CN117126232A - Enzymatic self-assembled molecular probes 68 Ga-Nap-Yp and preparation method and application thereof - Google Patents
Enzymatic self-assembled molecular probes 68 Ga-Nap-Yp and preparation method and application thereof Download PDFInfo
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- CN117126232A CN117126232A CN202311130705.0A CN202311130705A CN117126232A CN 117126232 A CN117126232 A CN 117126232A CN 202311130705 A CN202311130705 A CN 202311130705A CN 117126232 A CN117126232 A CN 117126232A
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
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Abstract
The invention relates to the field of radiopharmaceuticals, namely nuclear medicine, in particular to an enzymatic self-assembled molecular probe 68 Ga-Nap-Yp, a preparation method and application thereof. The invention provides an enzymatic self-assembled molecular probe 68 Ga-Nap-Yp with the structural formula of 2-naphthylacetic acid-Phe-Phe-Tyr (H) 2 PO 3 )‑Lys(DOTA‑ 68 Ga)‑OH( 68 Ga-Nap-Yp) can be recognized and cleaved by alkaline phosphatase highly expressed in tumor cells to produce a hydrophobic product 68 Ga-Nap-Y is enriched in a great amount at the tumor part and self-assembled to form nano fiber, so that PET imaging of the tumor is enhanced. In addition, the nanofibers are connected with each other to form a three-dimensional network structure and are attached to the periphery of tumor tissues, so that the elimination in the tumor is greatly delayed, and the tumor is caused 68 The residence of Ga at the tumor site eventually prolongs the tumor imaging time.
Description
Technical Field
The invention relates to the field of radiopharmaceuticals, namely nuclear medicine, in particular to an enzymatic self-assembled molecular probe 68 Ga-Nap-Yp, a preparation method and application thereof.
Background
Imaging technology is one of the common means of clinically screening cancer and monitoring the therapeutic effect of drugs on malignant tumors. The intuitive nature of tumor imaging, compared to other cancer detection methods, facilitates the assessment of results by a clinician, which is non-invasive in avoiding physical damage or even complications that may be caused to the patient by invasive examination (e.g., puncture or biopsy). In various tumor imaging methods, positron Emission Tomography (PET) is a molecular imaging technique that detects specific molecular level changes before changes in tumor cells evolve into functional changes and subsequent morphological changes. This meets the clinical requirements for early detection of cancer to a great extent. In addition, PET is capable of scanning multiple levels throughout the body at one time, and in addition to primary lesions, it is also capable of finding metastatic lesions in various parts of the body, and is therefore critical for determining tumor stage. Because of these advantages, PET imaging has become one of the most promising tumor imaging techniques in the clinic. However, existing PET probes are mainly small molecule probes, which are easily cleared from tumor sites, possibly resulting in a shorter imaging time window, which is detrimental to clinical transformation of the probe. Labeling on the surface of a nano-carrier or doping positron nuclides into a nano-material is a common strategy for constructing a tumor long-retention PET nano-probe, and the strategy can effectively prolong the imaging time. However, most of these nanoprobes lack active targeting, and the low tumor penetration force limits their application in tumor imaging. In addition, they face a number of problems to be solved in clinical transformations, including poor biocompatibility and reproducibility. Therefore, developing more efficient, more friendly PET probes to achieve accurate targeting and tumor length retention remains a challenging task.
In recent years, enzymatic self-assembly (EISA) has attracted considerable attention as a novel targeted delivery strategy. This strategy allows the introduction of exogenous small molecules into specific physiological and pathological environments for self-assembly within cells, tissues and even living organisms. The introduced small molecules can easily penetrate the tumor, and can self-assemble into a high-order nano structure at the tumor site in situ after contacting with the enzyme with high expression in the tumor cells. This strategy not only preserves the long retention of the nanomaterial to the tumor, but also improves the low tumor penetration of the nanoprobe by exploiting the high permeability of the small molecules. More importantly, unlike traditional nonspecific nanoprobes, this strategy utilizes enzymes as triggers for self-assembly, allowing the probe to gain the ability to actively target tumors. The polypeptide is a compound formed by connecting a plurality of amino acids (N is more than or equal to 2 and less than or equal to 50) through amide bonds, and the molecular size of the polypeptide is between a small molecular chemical drug and a macromolecular biological drug. Polypeptides have higher efficacy, selectivity and specificity and lower metabolic toxicity than small molecule chemicals. Compared with macromolecular biological medicine, the polypeptide has lower immunogenicity, can penetrate deeply into target tissue, and has lower continuous production cost. Based on these unique advantages, polypeptides are increasingly becoming widely used building blocks in enzymatic self-assembly systems. Therefore, the enzymatic polypeptide self-assembly shows good application prospect and great medical value in the fields of biological materials and biomedicine.
Alkaline phosphatase (ALP) is an enzyme capable of dephosphorylating a corresponding substrate, i.e., removing phosphate groups from the substrate molecule by hydrolysis of a phosphate monoester, and generating phosphate ions and free hydroxyl groups. Alkaline phosphatase has been reported to be highly expressed in various human malignancies (e.g., heLa, hepG2, saos-2 and MESSA/Dx 5), and is a valuable tumor marker for early diagnosis, therapeutic observation and recurrence monitoring of cancer. Recently, alkaline phosphatase has been combined with an enzymatic self-assembly strategy, and a number of ALP-triggered self-assembled polypeptide probes have been designed for biomedical imaging, greatly widening the approaches to early diagnosis and treatment of cancer. For example, beams et al use this strategy to develop an ALP-triggered self-assembled polypeptide photoacoustic probe for enhancing photoacoustic imaging and an ALP-triggered self-assembled polypeptide magnetic resonance contrast agent for enhancing nuclear magnetic resonance imaging. Unfortunately, the report of studying this strategy in vivo tumor PET imaging is very limited, and to our knowledge, there is no ALP-triggered self-assembled polypeptide PET probe for enhancing PET imaging.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide an enzymatic self-assembled molecular probe 68 Ga-Nap-Yp, a preparation method and application thereof.
For this purpose, the invention provides the following technical scheme:
enzymatic self-assembled molecular probe 68 Ga-Nap-Yp has the following structural formula:
an enzymatic self-assembled molecular probe precursor Nap-Yp has the following structural formula:
the preparation method of the enzymatic self-assembled molecular probe precursor Nap-Yp comprises the following steps:
s1, coupling a compound Fmoc-Lys (Dde) -OH to a solid-phase carrier 2-chlorotrityl chloride resin, then adding a blocking reagent for blocking, and then removing Fmoc protecting groups to obtain a compound 1;
s2, fmoc-Tyr (H) 2 PO 3 ) -OH is coupled to compound 1, followed by removal of Fmoc protecting groups to give compound 2;
s3, coupling a compound Fmoc-Phe-OH to the compound 2, and removing Fmoc protecting groups to obtain a compound 3;
s4, coupling a compound Fmoc-Phe-OH to the compound 3, and removing Fmoc protecting groups to obtain a compound 4;
s5, coupling the compound 2-naphthylacetic acid to the compound 4, and then removing Dde protecting groups to obtain a compound 5;
s6, DOTA (OtBu) 3 Coupling to the compound 5, and then adding a peptide cutting reagent to cut peptide to obtain a compound 6;
s7, adding a tBu group deprotection reagent into the compound 6, and removing the tBu protecting group to obtain a compound Nap-Yp; the synthetic route is as follows:
The preparation method of the precursor Nap-Yp of the enzymatic self-assembled molecular probe,
in the S1 step, a compound Fmoc-Lys (Dde) -OH and a pH regulator are dissolved in a DMF solvent, then the mixture is added into an activated solid-phase carrier 2-chlorotrityl chloride resin, inert gas is introduced to react for 1 to 1.5 hours for coupling, then the reaction liquid is emptied for washing, then a blocking reagent is added, inert gas is introduced to react for 0.5 to 1 hour for blocking, then the reaction liquid is emptied for washing, then a solution for removing Fmoc protective groups is added, inert gas is introduced to react for 0.5 to 1 hour for removing Fmoc protective groups, and then the reaction liquid is emptied for washing, thus obtaining the compound 1;
and/or, in step S2, the compound Fmoc-Tyr (H 2 PO 3 ) dissolving-OH, a coupling agent and a pH regulator in a DMF solvent, then adding the mixture into a compound 1, introducing inert gas to react for 1-1.5 hours for coupling, then evacuating the reaction liquid, washing, then adding a solution for removing Fmoc protecting groups, introducing inert gas to react for 0.5-1 hour for removing Fmoc protecting groups, evacuating the reaction liquid, and washing to obtain a compound 2;
and/or in the step S3, dissolving a compound Fmoc-Phe-OH, a coupling agent and a pH regulator in a DMF solvent, then adding the compound into a compound 2, introducing inert gas to react for 1-1.5 hours for coupling, then evacuating the reaction liquid, washing, then adding a solution for removing Fmoc protecting groups, introducing inert gas to react for 0.5-1 hour for removing Fmoc protecting groups, evacuating the reaction liquid, and washing to obtain a compound 3;
And/or in the step S4, dissolving a compound Fmoc-Phe-OH, a coupling agent and a pH regulator in a DMF solvent, then adding the compound into a compound 3, introducing inert gas to react for 1-1.5 hours for coupling, then evacuating the reaction liquid, washing, then adding a solution for removing Fmoc protecting groups, introducing inert gas to react for 0.5-1 hour for removing Fmoc protecting groups, evacuating the reaction liquid, and washing to obtain a compound 4;
and/or in the step S5, dissolving the compound 2-naphthylacetic acid, a coupling agent and a pH regulator in DMF solvent, then adding the mixture into the compound 4, introducing inert gas to react for 1-1.5 hours for coupling, then emptying the reaction liquid, washing, then adding the solution for removing Dde groups, introducing inert gas to react for 5-10 minutes for removing the Dde groups, then emptying the reaction liquid, and washing to obtain the compound 5;
and/or, in step S6, the compound DOTA (OtBu) 3 Dissolving coupling agent and pH regulator in DMF solvent, adding into compound 5, introducing inert gas to react for 6-8 hr for coupling, evacuating the reaction liquid, washing, andadding a peptide cutting reagent, introducing inert gas to react for 1-3 minutes, and repeating the peptide cutting step for 4-6 times;
And/or in the step S7, adding a tBu group deprotection reagent, introducing inert gas, performing tBu group deprotection in a water bath reaction at 20-30 ℃ for 3-4 hours, and repeating the tBu group deprotection step for 3-4 times.
In the step of removing Fmoc protecting groups, removing the Fmoc protecting groups by adopting DMF solution containing 20% of piperidine by volume percent;
and/or, the coupling reagent is HOBT and HBTU;
and/or, the pH adjuster is DIEA;
and/or, in the step of removing Dde groups, removing the Dde groups by using a DMF solution containing 2% of hydrazine hydrate by volume percent;
and/or, the tBu group deprotection reagent is a DCM solution containing 50% trifluoroacetic acid by volume;
and/or, the peptide-cleaving reagent is a DCM solution containing 1% by volume of trifluoroacetic acid;
and/or, the end capping reagent is 40% methanol in DMF by volume.
In the preparation method of the enzymatic self-assembled molecular probe precursor Nap-Yp, in the step S1, the proportion of 2-chlorotrityl chloride resin, a compound Fmoc-Lys (Dde) -OH, a pH regulator and a DMF solvent is (600-800): (450-550): (0.5-0.7): (15-25), the proportion relation is mg: mg: ml: ml;
And/or, in the step S1, the ratio of the 2-chlorotrityl chloride resin to the end capping reagent is (600-800): (15-25), the proportion relation is mg: ml;
and/or, in the step S1, the ratio of the Fmoc-Lys (Dde) -OH compound to the Fmoc protecting group-removing solution is (450-550): (15-25), the proportion relation is mg: ml;
and/or, in step S2, 2-chlorotrityl chloride resin, compound Fmoc-Tyr (H 2 PO 3 ) -OH, DIEA, DMF solvent, HOBT and HBTU in the ratio (600-800): (450-550):(0.5-0.7): (15-25): (120-160): (350-450), the proportion relation is mg: mg: ml: ml: mg: mg;
and/or, in step S2, the compound Fmoc-Tyr (H 2 PO 3 ) The ratio of the-OH to the Fmoc protecting group-removing solution is (450-550): (15-25), the proportion relation is mg: ml;
and/or in step S3 or S4, the ratio of the 2-chlorotrityl chloride resin, the Fmoc-Phe-OH, DIEA, DMF solvent, HOBT and HBTU is (600-800): (350-450): (0.5-0.7): (15-25): (120-160): (350-450), the proportion relation is mg: mg: ml: ml: mg: mg;
and/or, in step S3 or S4, the ratio of the compound Fmoc-Phe-OH to the solution for removing the Fmoc protecting group is (350 to 450): (15-25), the proportion relation is mg: ml;
And/or in step S5, the ratio of 2-chlorotrityl chloride resin, 2-naphthylacetic acid, DIEA, DMF solvent, HOBT and HBTU is (600-800): (160-200): (0.5-0.7): (15-25): (120-160): (350-450), the proportion relation is mg: mg: ml: ml: mg: mg;
and/or, in the step S5, the ratio of the 2-naphthylacetic acid to the Dde group-removing solution is (160-200): (15-25), the proportion relation is mg: ml;
and/or, in step S6, 2-chlorotrityl chloride resin, DOTA (OtBu) 3 The ratios of DIEA, DMF solvent, HOBT and HBTU are (600-800): (550-650): (0.5-0.7): (15-25): (120-160): (350-450), the proportion relation is mg: mg: ml: ml: mg: mg;
and/or, in the step S6, the ratio of the 2-chlorotrityl chloride resin to the peptide-cutting reagent is (600-800): (20-25), the proportion relation is mg: ml;
and/or, in step S7, the ratio of the compound 6 to the tBu group deprotection reagent is (180-240): (20-30), the proportion relation is mg: ml.
The enzymatic self-assembled molecular probe 68 The preparation method of Ga-Nap-Yp comprises the following steps:
the enzymatic self-assembly molecular probeThe needle precursor Nap-Yp or the enzymatic self-assembled molecular probe precursor Nap-Yp prepared by the preparation method of the enzymatic self-assembled molecular probe precursor Nap-Yp is carried out 68 Ga labeling to obtain a compound 68 Ga-Nap-Yp; the synthetic route is as follows:
the enzymatic self-assembled molecular probe 68 Method for preparing Ga-Nap-Yp, said method 68 The Ga marking method comprises the steps of 68 Adding acid solution into Ga eluent, and regulating pH value to 4-5 to obtain 68 Adding the Ga mixed solution into an aqueous solution of a compound Nap-Yp, and heating and reacting for 5-10 minutes at the temperature of 90-95 ℃;
optionally, the 68 The radioactivity intensity in the Ga mixed solution is 1.6-2.4 mCi;
optionally, the concentration of the aqueous solution of the compound Nap-Yp is 4-6 mg/mL;
optionally, the 68 The volume ratio of Ga solution to the aqueous solution of the compound Nap-Yp is (10-15): (0.8-1.2).
The enzymatic self-assembled molecular probe 68 Use of Ga-Nap-Yp in the preparation of a tumor targeted PET tracer.
A PET tracer agent contains the enzymatic self-assembled molecular probe 68 Ga-Nap-Yp；
Optionally, the precursor Nap-Yp of the enzymatic self-assembled molecular probe is also included.
The technical scheme of the invention has the following advantages:
1. the invention provides an enzymatic self-assembled molecular probe 68 Ga-Nap-Yp with the structural formula of 2-naphthylacetic acid-Phe-Phe-Tyr (H) 2 PO 3 )-Lys(DOTA- 68 Ga) -OH (abbreviated as 68 Ga-Nap-Yp), consisting of: (i) Positron nuclides as signaling reports 68 Ga, attached to the peptide chain by complexation with the chelating agent DOTA on lysine; (ii) Polypeptide sequences Nap-Phe-Phe as self-assembling moietiesThe pi-pi stacking action between the perbenzoic ring and naphthalene ring promotes aggregation of polypeptide molecules; (iii) Phosphorylated tyrosine Tyr (H) 2 PO 3 ) The polypeptide self-assembly can be triggered after the polypeptide self-assembly is cut by alkaline phosphatase (ALP), and the phosphate group endows the molecular probe with the function of precisely targeting tumors. After the molecular probe enters the body, the molecular probe can be recognized by ALP with high expression in tumor cells and can be used for enzymatic cleavage of phosphate groups to obtain a dephosphorylated product 2-naphthylacetic acid-Phe-Phe-Tyr-Lys (DOTA- 68 Ga)-OH( 68 Ga-Nap-Y). Hydrophilic due to the removal of phosphate groups 68 Conversion of Ga-Nap-Yp to hydrophobic 68 Ga-Nap-Y and is enriched in a great amount at the tumor site. Self-assembling into the water-containing component under the action of hydrophobic action and pi-pi stacking action 68 Ga nanofibers, thereby enhancing PET imaging of tumors. In addition, the nanofibers are connected with each other to form a three-dimensional network structure and are attached to the periphery of tumor tissues, so that the elimination in the tumor is greatly delayed, and the tumor is caused 68 The residence of Ga at the tumor site eventually prolongs the tumor imaging time.
2. The invention provides an enzymatic self-assembled molecular probe 68 Method for preparing Ga-Nap-Yp, said method 68 The Ga marking method comprises the steps of 68 Adding acid solution into Ga eluent, and regulating pH value to 4-5 to obtain 68 The Ga mixed solution is then added into the aqueous solution of the compound Nap-Yp, and the mixture is heated and reacts for 5 to 10 minutes at the temperature of between 90 and 95 ℃; the preparation method has simple steps and short time.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a high performance liquid chromatogram of the compound Nap-Yp in example 1 of the present invention;
FIG. 2 is a mass spectrum of the compound Nap-Yp in example 1 of the present invention;
FIG. 3 shows the Nap-Yp compound of example 1 of the present invention 1 H NMR chart;
FIG. 4 shows the Nap-Yp compound of example 1 of the present invention 13 C NMR chart;
FIG. 5 shows the Nap-Yp compound of example 1 of the present invention 31 P NMR map;
FIG. 6 is a diagram of embodiment 2 of the present invention 68 High performance liquid chromatography of Ga-Nap-Yp;
FIG. 7 is a high performance liquid chromatogram of the compound Nap-Y in comparative example 1 of the present invention;
FIG. 8 is a mass spectrum of the compound Nap-Y in comparative example 1 of the present invention;
FIG. 9 is a graph showing the Nap-Y compound of comparative example 1 of the present invention 1 H NMR chart;
FIG. 10 shows the Nap-Y compound of comparative example 1 of the present invention 13 C NMR chart;
FIG. 11 is a diagram of comparative example 2 of the present invention 68 High performance liquid chromatography of Ga-Nap-Y;
FIG. 12 shows a molecular probe according to experimental example 1 of the present invention 68 Stability detection results after 1, 2 and 3 hours of incubation of Ga-Nap-Yp in PBS;
FIG. 13 shows a molecular probe according to example 1 of the present invention 68 Stability detection results after 1, 2 and 3 hours of incubation of Ga-Nap-Yp in FBS;
FIG. 14 shows a molecular probe according to example 1 of the present invention 68 Stability detection results after 1, 2 and 3 hours of incubation of Ga-Nap-Y in PBS;
FIG. 15 shows a molecular probe according to example 1 of the present invention 68 Stability detection results after 1, 2 and 3 hours of incubation of Ga-Nap-Y in FBS;
FIG. 16 is a graph showing the results of the biological property test in Experimental example 2 of the present invention; panel a shows the results of hydrogel incubation for group a; b is the incubation result of the hydrogel of the group B; the hydrogel of the group A is subjected to dynamic stress scanning at the frequency of 1 Hz; dynamic frequency sweep of the hydrogels of group a at 1.0% stress; TEM images of hydrogels of group A (inset: inverted view of hydrogels); f is 68 Ga-Nap-Yp 68 Lipid fraction of Ga-Nap-Y; g is the HPLC monitoring result of the hydrogel incubation process of the A group, the B group and the control group; h is the cytotoxicity assay result of Nap-Yp and Nap-Y formulated with PBS at 200. Mu.M; i is HeLa cell pair molecular probe 68 Ga-Nap-Yp and control molecular probe 68 Uptake of Ga-Nap-Y;
FIG. 17 shows cytotoxicity test results of Nap-Yp and Nap-Y in 50. Mu.M in Experimental example 2 of the present invention using PBS;
FIG. 18 shows cytotoxicity test results of Nap-Yp and Nap-Y in 100. Mu.M in experimental example 2 of the present invention using PBS;
FIG. 19 is an in vivo PET imaging result in Experimental example 3 of the present invention; in the figure, A is intravenous administration 68 PET static scan images (6% ID/g) of HeLa tumor-bearing nude mice at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 hours, respectively, of Ga-Nap-Yp (Single injection group); b is intravenous administration 68 PET static scan images (6% ID/g) of HeLa tumor-bearing nude mice of Ga-Nap-Y (control) at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 hours, respectively; c is intravenous administration 68 PET static scan images (8% ID/g) of HeLa tumor-bearing nude mice of Ga-Nap-Yp+Nap-Yp (co-injected group) at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 hours, respectively; d is intravenous administration 68 PET static scan images (8% ID/g) of HeLa tumor-bearing nude mice at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 hours, respectively, of Ga-Nap-Yp (Single injection group); e is in the co-injection group% 68 Ga-Nap-Yp+Nap-Yp, single injection group 68 Ga-Nap-Yp) and control group 68 Tumor uptake obtained by delineating tumor regions on the PET image of Ga-Nap-Y); f is a single injection group 68 Ga-Nap-Yp) and control group 68 Ga-Nap-Y); g is the co-injection group 68 Ga-Nap-Yp+Nap-Yp) and single injection group 68 Ga-Nap-Yp);
FIG. 20 shows the result of the biodistribution study of the molecular probe in experimental example 3 of the present invention; panel a is 0.5 hours after injection, 68 Ga-Nap-Y (control group), 68 Ga-Nap-Yp (Single injection group) and 68 biodistribution of Ga-Nap-Yp+Nap-Yp (co-injection group) in HeLa tumor-bearing nude mice; b is the injection of a control group, a single injection group and a co-injection groupThe ratio of uptake of radioactivity into the muscle (T/M) for tumors 0.5 hours and 3 hours post-injection; c is the biological distribution of a control group, a single injection group and a co-injection group in HeLa tumor-bearing nude mice 3 hours after injection; d is the ratio of the uptake rate of tumors to liver (T/L) at 0.5 and 3 hours after injection in the control group, the single injection group and the co-injection group;
FIG. 21 is a graph showing the pharmacokinetic results of the molecular probe in Experimental example 3 of the present invention;
FIG. 22 shows a molecular probe according to the present invention 68 The operational schematic diagram of Ga-Nap-Yp.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
HOBT Chinese is named 1-hydroxybenzotriazole;
HBTU has the Chinese name O-benzotriazol-tetramethyluronium hexafluorophosphate;
DIEA chinese name N, N-diisopropylethylamine;
DMF chinese name N, N-dimethylformamide;
DCM chinese name dichloromethane.
Example 1
This example provides an enzymatic self-assembled molecular probe precursor Nap-Yp of the formula 2-naphthylacetic acid-Phe-Phe-Tyr (H) 2 PO 3 ) -Lys (DOTA) -OH (abbreviated as Nap-Yp) of the formula:
the synthetic route is as follows:
the preparation method comprises the following steps:
s1, coupling a compound Fmoc-Lys (Dde) -OH to a solid-phase carrier 2-chlorotrityl chloride resin, then adding a blocking reagent for blocking, and then removing Fmoc protecting groups to obtain a compound 1; the method comprises the following specific steps:
690.12mg of 2-chlorotrityl chloride resin and 20mL of DMF were added to a 50mL solid-phase polypeptide synthesis tube, and the mixture was swelled with nitrogen for 30 minutes to fully activate the 2-chlorotrityl chloride resin. After the activation was completed, the DMF in the synthesis tube was drained. 532.26mg Fmoc-Lys (Dde) -OH, 20mL DMF and 555. Mu.L DIEA were added and reacted for 1 hour with nitrogen. The reaction was drained and washed 3 times with 15mL DMF. 15mL of a DMF solution containing 40% by volume of methanol was added for capping, and the reaction was carried out by introducing nitrogen for 30 minutes to block the unreacted active sites on the 2-chlorotrityl chloride resin. After the end-capping was completed, the reaction solution was drained and washed 3 times with 15mL of DMF. 15mL of a DMF solution containing 20% (volume percent) piperidine was added and reacted for 30 minutes with nitrogen to remove Fmoc protecting groups. The reaction was emptied and washed 3 times with 15mL DMF to give compound 1.
S2, fmoc-Tyr (H) 2 PO 3 ) -OH is coupled to compound 1, followed by removal of Fmoc protecting groups to give compound 2; the method comprises the following specific steps:
to compound 1 obtained in step S1 was added 483.41mg of Fmoc-Tyr (H 2 PO 3 ) -OH, 20mL DMF, 555. Mu.L DIEA, 135mg HOBT and 379mg HBTU were reacted for 1 hour with nitrogen. The reaction was drained and washed 3 times with 15mL DMF. 15mL of a DMF solution containing 20% (volume percent) piperidine was added and reacted for 30 minutes with nitrogen to remove Fmoc protecting groups. The reaction was emptied and washed 3 times with 15mL DMF to give compound 2.
S3, coupling a compound Fmoc-Phe-OH to the compound 2, and removing Fmoc protecting groups to obtain a compound 3; the method comprises the following specific steps:
to compound 2 obtained in step S2, 387.43mg of Fmoc-Phe-OH, 20mL of DMF, 555. Mu.L of DIEA, 135mg of HOBT and 379mg of HBTU were added and reacted for 1 hour with nitrogen. The reaction was drained and washed 3 times with 15mL DMF. 15mL of a DMF solution containing 20% (volume percent) piperidine was added and reacted for 30 minutes with nitrogen to remove Fmoc protecting groups. The reaction was emptied and washed 3 times with 15mL DMF to give compound 3.
S4, coupling a compound Fmoc-Phe-OH to the compound 3, and removing Fmoc protecting groups to obtain a compound 4; the method comprises the following specific steps:
To compound 3 obtained in step S3, 387.43mg of Fmoc-Phe-OH, 20mL of DMF, 555. Mu.L of DIEA, 135mg of HOBT and 379mg of HBTU were added and reacted for 1 hour with nitrogen. The reaction was drained and washed 3 times with 15mL DMF. 15mL of a DMF solution containing 20% (volume percent) piperidine was added and reacted for 30 minutes with nitrogen to remove Fmoc protecting groups. The reaction was emptied and washed 3 times with 15mL DMF to give compound 4.
S5, coupling the compound 2-naphthylacetic acid to the compound 4, and then removing Dde protecting groups to obtain a compound 5; the method comprises the following specific steps:
to compound 4 obtained in step S4 was added 186.21mg of 2-naphthylacetic acid, 20mL of DMF, 555. Mu.L of DIEA, 135mg of HOBT and 379mg of HBTU, and the mixture was reacted under nitrogen for 1 hour. The reaction was drained and washed 3 times with 15mL DMF. 15mL of DMF solution containing 2% (volume percent) hydrazine hydrate was added, and the mixture was reacted for 5 minutes by introducing nitrogen gas to remove Dde protecting group. The reaction was emptied and washed 3 times with 15mL DMF to give compound 5.
S6, DOTA (OtBu) 3 Coupling to the compound 5, and then adding a peptide cutting reagent to cut peptide to obtain a compound 6;
to compound 5 obtained in step S5, 572.73mg DOTA (OtBu) was added 3 20mL DMF, 555. Mu.L DIEA, 135mg HOBT and 379mg HBTU. The reaction was carried out for 6 hours with nitrogen. The reaction was emptied and washed 3 times with 15mL DMF, 15mL isopropanol, 15mL n-hexane in this order. 20mL of DCM solution (peptide cutting reagent) containing 1% (volume percent) trifluoroacetic acid was added, the mixture was reacted for 1 minute by introducing nitrogen gas, and the peptide cutting solution was collected by a 300mL eggplant-shaped bottle . The peptide cleavage operation was repeated 5 times to obtain compound 6.
S7, adding a tBu group deprotection reagent into the compound 6, and removing the tBu protecting group to obtain a compound Nap-Yp. The method comprises the following specific steps:
to compound 6 obtained in step S6, 20mL of 50% (volume percent) trifluoroacetic acid in DCM was added, and the mixture was reacted in a water bath at 25 ℃ under nitrogen for 3 hours to remove the tBu protecting group, and the step of removing the tBu protecting group was repeated 3 times. The reaction solution was rotary evaporated to obtain an oily liquid. 100mL of glacial diethyl ether was added to precipitate a white precipitate, and the supernatant was removed by centrifugation to give a white solid. And freeze-drying for 6 hours to obtain the compound Nap-Yp.
Purification by HPLC gave pure Nap-Yp (902.72 mg,0.73mmol, yield: 72.9%), chemical purity greater than 98%, HPLC purification conditions: the chromatographic column is a Waters XBridge Peptide BEH C column 18; isocratic elution, water: acetonitrile=55%: 45% (volume percent) of acetonitrile contains 0.1% (volume percent) of trifluoroacetic acid, the detection wavelength is 268nm, the column temperature is 25 ℃, the flow rate is 3mL/min, the sample injection volume is 500 μl, and the high performance liquid chromatography is shown in figure 1. MS: C of said Compound Nap-Yp 61 H 76 N 9 O 17 P[(M+H) - ]1239.30; HR-MS: m/z 1238.5178, see FIG. 2. 1 The H NMR chart is shown in figure 3, 13 The C NMR chart is shown in figure 4, 31 the P NMR chart is shown in FIG. 5.
Example 2
This embodiment differs from embodiment 1 in that:
in step S1, 600mg of 2-chlorotrityl chloride resin was added; the ratio of 2-chlorotrityl chloride resin, fmoc-Lys (Dde) -OH, pH regulator and DMF solvent was 600:550:0.5:25, the proportional relationship is mg: mg: ml: ml; introducing nitrogen to react for 1.5 hours for coupling;
in the end capping process, 25mL of DMF solution containing 40% (volume percent) methanol is added for end capping, and nitrogen is introduced for reaction for 1 hour for end capping;
when removing Fmoc protecting groups, 25mL of DMF solution containing 20% (volume percent) piperidine was added and reacted for 1 hour by introducing nitrogen to remove Fmoc protecting groups.
In step S2, 2-chlorotrityl chloride resin, compound Fmoc-Tyr (H 2 PO 3 ) The ratio of OH, DIEA, DMF solvent, HOBT and HBTU is 600:550:0.5:25:120:450, the proportional relationship is mg: mg: ml: ml: mg: mg; introducing nitrogen to react for 1.5 hours;
when Fmoc protecting groups were removed, 25mL of a 20% (volume percent) piperidine in DMF was added and reacted for 1 hour with nitrogen to remove Fmoc protecting groups.
In step S3, the ratio of 2-chlorotrityl chloride resin, fmoc-Phe-OH, DIEA, DMF solvent, HOBT and HBTU was 600:450:0.5:25:120:450, the proportional relationship is mg: mg: ml: ml: mg: mg; introducing nitrogen to react for 1.5 hours;
When Fmoc protecting groups were removed, 25mL of a 20% (volume percent) piperidine in DMF was added and reacted for 1 hour with nitrogen to remove Fmoc protecting groups.
In step S4, the ratio of 2-chlorotrityl chloride resin, fmoc-Phe-OH, DIEA, DMF solvent, HOBT and HBTU was 600:450:0.5:25:120:450, the proportional relationship is mg: mg: ml: ml: mg: mg; introducing nitrogen to react for 1.5 hours;
when Fmoc protecting groups were removed, 25mL of a 20% (volume percent) piperidine in DMF was added and reacted for 1 hour with nitrogen to remove Fmoc protecting groups.
In step S5, the ratio of 2-chlorotrityl chloride resin, 2-naphthylacetic acid, DIEA, DMF solvent, HOBT and HBTU was 600:200:0.5:25:120:450, the proportional relationship is mg: mg: ml: ml: mg: mg; introducing nitrogen to react for 1.5 hours;
when the Dde protecting group is removed, 25mL of DMF solution containing 2% (volume percent) hydrazine hydrate is added, nitrogen is introduced for reaction for 10 minutes, and the Dde protecting group is removed.
In the S6 step, 2-chlorotrityl chloride resin, DOTA (OtBu) 3 The ratio of DIEA, DMF solvent, HOBT and HBTU was 600:650:0.5:25:120:450, the proportional relationship is mg: mg: ml: ml: mg: mg; introducing nitrogen to react for 8 hours;
In the cleavage, 25mL of DCM solution (cleavage reagent) containing 1% (volume percent) trifluoroacetic acid was added and the reaction was carried out under nitrogen for 3 minutes.
In step S7, 30mL of a DCM solution containing 50% (volume percent) trifluoroacetic acid was added to the compound 6 obtained in step S6, and the mixture was reacted in a water bath at 30℃for 4 hours under nitrogen protection to remove the tBu protecting group.
Example 3
This embodiment differs from embodiment 1 in that:
in step S1, 600mg of 2-chlorotrityl chloride resin was added; the ratio of 2-chlorotrityl chloride resin, fmoc-Lys (Dde) -OH, pH regulator and DMF solvent was 800:450:0.7:15, the ratio relationship is mg: mg: ml: ml; introducing nitrogen to react for 1.3 hours for coupling;
when in end capping, 20mL of DMF solution containing 40% (volume percent) methanol is added for end capping, and nitrogen is introduced for reaction for 1 hour for end capping;
when removing Fmoc protecting groups, 20mL of DMF solution containing 20% (volume percent) piperidine was added, and the reaction was conducted by introducing nitrogen for 1 hour to remove Fmoc protecting groups.
In step S2, 2-chlorotrityl chloride resin, compound Fmoc-Tyr (H 2 PO 3 ) The ratio of OH, DIEA, DMF solvent, HOBT and HBTU was 800:450:0.7:15:160:350, the ratio relationship is mg: mg: ml: ml: mg: mg; introducing nitrogen to react for 1.3 hours;
When Fmoc protecting groups were removed, 20mL of a 20% (volume percent) piperidine in DMF was added and the mixture was reacted with nitrogen for 0.8 hour to remove Fmoc protecting groups.
In step S3, the ratio of 2-chlorotrityl chloride resin, fmoc-Phe-OH, DIEA, DMF solvent, HOBT and HBTU was 800:350:0.7:15:160:350, the ratio relationship is mg: mg: ml: ml: mg: mg; introducing nitrogen to react for 1.3 hours;
when Fmoc protecting groups were removed, 20mL of a 20% (volume percent) piperidine in DMF was added and the mixture was reacted with nitrogen for 0.8 hour to remove Fmoc protecting groups.
In step S4, the ratio of 2-chlorotrityl chloride resin, fmoc-Phe-OH, DIEA, DMF solvent, HOBT and HBTU was 800:350:0.7:15:160:350, the ratio relationship is mg: mg: ml: ml: mg: mg; introducing nitrogen to react for 1.3 hours;
when Fmoc protecting groups were removed, 20mL of a 20% (volume percent) piperidine in DMF was added and the mixture was reacted with nitrogen for 0.8 hour to remove Fmoc protecting groups.
In step S5, the ratio of 2-chlorotrityl chloride resin, 2-naphthylacetic acid, DIEA, DMF solvent, HOBT and HBTU was 800:160:0.7:15:160:350, the ratio relationship is mg: mg: ml: ml: mg: mg; introducing nitrogen to react for 1.3 hours;
When the Dde protecting group is removed, 20mL of DMF solution containing 2% (volume percent) hydrazine hydrate is added, nitrogen is introduced for reaction for 10 minutes, and the Dde protecting group is removed.
In the S6 step, 2-chlorotrityl chloride resin, DOTA (OtBu) 3 The ratio of DIEA, DMF solvent, HOBT and HBTU was 800:550:0.7:15:160:350, the ratio relationship is mg: mg: ml: ml: mg: mg; introducing nitrogen to react for 7 hours;
at the time of peptide cleavage, 23mL of DCM solution (peptide cleavage reagent) containing 1% (volume percent) trifluoroacetic acid was added and the reaction was carried out under nitrogen for 3 minutes.
In step S7, 25mL of a DCM solution containing 50% (volume percent) trifluoroacetic acid was added to the compound 6 obtained in step S6, and the mixture was reacted in a water bath at 20℃for 3.5 hours under nitrogen protection to remove the tBu protecting group.
Example 4
The embodiment provides an enzymatic self-assembled molecular probe 68 Ga-Nap-Yp with the structural formula of 2-naphthylacetic acid-Phe-Phe-Tyr (H) 2 PO 3 )-Lys(DOTA- 68 Ga) -OH (abbreviated as 68 Ga-Nap-Yp) with the following structural formula:
the synthetic route is as follows:
the preparation method comprises the following steps:
rinsing with 0.05M hydrochloric acid 68 Ge/ 68 Ga generator is obtained 68 Ga eluate. At 800. Mu.L 68 200 mu L of 0.25M sodium acetate solution is added into Ga eluent, the pH value is adjusted to 4-5, and the radioactivity intensity of the obtained mixed solution is 1.6-2.4 mCi. Then 80. Mu.L of Nap-Yp (5 mg/mL) as in example 1 in water was added. The mixture was heated at 95℃for 5 minutes. The reaction solution was detected by using radioactive HPLC with a retention time of 6.0 minutes and a radiochemical purity of more than 97% under the following conditions: the chromatographic column is a Waters XBridge C18 column; gradient elution, water: acetonitrile= (60% -10%): (40-90%) (volume percent) and acetonitrile containing 0.1% (volume percent) trifluoroacetic acid, the column temperature is 25 ℃, the sample injection volume is 20 mu L, and the high performance liquid chromatography is shown in figure 6.
Example 5
This embodiment differs from embodiment 4 in that: rinsing with 0.05M hydrochloric acid 68 Ge/ 68 Ga generator is obtained 68 Ga eluate. At 800. Mu.L 68 200 mu L of 0.25M sodium acetate solution is added into Ga eluent, the pH value is adjusted to 4-5, and the radioactivity intensity of the obtained mixed solution is 1.6-2.4 mCi. Then 120. Mu.L of Nap-Yp (4 mg/mL) as in example 1 in water was added. The mixture was heated at 90℃for 10 minutes.
Example 6
This embodiment differs from embodiment 4 in that: rinsing with 0.05M hydrochloric acid 68 Ge/ 68 Ga generator is obtained 68 Ga eluate. At 1200. Mu.L 68 300 mu L of 0.25M sodium acetate solution is added into Ga eluent, the pH value is adjusted to 4-5, and the radioactivity intensity of the obtained mixed solution is 1.6-2.4 mCi. Then 100. Mu.L of Nap-Yp (6 mg/mL) as in example 1 in water was added. The mixture was heated at 93℃for 8 minutes.
Comparative example 1
This comparative example provides a structural formula of 2-naphthylacetic acid-Phe-Phe-Tyr-Lys (DOTA) -OH (abbreviated as Nap-Y) as follows:
the synthetic route is as follows:
the preparation method comprises the following steps:
the difference between this comparative example and example 1 is that: in step (2), fmoc-Tyr (H) as a reaction material 2 PO 3 ) Equimolar substitution of-OH was Fmoc-Tyr-OH. The finally obtained compound Nap-Y was purified by HPLC (purification method is the same as that of the compound Nap-Yp) to obtain pure Nap-Y (876.85 mg,0.76mmol, yield: 75.7%) and the high performance liquid chromatogram was shown in FIG. 7.MS: C 61 H 75 N 9 O 14 [(M+H) - ]1159.32; HR-MS, m/z 1158.5501, as shown in FIG. 8. Which is a kind of 1 The H NMR chart is shown in figure 9, 13 the C NMR chart is shown in FIG. 10.
Comparative example 2 68 Ga-Nap-Y
This comparative example was prepared using Nap-Y obtained in comparative example 1 68 Ga-Nap-Y is synthesized by the following route:
the preparation method comprises the following steps:
this comparative example differs from example 2 only in that: 20. Mu.L of Nap-Y (5 mg/mL) in water was added. The reaction solution was detected by using radioactive HPLC, and the method of detection by radioactive HPLC was the same as 68 Ga-Nap-Yp, retention time is 7.5 minutes, radiochemical purity is more than 97%, and high performance liquid chromatography is shown in figure 11.
Experimental example 1 chemical stability and proteolytic stability of molecular probes
Molecular probes of example 4 68 Ga-Nap-Yp and molecular probes of comparative example 2 68 Ga-Nap-Y was incubated with Phosphate Buffered Saline (PBS) and Fetal Bovine Serum (FBS), respectively, at 37 ℃. The method comprises the following steps: the molecular probes are respectively added with Phosphate Buffer (PBS) and fetal bovine serum (F)BS) and the final emission intensity or concentration of the molecular probe is 2-3 mCi/mL, and the molecular probe is incubated at 37 ℃ for 0, 1, 2 and 3 hours and detected by the method of radioactive HPLC detection in example 4 68 The results of Ga-Nap-Yp are shown in FIGS. 12 and 13, and molecular probes 68 The results of Ga-Nap-Y are shown in FIGS. 14 and 15, after incubation for 3 hours, 68 Ga-Nap-Yp 68 The radiochemical purity of Ga-Nap-Y is still higher than 97%, and no impurity peak appears, indicating 68 Ga-Nap-Yp 68 Ga-Nap-Y has reliable stability.
Experimental example 2 biological Properties
1. ALP-induced nanofiber and hydrogel formation
To verify the self-assembly ability of molecular probes, ALP-induced cleavage experiments were performed in vitro using the molecular probe precursor Nap-Yp. The specific method comprises the following steps: two Nap-Yp solutions (in PBS,1.0 wt%) were prepared in advance, one with 200U of ALP added (group A) and the other without ALP added (group B), and then incubated at 37℃for 3 hours. As a result, as shown in FIG. 16A (for ALP addition) and B (for ALP addition), the Nap-Yp solution with ALP addition formed a transparent hydrogel while the Nap-Yp solution without ALP addition was still in a flowable liquid state.
To evaluate the physical properties of the hydrogels formed by self-assembly of molecular probes, the present invention further rheologically tested the viscoelasticity of the hydrogels formed in FIG. 16A, above. First, to determine the appropriate conditions for dynamic frequency sweep, the hydrogel was subjected to dynamic stress sweep at a frequency of 1.0 Hz. As a result, as shown in fig. 16C, the storage modulus (G ') value and the loss modulus (G ") value of the hydrogel showed weak correlation in the stress range of 0.1% -10%, and the G' value was much larger than the G" value, indicating that the formed hydrogel had good viscoelasticity. The hydrogel was then subjected to dynamic frequency sweep at 1.0% stress. As a result, as shown in FIG. 16D, the G 'and G' values of the hydrogels increased slowly over the frequency range of 0.1Hz-10 Hz, with the G 'value exceeding 7 times the G' value, indicating that the hydrogels have good resistance to external shear stress.
To investigate the hydrogel formation process more deeply, the present invention captures the microstructure of a hydrogel (the hydrogel formed in a of fig. 16) at a nanoscale using a Transmission Electron Microscope (TEM). As a result, as shown in fig. 16E, the hydrogel is composed of dense fibers, which cross each other to form a complex network structure. In addition, the present invention also notes that there is a higher local density at the intersections between nanofibers, which may be due to interactions or entanglement between the fibers. Such a network-like cross-structure may provide good mechanical properties to the hydrogel, including elasticity, toughness, and strain response to external forces.
To confirm that self-assembly was initiated by cleavage of Nap-Yp by ALP, the present invention monitored the formation of hydrogels from groups a and B described above by HPLC (method of example 1) while setting a control group of Nap-Y. HPLC analysis showed that, within 10 minutes after addition of ALP, 95% of Nap-Yp was converted to dephosphorylated product Nap-Y, as shown in FIG. 16G.
In order to examine the change in hydrophilicity of the molecular probe before and after cleavage, the experiment was performed in a mixed solution of water and n-butanol. The specific experimental method comprises the following steps: 100. Mu.L was added to a 5mL centrifuge tube 68 Ga-Nap-Yp or 68 Ga-Nap-Y (radioactive dose 150-300. Mu. Ci), 1mL n-octanol and 900. Mu.L sterilized water. Ultrasonic shaking for 2 min and centrifuging for 5 min. 100. Mu.L of n-butanol and 100. Mu.L of sterilized water were taken and examined using a gamma counter. Lipid partition coefficient Log p=log (n-butanol gamma count)/(sterilized water gamma count).
As a result of measurement, as shown in F in FIG. 16, 68 Ga-Nap-Yp 68 The lipid partition coefficients Log P of Ga-Nap-Y are-1.416.+ -. 0.053 and-0.481.+ -. 0.038, respectively, which means that Nap-Y is much more hydrophobic than Nap-Yp.
In summary, nap-Yp can be efficiently cleaved by ALP to generate hydrophobic Nap-Y. Hydrophobic Nap-Y are interconnected to form nanofibers. The resulting nanofibers form a three-dimensional network, encapsulate large numbers of water molecules, and appear as hydrogels on a macroscopic scale.
2. Cytotoxicity test
In order to evaluate the biological properties of the probes, the present invention investigated the safety and specificity of molecular probes at the cellular level. For this purpose, the invention selects a human cervical cancer cell line HeLa as a representative model of ALP high-expression tumor cells. HeLa cells were cultured in an environment containing molecular probe precursor Nap-Yp or control molecular probe precursor Nap-Y, respectively, and the viability of HeLa cells was examined by CCK-8 method. The specific method comprises the following steps:
Nap-Yp (prepared in example 1) and Nap-Y (prepared in comparative example 1) solutions were formulated at different concentrations (50, 100 and 200. Mu.M) using PBS. HeLa cells were grown at 5X 10 4 The concentration of each/mL was seeded in 96-well plates and 100. Mu.L of cell suspension was added to each well. Cells were allowed to adhere by incubation in a cell incubator for 12 hours. 100. Mu.L of PBS or formulated sample solution was added to each well and incubated at 37℃for various times (6, 12 and 24 hours). The sample added per well and incubation time were recorded. After the corresponding incubation time has been reached, 10 μl of CCK-8 (5 mg/mL) is added followed by incubation for 4 hours to yield a highly water soluble yellow formazan product. Absorbance values of the formazan product were measured at a wavelength of 450nm using an enzyme-linked immunosorbent assay (BioTek). Cell viability = absorbance value added to sample solution/absorbance value added to PBS x 100%.
As shown in FIG. 16, H (corresponding to 200. Mu.M), heLa cells survived cultivation in 200. Mu.M Nap-Yp for 6, 12 and 24 hours at 100.77.+ -. 7.18%, 102.43.+ -. 1.27% and 101.00.+ -. 3.20%, respectively; cell viability in 200. Mu.M Nap-Y was 101.20.+ -. 5.71%, 101.17.+ -. 0.31% and 102.00.+ -. 0.70%, respectively. The results corresponding to 50. Mu.M are shown in FIG. 17 and those corresponding to 100. Mu.M are shown in FIG. 18, and it is shown by H in FIG. 16, FIG. 17 and FIG. 18 that the cell viability after treatment with Nap-Yp or Nap-Y is nearly 100% and does not significantly decrease within 24 hours. This indicates that Nap-Yp and Nap-Y have a weak effect on the normal physiological activities of HeLa cells, thus exhibiting good biocompatibility.
3. Cell uptake assay
The invention researches molecular probes of HeLa cells 68 Ga-Nap-Yp and control molecular probe 68 Uptake of Ga-Nap-Y to verify the probe 68 Ga-Nap-Yp targets ALP potential in cells. Tool withThe body method comprises the following steps:
HeLa cells were cultured to a log phase of growth with higher cell activity. HeLa cells were seeded in 24-well plates, each well seeded with about 30-50 ten thousand cells. Cells were allowed to adhere by incubation in a cell incubator for 12 hours. PBS was used to carry out 68 Ga-Nap-Yp (prepared in example 4) and 68 the concentration of the labeling reaction solution of Ga-Nap-Y (prepared in comparative example 2) was diluted to 10. Mu. Ci/mL. After sucking out the medium of the 24-well plate, 200. Mu.L of the medium was added to each well 68 Ga-Nap-Yp or 68 Diluted Ga-Nap-Y solution. Incubation was carried out at 37℃for different times (0.5, 1, 1.5, 2, 2.5 and 3 hours). The sample added per well and incubation time were recorded. After the corresponding incubation time was reached, the supernatant was removed and washed twice with PBS. Cells were detached from the wall with pancreatin digest and collected into gamma-counting tubes. Another 200. Mu.L of sample dilution solution was added to the gamma counter. The gamma counter containing the collected cells and the gamma counter to which the sample dilution solution was added were simultaneously measured with a gamma counter. Uptake of molecular probes = radioactivity of gamma counter containing the collected cells/radioactivity of gamma counter added to sample dilution solution x 100%.
The results are shown as I in FIG. 16, molecular probes 68 The cell uptake rate of Ga-Nap-Yp increased rapidly from 3.27.+ -. 0.12% for 0.5 hours to 5.93.+ -. 0.39% for 3 hours. In contrast, control molecular probes 68 The increase in cell uptake of Ga-Nap-Y was quite slow, increasing from 0.21.+ -. 0.08% for 0.5 hours to 0.40.+ -. 0.09% for 3 hours only. The experimental data clearly demonstrate that HeLa cell pairs 68 The uptake of Ga-Nap-Yp is far higher than 68 Ga-Nap-Y due to 68 Phosphate groups in the Ga-Nap-Yp structure. The phosphate radical can directly target ALP in cells, effectively increase 68 Aggregation of Ga-Nap-Yp in HeLa cells. The results of the cell uptake experiments well demonstrate that molecular probes 68 Ga-Nap-Yp compared with control molecular probe 68 Ga-Nap-Y has better specificity.
Experimental example 3 animal experiment
1. Construction of animal models
All experimental animals were kept in SPF class animal houses. Make the following stepsA tumor model was established with healthy female nude mice of 5-6 weeks of age and weight 20-25 g. HeLa cells were cultured to a log phase of growth with higher cell activity. HeLa cells were digested and transferred to a centrifuge tube, and the supernatant was removed. HeLa cells were diluted to 2.5X10 using PBS 7 Cell suspensions per mL. Each nude mouse was inoculated with 200. Mu.L of HeLa cell suspension in the right limb axilla. After 24 hours, 2mg cyclophosphamide was injected by tail vein. After 4-6 weeks of feeding, the HeLa tumor diameter reaches about 5-10 mm, and then corresponding animal experiments can be carried out.
2. In vivo PET imaging
Nude mice with tumor diameters of 5-10 mm were selected and randomly divided into three groups (n=3 per group) for PET imaging. Will be 5.6-9.3MBq 68 Ga-Nap-Y (prepared in comparative example 2), 68 Ga-Nap-Yp (prepared in example 4) or 68 Ga-Nap-Yp (prepared in example 4) +Nap-Yp (prepared in example 1) were injected into nude mice via the tail vein, respectively. Acquisition of PET imaging images was performed using an Inveon microPET scanner (siemens germany) at 0.5, 1, 1.5, 2, 2.5 and 3 hours, respectively. During the collection, the nude mice subjected to the experiment were anesthetized and supplied with 0.5L/min oxygen and 2% isoflurane. PET scan data was reconstructed by the OSEM 3D/MAP algorithm without attenuation correction and then processed by Inveon Research Workplace (siemens germany). Tumor regions of interest (ROIs) were delineated and calculated using ASIPro VM 6.8.6.9 software. Uptake rate is expressed as percentage of radioactive dose per gram of tissue (% ID/g).
The results are shown in FIG. 19, in which A, B, C, D shows, respectively 68 Ga-Nap-Y (control group), 68 Ga-Nap-Yp (Single injection group) or 68 Imaging results of Ga-Nap-Yp+Nap-Yp (Co-injected group). Separate injection 68 At the tumor site, a clear and bright PET signal (single shot group, a in fig. 19) was observed with high separation from surrounding muscle tissue, resulting in good imaging discrimination. In comparison, the molecular probes of the control group 68 The PET signal of Ga-Nap-Y at the tumor site was so dim that it was difficult to observe (control group, B in FIG. 19). Tumor regions of interest (ROIs) for each group are shown as E in figure 19, 68 Ga-Nap-Yp 68 Ga-NThe tumor uptake rate of ap-Y was highest at 0.5 hours, 2.95.+ -. 0.58% ID/g and 1.37.+ -. 0.34% ID/g, respectively, 68 tumor uptake of Ga-Nap-Yp was 68 More than twice of Ga-Nap-Y, illustrating a probe 68 Ga-Nap-Yp can be specifically absorbed by tumors with high ALP expression for specific imaging. After a period of 0.5 hour, the sample was taken, 68 Ga-Nap-Yp 68 The tumor uptake rate of Ga-Nap-Y gradually decreased, reaching the lowest point at 3 hours, 0.92+ -0.37% ID/g and 0.21+ -0.09% ID/g, respectively, indicating that they were cleared by the body's metabolism. Theoretically, when 68 After Ga-Nap-Yp is specifically ingested by tumor, the phosphate group is cleaved off to generate 68 Ga-Nap-Y. Thus, the first and second substrates are bonded together, 68 metabolism of Ga-Nap-Yp at tumor site should be compatible with 68 The metabolism of Ga-Nap-Y is consistent. However, the process is not limited to the above-described process, 68 Ga-Nap-Yp 68 The tumor uptake ratio of Ga-Nap-Y increased from 2.18.+ -. 0.13 at 0.5 hours to 4.44.+ -. 0.83 at 3 hours (F in FIG. 19), indicating that the clearance rates of the two molecular probes at the tumor sites were not uniform. The present invention speculates that this difference in clearance rate 68 Self-assembly of Ga-Nap-Yp is closely related. That is to say that the first and second, 68 Ga-Nap-Yp is subjected to in-situ self-assembly at a tumor site under the control of ALP, and then nanofibers which are not easy to metabolize are formed, so that in-vivo clearance is delayed. The experiment uses a co-injection strategy, i.e. at the probe 68 The addition of the precursor Nap-Yp to Ga-Nap-Yp to increase the self-assembly concentration of the tumor site (co-injected group) validated the above-described speculation, and the results are shown in FIG. 19C, where the image of the co-injected group showed that the PET signal of the tumor was not only weakened over time, but rather significantly enhanced, indicating that the aggregation and retention of the probe at the tumor site exceeded clearance over the imaging time window, and the tumor uptake rate of the co-injected group increased from 3.05.+ -. 0.40% ID/g for 0.5 hours to 4.67.+ -. 1.16% ID/g for 3 hours, as shown in FIG. 19E. The tumor uptake rates of the co-injected group and the single injected group were then compared, and as a result, as shown in fig. 19G, the tumor uptake rate ratio of the co-injected group to the single injected group increased from 1.05±0.21 for 0.5 hours to 5.44±1.51 for 3 hours. The above results reveal a probe 68 Ga-Nap-Yp under enzyme-induced conditionsThe probe has good in-vivo self-assembly capability, and simultaneously, the self-assembly performance can be greatly promoted by improving the self-assembly concentration, and the long retention of the probe at the tumor part is realized, so that the aim of enhancing PET imaging is fulfilled.
3. Biodistribution and pharmacokinetic studies
In order to accurately quantify the distribution and metabolism of molecular probes in tumor-bearing nude mice, the invention further researches tissue distribution and pharmacokinetics. Nude mice with tumor diameters of 5-10 mm were selected and randomly divided into three groups (n=3 per group) for tissue distribution study or pharmacokinetic study. Control group of 5.6-9.3MBq 68 Ga-Nap-Y (prepared in comparative example 2), single injection group 68 Ga-Nap-Yp (prepared in example 4) or Co-injection group 68 Ga-Nap-Yp (prepared in example 4) +Nap-Yp (prepared in example 1) were injected into nude mice via the tail vein, respectively.
Biodistribution: mice were dissected at 0.5 and 3 hours after dosing, respectively. The weight and the radiation dose of each tissue (heart, liver, spleen, lung, kidney, tumor, muscle, bone, stomach, intestine) were measured. Pharmacokinetic studies: blood was collected 1, 5, 10, 30, 60, 120 and 240 minutes after dosing. Blood weight and radiation dose were measured. Uptake rate is expressed as percentage of radioactive dose per gram of tissue (% ID/g).
The biodistribution results, corresponding to the results for 0.5 hours, are shown in FIG. 20A, and tumor uptake rates of the co-injection group and the single injection group at 0.5 hour are 3.90.+ -. 0.62% ID/g and 3.06.+ -. 0.21% ID/g, respectively, which are significantly higher than those of the control group (1.96.+ -. 0.32% ID/g), indicating molecular probes 68 Ga-Nap-Yp is taken up specifically by the tumor. The results for 3 hours are shown in FIG. 20C, and it was found that the tumor uptake rates of the single injection group and the control group were significantly reduced to 1.06.+ -. 0.23% ID/g and 0.30.+ -. 0.06% ID/g, respectively. In this way, 68 Ga-Nap-Yp is metabolized by the body by about 65%, ratio 68 Ga-Nap-Y (85%) is small, showing 68 Ga-Nap-Yp is retained by self-assembly to some extent. Notably, the tumor uptake rate of the co-injection group is greatly increased, and the tumor uptake rate is 5.04+/-0.38% ID/g in 3 hours, which effectively proves that the in-situ self-assembly of tumor sites can effectively counteract the generation of organismsXie Qingchu.
As one of important indexes of molecular probe imaging performance, target background ratios (B and D in fig. 20) were calculated with muscle and liver as backgrounds, respectively. As shown in B in fig. 20, 0.5 hours 68 The tumor/muscle (T/M) uptake ratio of Ga-Nap-Yp is 5.14+/-0.69, which is obviously higher than that of Ga-Nap-Yp 68 Ga-Nap-Y (1.59.+ -. 0.12) and 3 hours 2.03.+ -. 0.20, slightly higher 68 Ga-Nap-Y (1.75.+ -. 0.55). This indicates that when molecular probes are used 68 Ga-Nap-Yp single injection is most effective in tumor imaging within 0.5 hour. In contrast to this, the process is performed, 68 tumor/muscle (T/M) uptake ratio of Ga-Nap-yp+nap-Yp (co-injected group) was 3.92±0.62 for 0.5 hours and 3.68±0.56 for 3 hours, respectively, maintaining a constant target background ratio (B in fig. 20) throughout the imaging time window. This is more pronounced in tumor/liver (T/L) uptake ratio (D in fig. 20). As shown in the drawing, the liquid crystal display device, 68 The tumor/liver (T/L) uptake ratio of Ga-Nap-Yp+Nap-Yp increased from 0.91.+ -. 0.12 for 0.5 hour to 1.89.+ -. 0.39 for 3 hours, approximately 68 17 times the sum of Ga-Nap-Yp (0.11+ -0.01) 68 Ga-Nap-Y (0.16.+ -. 0.03). Notably, the T/L uptake ratio of the co-injected group remained above 1 after 3 hours, effectively highlighting the molecular probe under the co-injection strategy 68 Ga-Nap-Yp has excellent tumor imaging performance.
The results of the pharmacokinetics showed that, 68 Ga-Nap-Yp 68 The plasma concentration-time curves for Ga-Nap-Y were not greatly different, and the half-lives were 21.1 minutes and 20.3 minutes, respectively (see FIG. 21). Notably, after co-injection with Nap-Yp, 68 the half-life of Ga-Nap-Yp is prolonged to 61.2 minutes, which is 68 The half-life of Ga-Nap-Yp single injection is 3 times. Under the co-injection strategy, the self-assembly concentration of the molecular probe is increased, more nanofibers are generated in situ, and the nanofibers play a role in resisting the metabolism of organisms, so that the clearance of the medicine in the body is greatly delayed.
In conclusion, the invention reasonably designs a novel PET probe for tumor accurate imaging based on an enzymatic self-assembly strategy 68 Ga-Nap-Yp, the molecular probe shows unique advantages in prolonging the retention time of signals and improving the specificity Potential of the material. The molecular probe accords with ideal standards of some production and use links, such as easily available raw materials, simple synthesis, rapid radiolabeling and the like. In the aspect of in vitro stability, the molecular probe has reliable stability. Shows good biocompatibility in the aspect of in vitro and in vivo safety. The molecular probe can self-assemble to form hydrogel under the induction of alkaline phosphatase, the hydrogel has good tolerance to external shearing stress, and the hydrogel has good mechanical properties. More importantly, higher PET signals and longer imaging time windows were observed at the tumor site (in vivo) by co-injection strategy. These experimental results strongly demonstrate that the designed molecular probes can achieve targeted enrichment, in-situ self-assembly and long-time retention under the control of ALP, and finally enhance PET imaging of tumors. Studies of tissue distribution and pharmacokinetics further confirm the effectiveness of co-injection strategies. The efficient self-assembly not only enhances the intensity of radioactive signals, but also brings ideal long retention, thereby providing a longer time window for PET imaging of tumors and being more beneficial to clinically accurately diagnosing the tumors. Thus, the molecular probe of the present invention can operate on the principle as shown in FIG. 22, in A, 68 Ga-Nap-Yp under the action of alkaline phosphatase to obtain the dephosphorylated product 68 Ga-Nap-Y, made of hydrophilic material 68 Ga-Nap-Yp becomes hydrophobic 68 Ga-Nap-Y, hydrophobic 68 Ga-Nap-Y self-assembles to form hydrogel under the action of hydrophobic effect and pi-pi stacking effect. In B, when the molecular probe is applied 68 Ga-Nap-Yp is injected into a mouse body, enters the blood circulation of the mouse, is distributed to tumors, 68 Ga-Nap-Yp is recognized and cleaved by alkaline phosphatase highly expressed in tumor cells to produce a hydrophobic product 68 Ga-Nap-Y is enriched in a great amount at the tumor part and self-assembled to form nano fiber, so that PET imaging of the tumor is enhanced. In addition, the nanofibers are connected with each other to form a three-dimensional network structure and are attached to the periphery of tumor tissues, so that the elimination in the tumor is greatly delayed, and the tumor is caused 68 The residence of Ga in the tumor part eventually prolongs the tumor imaging time.
Furthermore, for probe-expandable repairThe decoration has great flexibility and diversity. For example: 1) Different amino acids, gelators (PATT and LAFF), chelating ligands (NOTA and DTPA), enzyme-related tumor targets (ALP and beta-galactosidase) and the like are matched. These can play a certain role in regulating the self-assembly performance of molecules and the dynamic distribution of targets, and can provide different or better imaging performance; 2) Will be 68 Ga is replaced by medical isotope with other long half life period 89 Sr、 131 I) A. The invention relates to a method for producing a fibre-reinforced plastic composite With the aid of co-injection strategy, the method can bring new opportunities for developing long-acting radioactive therapeutic drugs, and can possibly realize new progress in diagnosis and treatment integration. In summary, we believe that this study provides a new perspective for the development of radiopharmaceuticals in addition to exhibiting a novel radiotracer with high specificity and long retention.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (10)
1. Enzymatic self-assembled molecular probe 68 Ga-Nap-Yp is characterized by the following structural formula:
2. an enzymatic self-assembled molecular probe precursor Nap-Yp is characterized by having the following structural formula:
3. a method for preparing the precursor Nap-Yp of the enzymatic self-assembled molecular probe according to claim 2, comprising the steps of:
S1, coupling a compound Fmoc-Lys (Dde) -OH to a solid-phase carrier 2-chlorotrityl chloride resin, then adding a blocking reagent for blocking, and then removing Fmoc protecting groups to obtain a compound 1;
s2, fmoc-Tyr (H) 2 PO 3 ) -OH is coupled to compound 1, followed by removal of Fmoc protecting groups to give compound 2;
s3, coupling a compound Fmoc-Phe-OH to the compound 2, and removing Fmoc protecting groups to obtain a compound 3;
s4, coupling a compound Fmoc-Phe-OH to the compound 3, and removing Fmoc protecting groups to obtain a compound 4;
s5, coupling the compound 2-naphthylacetic acid to the compound 4, and then removing Dde protecting groups to obtain a compound 5;
s6, DOTA (OtBu) 3 Coupling to the compound 5, and then adding a peptide cutting reagent to cut peptide to obtain a compound 6;
s7, adding a tBu group deprotection reagent into the compound 6, and removing the tBu protecting group to obtain a compound Nap-Yp; the synthetic route is as follows:
4. the method for preparing the precursor Nap-Yp of the enzymatic self-assembled molecular probe according to claim 3,
in the S1 step, a compound Fmoc-Lys (Dde) -OH and a pH regulator are dissolved in a DMF solvent, then the mixture is added into an activated solid-phase carrier 2-chlorotrityl chloride resin, inert gas is introduced to react for 1 to 1.5 hours for coupling, then the reaction liquid is emptied for washing, then a blocking reagent is added, inert gas is introduced to react for 0.5 to 1 hour for blocking, then the reaction liquid is emptied for washing, then a solution for removing Fmoc protective groups is added, inert gas is introduced to react for 0.5 to 1 hour for removing Fmoc protective groups, and then the reaction liquid is emptied for washing, thus obtaining the compound 1;
And/or, in step S2, the compound Fmoc-Tyr (H 2 PO 3 ) dissolving-OH, a coupling agent and a pH regulator in a DMF solvent, then adding the mixture into a compound 1, introducing inert gas to react for 1-1.5 hours for coupling, then evacuating the reaction liquid, washing, then adding a solution for removing Fmoc protecting groups, introducing inert gas to react for 0.5-1 hour for removing Fmoc protecting groups, evacuating the reaction liquid, and washing to obtain a compound 2;
and/or in the step S3, dissolving a compound Fmoc-Phe-OH, a coupling agent and a pH regulator in a DMF solvent, then adding the compound into a compound 2, introducing inert gas to react for 1-1.5 hours for coupling, then evacuating the reaction liquid, washing, then adding a solution for removing Fmoc protecting groups, introducing inert gas to react for 0.5-1 hour for removing Fmoc protecting groups, evacuating the reaction liquid, and washing to obtain a compound 3;
and/or in the step S4, dissolving a compound Fmoc-Phe-OH, a coupling agent and a pH regulator in a DMF solvent, then adding the compound into a compound 3, introducing inert gas to react for 1-1.5 hours for coupling, then evacuating the reaction liquid, washing, then adding a solution for removing Fmoc protecting groups, introducing inert gas to react for 0.5-1 hour for removing Fmoc protecting groups, evacuating the reaction liquid, and washing to obtain a compound 4;
And/or in the step S5, dissolving the compound 2-naphthylacetic acid, a coupling agent and a pH regulator in DMF solvent, then adding the mixture into the compound 4, introducing inert gas to react for 1-1.5 hours for coupling, then emptying the reaction liquid, washing, then adding the solution for removing Dde groups, introducing inert gas to react for 5-10 minutes for removing the Dde groups, then emptying the reaction liquid, and washing to obtain the compound 5;
and/or, in step S6, the compound DOTA (OtBu) 3 Dissolving coupling agent and pH regulator in DMF solvent, adding into compound 5, introducing inert gas to react for 6-8 hr for coupling, evacuating reaction liquid, washing, and adding peptide cutting reagentIntroducing inert gas to react for 1-3 minutes, and repeating the peptide cutting step for 4-6 times;
and/or in the step S7, adding a tBu group deprotection reagent, introducing inert gas, performing tBu group deprotection in a water bath reaction at 20-30 ℃ for 3-4 hours, and repeating the tBu group deprotection step for 3-4 times.
5. The method for preparing the precursor Nap-Yp of the enzymatic self-assembled molecular probe according to claim 4, wherein in the Fmoc protecting group removal step, fmoc protecting groups are removed by using a DMF solution containing 20% by volume of piperidine;
And/or, the coupling reagent is HOBT and HBTU;
and/or, the pH adjuster is DIEA;
and/or, in the step of removing Dde groups, removing the Dde groups by using a DMF solution containing 2% of hydrazine hydrate by volume percent;
and/or, the tBu group deprotection reagent is a DCM solution containing 50% trifluoroacetic acid by volume;
and/or, the peptide-cleaving reagent is a DCM solution containing 1% by volume of trifluoroacetic acid;
and/or, the end capping reagent is 40% methanol in DMF by volume.
6. The method for preparing the precursor Nap-Yp of the enzymatic self-assembled molecular probe according to claim 4 or 5, wherein in the step S1, the ratio of 2-chlorotrityl chloride resin, the compound Fmoc-Lys (Dde) -OH, the pH regulator and the DMF solvent is (600-800): (450-550): (0.5-0.7): (15-25), the proportion relation is mg: mg: ml: ml;
and/or, in the step S1, the ratio of the 2-chlorotrityl chloride resin to the end capping reagent is (600-800): (15-25), the proportion relation is mg: ml;
and/or, in the step S1, the ratio of the Fmoc-Lys (Dde) -OH compound to the Fmoc protecting group-removing solution is (450-550): (15-25), the proportion relation is mg: ml;
And/or, in step S2, 2-chlorotritylMethyl chloride resin, fmoc-Tyr (H) 2 PO 3 ) -OH, DIEA, DMF solvent, HOBT and HBTU in the ratio (600-800): (450-550): (0.5-0.7): (15-25): (120-160): (350-450), the proportion relation is mg: mg: ml: ml: mg: mg;
and/or, in step S2, the compound Fmoc-Tyr (H 2 PO 3 ) The ratio of the-OH to the Fmoc protecting group-removing solution is (450-550): (15-25), the proportion relation is mg: ml;
and/or in step S3 or S4, the ratio of the 2-chlorotrityl chloride resin, the Fmoc-Phe-OH, DIEA, DMF solvent, HOBT and HBTU is (600-800): (350-450): (0.5-0.7): (15-25): (120-160): (350-450), the proportion relation is mg: mg: ml: ml: mg: mg;
and/or, in step S3 or S4, the ratio of the compound Fmoc-Phe-OH to the solution for removing the Fmoc protecting group is (350 to 450): (15-25), the proportion relation is mg: ml;
and/or in step S5, the ratio of 2-chlorotrityl chloride resin, 2-naphthylacetic acid, DIEA, DMF solvent, HOBT and HBTU is (600-800): (160-200): (0.5-0.7): (15-25): (120-160): (350-450), the proportion relation is mg: mg: ml: ml: mg: mg;
And/or, in the step S5, the ratio of the 2-naphthylacetic acid to the Dde group-removing solution is (160-200): (15-25), the proportion relation is mg: ml;
and/or, in step S6, 2-chlorotrityl chloride resin, DOTA (OtBu) 3 The ratios of DIEA, DMF solvent, HOBT and HBTU are (600-800): (550-650): (0.5-0.7): (15-25): (120-160): (350-450), the proportion relation is mg: mg: ml: ml: mg: mg;
and/or, in the step S6, the ratio of the 2-chlorotrityl chloride resin to the peptide-cutting reagent is (600-800): (20-25), the proportion relation is mg: ml;
and/or, in step S7, the ratio of the compound 6 to the tBu group deprotection reagent is (180-240): (20-30), the proportion relation is mg: ml.
7. An enzymatically self-assembled molecular probe of claim 1 68 The preparation method of Ga-Nap-Yp is characterized by comprising the following steps:
the enzymatic self-assembled molecular probe precursor Nap-Yp prepared by the preparation method of the enzymatic self-assembled molecular probe precursor Nap-Yp of claim 2 or any one of claims 3-6 is subjected to 68 Ga labeling to obtain a compound 68 Ga-Nap-Yp; the synthetic route is as follows:
8. The enzymatic self-assembled molecular probe of claim 7 68 A process for the preparation of Ga-Nap-Yp characterized in that 68 The Ga marking method comprises the steps of 68 Adding acid solution into Ga eluent, and regulating pH value to 4-5 to obtain 68 Adding the Ga mixed solution into an aqueous solution of a compound Nap-Yp, and heating and reacting for 5-10 minutes at the temperature of 90-95 ℃;
optionally, the 68 The radioactivity intensity in the Ga mixed solution is 1.6-2.4 mCi;
optionally, the concentration of the aqueous solution of the compound Nap-Yp is 4-6 mg/mL;
optionally, the 68 The volume ratio of the Ga mixed solution to the aqueous solution of the compound Nap-Yp is (10-15): (0.8-1.2).
9. An enzymatically self-assembled molecular probe of claim 1 68 Use of Ga-Nap-Yp in the preparation of a tumor targeted PET tracer.
10. A PET tracer comprising the enzymatically self-assembled molecular probe of claim 1 68 Ga-Nap-Yp；
Optionally, the enzymatic self-assembled molecular probe precursor Nap-Yp of claim 2 is also included.
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