Polypeptide radionuclide carriers, radionuclide probes, their preparation methods and applications

Abstract

This invention relates to the field of biomedicine, specifically disclosing a polypeptide nuclide carrier, a nuclide probe, its preparation method, and its uses. The polypeptide nuclide carrier of this invention is made from the following raw materials: human type I collagen polypeptide, a double-bond functional group compound, and a chelating agent; wherein the human type I collagen polypeptide is selected from at least one polypeptide with an amino acid sequence as shown in SEQ ID No. 1-4; the double-bond functional group compound has a substitution degree of 45-80% for the amino group of lysine in the human type I collagen polypeptide; the chelating agent has a substitution degree of 20-55% for the amino group of lysine in the human type I collagen polypeptide; the double-bond functional group compound is selected from at least one of acrylic acid, methacrylic acid, acrylic anhydride, and methacrylic anhydride; and the chelating agent is selected from at least one of DOTA, NOA, EDTA, and DTPA. The polypeptide nuclide carrier of this invention can simultaneously bind metal nuclides and non-metal nuclides, can be photocured, and has good targeting properties.

Description

Technical Field

[0001] This invention relates to a polypeptide nuclide carrier, a nuclide probe, its preparation method and uses. Background Technology

[0002] Radionuclide probes are tools or techniques used to detect, measure, or label specific radionuclides, and have wide applications in nuclear medicine, radiopharmaceutical research, environmental monitoring, and the nuclear energy industry. Radionuclide probes typically consist of tiny particles or labels made from radionuclides with known radioactive decay characteristics. Once introduced into a substance, these probes can be used to study the substance's properties, distribution, and behavior by measuring its radioactive decay. The core principle lies in utilizing the decay characteristics of radionuclides to obtain target information by detecting the rays (such as alpha rays, beta rays, and gamma rays) released during the decay process.

[0003] In recent years, radionuclide probes have been widely used in nuclear medicine imaging and targeted therapy. Radionuclide probes can be used to diagnose and monitor various diseases, such as cardiovascular diseases, neurological diseases, and tumors. Using probes labeled with radionuclides, doctors can non-invasively observe metabolic activity, blood flow, and receptor expression in diseased tissues, providing crucial information for disease diagnosis and treatment. Radionuclide probes can also be used for targeted therapy, such as radioimmunotherapy and radioactive particle implantation. By labeling specific antibodies or drugs with radionuclides, precise targeting of diseased tissues can be achieved, improving treatment efficacy and reducing side effects. However, existing radionuclide probes generally only carry one type of radionuclide, either metallic or non-metallic, resulting in poor versatility. Furthermore, existing radionuclide probes are administered intravenously, requiring blood circulation to reach the target organ or tissue. Since the probes exist in a free state in the blood, the risk of radiation damage to non-target organs or tissues increases. Additionally, existing radionuclide probes cannot cross the blood-brain barrier, limiting their use in the diagnosis and treatment of intracranial diseases.

[0004] CN120518717A discloses a radionuclide-labeled collagen hybridization peptide probe. The general chemical formula of this collagen hybridization peptide probe is as follows: (metal nuclide-chelating agent)-Linker-CHP; wherein the metal nuclide is selected from Al. 18 F, 64 Cu、 67 Cu、 67 Ga、 68 Ga、 99m Tc, 89 Zr、 111 In、 177 Lu、 186 Re、 225The chelating agent is selected from any one of Ac; the linker is selected from any one of NODAGA, Nota2, and DOTA; the linker is selected from any one of 6-aminocaproic acid, polyethylene glycol, and oligoglycine residues; and the CHP is a collagen hybrid peptide probe with a polypeptide sequence of (GfO)n or (GPO)n, where n is any positive integer from 6 to 12. The polypeptide of this collagen hybrid peptide probe has a linear structure, and the chelating agent can only be grafted onto the end of the polypeptide body, resulting in the collagen hybrid peptide probe binding to only one metal nuclide, thus lacking versatility. Furthermore, this collagen hybrid peptide probe needs to be injected intravenously into the bloodstream, which can easily cause radiation damage to non-target organs or tissues.

[0005] CN120550151A discloses a radionuclide-labeled molecular probe. The general chemical formula of this molecular probe is as follows: K(chelating agent-nucleoside)-K-Linker-CHP; where K(chelating agent-nucleoside) is a side-chain amino-coupled chelating agent and a radionuclide-labeled lysine residue; Linker is a linker; CHP is a collagen hybrid peptide, the polypeptide sequence of which is (GfO)n or (GPO)n, where n is a positive integer between 6 and 10. The radionuclide is selected from... 11 C 13 N、 15 O, Al 18 F, 64 Cu、 67 Ga、 68 Ga、 75 Br、 76 Br、 77 Br、 99m Tc, 111 In、 123 I, 124 I, 125 I, 131 I, 18 The chelating agent is selected from any of the following: Re; and / or, the chelating agent includes a metal chelating agent selected from any of Nota, Nodaga, Hynic, Dota, and DTPA. The linker includes an amino acid and / or an amino acid derivative. The polypeptide of this molecular probe has a linear structure, and the chelating agent can only be grafted to the end of the polypeptide body, resulting in the molecular probe binding to only one nuclide, thus limiting its versatility. Furthermore, this molecular probe needs to be injected intravenously into the bloodstream, which can easily cause radiation damage to non-target organs or tissues. Summary of the Invention

[0006] In view of this, one object of the present invention is to provide a polypeptide nuclide carrier that can simultaneously bind metal nuclides and non-metal nuclides, exhibiting high versatility, and the polypeptide nuclide carrier can be photocured. Another object of the present invention is to provide a method for preparing the above-mentioned polypeptide nuclide carrier. A further object of the present invention is to provide the uses of the above-mentioned polypeptide nuclide carrier. Yet another object of the present invention is to provide a nuclide probe. A further object of the present invention is to provide a method for preparing the above-mentioned nuclide probe. A further object of the present invention is to provide the uses of the above-mentioned nuclide probe.

[0007] The present invention achieves the above objectives using the following technical solutions.

[0008] On one hand, the present invention provides a polypeptide nuclide carrier made from raw materials comprising:

[0009] Human-derived type I collagen peptides, compounds with double bond functional groups, and chelating agents; among which,

[0010] The human type I collagen polypeptide is selected from at least one polypeptide with an amino acid sequence as shown in SEQ ID No. 1 to 4;

[0011] The degree of substitution of the amino group of lysine in the human type I collagen polypeptide by the double-bonded functional group compound is 45-80%; the degree of substitution of the amino group of lysine in the human type I collagen polypeptide by the chelating agent is 20-55%.

[0012] The double-bonded functional group compound is selected from at least one of acrylic acid, methacrylic acid, acrylic anhydride, and methacrylic anhydride;

[0013] The chelating agent is selected from at least one of DOTA, NOTA, EDTA, and DTPA.

[0014] On the other hand, the present invention also provides a method for preparing the above-mentioned polypeptide nuclide carrier, comprising the following steps:

[0015] A) Using swollen Rink Amide resin as a solid-phase support, amino acids were cyclically coupled and assembled using a solid-phase synthesis method to obtain human type I collagen peptides. In each cycle of amino acid coupling and assembly, the Fmoc protecting groups on the Rink Amide resin were removed using an Fmoc protecting group elution buffer to obtain deprotected resin. Then, the Fmoc-protected amino acids were coupled and assembled onto the deprotected resin.

[0016] B) Mix human type I collagen peptides with compounds containing double bond functional groups and react at 40–65 °C to obtain peptides modified with double bond functional groups.

[0017] C) The double-bonded functional group modified peptide is mixed with the activating precursor of the chelating agent and reacted at 20-45℃ to obtain the peptide nuclide carrier.

[0018] According to the preparation method of the present invention, preferably:

[0019] In step A), the Fmoc protecting group eluent is selected from at least one of piperidine DMF solution, piperidine dichloromethane solution, diethylamine DMF solution, and diethylamine dichloromethane solution; wherein the mass concentration of the Fmoc protecting group eluent is 10-30 wt%.

[0020] In step C), the activating precursor of the chelating agent is an NHS ester of the chelating agent.

[0021] According to the preparation method of the present invention, preferably:

[0022] In step B), the reaction time is 1 to 5 hours;

[0023] In step C), the reaction time is 8 to 20 hours.

[0024] In another aspect, the present invention also provides the use of the above-mentioned polypeptide nuclide carrier in the preparation of nuclear medicine detection products or radiotherapy products.

[0025] In another aspect, the present invention also provides a radionuclide probe, which is made of the above-mentioned polypeptide radionuclide carrier and radionuclide; wherein the molar ratio of the polypeptide radionuclide carrier and radionuclide is 1:1 to 5;

[0026] The nuclide is selected from at least one of metallic nuclides and non-metallic nuclides; the metallic nuclide is selected from... 177 Lu、 90 Y、 67 Ga、 64 Cu、 99m Tc, 89 At least one of Sr; the nonmetallic nuclide is selected from... 125 I, 131 I, 18 F, 32 At least one of P.

[0027] According to the nuclide probe of the present invention, preferably, the nuclide is a metallic nuclide and a non-metallic nuclide, and the molar ratio of the metallic nuclide to the non-metallic nuclide is 0.25 to 4:1.

[0028] In another aspect, the present invention also provides a method for preparing the above-mentioned radionuclide probe, comprising the following steps:

[0029] The above-mentioned polypeptide nuclide carrier is mixed with the nuclide and reacted at 20–60 °C to obtain the nuclide probe.

[0030] In another aspect, the present invention also provides the use of the above-mentioned radionuclide probe in the preparation of nuclear medicine detection products or radiotherapy products.

[0031] According to the intended use of the present invention, preferably, the product comprises a nuclide probe and a photoinitiator; the weight ratio of the nuclide probe to the photoinitiator is 100 to 1000:1; the photoinitiator is selected from at least one of benzophenone, LAP, and TPO;

[0032] The nuclide probe solidifies into a gel in the presence of a photoinitiator and under irradiation by a light source with a power of 5–40 W and a wavelength of 300–500 nm.

[0033] The polypeptide nuclide carrier of the present invention can simultaneously bind metal nuclides and non-metal nuclides, exhibiting high versatility. Furthermore, the polypeptide nuclide carrier can be photocured. The polypeptide nuclide carrier of the present invention contains an RGD tripeptide structure, which has good targeting properties. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the amino acid sequence of human type I collagen polypeptide I; in which, the box contains the RGD tripeptide structure, ◇ is marked as lysine, and △ is marked as tyrosine.

[0035] Figure 2 This is a schematic diagram of the amino acid sequence of human type I collagen polypeptide II; the box contains the RGD tripeptide structure, ◇ is marked as lysine, and △ is marked as tyrosine.

[0036] Figure 3 This is a schematic diagram of the amino acid sequence of human type I collagen polypeptide III; the box contains the RGD tripeptide structure, ◇ indicates lysine, and △ indicates tyrosine.

[0037] Figure 4 This is a schematic diagram of the amino acid sequence of human type I collagen polypeptide IV; in which the box represents the RGD tripeptide structure, ◇ is marked as lysine and △ is marked as tyrosine.

[0038] Figure 5 This is the concentration-time curve of radioactive substances in the blood of rats after local administration in Experiment Example 1.

[0039] Figure 6 The cumulative excretion rate of radioactive substances in the feces and urine of rats in Experiment Example 1 after local administration is shown.

[0040] Figure 7 The concentrations of reflective substances in different tissues of rats at different times after local administration in Experiment Example 1.

[0041] Figure 8 PET images of rats at different times after local administration in Experiment Example 2. Detailed Implementation

[0042] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0043] polypeptide nuclide carrier

[0044] This invention provides a polypeptide nuclide carrier, made from raw materials comprising the following parts by weight:

[0045] Human type I collagen peptides, double-bonded functional group compounds, and chelating agents.

[0046] According to one embodiment of the present invention, the human type I collagen polypeptide may be selected from at least one polypeptide with an amino acid sequence as shown in SEQ ID No. 1 to 4.

[0047] In this invention, SEQ ID No. 1 is GKSGDRGEYG PAGPAGPVGP VGARGPAGPQ GPRGDKGEYGEQGDRGIKGH. SEQ ID No. 2 is PKGDRGDAGP KGADGSPGKD GVRGLYGPIG PPGPAGAPGDKGESGPSGPA. SEQ ID No. 3 is AAGQPGAKGE RGAKGPKGEN GVVGPYGPVG AAGPAGPNGPPGPAGSRGDG. SEQ ID No. 4 is PAGKHGNRGEYGPSGPVGPA GAVGPRGPSG PQGIRGDKGEPGEKGPRGLP.

[0048] According to a preferred embodiment of the present invention, the human type I collagen polypeptide is preferably one or two of the polypeptides with amino acid sequences as shown in SEQ ID No. 1 to 4, more preferably one of the polypeptides with amino acid sequences as shown in SEQ ID No. 1 to 4.

[0049] The human type I collagen polypeptide of this invention contains an RGD tripeptide structure, which exhibits good targeting properties. The human type I collagen polypeptide of this invention also contains at least three lysines (K) and at least one tyrosine (Y). Lysine can be grafted onto double-bond functional groups and chelating agents, while tyrosine can be grafted onto non-metallic nuclides. This process forms a branched structure, allowing for greater binding of double-bond functional groups and chelating agents. In this invention, double-bond functional groups can be cross-linked under photoinitiator and light irradiation conditions, while chelating agents can bind to metallic nuclides. Therefore, the structure of the human type I collagen polypeptide of this invention is highly targeted and can simultaneously bind to both metallic and non-metallic nuclides, exhibiting strong versatility. Furthermore, it can be photocured to form a gel.

[0050] The human type I collagen peptides of the present invention can be screened and designed using any protein database or protein design tool known in the art, without any particular limitation. For example, it can be the UniProt database, the SabDab database, the BLAST tool, the Alignment Viewer tool, etc., preferably the UniProt database or the BLAST tool.

[0051] According to one embodiment of the present invention, the degree of substitution of the amino group of lysine in the human type I collagen polypeptide by the double-bonded functional group compound can be 45-80%, preferably 50-80%, and more preferably 50-75%. The degree of substitution of the amino group of lysine in the human type I collagen polypeptide by the chelating agent can be 20-55%, preferably 20-50%, and more preferably 25-50%. A reasonable degree of substitution can ensure that the double-bonded functional group and the chelating agent are grafted onto the human type I collagen polypeptide in a better ratio, thereby benefiting the versatility and photocuring properties of the human type I collagen polypeptide.

[0052] According to one embodiment of the present invention, the double-bonded functional group compound may be selected from at least one of acrylic acid, methacrylic acid, acrylic anhydride, and methacrylic anhydride, preferably at least one of methacrylic acid and methacrylic anhydride, and more preferably methacrylic acid or methacrylic anhydride. Such a double-bonded functional group compound is beneficial for grafting onto the lysine residues of human type I collagen peptides, and is more beneficial for the photocuring properties of human type I collagen peptides.

[0053] According to one embodiment of the present invention, the chelating agent may be selected from at least one of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), EDTA (ethylenediaminetetraacetic acid), and DTPA (diethyltriaminepentaacetic acid), preferably at least one of DOTA, NOTA, and DTPA, and more preferably at least one of DOTA and NOTA. Such a chelating agent is beneficial for grafting onto the lysine residues of human type I collagen peptides, and even more beneficial for the binding of human type I collagen peptides to metal nuclides.

[0054] Preparation method of polypeptide nuclide carrier

[0055] This invention also provides a method for preparing the polypeptide nuclide carrier as described above, comprising a solid-phase polypeptide synthesis step, a double-bond functional group modification step, and a chelating agent binding step. This is described in detail below.

[0056] Solid-phase synthesis of peptides

[0057] Using swollen Rink Amide resin as a solid-phase support, amino acids were cyclically coupled and assembled using a solid-phase synthesis method to obtain human type I collagen peptides.

[0058] According to one embodiment of the present invention, in each cycle of amino acid coupling assembly, the Fmoc (9-fluorenylmethoxycarbonyl) protecting group elution buffer is used to remove the Fmoc protecting group from the Rink Amide resin, resulting in a deprotected resin. Then, the Fmoc-protected amino acids are coupled and assembled onto the deprotected resin to obtain a human type I collagen polypeptide.

[0059] In this invention, swollen Rink Amide resin can be added to the reactor of a solid-phase synthesis apparatus as a solid-phase support for synthesis. According to one embodiment of the invention, the loading of Rink Amide resin can be 0.05–0.5 mmol / g, preferably 0.08–0.45 mmol / g, and more preferably 0.1–0.4 mmol / g. The solid-phase synthesis apparatus of this invention can be any type of solid-phase apparatus for peptide synthesis known in the art, and is not particularly limited herein. For example, it can be a solid-phase peptide synthesizer.

[0060] According to one embodiment of the present invention, the swelling agent of the swollen Rink Amide resin may be selected from at least one of DMF, dichloromethane, and dichloroethane, preferably at least one of DMF and dichloromethane, and more preferably DMF. Based on 1g of Rink Amide resin, the amount of swelling agent may be 10-50mL, preferably 13-45mL, and more preferably 15-40mL.

[0061] According to one embodiment of the present invention, the Fmoc protecting group eluent may be selected from at least one of piperidine DMF (N,N-dimethylformamide) solution, piperidine dichloromethane solution, diethylamine DMF solution, and diethylamine dichloromethane solution, preferably at least one of piperidine DMF solution and piperidine dichloromethane solution, more preferably piperidine DMF solution or piperidine dichloromethane solution. The mass concentration of the Fmoc protecting group eluent may be 10-30 wt%, preferably 12-28 wt%, more preferably 15-25 wt%. The amount of Fmoc protecting group eluent may be added according to the elution volume known in the art, and is not particularly limited in the art. For example, based on 1 g of swollen Rink Amide resin, the amount of Fmoc protecting group eluent may be 20-80 mL, preferably 30-75 mL, more preferably 35-70 mL.

[0062] A suitable Fmoc protecting eluent can ensure the effective removal of Fmoc protecting groups from Rink Amide resin, which is beneficial for amino acids to bind to Rink Amide resin, thereby effectively synthesizing peptides.

[0063] In this invention, during each cycle of amino acid coupling assembly, the swollen Rink Amide resin can be eluted 1 to 5 times, preferably 1 to 3 times, and more preferably 2 to 3 times, with an Fmoc protecting group eluent to deprotect the resin, obtaining a deprotected resin. Next, the deprotected resin is thoroughly washed with an eluent to remove residual Fmoc protecting group eluent. The eluent can be selected from at least one of DMF and dichloromethane, preferably DMF or dichloromethane, and more preferably DMF. The number of washes can be 1 to 5 times, preferably 1 to 3 times, and more preferably 2 to 3 times. The amount of eluent used each time can be 20 to 80 mL, preferably 25 to 70 mL, and more preferably 30 to 65 mL. Such washing conditions are more conducive to removing residual Fmoc protecting group eluent.

[0064] In this invention, after washing the deprotected resin, an activation solution of Fmoc-based protected amino acids is added to the deprotected resin to carry out a coupling reaction, thereby coupling and assembling the Fmoc-based protected amino acids onto the deprotected resin. The activation solution of Fmoc-based protected amino acids is mainly prepared by dissolving Fmoc-based protected amino acids, HBTU (benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate), and N,N-diisopropylethylamine in DMF or dichloromethane. The molar ratio of Fmoc-based protected amino acids, HBTU, and N,N-diisopropylethylamine can be 1:1 to 3:1 to 5, preferably 1:1 to 2:2 to 4, and more preferably 1:1:2 to 3. Based on 1 mmol of Fmoc-based protected amino acids, the amount of DMF or dichloromethane can be 8 to 20 mL, preferably 10 to 18 mL, and more preferably 12 to 15 mL. The coupling reaction time can be 30 to 120 min, preferably 40 to 90 min, and more preferably 45 to 60 min. Such coupling reactions are more conducive to the coupling and assembly of Fmoc-based protected amino acids onto the deprotected resin and the further binding of new amino acids. In this invention, after each coupling reaction, the completeness of the reaction can be detected. The detection method can be any method known in the art, and is not particularly limited here. For example, it could be the ninhydrin detection method, etc.

[0065] In this invention, the above-described deprotection, washing, and coupling steps are repeated until the target amino acid sequence of the human type I collagen polypeptide is assembled. According to one embodiment of the invention, after the human type I collagen polypeptide is assembled, a cleavage solution can be used to cleave the polypeptide from the deprotection resin to obtain the human type I collagen polypeptide. The cleavage solution mainly consists of trifluoroacetic acid, water, phenol, and triisopropylsilane. The volume ratio of trifluoroacetic acid, water, phenol, and triisopropylsilane can be 30–50:1–5:1–5:1–5, preferably 32–48:1–3:1–3:1–3, more preferably 35–45:1–3:1–3:1–3. The cleavage temperature can be 15–60°C, preferably 20–50°C, more preferably 22–40°C. The cleavage time can be 20–80 min, preferably 30–70 min, more preferably 40–65 min. Appropriate cutting solutions and cutting conditions are beneficial for cutting the synthesized human type I collagen peptides from the deprotected resin and can ensure the removal of amino acid protecting groups from the peptide side chains.

[0066] This invention may further include steps of performing a first solid-liquid separation, precipitation, a second solid-liquid separation, and purification on the cleaved human type I collagen peptides. Both the first and second solid-liquid separations can be performed using solid-liquid separation methods and equipment known in the art, and are not particularly limited herein. For example, they can be vacuum filtration, centrifugation, etc. Precipitation can be performed using various ether solvents known in the art for precipitating peptides, and are not particularly limited herein. For example, they can be methyl tert-butyl ether, diethyl ether, petroleum ether, isopropyl ether, etc. The ether solvents are all cold ether solvents, with a temperature of up to 4°C, preferably -20 to 4°C, more preferably -20 to 0°C. Purification can be performed using any purification method known in the art, and is not particularly limited herein. For example, high-performance liquid chromatography (HPLC) can be used.

[0067] According to a preferred embodiment of the present invention, the cleaved human type I collagen peptide can be subjected to a single solid-liquid separation to obtain a crude peptide. Next, the crude peptide is precipitated using a cold ether solvent. Then, the precipitated crude peptide undergoes a second solid-liquid separation to obtain the human type I collagen peptide, which is then purified using HPLC.

[0068] According to one embodiment of the present invention, the molecular weight of the purified human type I collagen polypeptide can also be determined to confirm whether the product was correctly synthesized. Molecular weight determination can be performed using any identification method known in the art, and is not particularly limited herein. For example, mass spectrometry can be used.

[0069] The water used in each step of the present invention can be selected from at least one of deionized water, ultrapure water, and distilled water, preferably at least one of deionized water and ultrapure water, and more preferably deionized water or ultrapure water.

[0070] Double bond functional group modification steps

[0071] Human type I collagen peptides were mixed with compounds with double bond functional groups and reacted at 40–65 °C to obtain peptides modified with double bond functional groups.

[0072] In this step, the double-bonded functional group compound is the same as the double-bonded functional group compound in the above-mentioned polypeptide nuclide carrier, which will not be described again here.

[0073] In this invention, the degree of substitution of the amino group of lysine in the human type I collagen polypeptide by the double-bonded functional group compound is the same as that of the double-bonded functional group compound in the polypeptide nuclide carrier, and will not be repeated here. Based on the degree of substitution, the molar ratio of the human type I collagen polypeptide to the double-bonded functional group compound can be 1:1.3 to 3.5, preferably 1:2 to 3.5, and more preferably 1:2 to 3.

[0074] According to one embodiment of the present invention, the reaction temperature can be 40–65°C, preferably 45–65°C, more preferably 45–60°C. The reaction time can be 1–5 h, preferably 1.5–4.5 h, more preferably 2–4 h. Such reaction conditions are more conducive to the substitution of the amino group of lysine in the polypeptide by the double bond functional group.

[0075] In this invention, human type I collagen peptides and double-bonded functional group compounds are reacted in water. Based on 1 mmol of human type I collagen peptides, the amount of water used can be 150-400 mL, preferably 200-350 mL, and more preferably 250-300 mL.

[0076] In this invention, the reaction product solution obtained after the reaction can be diluted with water, and then placed in a dialysis bag for dialysis to obtain a dialysate. The dialysate in the dialysis bag is then filtered to obtain a filtrate. The filtrate is then dried to obtain a polypeptide modified with double bond functional groups.

[0077] According to one embodiment of the present invention, the amount of water added for dilution can be 1 to 5 times the amount of water used in the reaction, preferably 2 to 5 times, and more preferably 2 to 4 times. The molecular weight cutoff of the dialysis bag can be 800 to 3000 Da, preferably 900 to 2500 Da, and more preferably 1000 to 2000 Da. The dialysis temperature can be 20 to 60°C, preferably 22 to 50°C, and more preferably 24 to 45°C. The dialysis time can be 1 to 5 days, preferably 2 to 5 days, and more preferably 2 to 4 days. Such dialysis conditions are beneficial for removing residual double-bonded functional group compounds.

[0078] According to one embodiment of the present invention, filtration can be achieved using any filtration method and filtration device known in the art, without particular limitation. For example, it can be vacuum filtration, etc. Drying can be achieved using any drying method known in the art, without particular limitation. For example, it can be freeze drying, vacuum drying, rotary evaporation, etc.

[0079] Combined chelating agent steps

[0080] The double-bonded functional group-modified peptide is mixed with the activating precursor of the chelating agent and reacted at 20–45 °C to obtain the peptide nuclide carrier.

[0081] In this step, the chelating agent is the same as the chelating agent in the polypeptide nuclide carrier mentioned above, and will not be described again here.

[0082] According to one embodiment of the present invention, the activating precursor of the chelating agent may be an NHS ester of the chelating agent.

[0083] In this invention, the degree of substitution of the amino group of lysine in the human type I collagen polypeptide by the chelating agent is the same as that of the chelating agent in the polypeptide nuclide carrier described above, and will not be repeated here. Based on the degree of substitution, the molar ratio of the human type I collagen polypeptide to the activating precursor of the chelating agent can be 1:0.5 to 3, preferably 1:1 to 3, and more preferably 1:1 to 2.

[0084] According to one embodiment of the present invention, the reaction temperature can be 20–45°C, preferably 22–40°C, and more preferably 25–35°C. The reaction time can be 8–20 h, preferably 10–19 h, and more preferably 12–18 h. Such reaction conditions are more conducive to the chelating agent replacing the amino group of lysine in the polypeptide.

[0085] In this invention, the human type I collagen polypeptide reacts with the activating precursor of the chelating agent in an organic solvent. The organic solvent can be selected from at least one of DMSO (dimethyl sulfoxide), DMF, and dichloromethane, preferably at least one of DMSO and DMF, and more preferably DMSO or DMF. Based on 1 mmol of double-bonded functional group-modified polypeptide, the amount of organic solvent can be 30–100 mL, preferably 40–80 mL, and more preferably 45–70 mL. According to a preferred embodiment of the invention, an acid-binding agent can also be added to the reaction to provide an alkaline environment and promote the reaction. The acid-binding agent can be selected from at least one of triethylamine, DIPEA (N,N-diisopropylethylamine), pyridine, and potassium carbonate, preferably at least one of triethylamine, DIPEA, and pyridine, and more preferably at least one of triethylamine and DIPEA. The molar ratio of the acid-binding agent to the double-bonded functional group-modified polypeptide can be 2–8:1, preferably 2.5–7:1, and more preferably 3–6:1.

[0086] In this invention, the reaction process may further include a stirring step. Stirring can be achieved using any stirring equipment known in the art, without particular limitation. For example, it can be a vortex stirrer, ultrasonic stirrer, etc. The stirring speed can be 100–800 r / min, preferably 200–600 r / min, more preferably 250–500 r / min. The reaction product solution obtained after the reaction can also be placed in a dialysis bag for dialysis to obtain a dialysate. The dialysate in the dialysis bag is then filtered to obtain a filtrate. The filtrate is then dried to obtain a polypeptide nuclide carrier.

[0087] According to one embodiment of the present invention, the molecular weight cutoff of the dialysis bag can be 800–3000 Da, preferably 900–2500 Da, more preferably 1000–2000 Da. The dialysis temperature is 20–60°C, preferably 22–50°C, more preferably 24–45°C. The dialysis time can be 1–5 days, preferably 2–5 days, more preferably 2–4 days. Such dialysis conditions are beneficial for removing residual chelating agent activation precursors.

[0088] According to one embodiment of the present invention, filtration can be achieved using filtration methods and equipment known in the art, without particular limitation. For example, it can be vacuum filtration, etc. Drying can be achieved using drying methods known in the art, without particular limitation. For example, it can be freeze drying, vacuum drying, rotary evaporation, etc.

[0089] Applications of polypeptide nuclide carriers

[0090] The present invention also provides the use of the above-mentioned polypeptide nuclide carrier in the preparation of nuclear medicine detection products or radiotherapy products.

[0091] According to one embodiment of the present invention, the nuclear medicine testing product may be selected from tracers or imaging agents used in any nuclear medicine testing equipment known in the art, preferably tracers or imaging agents used in at least one of PET (positron emission tomography), PET / CT (positron emission tomography and computed tomography), and PET / MRI (positron emission tomography and magnetic resonance imaging).

[0092] According to one embodiment of the present invention, the radiotherapy product may be selected from any radiotherapy drug known in the art, preferably a radiotherapy drug for treating at least one of the following diseases: glioma, breast cancer, liver cancer, lung cancer, and thyroid disease.

[0093] Nuclide probe

[0094] The present invention also provides a radionuclide probe, which is made of the above-mentioned polypeptide radionuclide carrier and radionuclide.

[0095] According to one embodiment of the present invention, the molar ratio of the polypeptide nuclide carrier and the nuclide can be 1:1 to 5, preferably 1:1 to 4, and more preferably 1:1.5 to 4.

[0096] According to one embodiment of the present invention, the nuclide can be selected from at least one of metallic nuclides and non-metallic nuclides, preferably metallic nuclides and non-metallic nuclides. According to a preferred embodiment of the present invention, the molar ratio of metallic nuclides to non-metallic nuclides can be 0.25 to 4:1, preferably 0.4 to 3:1, and more preferably 0.5 to 2:1. Such a ratio ensures that the ratio of metallic nuclides to non-metallic nuclides in the nuclide probe is clear and that both can be stably bound to the polypeptide nuclide carrier.

[0097] According to one embodiment of the present invention, the metal nuclide can be selected from... 177 Lu、 90 Y、 67 Ga、 64 Cu、 99m Tc, 89 At least one of Sr, preferably 177 Lu、 90 Y、 99m Tc, 89 At least one of Sr, more preferably Sr 177 Lu、 90 Y、 99m At least one of Tc. Non-metallic nuclides may be selected from... 125 I, 131 I, 18 F, 32 At least one of P, preferably 125 I, 131 I, 18 At least one of F, more preferably, 125 I, 131 At least one of I.

[0098] The nuclide probe of this invention simultaneously carries metallic and non-metallic nuclides in a clearly defined ratio, making it highly versatile. It can be used to prepare both nuclear medicine diagnostic products and radiotherapy products.

[0099] Preparation methods of radionuclide probes

[0100] The present invention also provides a method for preparing the radionuclide probe as described above, including a mixing reaction step. This is described in detail below.

[0101] Mixed reaction steps

[0102] The above-mentioned polypeptide nuclide carrier is mixed with the nuclide and reacted at 20–60 °C to obtain the nuclide probe.

[0103] In this step, the nuclide is the same as the nuclide in the aforementioned nuclide probe, and will not be repeated here. During the reaction, the corresponding salt of the nuclide can be used. According to one embodiment of the present invention, the salt of the metallic nuclide can be selected from at least one of the following: halides, nitrates, sulfates, phosphates, and acetates of the metallic nuclide, preferably at least one of the following: halides and nitrates of the metallic nuclide, more preferably at least one of the following: chlorides and bromides of the metallic nuclide. The salt of the non-metallic nuclide can be selected from at least one of the following: alkali metal salts of the non-metallic nuclide, preferably at least one of the following: sodium salt or potassium salt of the non-metallic nuclide, more preferably sodium salt or potassium salt of the non-metallic nuclide.

[0104] According to one embodiment of the present invention, the reaction temperature can be 20–60°C, preferably 22–55°C, more preferably 24–50°C. The reaction time can be 0.5–5 h, preferably 0.8–4 h, more preferably 1–3 h.

[0105] According to a preferred embodiment of the present invention, when the nuclide is a metal nuclide, the reaction temperature between the polypeptide nuclide carrier and the metal nuclide can be 30–60°C, preferably 35–55°C, and more preferably 40–50°C. The reaction time between the polypeptide nuclide carrier and the metal nuclide can be 0.5–5 h, preferably 0.8–4 h, and more preferably 1–3 h. Such reaction conditions are beneficial for the metal nuclide to bind to the chelating agent of the polypeptide nuclide carrier.

[0106] According to a preferred embodiment of the present invention, when the nuclide is a metal nuclide, the molar ratio of the polypeptide nuclide carrier to the metal nuclide can be 1:1 to 3, preferably 1:1.5 to 3, and more preferably 1:1.5 to 2.5.

[0107] In this invention, when the polypeptide nuclide carrier reacts with the metal nuclide, the polypeptide nuclide carrier can be dissolved in ammonium acetate buffer solution, and then the metal nuclide can be added. Stirring can be performed during the reaction to promote uniform mixing of the reaction system.

[0108] According to one embodiment of the present invention, the pH value of the ammonium acetate buffer solution can be 3.5-6, preferably 3.8-5.5, and more preferably 4-5. The concentration of the ammonium acetate buffer solution can be 0.1-1M, preferably 0.2-0.8M, and more preferably 0.3-0.7M. Based on 1g of polypeptide nuclide carrier, the volume of ammonium acetate buffer solution can be 5-30mL, preferably 6.5-25mL, and more preferably 8-20mL.

[0109] According to one embodiment of the present invention, stirring can be achieved using any stirring method known in the art, without particular limitation. For example, it can be vortex stirring, ultrasonic stirring, etc. The stirring speed can be 100-800 r / min, preferably 200-600 r / min, and more preferably 250-500 r / min.

[0110] In this invention, when the polypeptide nuclide carrier reacts with the metal nuclide, the reaction product obtained after the reaction can be separated and purified by ultrafiltration centrifugation to obtain the nuclide probe. The molecular weight cutoff of the ultrafiltration membrane used for ultrafiltration centrifugation can be 500-2000 Da, preferably 800-1500 Da, and more preferably 900-1200 Da. The rotation speed of ultrafiltration centrifugation can be 2000-5000 r / min, preferably 2200-4500 r / min, and more preferably 2500-4000 r / min. Such ultrafiltration centrifugation conditions can ensure that unreacted metal nuclide ions smaller than the molecular weight cutoff of the ultrafiltration membrane and impurities such as buffer solution can pass through the ultrafiltration membrane smoothly into the filtrate, while the nuclide probe loaded with the metal nuclide is effectively retained in the ultrafiltration tube, thereby achieving efficient separation and purification of the metal nuclide probe.

[0111] According to a preferred embodiment of the present invention, when the nuclide is a non-metallic nuclide, the reaction temperature between the polypeptide nuclide carrier and the non-metallic nuclide can be 20–45°C, preferably 22–40°C, and more preferably 25–35°C. The reaction time between the polypeptide nuclide carrier and the metallic nuclide can be 0.5–5 h, preferably 0.8–4 h, and more preferably 1–3 h. Such reaction conditions are conducive to the substitution reaction between the non-metallic nuclide and the tyrosine of the polypeptide nuclide carrier, thereby binding to the polypeptide nuclide carrier.

[0112] According to a preferred embodiment of the present invention, when the nuclide is a non-metallic nuclide, the molar ratio of the polypeptide nuclide carrier to the metallic nuclide can be 1:1 to 3, preferably 1:1.5 to 3, and more preferably 1:1.5 to 2.5.

[0113] In this invention, when the polypeptide nuclide carrier reacts with a non-metallic nuclide, the polypeptide nuclide carrier can be dissolved in phosphate buffered saline (PBS), and then the non-metallic nuclide and oxidant solution are added. Stirring can be performed during the reaction to promote uniform mixing of the reaction system. The oxidant can activate the non-metallic nuclide, thereby causing the activated non-metallic nuclide to undergo a substitution reaction with tyrosine in the polypeptide nuclide carrier. The oxidant can be selected from at least one of N-chloro-4-toluenesulfonamide sodium, hydrogen peroxide, and sodium nitrite, preferably at least one of N-chloro-4-toluenesulfonamide sodium and hydrogen peroxide, and more preferably N-chloro-4-toluenesulfonamide sodium.

[0114] According to one embodiment of the present invention, the pH value of the phosphate buffer can be 5-8, preferably 5.5-7.5, and more preferably 6-7. The concentration of the phosphate buffer can be 0.01-0.1M, preferably 0.02-0.08M, and more preferably 0.03-0.07M. Based on 1g of polypeptide nuclide carrier, the volume of phosphate buffer used can be 5-30mL, preferably 6.5-25mL, and more preferably 8-20mL.

[0115] According to one embodiment of the present invention, the oxidant solution can be prepared by dissolving an oxidant in PBS. The concentration of the oxidant can be 0.5–5 mg / mL, preferably 0.8–4 mg / mL, more preferably 1–3 mg / mL. The pH value of the PBS can be 5–8, preferably 5.5–7.5, more preferably 6–7. The concentration of the PBS can be 0.01–0.1 M, preferably 0.02–0.08 M, more preferably 0.03–0.07 M. Based on 1 g of polypeptide nuclide carrier, the volume of the oxidant solution can be 5–30 mL, preferably 6.5–25 mL, more preferably 8–20 mL.

[0116] According to one embodiment of the present invention, stirring can be achieved using any stirring method known in the art, without particular limitation. For example, it can be vortex stirring, ultrasonic stirring, etc. The stirring speed can be 100-800 r / min, preferably 200-600 r / min, and more preferably 250-500 r / min.

[0117] According to one embodiment of the present invention, after the reaction is completed, a terminator solution can be added to the reaction system to terminate the reaction. Then, a salt solution of a non-metallic nuclide is added to compete with the unreacted activated non-metallic nuclide, thereby reducing the adsorption of the remaining activated non-metallic nuclide onto the reaction vessel.

[0118] According to a preferred embodiment of the present invention, the terminator may be selected from at least one of sodium thiosulfate, ascorbic acid, cysteine, and glucose, preferably at least one of sodium thiosulfate and ascorbic acid, and more preferably sodium thiosulfate. The concentration of the terminator solution may be 5–20 mg / mL, preferably 6–15 mg / mL, and more preferably 8–12 mg / mL. Based on 1 g of polypeptide nuclide carrier, the amount of terminator solution may be 0.5–10 mL, preferably 0.7–8 mL, and more preferably 1–5 mL.

[0119] According to a preferred embodiment of the present invention, the salt of the non-metallic nuclide can be selected from at least one of the alkali metal salts of the non-metallic nuclide, preferably at least one of the lithium, sodium, and potassium salts of the non-metallic nuclide, and more preferably at least one of the sodium and potassium salts of the non-metallic nuclide. The concentration of the non-metallic nuclide salt solution can be 5–20 mg / mL, preferably 7–18 mg / mL, and more preferably 9–15 mg / mL. Based on 1 g of polypeptide nuclide carrier, the amount of non-metallic nuclide salt solution used can be 0.5–10 mL, preferably 0.8–7 mL, and more preferably 1–5 mL.

[0120] In this invention, when the polypeptide nuclide carrier reacts with a non-metallic nuclide, the reaction product obtained after the reaction can be separated and purified by ultrafiltration centrifugation to obtain the nuclide probe. The molecular weight cutoff of the ultrafiltration membrane used for ultrafiltration centrifugation can be 500–2000 Da, preferably 800–1500 Da, and more preferably 900–1200 Da. The rotation speed of ultrafiltration centrifugation can be 2000–5000 r / min, preferably 2200–4500 r / min, and more preferably 2500–4000 r / min. Such ultrafiltration centrifugation conditions can ensure that unreacted non-metallic nuclide ions smaller than the molecular weight cutoff of the ultrafiltration membrane and impurities such as buffer solution can pass smoothly through the ultrafiltration membrane into the filtrate, while the nuclide probe loaded with non-metallic nuclide is effectively retained in the ultrafiltration tube, thereby achieving efficient separation and purification of the non-metallic nuclide probe.

[0121] According to a preferred embodiment of the present invention, when the nuclide is a metallic nuclide or a non-metallic element, the method for binding metallic or non-metallic elements to a polypeptide nuclide carrier described above can be referred to, so that metallic or non-metallic elements are alternately bound to the polypeptide nuclide carrier. Specific steps will not be repeated here. The order of binding of metallic and non-metallic elements is not important.

[0122] Applications of radionuclide probes

[0123] The present invention also provides the use of the above-mentioned radionuclide probe in the preparation of nuclear medicine detection products or radiotherapy products.

[0124] According to one embodiment of the present invention, the nuclear medicine testing product may be selected from tracers or imaging agents used in any nuclear medicine testing device known in the art, preferably tracers or imaging agents used in at least one of PET, PET / CT, and PET / MRI.

[0125] According to one embodiment of the present invention, the radiotherapy product may be selected from any radiotherapy drug known in the art, preferably a radiotherapy drug for treating at least one of the following diseases: glioma, breast cancer, liver cancer, lung cancer, and thyroid disease.

[0126] According to a preferred embodiment of the present invention, the product may include a radionuclide probe and a photoinitiator. The weight ratio of the radionuclide probe to the photoinitiator may be 100–1000:1, preferably 200–800:1, and more preferably 300–600:1. The photoinitiator may be selected from at least one of benzophenone, lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), and 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO), preferably at least one of LAP and TPO, and more preferably LAP. Such a photoinitiator and dosage are more conducive to the curing of the radionuclide probe into a gel under light irradiation. According to a preferred embodiment of the present invention, the radionuclide probe and the photoinitiator may be separately prepared into solutions before light irradiation. The concentration and dosage of the solutions need only ensure that the weight ratio of the radionuclide probe to the photoinitiator conforms to the above relationship, and will not be elaborated further here.

[0127] According to one embodiment of the present invention, the nuclide probe solidifies into a gel in the presence of a photoinitiator and under the irradiation of a light source.

[0128] According to one embodiment of the present invention, the power of the light source can be 5-40W, preferably 8-30W, and more preferably 10-25W. The wavelength of the light source can be 300-500nm, preferably 350-480nm, and more preferably 380-450nm. The irradiation time can be 5-60s, preferably 7-45s, and more preferably 10-30s. Such light irradiation conditions are more conducive to the solidification of the radionuclide probe into a gel.

[0129] Test methods

[0130] HPLC purification

[0131] The synthesized peptides were purified using reversed-phase high-performance liquid chromatography (RP-HPLC). First, the crude product was pretreated by dissolving it in an aqueous solution containing 0.1% trifluoroacetic acid to obtain a 10 mg / mL solution, which was then filtered through a 0.22 μm filter for later use. Purification was then performed using a preparative-grade HPLC system (Agilent 1260 Infinity II, 4.6 mm × 250 mm, 5 μm particle size, 300 Å pore size C18 column). The mobile phase consisted of chromatographically pure 0.1% trifluoroacetic acid-water solution (phase A) and 0.1% trifluoroacetic acid-acetonitrile solution (phase B), followed by ultrasonic degassing for 15 min and filtration through a 0.22 μm filter. The purification process was monitored in real-time using a UV detector (214 nm), and fractional fractions were collected using an automated fraction collector. Finally, the collected purified fractions were combined and freeze-dried (-50 °C, 72 h) to obtain high-purity peptide powder.

[0132] Molecular weight identification by mass spectrometry

[0133] The molecular weight of the purified peptides was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF, manufactured by Bruker GmbH, Germany). A 2 mg / mL aqueous solution of the peptides was prepared, and the sample was added to the detection plate using a spotting method. After drying at room temperature, the sample was analyzed.

[0134] Detection of radioactive substance concentration

[0135] After administration, rat samples were collected at different time points and radioactive samples were detected using a gamma counter (model 2470, manufactured by PerkinElmer).

[0136] Raw material description

[0137] Unless otherwise specified, all raw materials used in the following examples are commercially available products.

[0138] The Fmoc-protected amino acids were purchased from Gir Biochemical Company. Methacrylic anhydride was purchased from Aladdin Company. DOTA-NHS was purchased from Aladdin Company. 177 LuCl3 was purchased from PerkinElmer. 125 I was purchased from PerkinElmer.

[0139] The rats were SD (Sprague-Pawley) type rats, purchased from Beijing Vital River Laboratory Animal Co., Ltd.

[0140] The PET sensor used is the Mediso NanoScan PET / CT.

[0141] Preparation Examples 1-4

[0142] 1. Screening and design of human type I collagen peptides

[0143] Human type I collagen peptides were screened and designed using the UniProt database. Four human type I collagen peptides containing RGD tripeptide structure, lysine (K), and tyrosine (Y) were screened and designed. Specific information is as follows:

[0144] Preparation Example 1: The amino acid sequence of human type I collagen polypeptide I (denoted as polypeptide I) is shown in SEQ ID No. 1: GKSGDRGEYG PAGPAGPVGP VGARGPAGPQ GPRGDKGEYG EQGDRGIKGH.

[0145] Polypeptide I is derived from the Collagen alpha-1(I) chain protein of the α1 chain of human type I collagen. This protein has a total of 1464 amino acids, and the gene encoding this protein is COL1A1.

[0146] Depend on Figure 1 It is known that polypeptide I contains one RGD tripeptide structure, three lysines and one tyrosine.

[0147] Preparation Example 2: The amino acid sequence of human type I collagen polypeptide II (denoted as polypeptide II) is shown in SEQ ID No. 2: PKGDRGDAGP KGADGSPGKD GVRGLYGPIG PPGPAGAPGD KGESGPSGPA.

[0148] Polypeptide II is derived from the Collagen alpha-1(I) chain preproprotein of the α1 chain of human type I collagen. This protein has a total of 1091 amino acids, and the gene encoding this protein is COL1A1.

[0149] Depend on Figure 2 It is known that polypeptide II contains an RGD tripeptide structure, four lysines and one tyrosine.

[0150] The amino acid sequence of human type I collagen polypeptide III (denoted as polypeptide III) is shown in SEQ ID No. 3: AAGQPGAKGE RGAKGPKGEN GVVGPYGPVG AAGPAGPNGP PGPAGSRGDG.

[0151] Preparation Example 3: Peptide III is derived from the Collagen alpha-2(I) chain protein of the α2 chain of human type I collagen. This protein has a total of 1366 amino acids, and the gene encoding this protein is COL1A2.

[0152] Depend on Figure 3 It is known that polypeptide III contains an RGD tripeptide structure, three lysines, and one tyrosine.

[0153] Preparation Example 4: The amino acid sequence of human type I collagen peptide IV (denoted as peptide IV) is shown in SEQ ID No. 4: PAGKHGNRGE YGPSGPVGPA GAVGPRGPSG PQGIRGDKGE PGEKGPRGLP.

[0154] Polypeptide IV is derived from the Collagen alpha-2(I) chain protein of the α2 chain of human type I collagen. This protein has a total of 1366 amino acids, and the gene encoding this protein is COL1A2.

[0155] Depend on Figure 4 It is known that polypeptide III contains an RGD tripeptide structure, three lysines and one tyrosine.

[0156] 2. Synthesis of human type I collagen peptides

[0157] Peptides I–IV, with a target size of 0.2 mmol (equivalent to 1 eq), were prepared using a solid-phase synthesis method and a Fmoc (9-fluorenylmethoxycarbonyl) protection strategy, following the steps below:

[0158] 1) 20 mL of DMF was used to pre-swell Rink Amide resin with a loading of 0.2 mmol / g. Then, 1 g of pre-swollen Rink Amide resin with a loading of 0.2 mmol / g was loaded into the reactor of the solid-phase peptide synthesizer as a solid support for synthesizing peptides.

[0159] 2) Cyclic Coupling of Amino Acids: In each cycle, Rink Amide resin was eluted twice with 60 mL of 20 wt% piperidine DMF solution to obtain deprotected resin. Then, the deprotected resin was washed twice with 60 mL of DMF each time. Next, 4 eq of Fmoc-based protected amino acids (0.8 mmol), 4 eq of HBTU (0.8 mmol), and 8 eq of N,N-diisopropylethylamine (1.6 mmol) were dissolved in 10 mL of DMF to obtain an activated solution of Fmoc-based protected amino acids. The activated solution of Fmoc-based protected amino acids was added to the deprotected resin, and the coupling reaction was carried out at room temperature (25 °C, the same below) for 60 min. After the reaction, the completeness of the reaction was confirmed by ninhydrin detection.

[0160] After coupling, the deprotecting resin was washed twice with 60 mL of DMF each time. Then, the above deprotection-coupling cycle was repeated until the target amino acid sequence of the peptide was assembled.

[0161] 3) Mix 9.25 mL of trifluoroacetic acid, 25 mL of water, 0.25 mL of phenol, and 0.25 mL of triisopropylsilane to obtain a cleavage solution. Then, use the cleavage solution to cleave the deprotected resin with the peptide attached at room temperature for 3 hours to obtain a reaction solution of the peptide and remove the side chain protecting groups of the peptide.

[0162] 4) Filter the peptide reaction solution using a sintered glass funnel and collect the residue. Add the residue to methyl tert-butyl ether at -20℃ to precipitate the crude peptide. Then, at 3000 r / min...

[0163] Centrifuge the crude peptide for 5 min, discard the supernatant to obtain the crude polypeptide. Purify the crude polypeptide using HPLC and determine its molecular weight by mass spectrometry to obtain the final polypeptide.

[0164] Examples 1-6

[0165] 1. Double bond functional group modification

[0166] 1 g of each of the polypeptides I-IV (0.18 mmol, 1 eq) prepared in Examples 1-4 was weighed and dissolved in 50 mL of deionized water to obtain polypeptide solutions. Then, 55.5 mg (0.36 mmol, 2 eq) or 83.3 mg (0.54 mmol, 3 eq) of MAAH (methacrylic anhydride) was added to the polypeptide solutions, and the mixture was reacted at 55 °C for 3 h to obtain a reaction product solution. 150 mL of deionized water was added to the reaction product solution for dilution. The diluted reaction product solution was then placed in a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed at room temperature for 3 days to remove residual MAA, yielding the dialysate. The dialysate in the dialysis bag was vacuum filtered, and the filtrate was collected. The filtrate was freeze-dried at -50 °C for 72 h to obtain double-bond functionalized polypeptides (methacrylamide polypeptides). The degree of substitution of the double-bond functional groups (MA, methacrylate) in the prepared double-bond functionalized polypeptides is shown in Table 1 below.

[0167] Table 1. Degree of substitution of double bond functional groups in peptides modified with different double bond functional groups

[0168]

[0169] 2. Combined chelating agents

[0170] 1.0 g of each of the double-bonded functional group modified peptides I–V (0.18 mmol, 1 eq) prepared above was dissolved in 10 mL of DMSO (dimethyl sulfoxide). Then, 90 mg (0.18 mmol, 1 eq) or 180 mg (0.36 mmol, 2 eq) of DOTA-NHS ester (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-1-(2,5-dioxo-1-pyrrolyl) ester) and 73 mg (0.72 mmol, 4 eq) of triethylamine were added to obtain a mixed solution. The mixed solution was then reacted overnight (approximately 12 h) at 30 °C and 300 rpm with stirring to obtain a reaction product solution. The reaction product solution was placed in a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed for 3 days to remove residual DOTA-NHS ester. The dialyzed solution was then vacuum filtered, and the filtrate was collected. The filtrate was freeze-dried to obtain the polypeptide nuclide carrier. The degree of DOTA substitution of different polypeptide nuclide carriers is shown in Table 2 below.

[0171] Table 2. Degree of DOTA substitution in different polypeptide nuclide carriers

[0172]

[0173] Examples 7-12

[0174] Using the polypeptide nuclide carriers I-VI prepared in Examples 1-6 as carriers, metal nuclides were prepared respectively. 177 The specific steps for creating a Lu nuclide probe are as follows:

[0175] Weigh 1 g (0.18 mmol, 1 eq) of each of the polypeptide radionuclide carriers I-VI prepared in Examples 1-6, dissolve them in 10 mL of 0.5 M ammonium acetate buffer at pH 5, and then add... 177 LuCl3 was used to obtain a mixed solution. The mixed solution was reacted at 45℃ and 300 r / min for 1 h to obtain a reaction product solution. The reaction product solution was ultrafiltered and centrifuged at 3000 r / min for 5 min using an ultrafiltration membrane with a molecular weight cutoff of 1000 Da. The retentate in the ultrafiltration tube was collected to obtain the loaded product. 177 Lu nuclide probes. 177 LuCl3 dosage and each nuclide probe 177 The Lu load is shown in Table 3 below.

[0176] Table 3 177 LuCl3 dosage and each nuclide probe 177 Lu load

[0177]

[0178] Examples 13-19

[0179] Using the polypeptide nuclide carriers I-VI prepared in Examples 1-6 as carriers, non-metallic nuclides were prepared respectively. 125 The specific steps for using the I-type nuclide probe are as follows:

[0180] Weigh 1 g (0.18 mmol, 1 eq) of the polypeptide radionuclide carriers I-VI prepared in Examples 1-6, dissolve them in 10 mL of 0.5 M PBS buffer (pH=7), and then add Na... 125 I and 10 mL of a PBS solution containing 1 mg / mL N-chloro-4-toluenesulfonamide sodium salt (PBS buffer concentration: 0.5 M, pH=7) were added to obtain a mixed solution. The mixed solution was reacted at room temperature and vortexed at 300 rpm for 1 h to obtain a reaction product solution. Next, 1 mL of a 10 mg / mL sodium thiosulfate aqueous solution was added to the reaction product solution to terminate the reaction, followed by the addition of 1 mL of a 10 mg / mL potassium iodide aqueous solution to compete for unreacted reactivity. 125 I, to obtain the inactivated reaction product solution.

[0181] The inactivated reaction product solution was ultrafiltered and centrifuged for 5 min using an ultrafiltration membrane with a molecular weight cutoff of 1000 Da at 3000 r / min. The retentate in the ultrafiltration tube was collected to obtain the loaded product. 125 I nuclide probe. Na 125 I and each nuclide probe 125 The load is shown in Table 4 below.

[0182] Table 4 Na 125 I and each nuclide probe 125 I load

[0183]

[0184] Examples 20-26

[0185] Using the polypeptide nuclide carriers I-VI prepared in Examples 1-6 as carriers, simultaneous loading was prepared. 177 Lu and 125 The specific steps for using the I-type nuclide probe are as follows:

[0186] 1) Loaded with metallic nuclides 177 Lu

[0187] Referring to the preparation method in Examples 7-12, the following were prepared: 177 Lu was loaded onto a polypeptide nuclide vector to obtain the loaded... 177 Lu nuclide probes.

[0188] 2) Loaded with non-metallic nuclides 125 I

[0189] Referring to the preparation method in Examples 13-19, the following were prepared: 125 I load on load 177 Simultaneous loading of Lu's nuclide probes was obtained. 177 Lu and 125 I nuclide probe.

[0190] The order of steps 1) and 2) above can be interchanged.

[0191] 177 The amount of LuCl3 used, Na 125 The amount of I used and the amount loaded in the nuclide probe 177 Lu and 125 The molar amounts of I are shown in Table 5 below.

[0192] Table 5 77 The amount of LuCl3 used, Na 125 The amount of I used and the amount loaded in the nuclide probe 177 Lu and 125 molar ratio of I

[0193]

[0194] Application Examples

[0195] The radionuclide probe XIV prepared in Example 20 was placed in deionized water to prepare a radionuclide probe solution with a concentration of 10 mg / mL. The photoinitiator LAP was placed in deionized water to prepare a LAP solution with a concentration of 10 mg / mL. 5 mL of the radionuclide probe solution and 1 mL of the LAP solution were taken and mixed thoroughly to obtain a mixed solution. The mixed solution was irradiated for 10 s under a light source with a power of 10 W and a wavelength of 405 nm, and the radionuclide probe solidified to form a gel.

[0196] Experimental Example 1

[0197] Thirty-two 6-week-old SD rats (half male and half female), weighing 180–200 g, were selected as experimental subjects. After anesthetizing the rats, craniotomy was performed, and 1 mL of a mixed solution containing LAP and radionuclide probe XIV (obtained in the application experiment) was sprayed onto the dura mater. The solution was then irradiated with 10 W of blue light at a wavelength of 405 nm for 10 seconds to solidify. After solidification, craniotomy was performed.

[0198] 1. Detection of drug concentration in blood

[0199] Postoperatively, blood samples were collected from all rats at different time points. 120 μL of whole blood samples were taken to detect the radionuclide probe's radioactivity. The average value was used to assess blood drug concentration. The results are as follows: Figure 5 As shown. By Figure 5 It is known that a small amount of the radionuclide in the probe enters the bloodstream and is slowly absorbed. The concentration of blood metabolites reaches its peak at 16 days, then slowly decreases, reaching near baseline at 90 days. This indicates that the radionuclide probe of this invention can be used for local drug administration with low post-administration blood drug concentrations, effectively reducing systemic radiation damage.

[0200] 2. In vivo metabolic experiments

[0201] Postoperatively, urine and feces were collected from all rats at different time points for radioactivity testing, and the average value was calculated. Simultaneously, the entire cage was cleaned with 30 mL of a 50 vol% ethanol solution, and 1 mL of the cleaning solution was used to measure the radioactivity concentration, thereby calculating the total radioactivity of the entire cleaning solution. Results are as follows: Figure 6 As shown, the radionuclides in the radionuclide probe of this invention are mainly excreted in urine, with a small amount excreted in feces. 97 days after administration, the total recovery rate in feces and urine was 67.3%, and the radioactivity of the washing solution accounted for 7.4% of the total administered radioactivity, resulting in a total recovery rate of 74.7%. Based on the trend of the excretion rate, it can be inferred that the radionuclide probe of this invention allows for local drug application, and the radionuclides can be metabolized relatively completely in vivo.

[0202] 3. Distribution of radionuclide probes in vivo tissues

[0203] Postoperatively, whole blood and organ tissues were collected from all rats at different time points for radioactive analysis, and the average value was calculated. The results are as follows: Figure 7 As shown. By Figure 7 It is known that the radionuclides in the radionuclide probe of the present invention are mainly distributed at the administration site and in the kidneys, with very few distributed in other tissues such as fat and muscle. This indicates that the radionuclide probe of the present invention has low ionization, effectively reducing radiation damage to other tissues.

[0204] Experimental Example 2

[0205] Referring to Experiment 1, one rat was given intracranial nuclear cinnamic probe.

[0206] Postoperative PET imaging was performed on rats at different time points, and the results are as follows: Figure 8 As shown. By Figure 8 It was observed that the radioactive signal at the administration site was strong on days 4 and 16 after drug administration, and significantly weakened on days 60 and 90. The radioactive signal in other sites such as the kidneys and liver was extremely low or almost nonexistent. This indicates that the radionuclide probe of the present invention can be effectively fixed at the administration site, has low mobility, and can effectively reduce radiation damage to other tissues. Simultaneously, from... Figure 8 It is also understood that the radionuclide probe of the present invention can be effectively used for intracranial imaging. The radionuclide probe of the present invention can be used to prepare nuclear medicine detection products or radiotherapy products.

[0207] This invention is not limited to the above-described embodiments. Any modifications, improvements, or substitutions that can be conceived by those skilled in the art without departing from the essential content of this invention fall within the scope of this invention.

Claims

1. A polypeptide nucleic acid carrier, characterized by, The polypeptide radionuclide carrier is made of raw materials comprising: a human type I collagen polypeptide, a double-bond functional group compound, and a chelating agent, wherein the human type I collagen polypeptide is selected from at least one of polypeptides with amino acid sequences as shown in SEQ ID No. 1-4; the substitution degree of the double-bond functional group compound to the amino group of lysine in the human type I collagen polypeptide is 45%-80%; and the substitution degree of the chelating agent to the amino group of lysine in the human type I collagen polypeptide is 20%-55%; the double-bond functional group compound is selected from at least one of methacrylic acid and methacrylic anhydride; the chelating agent is DOTA; the polypeptide radionuclide carrier is prepared by the following method: A) using a swelled Rink Amide resin as a solid-phase carrier, and using a solid-phase synthesis method to cyclically couple and assemble amino acids to obtain a human type I collagen polypeptide; wherein, in each cycle of coupling and assembling amino acids, a Fmoc protecting group eluent is used to remove the Fmoc protecting group on the Rink Amide resin to obtain a deprotection resin; then, a Fmoc group-protected amino acid is coupled and assembled on the deprotection resin; B) mixing the human type I collagen polypeptide with a double-bond functional group compound, and reacting at 40-65°C to obtain a double-bond functional group-modified polypeptide; C) mixing the double-bond functional group-modified polypeptide with an activated precursor of a chelating agent, and reacting at 20-45°C to obtain a polypeptide radionuclide carrier.

2. The polypeptide radionuclide carrier according to claim 1, wherein: in step A), the Fmoc protecting group eluent is selected from at least one of a piperidine DMF solution, a piperidine dichloromethane solution, a diethylamine DMF solution, and a diethylamine dichloromethane solution; wherein the mass concentration of the Fmoc protecting group eluent is 10wt%-30wt%; in step C), the activated precursor of the chelating agent is an NHS ester of the chelating agent.

3. The polypeptide radionuclide carrier according to claim 1, wherein: in step B), the reaction time is 1-5h; in step C), the reaction time is 8-20h.

4. A nuclear species probe characterized by, The radionuclide probe is made of the polypeptide radionuclide carrier and a radionuclide according to claim 1; wherein the molar ratio of the polypeptide radionuclide carrier to the radionuclide is 1:1-5. The nuclide is selected from at least one of a metal nuclide, a non-metal nuclide; the metal nuclide is 177 Lu; the non-metal nuclide is 125 I.

5. The isotope probe of claim 4, wherein, The radionuclide is a metal radionuclide and a non-metal radionuclide, and the molar ratio of the metal radionuclide to the non-metal radionuclide is 0.25-4:

1.

6. A preparation method of the radionuclide probe according to claim 4 or 5, comprising the following steps: mixing the polypeptide radionuclide carrier according to claim 1 with a radionuclide, and reacting at 20-60°C to obtain a radionuclide probe.

7. Use of the radionuclide probe according to claim 4 or 5 in the preparation of a nuclear medicine detection product.

8. Use according to claim 7, characterized in that, The product comprises the radionuclide probe and a photoinitiator; the weight ratio of the radionuclide probe to the photoinitiator is 100-1000:1; and the photoinitiator is selected from at least one of benzophenone, LAP, and TPO. The radionuclide probe is cured into a gel in the presence of the photoinitiator and under the irradiation of a light source with a power of 5-40W and a wavelength of 300-500nm.

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