into 3 tumor-bearing nude mice with pancreatic cancer SW1190, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration, respectively. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The results of 1h SPECT-CT imaging are shown in FIG. 14, from which it can be seen that the probe can target and recognize pancreatic cancer SW1190 tumor.
The radioactive compound prepared in example 30 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-11 in liver cancer HepG2 tumor-bearing mice
Preparation of radioactive Compounds by the method of reference example 2 99m Tc-HYNIC-PEG 4 -YQGA-11(HYNIC-PEG 4 The YQGA-11 is confirmed by mass spectrum, ESI-MS: [ M+2H ]] 2+ = 680.5 and [ m+3h] 3+ =453.6). Will be described in the same manner as in example 3 99m Tc-HYNIC-PEG 4 YQGA-11 was injected into 3 liver cancer HepG2 tumor-bearing nude mice, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration, respectively. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The 1h SPECT-CT imaging result is shown in FIG. 15, and the probe can be used for target recognition of liver cancer HepG2 tumor.
Radioactive compound prepared in example 31 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of YQGA-12 in breast cancer MCF-7 tumor-bearing mice
Preparation of radioactive Compounds by the method of reference example 2 99m Tc-HYNIC-PEG 4 -YQGA-12(HYNIC-PEG 4 The YQGA-12 is confirmed by mass spectrum, ESI-MS: [ M+2H ]] 2+ = 680.5 and [ m+3h] 3+ = 453.5). Will be described in the same manner as in example 3 99m Tc-HYNIC-PEG 4 YQGA-12 was injected into 3 breast cancer MCF-7 tumor-bearing nude mice, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration. The distribution of the probe in the mice was observed and the enrichment in the tumor area was observed. The results of 1h SPECT-CT imaging are shown in FIG. 16, from which it can be seen that the probe can target and recognize breast cancer MCF-7 tumor.

Claims (11)

1. Use of a tumor-specific targeting polypeptide for the preparation of a tumor diagnostic agent, said tumor-specific targeting polypeptide:
YQGA-5：D-Asp-Arg-Val-Tyr-NH 2 ;
wherein: D-Asp: d-aspartic acid;
the tumor is liver cancer and breast cancer.
2. Use according to claim 1, characterized in that the tumor-specific targeting polypeptide according to claim 1 is used for the preparation of a tumor diagnostic imaging agent.
3. The use according to claim 2, characterized in that the tumor-specific targeted polypeptide according to claim 1 is used for the preparation of tumor boundary-specific localization and intra-operative image-guided imaging agents or for the preparation of radionuclide imaging agents.
4. The polypeptide compound with the tumor fluorescence targeting imaging function is characterized in that the structure of the polypeptide compound contains the tumor specific targeting polypeptide in claim 1 and an infrared fluorescent dye structure for optical imaging, and the structural general formula of the polypeptide compound is shown as the following formula (I):
，
wherein R is selected from the tumor-specific targeted polypeptide YQGA-5 of claim 1; l is selected from any one of four types shown in the following II;
。
5. the application of the polypeptide compound with tumor-targeted fluorescence imaging function in preparing tumor diagnosis reagents, wherein the tumor is liver cancer or breast cancer.
6. The use according to claim 5, characterized in that the polypeptide compound with tumor-targeted fluorescence imaging function according to claim 4 is used for preparing tumor diagnostic imaging agents.
7. The use according to claim 6, characterized in that the polypeptide compound with tumor-targeted fluorescence imaging function according to claim 4 is used for preparing tumor boundary accurate localization and intraoperative image navigation imaging reagent or radionuclide imaging reagent.
8. A radionuclide probe characterized by a radionuclide technetium labeled polypeptide monomer complex according to claim 1 or a polypeptide dimer complex according to claim 1; the structural formula is shown as (III) or (IV) or (V):
，
or the bifunctional chelating agent HYNIC in the formulas (III), (IV) and (V) is replaced by DOTA, NOTA, MAG 3 Or DTPA, radionuclide 99m Tc is replaced by 68 Ga， 64 Cu， 67 Ga， 90 Y， 111 In or In 177 Lu; the YQGA-X is YQGA-5 as recited in claim 1, wherein L is any one or more of L1, L2, L3 or L4 as recited in claim 4.
9. The use of the radionuclide probe according to claim 8 for preparing a tumor diagnostic reagent, wherein the tumor is liver cancer or breast cancer.
10. The use according to claim 9, characterized in that the radionuclide probe according to claim 8 is used in the preparation of a diagnostic imaging agent for tumors.
11. The use according to claim 10, characterized in that the radionuclide probe according to claim 8 is used for preparing tumor border accurate localization and intra-operative image navigation imaging agent or radionuclide imaging agent.
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