Polypeptide nuclide carrier, nuclide probe and preparation method and application thereof

Abstract

The invention relates to the field of biological medicine, and particularly discloses a polypeptide nuclide carrier, a nuclide probe and a preparation method and application of the polypeptide nuclide carrier and the nuclide probe. The polypeptide nuclide carrier is prepared from the following raw materials: human I-type collagen polypeptide, a double-bond functional group compound and a chelating agent, wherein the human I-type 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 on amino of lysine in the human I-type collagen polypeptide is 45-80%; the substitution degree of the chelating agent on amino of lysine in the human I-type collagen polypeptide is 20-55%; the double-bond functional group compound is selected from at least one of acrylic acid, methacrylic acid, acrylic anhydride and methacrylic anhydride; the chelating agent is selected from at least one of DOTA, NOTA, EDTA and DTPA. The polypeptide nuclide carrier can be combined with metal nuclide and non-metal nuclide at the same time, can be photocured and has good targeting performance.

Description

TECHNICAL FIELD

[0001] The present application relates to a polypeptide nuclide carrier, a nuclide probe and a preparation method and use thereof. BACKGROUND

[0002] A nuclide probe is a tool or technique for detecting, measuring or labeling a specific radionuclide, which has a wide range of applications in the fields of nuclear medicine, radiopharmaceutical research, environmental monitoring, nuclear energy industry, etc. A nuclide probe is usually composed of a small particle or label made of a radionuclide with known radioactive decay characteristics. After being introduced into a substance, these probes can be used to study the properties, distribution and behavior of the substance by measuring its radioactive decay. The core principle is to use the decay characteristics of radionuclides to obtain target information by detecting the radiation (such as alpha, beta, gamma rays, etc.) released during the decay process.

[0003] In recent years, nuclide probes have been widely used in the fields of nuclear medical imaging and targeted therapy. Nuclide probes can be used to diagnose and monitor a variety of diseases, such as cardiovascular diseases, neurological diseases and tumors, etc. Through the use of probes labeled with radionuclides, doctors can non-invasively observe the metabolic activity, blood flow and receptor expression of diseased tissues, thereby providing important evidence for the diagnosis and treatment of diseases. Nuclide probes can also be used for targeted therapy, such as radioimmunotherapy and radioactive particle implantation therapy, etc. By labeling specific antibodies or drugs with radionuclides, precise attacks on diseased tissues can be achieved, improving treatment efficacy and reducing side effects. However, existing nuclide probes can only carry one type of nuclide, either a metal nuclide or a non-metal nuclide, which has poor versatility. Moreover, existing nuclide probes are introduced into the body through intravenous injection, and the nuclide probes need to circulate through the blood to reach the target organs or tissues. Since the nuclide probes exist in a free state in the blood, the risk of radiation damage to non-target organs or tissues is increased. Furthermore, existing nuclide probes cannot cross the blood-brain barrier and cannot be used for the diagnosis and treatment of intracranial diseases.

[0004] CN120518717A discloses a radionuclide-labeled collagen hybrid peptide probe. The chemical structure of the collagen hybrid 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, 225Any one of Ac; the chelator is selected from any one of NODAGA, NOTA2, DOTA; the Linker is selected from one of 6-aminohexanoic acid, polyethylene glycol, oligoglycine residues; the CHP is a collagen-hybrid peptide probe with a polypeptide sequence of (GfO)n or (GPO)n, wherein n is any positive integer from 6 to 12. The polypeptide of the collagen-hybrid peptide probe is in a linear structure, and the chelator can only be grafted at the end of the polypeptide main body, resulting in that the collagen-hybrid peptide probe can only bind one metal radionuclide, and the versatility is not strong. Moreover, the collagen-hybrid peptide probe needs to enter the blood circulation system through intravenous injection, and is easy to cause radioactive damage to non-target organs or tissues.

[0005] CN120550151A discloses a radionuclide-labeled molecular probe. The chemical structure of the molecular probe is as follows: K (chelator-nuclide)-K-Linker-CHP; wherein K (chelator-nuclide) is a lysine residue coupled with a side chain amino group and a radionuclide label; Linker is a connecting linker; and CHP is a collagen-hybrid peptide, and the polypeptide sequence of the collagen-hybrid peptide is (GfO)n or (GPO)n, wherein n is a positive integer from 6 to 10. The radionuclide is selected from any one of 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 Re. And / or, the chelator comprises a metal chelator, and the metal chelator is selected from any one of NOTA, NODAGA, HYNIC, DOTA, and DTPA. The Linker comprises an amino acid and / or an amino acid derivative. The polypeptide of the molecular probe is in a linear structure, and the chelator can only be grafted at the end of the polypeptide main body, resulting in that the molecular probe can only bind one radionuclide, and the versatility is not strong. Moreover, the molecular probe needs to enter the blood circulation system through intravenous injection, and is easy to cause radioactive damage to non-target organs or tissues. SUMMARY

[0006] In view of the above, an object of the present application is to provide a polypeptide radionuclide carrier which can simultaneously bind metal radionuclides and non-metal radionuclides, has high versatility, and can be photocured. Another object of the present application is to provide a method for preparing the polypeptide radionuclide carrier. Still another object of the present application is to provide a use of the polypeptide radionuclide carrier. Yet another object of the present application is to provide a radionuclide probe. Yet another object of the present application is to provide a method for preparing the radionuclide probe. Yet another object of the present application is to provide a use of the radionuclide probe.

[0007] The present application achieves the above objects by adopting the following technical solutions.

[0008] In one aspect, the present application provides a polypeptide radionuclide carrier made from 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 having an amino acid sequence as shown in SEQ ID Nos. 1-4; the substitution degree of the double-bond functional group compound for the amino group of lysine in the human type I collagen polypeptide is 45-80%, and the substitution degree of the chelating agent for 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 acrylic acid, methacrylic acid, acrylic anhydride, and methacrylic anhydride; the chelating agent is selected from at least one of DOTA, NOTA, EDTA, and DTPA.

[0009] In another aspect, the present application also provides a method for preparing the polypeptide radionuclide carrier, comprising the following steps: A) using a swollen 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 deprotected resin; then, a Fmoc group-protected amino acid is coupled and assembled on the deprotected 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.

[0010] According to the preparation method of the present application, preferably: In the 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 10-30 wt%. In the step C), the activated precursor of the chelating agent is an NHS ester of the chelating agent.

[0011] According to the preparation method of the present application, preferably: In the step B), the reaction time is 1-5 h. In the step C), the reaction time is 8-20 h.

[0012] In another aspect, the present application also provides the use of the polypeptide radionuclide carrier in the preparation of a nuclear medical detection product or a radiotherapy product.

[0013] In another aspect, the present application also provides a radionuclide probe made of the polypeptide radionuclide carrier and a radionuclide; wherein the molar ratio of the polypeptide radionuclide carrier and the radionuclide is 1:1-5. The radionuclide is selected from at least one of a metal radionuclide and a non-metal radionuclide; the metal radionuclide is selected from at least one of 177 Lu, 90 Y, 67 Ga, 64 Cu, 99m Tc, 89 Sr; and the non-metal radionuclide is selected from at least one of 125 I, 131 I, 18 F, 32 P.

[0014] According to the radionuclide probe of the present application, preferably, 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.

[0015] In another aspect, the present application also provides a preparation method of the radionuclide probe, comprising the following steps: Mixing the polypeptide radionuclide carrier and the radionuclide, and reacting at 20-60°C to obtain the radionuclide probe.

[0016] In another aspect, the present application also provides the use of the radionuclide probe in the preparation of a nuclear medical detection product or a radiotherapy product.

[0017] According to the use of the present application, preferably, 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 a photoinitiator and under the irradiation of a light source with a power of 5-40 W and a wavelength of 300-500 nm.

[0018] The polypeptide radionuclide carrier of the present application can simultaneously bind metal radionuclides and non-metal radionuclides, has high versatility, and can be photocured. The polypeptide radionuclide carrier of the present application comprises an RGD tripeptide structure and has good targeting properties. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is a schematic diagram of the amino acid sequence of human type I collagen polypeptide I; the RGD tripeptide structure is in the box, and the lysine is marked with a circle (◇) and the tyrosine is marked with a triangle (△).

[0020] Figure 2 It is a schematic diagram of the amino acid sequence of human type I collagen polypeptide II; the RGD tripeptide structure is in the box, and the lysine is marked with a circle (◇) and the tyrosine is marked with a triangle (△).

[0021] Figure 3 It is a schematic diagram of the amino acid sequence of human type I collagen polypeptide III; the RGD tripeptide structure is in the box, and the lysine is marked with a circle (◇) and the tyrosine is marked with a triangle (△).

[0022] Figure 4 It is a schematic diagram of the amino acid sequence of human type I collagen polypeptide IV; the RGD tripeptide structure is in the box, and the lysine is marked with a circle (◇) and the tyrosine is marked with a triangle (△).

[0023] Figure 5 It is a concentration-time curve of radioactive substances in the blood of rats after local administration in Experimental Example 1.

[0024] Figure 6 It is the cumulative excretion rate of radioactive substances in the feces and urine of rats after local administration in Experimental Example 1.

[0025] Figure 7 It is the concentration of radioactive substances in various tissues of rats at different times after local administration in Experimental Example 1.

[0026] Figure 8 It is PET imaging of rats at different times after local administration in Experimental Example 2. DETAILED DESCRIPTION

[0027] The present application will be further described below in conjunction with specific embodiments, but the scope of protection of the present application is not limited thereto.

[0028] Polypeptide radionuclide carrier The present application provides a polypeptide radionuclide carrier made from raw materials including the following weight parts: Human type I collagen polypeptide, double-bond functional group compound, and chelating agent.

[0029] According to one embodiment of the present application, the human-derived type I collagen polypeptide can be selected from at least one of polypeptides with amino acid sequences as shown in SEQ ID No. 1-4.

[0030] In the present application, SEQ ID No. 1 is GKSGDRGEYG PAGPAGPVGP VGARGPAGPQ GPRGDKGEYGEQGDRGIKGH. SEQ ID No. 2 is PKGDRGDAGP KGADGSPGKD GVRGLYGPIG PPGPAGAPGDKGESGPSGPA. SEQ ID No. 3 is AAGQPGAKGERGAKGPKGEN GVVGPYGPVG AAGPAGPNGPPGPAGSRGDG. SEQ ID No. 4 is PAGKHGNRGEYGPSGPVGPA GAVGPRGPSG PQGIRGDKGEPGEKGPRGLP.

[0031] According to one preferred embodiment of the present application, the human-derived type I collagen polypeptide is preferably one or two of polypeptides with amino acid sequences as shown in SEQ ID No. 1-4, and more preferably one of polypeptides with amino acid sequences as shown in SEQ ID No. 1-4.

[0032] The human-derived type I collagen polypeptide of the present application contains an RGD tripeptide structure, and such tripeptide structure has good targeting property. The human-derived type I collagen polypeptide of the present application also contains at least three lysines (K) and at least one tyrosine (Y), the lysine can be grafted with a double bond functional group and a chelating agent, and the tyrosine can be grafted with a non-metallic nuclide, and meanwhile, such structure can form a branched structure, and more double bond functional groups and chelating agents can be combined. In the present application, the double bond functional group can be cross-linked under the condition of a photoinitiator and light, and the chelating agent can combine with a metallic nuclide. Thus, the structure of the human-derived type I collagen polypeptide of the present application has targeting property, and can simultaneously combine with a metallic nuclide and a non-metallic nuclide, and has strong versatility; meanwhile, it can also be photo-cured to form a gel.

[0033] The human-derived type I collagen polypeptide of the present application can be screened and designed by any protein database or protein design tool known in the art, which is not particularly limited herein. For example, it can be UniProt database, SabDab database, BLAST tool, Alignment Viewer tool, etc., and preferably UniProt database and BLAST tool.

[0034] According to one embodiment of the present application, the degree of substitution of the double bond functional group compound to the amino group of lysine in the human type I collagen polypeptide can be 45-80%, preferably 50-80%, and more preferably 50-75%. The degree of substitution of the chelating agent to the amino group of lysine in the human type I collagen polypeptide can be 20-55%, preferably 20-50%, and more preferably 25-50%. A reasonable degree of substitution can ensure that the double bond functional group and the chelating agent are grafted to the human type I collagen polypeptide in a better ratio, thereby facilitating the versatility and photocuring performance of the human type I collagen polypeptide.

[0035] According to one embodiment of the present application, the double bond functional group compound can 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 bond functional group compound is conducive to grafting to lysine of the human type I collagen polypeptide and is more conducive to the photocuring performance of the human type I collagen polypeptide.

[0036] According to one embodiment of the present application, the chelating agent can 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 (diethylenetriaminepentaacetic 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 conducive to grafting to lysine of the human type I collagen polypeptide and is more conducive to the binding of the human type I collagen polypeptide to a metal radionuclide.

[0037] Method for preparing polypeptide radionuclide carrier The present application also provides a method for preparing the polypeptide radionuclide carrier as described above, which comprises a solid-phase synthesis polypeptide step, a double bond functional group modification step, and a chelating agent binding step. Details are described below.

[0038] Solid-phase synthesis polypeptide step The human type I collagen polypeptide is obtained by using a solid-phase synthesis method to cyclically couple and assemble amino acids, with the swollen Rink Amide resin as a solid-phase carrier.

[0039] According to one embodiment of the present application, in each cyclic coupling and assembly of amino acids, the Fmoc (9-fluorenylmethoxycarbonyl) protecting group eluent is used to remove the Fmoc protecting group on the Rink Amide resin to obtain a deprotection resin. Then, the Fmoc group-protected amino acid is coupled and assembled on the deprotection resin to obtain the human type I collagen polypeptide.

[0040] In the present application, the swollen Rink Amide resin can be added into the reactor of the solid phase synthesis device as the solid phase carrier for synthesis. According to one embodiment of the present application, the loading of the 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 device of the present application can be any type of solid phase device for polypeptide synthesis known in the art, which is not particularly limited herein. For example, it can be a solid phase polypeptide synthesizer.

[0041] According to one embodiment of the present application, the swelling agent for the swollen Rink Amide resin can be selected from at least one of DMF, dichloromethane, dichloroethane, preferably at least one of DMF and dichloromethane, and more preferably DMF. The amount of the swelling agent can be 10-50 mL, preferably 13-45 mL, and more preferably 15-40 mL, based on 1 g of the Rink Amide resin.

[0042] According to one embodiment of the present application, the Fmoc protecting group eluent can be selected from at least one of a DMF (N,N-dimethylformamide) solution of piperidine, a dichloromethane solution of piperidine, a DMF solution of diethylamine, and a dichloromethane solution of diethylamine, preferably at least one of a DMF solution of piperidine and a dichloromethane solution of piperidine, and more preferably a DMF solution of piperidine or a dichloromethane solution of piperidine. The mass concentration of the Fmoc protecting group eluent can be 10-30 wt%, preferably 12-28 wt%, and more preferably 15-25 wt%. The amount of the Fmoc protecting group eluent can be added according to the elution amount known in the art, which is not particularly limited in the art. For example, the amount of the Fmoc protecting group eluent can be 20-80 mL, preferably 30-75 mL, and more preferably 35-70 mL, based on 1 g of the swollen Rink Amide resin.

[0043] The reasonable Fmoc protecting group eluent can ensure the effective removal of the Fmoc protecting group on the Rink Amide resin, which is beneficial to the combination of the amino acid on the Rink Amide resin, thereby effectively synthesizing the polypeptide.

[0044] In the present application, the swelled Rink Amide resin is deprotected by washing with Fmoc protecting group eluent for 1 to 5 times, preferably 1 to 3 times, more preferably 2 to 3 times, to obtain a deprotected resin. Then, the deprotected resin is washed with a washing solution to remove the residual Fmoc protecting group eluent. The washing solution can be selected from at least one of DMF and dichloromethane, preferably DMF or dichloromethane, more preferably DMF. The washing can be performed for 1 to 5 times, preferably 1 to 3 times, more preferably 2 to 3 times. The amount of the washing solution can be 20 to 80 mL, preferably 25 to 70 mL, more preferably 30 to 65 mL. Such washing condition is more favorable for removing the residual Fmoc protecting group eluent.

[0045] In the present application, after washing the deprotected resin, an activation solution of Fmoc group protected amino acid is added to the deprotected resin to perform a coupling reaction, and the Fmoc group protected amino acid is coupled and assembled on the deprotected resin. The activation solution of Fmoc group protected amino acid is prepared by dissolving Fmoc group protected amino acid, HBTU (benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate) and N,N-diisopropyl ethylamine in DMF or dichloromethane. The molar ratio of Fmoc group protected amino acid, HBTU and N,N-diisopropyl ethylamine can be 1:1 to 3:1 to 5, preferably 1:1 to 2:2 to 4, more preferably 1:1:2 to 3. The amount of DMF or dichloromethane can be 8 to 20 mL, preferably 10 to 18 mL, more preferably 12 to 15 mL, based on 1 mmol of Fmoc group protected amino acid. The coupling reaction can be performed for 30 to 120 min, preferably 40 to 90 min, more preferably 45 to 60 min. Such coupling reaction is more favorable for coupling and assembling Fmoc group protected amino acid on the deprotected resin and further combining new amino acid. In the present application, after each coupling reaction, the reaction can be detected to determine whether the reaction is complete. The detection method can be any detection method known in the art, which is not particularly limited herein. For example, it can be an indantrione detection method.

[0046] In the present application, the above-mentioned 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 present application, after the human type I collagen polypeptide is assembled, a cleavage solution can be used to cleave the polypeptide from the deprotection resin, thereby obtaining the human type I collagen polypeptide. The cleavage solution mainly consists of trifluoroacetic acid, water, phenol and triisopropylsilane, and 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, and more preferably 35-45: 1-3: 1-3: 1-3. The cleavage temperature can be 15-60°C, preferably 20-50°C, and more preferably 22-40°C. The cleavage time can be 20-80 min, preferably 30-70 min, and more preferably 40-65 min. Reasonable cleavage solution and cleavage conditions are conducive to cleaving the synthesized human type I collagen polypeptide from the deprotection resin and can ensure the removal of the amino acid protecting group of the side chain of the polypeptide.

[0047] In the present application, the step of subjecting the cleaved human type I collagen polypeptide to solid-liquid separation, precipitation, secondary solid-liquid separation and purification can also be included. Both the primary solid-liquid separation and the secondary solid-liquid separation can be achieved using the solid-liquid separation methods and equipment known in the art, which are not particularly limited here. For example, it can be suction filtration, centrifugal separation, etc. The precipitation can use various ether solvents known in the art for precipitating peptides, which are not particularly limited here. For example, it can be methyl tert-butyl ether, diethyl ether, petroleum ether, isopropyl ether, etc. The ether solvents are all cold ether solvents, and the temperature can be up to 4°C, preferably -20-4°C, and more preferably -20-0°C. The purification can be achieved using any purification method known in the art, which is not particularly limited here. For example, high performance liquid chromatography (HPLC) can be used, etc.

[0048] According to one preferred embodiment of the present application, the cleaved human type I collagen polypeptide can be subjected to primary solid-liquid separation to obtain a crude peptide. Then, the crude peptide is precipitated using a cold ether solvent. Then, the precipitated crude peptide is subjected to secondary solid-liquid separation to obtain the human type I collagen polypeptide, and the human type I collagen polypeptide is purified using HPLC.

[0049] According to one embodiment of the present application, the purified human type I collagen polypeptide can also be subjected to molecular weight identification, thereby confirming whether the product is correctly synthesized. The molecular weight identification can be achieved using any identification method known in the art, which is not particularly limited here. For example, mass spectrometry can be used, etc.

[0050] The water used in each step of the present application 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.

[0051] double bond functional group modification step The human type I collagen polypeptide is mixed with the double bond functional group compound, and the reaction is carried out at 40-65°C to obtain a double bond functional group modified polypeptide.

[0052] In this step, the double bond functional group compound is the same as the double bond functional group compound in the polypeptide carrier as described above, and will not be described here.

[0053] In the present application, the degree of substitution of the double bond functional group compound for the amino group of lysine in the human type I collagen polypeptide is the same as the double bond functional group compound in the polypeptide carrier as described above, and will not be described here. Based on the degree of substitution, the molar ratio of the human type I collagen polypeptide to the double bond functional group compound can be 1:1.3-3.5, preferably 1:2-3.5, and more preferably 1:2-3.

[0054] According to one embodiment of the present application, the reaction temperature can be 40-65°C, preferably 45-65°C, and more preferably 45-60°C. The reaction time can be 1-5h, preferably 1.5-4.5h, and more preferably 2-4h. Such reaction conditions are more conducive to the substitution of the double bond functional group for the amino group of lysine in the polypeptide.

[0055] In the present application, the human type I collagen polypeptide and the double bond functional group compound are reacted in water. Based on 1 mmol of human type I collagen polypeptide, the amount of water used can be 150-400 mL, preferably 200-350 mL, and more preferably 250-300 mL.

[0056] In the present application, the reaction product solution obtained after the reaction can be diluted with water, and the diluted solution can be loaded into 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 double bond functional group modified polypeptide.

[0057] According to one embodiment of the present application, the amount of water used for dilution can be 1-5 times, preferably 2-5 times, and more preferably 2-4 times the amount of water used in the reaction. The molecular weight cut-off of the dialysis bag can be 800-3000 Da, preferably 900-2500 Da, and more preferably 1000-2000 Da. The dialysis temperature can be 20-60°C, preferably 22-50°C, and more preferably 24-45°C. The dialysis time can be 1-5d, preferably 2-5d, and more preferably 2-4d. Such dialysis conditions are conducive to the removal of residual double bond functional group compounds.

[0058] According to one embodiment of the present application, the filtration can be achieved by using any filtration method and filtration device known in the art, which is not particularly limited here. For example, it can be suction filtration or the like. The drying can be achieved by using any drying method known in the art, which is not particularly limited here. For example, it can be freeze-drying, vacuum drying, rotary evaporation or the like.

[0059] Chelator step The double-bond functional group modified polypeptide is mixed with the activated precursor of the chelator, and reacted at 20-45°C to obtain the polypeptide radionuclide carrier.

[0060] In this step, the chelator is the same as the chelator in the polypeptide radionuclide carrier described above, which is not repeated here.

[0061] According to one embodiment of the present application, the activated precursor of the chelator can be an NHS ester of the chelator.

[0062] In the present application, the degree of substitution of the chelator to the amino group of the lysine in the human type I collagen polypeptide is the same as the chelator in the polypeptide radionuclide carrier described above, which is not repeated here. Based on the degree of substitution, the molar ratio of the human type I collagen polypeptide to the activated precursor of the chelator can be 1:0.5-3, preferably 1:1-3, and more preferably 1:1-2.

[0063] According to one embodiment of the present application, the temperature of the reaction can be 20-45°C, preferably 22-40°C, and more preferably 25-35°C. The time of the reaction can be 8-20h, preferably 10-19h, and more preferably 12-18h. Such reaction conditions are more conducive to the substitution of the chelator to the amino group of the lysine in the polypeptide.

[0064] In the present application, the reaction of the human type I collagen polypeptide and the activated precursor of the chelator is carried out in an organic solvent. The organic solvent can be selected from at least one of DMSO (dimethyl sulfoxide), DMF, dichloromethane, preferably at least one of DMSO and DMF, and more preferably DMSO or DMF. The amount of the organic solvent can be 30-100mL, preferably 40-80mL, and more preferably 45-70mL, based on 1mmol of the double-bond functional group modified polypeptide. According to one preferred embodiment of the present application, an acid-binding agent can also be added to the reaction to provide an alkaline environment for the reaction and promote the progress of the reaction. The acid-binding agent can be selected from at least one of triethylamine, DIPEA (N,N-diisopropylethylamine), pyridine, 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-bond functional group modified polypeptide can be 2-8:1, preferably 2.5-7:1, and more preferably 3-6:1.

[0065] In the present application, a stirring step can also be included in the reaction process. The stirring can be achieved using any stirring device known in the art, which is not particularly limited here. For example, vortex stirring, ultrasonic stirring, etc. can be used. The stirring speed can be 100-800 r / min, preferably 200-600 r / min, and more preferably 250-500 r / min. The reaction product solution obtained after the reaction can also be loaded into 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 the polypeptide radionuclide carrier.

[0066] According to one embodiment of the present application, the molecular weight cut-off of the dialysis bag can be 800-3000 Da, preferably 900-2500 Da, and more preferably 1000-2000 Da. The dialysis temperature can be 20-60°C, preferably 22-50°C, and more preferably 24-45°C. The dialysis time can be 1-5 d, preferably 2-5 d, and more preferably 2-4 d. Such dialysis conditions are conducive to removing the activated precursors of the residual chelating agent.

[0067] According to one embodiment of the present application, the filtration can be achieved using any filtration method and filtration device known in the art, which is not particularly limited here. For example, suction filtration, etc. can be used. The drying can be achieved using any drying method known in the art, which is not particularly limited here. For example, freeze-drying, vacuum drying, rotary evaporation, etc. can be used.

[0068] Use of the polypeptide radionuclide carrier The present application also provides the use of the above polypeptide radionuclide carrier in the preparation of a nuclear medical detection product or a radiotherapy product.

[0069] According to one embodiment of the present application, the nuclear medical detection product can be selected from any tracer or imaging agent for any nuclear medical detection device known in the art, and is preferably a tracer or imaging agent for at least one of PET (positron emission computed tomography), PET / CT (positron emission computed tomography and computed tomography integrated machine), and PET / MRI (positron emission computed tomography and nuclear magnetic resonance imaging integrated machine).

[0070] According to one embodiment of the present application, the radiotherapy product can be selected from any radiotherapy drug known in the art, and is preferably a radiotherapy drug for treating at least one of brain glioma, breast cancer, liver cancer, lung cancer, and thyroid disease.

[0071] Radionuclide probe The present application also provides a radionuclide probe made of the above polypeptide radionuclide carrier and a radionuclide.

[0072] According to one embodiment of the present application, the molar ratio of the polypeptide radionuclide carrier and the radionuclide can be 1:1-5, preferably 1:1-4, and more preferably 1:1.5-4.

[0073] According to one embodiment of the present application, the radionuclide can be selected from at least one of metal radionuclides and non-metal radionuclides, preferably metal radionuclides and non-metal radionuclides. According to one preferred embodiment of the present application, the molar ratio of the metal radionuclides and the non-metal radionuclides can be 0.25-4:1, preferably 0.4-3:1, and more preferably 0.5-2:1. Such a ratio can ensure that the proportion of the metal radionuclides and the non-metal radionuclides in the radionuclide probe is clear and both can be stably combined on the polypeptide radionuclide carrier.

[0074] According to one embodiment of the present application, the metal radionuclides can be selected from at least one of Lu, Y, Ga, Cu, Tc, and Sr, preferably at least one of Lu, Y, Tc, and Sr, and more preferably at least one of Lu, Y, and Tc. 177 Lu, 90 Y, 67 Ga, 64 Cu, 99m Tc, 89 Sr. 177 Lu, 90 Y, 99m Tc, 89 Sr. 177 Lu, 90 Y, 99m Tc. 125 I, 131 I, 18 F, 32 P. 125 I, 131 I, 18 F. 125 I, 131 I.

[0075] The radionuclide probe of the present application simultaneously carries metal radionuclides and non-metal radionuclides in a clear proportion, and has strong versatility. It can be used to prepare both nuclear medical detection products and radiotherapy products.

[0076] Preparation method of the radionuclide probe The present application also provides a preparation method of the radionuclide probe as described above, which comprises a mixing reaction step. The following will be described in detail.

[0077] Mixing reaction step The polypeptide radionuclide carrier and the radionuclide are mixed and reacted at 20-60°C to obtain the radionuclide probe.

[0078] In the present step, the nuclide is the same as the nuclide in the above-mentioned nuclide probe, which will not be described again. In the reaction, a salt corresponding to the nuclide can be used. According to an embodiment of the present application, the salt of the metal nuclide can be at least one selected from the group consisting of halide, nitrate, sulfate, phosphate, acetate of the metal nuclide, preferably at least one selected from the group consisting of halide, nitrate of the metal nuclide, and more preferably at least one selected from the group consisting of chloride, bromide of the metal nuclide. The salt of the non-metal nuclide can be at least one selected from the group consisting of alkali metal salt of the non-metal nuclide, preferably at least one selected from the group consisting of sodium salt of the non-metal nuclide, potassium salt of the non-metal nuclide, and more preferably sodium salt of the non-metal nuclide or potassium salt of the non-metal nuclide.

[0079] According to an embodiment of the present application, the temperature of the reaction can be 20-60°C, preferably 22-55°C, and more preferably 24-50°C. The time of the reaction can be 0.5-5h, preferably 0.8-4h, and more preferably 1-3h.

[0080] According to a preferred embodiment of the present application, when the nuclide is a metal nuclide, the temperature of the reaction of the polypeptide nuclide carrier with the metal nuclide can be 30-60°C, preferably 35-55°C, and more preferably 40-50°C. The time of the reaction of the polypeptide nuclide carrier with the metal nuclide can be 0.5-5h, preferably 0.8-4h, and more preferably 1-3h. Such reaction conditions are favorable for the binding of the metal nuclide to the chelator of the polypeptide nuclide carrier.

[0081] According to a preferred embodiment of the present application, when the nuclide is a metal nuclide, the molar ratio of the polypeptide nuclide carrier to the metal nuclide can be 1:1-3, preferably 1:1.5-3, and more preferably 1:1.5-2.5.

[0082] In the present application, when the polypeptide nuclide carrier is reacted with the metal nuclide, the polypeptide nuclide carrier can be dissolved in the ammonium acetate buffer, and then the metal nuclide is added. The reaction system can be stirred to promote the mixing of the reaction system.

[0083] According to an embodiment of the present application, the pH of the ammonium acetate buffer can be 3.5-6, preferably 3.8-5.5, and more preferably 4-5. The concentration of the ammonium acetate buffer can be 0.1-1M, preferably 0.2-0.8M, and more preferably 0.3-0.7M. The amount of the ammonium acetate buffer can be 5-30mL, preferably 6.5-25mL, and more preferably 8-20mL, based on 1g of the polypeptide nuclide carrier.

[0084] According to one embodiment of the present application, the stirring can be achieved by using any stirring mode known in the art, which is not particularly limited herein. For example, the stirring can be vortex stirring, ultrasonic stirring, or the like. The stirring speed can be 100-800 r / min, preferably 200-600 r / min, and more preferably 250-500 r / min.

[0085] In the present application, when the polypeptide radionuclide carrier is reacted with the metal radionuclide, the reaction product obtained after the reaction can also be separated and purified by using an ultrafiltration centrifugation mode to obtain the radionuclide probe. The ultrafiltration membrane used in the ultrafiltration centrifugation can have a molecular weight cut-off of 500-2000 Da, preferably 800-1500 Da, and more preferably 900-1200 Da. The rotation speed of the 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 the unreacted metal radionuclide ions and impurities such as buffer solution having a molecular weight less than the molecular weight cut-off of the ultrafiltration membrane can smoothly pass through the ultrafiltration membrane into the filtrate, while the radionuclide probe loaded with the metal radionuclide is effectively intercepted in the ultrafiltration tube, thereby achieving efficient separation and purification of the metal radionuclide probe.

[0086] According to one preferred embodiment of the present application, when the radionuclide is a non-metal radionuclide, the polypeptide radionuclide carrier can be reacted with the non-metal radionuclide at a temperature of 20-45°C, preferably 22-40°C, and more preferably 25-35°C. The polypeptide radionuclide carrier can be reacted with the metal radionuclide for a time period of 0.5-5 h, preferably 0.8-4 h, and more preferably 1-3 h. Such reaction conditions are conducive to the substitution reaction of the tyrosine in the polypeptide radionuclide carrier with the non-metal radionuclide, thereby binding to the polypeptide radionuclide carrier.

[0087] According to one preferred embodiment of the present application, when the radionuclide is a non-metal radionuclide, the molar ratio of the polypeptide radionuclide carrier to the metal radionuclide can be 1:1-3, preferably 1:1.5-3, and more preferably 1:1.5-2.5.

[0088] In the present application, when the polypeptide radionuclide carrier is reacted with the non-metal radionuclide, the polypeptide radionuclide carrier can be dissolved in a phosphate buffer solution (PBS), and then the non-metal radionuclide and an oxidizing agent solution can be added. The reaction system can be stirred to promote uniform mixing. The oxidizing agent can activate the non-metal radionuclide, so that the activated non-metal radionuclide can undergo a substitution reaction with the tyrosine in the polypeptide radionuclide carrier. The oxidizing agent can be selected from at least one of N-chloro-4-methylbenzenesulfonamide sodium, hydrogen peroxide, and sodium nitrite, preferably at least one of N-chloro-4-methylbenzenesulfonamide sodium and hydrogen peroxide, and more preferably N-chloro-4-methylbenzenesulfonamide sodium.

[0089] According to one embodiment of the present application, the pH 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 0.01-0.1 M, preferably 0.02-0.08 M, and more preferably 0.03-0.07 M. The amount of the phosphate buffer can be 5-30 mL, preferably 6.5-25 mL, and more preferably 8-20 mL, based on 1 g of the polypeptide nuclide carrier.

[0090] According to one embodiment of the present application, the oxidizing agent solution can be prepared by dissolving an oxidizing agent in PBS. The concentration of the oxidizing agent can be 0.5-5 mg / mL, preferably 0.8-4 mg / mL, and more preferably 1-3 mg / mL. The pH of the PBS can be 5-8, preferably 5.5-7.5, and more preferably 6-7. The concentration of the PBS can be 0.01-0.1 M, preferably 0.02-0.08 M, and more preferably 0.03-0.07 M. The amount of the oxidizing agent solution can be 5-30 mL, preferably 6.5-25 mL, and more preferably 8-20 mL, based on 1 g of the polypeptide nuclide carrier.

[0091] According to one embodiment of the present application, the stirring can be achieved by using any stirring method known in the art, which is not particularly limited herein. For example, the stirring can be vortex stirring, ultrasonic stirring, or the like. The stirring speed can be 100-800 r / min, preferably 200-600 r / min, and more preferably 250-500 r / min.

[0092] According to one embodiment of the present application, after the reaction is completed, a terminating agent solution can be further added to the reaction system after the reaction to terminate the reaction. Then, a salt solution of the 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 to the reaction container.

[0093] According to one preferred embodiment of the present application, the terminating agent can be at least one selected from the group consisting of sodium thiosulfate, ascorbic acid, cysteine, and glucose, preferably at least one selected from the group consisting of sodium thiosulfate and ascorbic acid, and more preferably sodium thiosulfate. The concentration of the terminating agent solution can be 5-20 mg / mL, preferably 6-15 mg / mL, and more preferably 8-12 mg / mL. The amount of the terminating agent solution can be 0.5-10 mL, preferably 0.7-8 mL, and more preferably 1-5 mL, based on 1 g of the polypeptide nuclide carrier.

[0094] According to a preferred embodiment of the present application, the salt of non-metallic element can be at least one selected from alkali metal salt of non-metallic element, preferably at least one selected from lithium salt, sodium salt and potassium salt of non-metallic element, more preferably at least one selected from sodium salt and potassium salt of non-metallic element. The concentration of the salt solution of non-metallic element can be 5-20 mg / mL, preferably 7-18 mg / mL, more preferably 9-15 mg / mL. The amount of the salt solution of non-metallic element can be 0.5-10 mL, preferably 0.8-7 mL, more preferably 1-5 mL, based on 1 g of polypeptide element carrier.

[0095] In the present application, when the polypeptide element carrier is reacted with the non-metallic element, the reaction product obtained after the reaction can also be separated and purified by ultrafiltration centrifugation to obtain the element probe. The molecular weight cut-off of the ultrafiltration membrane used in the ultrafiltration centrifugation can be 500-2000 Da, preferably 800-1500 Da, more preferably 900-1200 Da. The rotation speed of the ultrafiltration centrifugation can be 2000-5000 r / min, preferably 2200-4500 r / min, more preferably 2500-4000 r / min. Such ultrafiltration centrifugation conditions can ensure that the unreacted non-metallic element ions and impurities such as buffer which are smaller than the molecular weight cut-off of the ultrafiltration membrane can smoothly pass through the ultrafiltration membrane into the filtrate, while the element probe loaded with the non-metallic element is effectively intercepted in the ultrafiltration tube, thereby realizing efficient separation and purification of the non-metallic element probe.

[0096] According to a preferred embodiment of the present application, when the element is a metallic element and a non-metallic element, the metallic element or the non-metallic element can be alternately combined on the polypeptide element carrier by referring to the above-mentioned combination method of the metallic element or the non-metallic element with the polypeptide element carrier, and the specific steps are not described here. The combination order of the metallic element and the non-metallic element is not sequential.

[0097] Use of element probe The present application also provides the use of the above-mentioned element probe in the preparation of a nuclear medical detection product or a radiotherapy product.

[0098] According to an embodiment of the present application, the nuclear medical detection product can be selected from any tracer or imaging agent for the nuclear medical detection equipment known in the art, preferably a tracer or imaging agent for at least one of PET, PET / CT and PET / MRI equipment.

[0099] According to an embodiment of the present application, the radiotherapy product can be selected from any radiotherapy drug known in the art, preferably a radiotherapy drug for treating at least one of brain glioma, breast cancer, liver cancer, lung cancer and thyroid disease.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] Test methods HPLC purification 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.

[0104] Molecular weight identification by mass spectrometry The purified polypeptide molecular weight was identified by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF, produced by Bruker, Germany). The polypeptide aqueous solution with a concentration of 2 mg / mL was prepared, and the sample was dropped onto the detection plate by spotting method. After air-drying at room temperature, the detection was performed.

[0105] Detection of radioactive substance concentration After administration, the samples of rats at different time points were collected for detection, and the radioactivity was detected by using a gamma counter (2470, produced by PerkinElmer).

[0106] Raw material description The raw materials of the following examples were commercially available products unless otherwise specified.

[0107] Among them, the Fmoc-protected amino acid was purchased from Genview Biotech Co., Ltd. Methyl acrylate was purchased from Aladdin. DOTA-NHS was purchased from Aladdin. 177 LuCl3 was purchased from PerkinElmer. Na 125 I was purchased from PerkinElmer.

[0108] The rats were SD (Sprague-Pawley) type mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0109] The PET was NanoScan PET / CT type of Mediso.

[0110] Preparation examples 1-4 1. Screening and design of human type I collagen polypeptide The human type I collagen polypeptide was screened and designed through the UniProt database, and four human type I collagen polypeptides containing RGD tripeptide structure, lysine (K) and tyrosine (Y) were designed, and the specific information is as follows: 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.

[0111] The polypeptide I is derived from the Collagen alpha-1 (I) chain protein of the alpha 1 chain of human type I collagen protein, which has a total of 1464 amino acids, and the gene encoding the protein is COL1A1.

[0112] It can be seen that the polypeptide I contains one RGD tripeptide structure, three lysines and one tyrosine. Figure 1

[0113] ​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.

[0114] Polypeptide II is derived from the Collagen alpha-1 (I) chain preproprotein protein of the alpha 1 chain of human type I collagen protein, which has 1091 amino acids, and the gene encoding the protein is COL1A1.

[0115] As can be seen from Figure 2 Polypeptide II contains an RGD tripeptide structure, 4 lysines and 1 tyrosine.

[0116] 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.

[0117] Preparation Example 3: Polypeptide III is derived from the Collagen alpha-2 (I) chain protein of the alpha 2 chain of human type I collagen protein, which has 1366 amino acids, and the gene encoding the protein is COL1A2.

[0118] As can be seen from Figure 3 Polypeptide III contains an RGD tripeptide structure, 3 lysines and 1 tyrosine.

[0119] Preparation Example 4: The amino acid sequence of human type I collagen polypeptide IV (denoted as polypeptide IV) is shown in SEQ ID No. 4: PAGKHGNRGE YGPSGPVGPA GAVGPRGPSG PQGIRGDKGE PGEKGPRGLP.

[0120] Polypeptide IV is derived from the Collagen alpha-2 (I) chain protein of the alpha 2 chain of human type I collagen protein, which has 1366 amino acids, and the gene encoding the protein is COL1A2.

[0121] As can be seen from Figure 4 Polypeptide III contains an RGD tripeptide structure, 3 lysines and 1 tyrosine.

[0122] 2. Synthesis of human type I collagen protein peptides The polypeptides I-IV with a target scale of 0.2 mmol (counted as 1 eq) were prepared by using a solid-phase synthesis method and a Fmoc (9-fluorenylmethoxycarbonyl) protection strategy, using the following steps: 1) A Rink Amide resin with a loading of 0.2 mmol / g was pre-swelled with 20 mL of DMF. Then, the pre-swelled Rink Amide resin with a mass of 1 g and a loading of 0.2 mmol / g was loaded into a reactor of a solid-phase polypeptide synthesizer as a solid-phase carrier for synthesizing the polypeptide.

[0123] 2) Amino acid coupling cycle: In each cycle, the Rink Amide resin was eluted twice with 60 mL of a 20 wt% piperidine DMF solution to obtain a deprotected resin. Then, the deprotected resin was washed twice with 60 mL of DMF each time. Then, 4 eq of a Fmoc group-protected amino acid (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 activation solution of the Fmoc group-protected amino acid. The activation solution of the Fmoc group-protected amino acid was added to the deprotected resin, and a coupling reaction was performed at room temperature (25°C, the same below) for 60 min. After the reaction was completed, the reaction was confirmed to be complete by a ninhydrin detection method.

[0124] After the coupling was completed, the deprotected 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 polypeptide was assembled.

[0125] 3) 9.25 mL of trifluoroacetic acid, 25 mL of water, 0.25 mL of phenol, and 0.25 mL of triisopropylsilane were mixed to obtain a cleavage solution. Then, the deprotected resin with the polypeptide connected thereto was cleaved using the cleavage solution at room temperature for 3 h to obtain a reaction solution of the polypeptide and remove side chain protection groups of the polypeptide.

[0126] 4) The reaction solution of the polypeptide was suction-filtered using a sand core funnel, and the filter residue was collected. The filter residue was added to methyl tert-butyl ether at -20°C to precipitate a crude polypeptide. Then, the crude polypeptide was centrifuged at 3000 r / min for 5 min, and the supernatant was discarded to obtain a crude product of the polypeptide. The crude product of the polypeptide was purified by HPLC, and the molecular weight was identified by mass spectrometry to prepare the polypeptide.

[0127] Examples 1-6 1. Modification of double bond functional groups 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.

[0128] Table 1. Degree of substitution of double bond functional groups in peptides modified with different double bond functional groups

[0129] 2. Combined chelating agents 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.

[0130] Table 2. Degree of DOTA substitution in different polypeptide nuclide carriers

[0131] Examples 7-12 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: Take 1 g (0.18 mmol, 1 eq) of polypeptide nuclide carrier I-VI prepared in Examples 1-6, respectively, and dissolve in 10 mL of 0.5 M ammonium acetate buffer solution with pH=5, then add 177 LuCl3, to obtain a mixed solution. The mixed solution is reacted at 45°C and 300 r / min vortex stirring for 1 h to obtain a reaction product solution. The reaction product solution is ultrafiltration centrifuged at 3000 r / min for 5 min under the condition of an ultrafiltration membrane with a molecular weight cut-off of 1000 Da, and the retentate in the ultrafiltration tube is taken to obtain a nuclide probe loaded with 177 Lu. 177 The amount of LuCl3and the 177 Lu loading amount of each nuclide probe is shown in Table 3.

[0132] Table 3 177 The amount of LuCl3and the 177 Lu loading amount

[0133] Examples 13-19 Polypeptide nuclide carriers I-VI prepared in Examples 1-6 are taken as carriers, respectively, to prepare nuclide probes loaded with non-metallic nuclides 125 I, and the specific steps are as follows: Take 1 g (0.18 mmol, 1 eq) of polypeptide nuclide carrier I-VI prepared in Examples 1-6, respectively, and dissolve in 10 mL of 0.5 M PBS buffer solution with pH=7, then add Na 125 I and 10 mL of 1 mg / mL N-chloro-4-toluenesulfonamide sodium salt PBS solution (0.5 M PBS buffer solution, pH=7) to obtain a mixed solution. The mixed solution is reacted at room temperature and 300 r / min vortex stirring for 1 h to obtain a reaction product solution. Then, 1 mL of 10 mg / mL sodium thiosulfate aqueous solution is added to the reaction product solution to terminate the reaction, and 1 mL of 10 mg / mL potassium iodide aqueous solution is added to compete for the unreacted active 125 I, to obtain an inactivated reaction product solution.

[0134] The inactivated reaction product solution is ultrafiltration centrifuged at 3000 r / min for 5 min under the condition of an ultrafiltration membrane with a molecular weight cut-off of 1000 Da, and the retentate in the ultrafiltration tube is taken to obtain a nuclide probe loaded with 125 I. The amount of Na 125 I and the 125 I loading amount of each nuclide probe is shown in Table 4.

[0135] Table 4 Na 125I and each nuclide probe 125 I loading amount

[0137] Examples 20-26 The polypeptide nuclide carriers I-VI prepared in Examples 1-6 were used as carriers respectively to prepare nuclide probes simultaneously loaded with 177 Lu and 125 I, and the specific steps were as follows: 1) Loading metal nuclide 177 Lu Referring to the preparation method of Examples 7-12, the 177 Lu was loaded on the polypeptide nuclide carrier to obtain a nuclide probe loaded with 177 Lu.

[0138] 2) Loading non-metal nuclide 125 I

[0139] Referring to the preparation method of Examples 13-19, the 125 I was loaded on the nuclide probe loaded with 177 Lu to obtain a nuclide probe simultaneously loaded with 177 Lu and 125 I.

[0140] The order of the above steps 1) and 2) can be interchanged.

[0141] 177 The amount of LuCl3, the amount of Na 125 I, and the molar ratio of 177 Lu and 125 I loaded in the nuclide probe are shown in Table 5 below.

[0142] Table 5 77 The amount of LuCl3, the amount of Na 125 I, and the molar ratio of 177 Lu and 125 I loaded in the nuclide probe

[0143] Application Example The nuclide probe XIV prepared in Example 20 was placed in deionized water to prepare a nuclide 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 nuclide probe solution and 1 mL of the LAP solution were taken respectively and mixed uniformly to obtain a mixed solution. The mixed solution was irradiated under a light source with a power of 10 W and a wavelength of 405 nm for 10 s, and the nuclide probe was solidified to form a gel.

[0144] Experimental Example 1 Thirty-two 6-week-old SD rats, half male and half female, weighing 180-200 g were selected as experimental subjects. After the rats were anesthetized, a craniotomy was performed, and 1 mL of a mixed solution containing LAP and the nuclide probe XIV obtained in the experimental example was sprayed on the dura mater. Then, irradiation was performed for 10 s under the condition of blue light with a power of 10 W and a wavelength of 405 nm, and curing was performed. After curing, a craniotomy was performed.

[0145] 1. In vivo blood concentration detection The blood of all rats at different time points after the operation was collected, 120 μL of whole blood sample was taken to detect the radioactivity of the nuclide probe, and the average value was evaluated to assess the blood concentration, and the results are shown in Table 1. Figure 5 As can be seen from Table 1, the nuclide in the nuclide probe is absorbed into the blood in a small amount and is slowly absorbed. The concentration of the blood metabolite reaches a peak at 16 days, and then is slowly eliminated, and the concentration is close to the baseline at 90 days. This shows that the nuclide probe of the present application can be used for local administration and the blood concentration after administration is low, which can effectively reduce the systemic radioactive injury. Figure 5 2. In vivo metabolism experiment

[0146] The urine and feces of all rats at different time points after the operation were collected for radioactivity detection and the average value was taken. At the same time, 30 mL of 50 vol% ethanol aqueous solution was used to clean the entire cage, and 1 mL of the cleaning solution was measured for radioactivity concentration, so as to calculate the total radioactivity of all cleaning solutions. The results are shown in Table 2. As shown in Table 2, the nuclide in the nuclide probe of the present application is mainly excreted through urine and a small amount is excreted through feces. 97 days after administration, the total recovery rate of feces and urine is 67.3%, the radioactivity of the cleaning solution accounts for 7.4% of the total administered radioactivity, and the total recovery rate is 74.7%. According to the trend of the excretion rate, it can be inferred that the nuclide probe of the present application can be used for local administration and the nuclide can be completely metabolized in the body. Figure 6 3. In vivo tissue distribution of the nuclide probe

[0147] The whole blood and organ tissues of all rats at different time points after the operation were collected for radioactivity detection and the average value was taken, and the results are shown in Table 3. As can be seen from Table 3, the nuclide in the nuclide probe of the present application is mainly distributed in the administration site and the kidneys, and is rarely distributed in other tissues such as fat and muscle. This shows that the nuclide probe of the present application has low free nature, which effectively reduces the radioactive injury to other tissues. Figure 7 Figure 7 Experimental Example 2 Referring to Experimental Example 1, one rat was administered with the intracranial nuclide probe.

[0148] Experimental Example 2

[0149] ​The rats were subjected to PET imaging at different time points after the operation, and the results are shown in Figure 8 As can be seen from Figure 8 , the radioactive signal at the administration site was strong on the 4th and 16th day after administration, and the radioactive signal was significantly weakened on the 60th and 90th day. The radioactive signal of other parts such as the kidney and liver was extremely low or almost none. This shows that the nuclide probe of the application can be effectively fixed at the administration site, has low free nature, and can effectively reduce the radioactive damage to other tissues. At the same time, from Figure 8 , it can also be known that the nuclide probe of the application can be effectively used for intracranial imaging. The nuclide probe of the application can be used for preparing a nuclear medicine detection product or a radiotherapy product.

[0150] The application is not limited to the above-mentioned embodiments, and any modification, improvement or replacement that can be conceived by those skilled in the art without departing from the essential content of the application falls within the scope of the application.

Claims

1. A polypeptide nuclide carrier, characterized in that, The polypeptide nuclide carrier is made from the following raw materials: Human-derived type I collagen peptides, compounds with double bond functional groups, and chelating agents; among which, 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; 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%. The double-bonded functional group compound is selected from at least one of acrylic acid, methacrylic acid, acrylic anhydride, and methacrylic anhydride; The chelating agent is selected from at least one of DOTA, NOTA, EDTA, and DTPA.

2. A method for preparing a polypeptide nuclide carrier according to claim 1, comprising the following steps: 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. 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. 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.

3. The preparation method according to claim 2, characterized in that: 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%. In step C), the activating precursor of the chelating agent is an NHS ester of the chelating agent.

4. The preparation method according to claim 2, characterized in that: In step B), the reaction time is 1 to 5 hours; In step C), the reaction time is 8 to 20 hours.

5. The use of the polypeptide nuclide carrier according to claim 1 in the preparation of nuclear medicine detection products or radiotherapy products.

6. A radionuclide probe, characterized in that, The nuclide probe is made from a polypeptide nuclide carrier and a nuclide as described in claim 1; wherein the molar ratio of the polypeptide nuclide carrier and the nuclide is 1:1 to 5; 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.

7. The radionuclide probe according to claim 6, characterized in that, The nuclides are metallic nuclides and non-metallic nuclides, with a molar ratio of metallic nuclides to non-metallic nuclides of 0.25 to 4:

1.

8. A method for preparing a radionuclide probe according to claim 6 or 7, comprising the following steps: The polypeptide nuclide carrier described in claim 1 is mixed with a nuclide and reacted at 20–60°C to obtain a nuclide probe.

9. Use of the radionuclide probe according to claim 6 or 7 in the preparation of nuclear medicine detection products or radiotherapy products.

10. The use according to claim 9, characterized in that, 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. 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.

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