CN116023438A - CXCR4 targeting polypeptide and application thereof - Google Patents
CXCR4 targeting polypeptide and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of medicines, and particularly relates to a CXCR4 targeting polypeptide and application thereof. The invention first provides a CXCR4 targeting polypeptide selected from any one of SEQ ID NO. 1-10; next, a molecular probe is provided, wherein the molecular probe consists of a fluorescent label or radionuclide coupling CXCR4 targeting polypeptide; finally, there is also provided a CXCR4 targeting polypeptide as defined in any one of the preceding claims, a molecular probe as defined in any one of the preceding claims, for use in the manufacture of a reagent for the diagnosis or treatment of cancer. The CXCR4 targeting polypeptide provided by the invention can specifically target CXCR4, is coupled with fluorescent markers or radionuclides, and realizes high-sensitivity in vivo imaging of tumors; can be used for screening and early diagnosis of tumors, and can also realize treatment monitoring of tumors. The polypeptide provided by the invention has good biocompatibility and safety. The polypeptide of the invention is reported for the first time, and is convenient to obtain and popularize.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a CXCR4 targeting polypeptide and application thereof.
Background
Malignant tumors are one of the main causes of abnormal death of human beings worldwide, and pose a great threat to human health. Traditional tumor treatment methods mainly comprise operation, chemotherapy, radiotherapy and the like, but the traditional treatment methods generally bring great toxic and side effects to patients and increase the burden of the patients.
Tumor targeted therapies are methods of inhibiting tumor growth at the cellular and molecular level by interfering with molecular markers associated with tumor cell development, progression and metastasis.
Methods of treatment for inhibiting tumor progression by interfering with key molecules involved in tumor cell development, progression and spread. Compared with the traditional treatment means, the tumor targeting treatment has high selectivity to tumor cells, good curative effect and small side effect, and has good application prospect in the aspect of tumor treatment. Researchers are also developing various tumor-targeted therapies. Among the various targeted therapies, treatment with a targeted polypeptide is a desirable approach, which has the following advantages: 1) Has good penetrability, and is easy to be taken up by tumor cells; 2) High plasma eliminating speed, high selectivity, high affinity and long or short binding time. 3) Easy chemical synthesis, low immunogenicity and can avoid the shortage of monoclonal antibody treatment. Thus, specific targeting polypeptides are ideal and attractive targets for medical research, clinical applications, and molecular imaging.
Chemokines are proteins belonging to the cytokine superfamily with relatively low molecular mass (8,000-10,000) and are key mediators of cell migration during development, homeostasis and immune monitoring. Chemokines are involved in a variety of cancer progression processes, such as angiogenesis, cancer cell proliferation, invasion and metastasis, and are key determinants of disease progression. Chemokine12 (CXCL 12), also known as stromal cell derived factor-1 (stromal cell derived factor-1, SDF-1), belongs to the family of chemokine proteins.
CXCR4 (CXC motif chemokine receptor 4), also known as a fusion or CD184, is a seven transmembrane G Protein Coupled Receptor (GPCR) that is activated upon binding of the extracellular domain to a ligand. CXCR4 is overexpressed in more than 20 human tumor types, including ovarian cancer, prostate cancer, esophageal cancer, lung cancer, melanoma, neuroblastoma, renal cell carcinoma, and the like.
CXCR4 is a specific receptor of CXCL12, and after the combination of the two, the CXCL12 is involved in embryo growth and development processes such as embryo hematopoiesis, organogenesis, angiogenesis and the like, and a series of physiological and pathological processes such as inflammation, tissue immune monitoring and the like. Activation of CXCL12/CXCR4 signaling pathways can lead to a range of biological processes such as tumor proliferation, angiogenesis, invasion and migration, and tumor resistance.
In the existing detection method, immunohistochemical (IHC) detection is mainly adopted, but the IHC detection has fundamental limitation due to the heterogeneity of tumor time and space, so that the dynamic detection of tumors can not be carried out.
In contrast, molecular imaging (molecular imaging) allows for personalized diagnosis and treatment of tumors. After entering the body, the molecular probe is combined with a specific marker, and signal conversion is carried out in vitro through a specific imaging device, such as positron emission tomography (PET-CT), single Photon Emission Computed Tomography (SPECT), magnetic Resonance Imaging (MRI), fluorescence imaging (FL) and the like.
Based on the above considerations, the applicant has devised a novel class of CXCR4 targeting polypeptides, with high selectivity and affinity for CXCR4, which can achieve accurate excision of tumors during surgery by coupling fluorescent dyes and using molecular imaging surgical navigation equipment; furthermore, CXCR4 targeting polypeptide can also be used for nuclide imaging through coupling radionuclides so as to achieve the purposes of early and accurate diagnosis and treatment of tumors.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a CXCR4 targeting polypeptide, a molecular probe and application thereof.
The technical scheme adopted for solving the technical problems is as follows:
in a first aspect, the invention claims a CXCR4 targeting polypeptide selected from any one of the following amino acid sequences:
YQ-X-1: G-G-P-Y-R-R-G-R-G-P, as shown in SEQ ID NO. 1;
YQ-X-2: G-G-P-A-F-X-S-R-G-P, as shown in SEQ ID NO. 2;
YQ-X-3: G-G-G-H-R-R-G-R-G-P, as shown in SEQ ID NO. 3;
YQ-X-4: P-Y-R-X-S-R-G-P is shown as SEQ ID NO. 4;
YQ-X-5: G-G-P-Y-R-R-P-R-G-P, as shown in SEQ ID NO. 5;
YQ-X-6: G-G-P-Y-R-V-G-R-W-P, as shown in SEQ ID NO. 6;
YQ-X-7: G-G-P-Y-R-R-G-R-X-P, as shown in SEQ ID NO. 7;
YQ-X-8: G-G-P-Y-R-R-G-R-W-P, as shown in SEQ ID NO. 8;
YQ-X-9: G-G-P-Y-R-V-G-R-X-P, as shown in SEQ ID NO. 9;
YQ-X-10: G-G-P-Y-R-V-G-R-X-P, as shown in SEQ ID NO. 10.
Wherein, X in the polypeptides YQ-X-2 and YQ-X-4 is citrulline, X in the polypeptides YQ-X-7 and YQ-X-9 is D-2-naphthylalanine, and X in the polypeptides YQ-X-10 is L-2-naphthylalanine.
The amino acid residues may be L-type, D-type, or a mixture of L, D-types.
Further, the CXCR4 targeting polypeptide is selected from any one of the following amino acid sequences:
YQ-X-1：G-G-P-Y-R-R-G-R-G-P；
YQ-X-2：G-G-p-A-F-X 1 -S-R-G-p；
YQ-X-3：G-G-G-H-R-R-G-R-G-p；
YQ-X-4：P-Y-R-X 1 -S-R-G-p；
YQ-X-5：G-G-P-y-R-R-p-R-G-P；
YQ-X-6：G-G-P-y-R-v-G-R-W-P；
YQ-X-7：G-G-P-Y-r-R-G-R-X 2 -P；
YQ-X-8：G-G-P-Y-r-R-G-R-W-P；
YQ-X-9：G-G-P-y-R-v-G-R-X 2 -P；
YQ-X-10：G-G-P-y-R-v-G-R-X 3 -P；
the lower case letters in the above sequences represent D-amino acids, X 1 Represents L-citrulline, X 2 Represents D-2-naphthylalanine, X 3 Represents L-2-naphthylalanine.
In some specific embodiments, proline (Pro) may also be replaced with hydroxyproline (Hyp), and arginine (Arg) may be replaced with homoarginine (homoArg) or citrulline.
In a second aspect, the invention also provides a method for preparing a CXCR4 targeting polypeptide as described above, wherein the CXCR4 targeting polypeptide is prepared by an Fmoc solid phase polypeptide synthesis method.
In some specific embodiments, the method of making comprises:
(1) According to the designed polypeptide sequence, the amino acid is European to Rink Amide MBHA resin one by one, in the reaction process, the solvent adopts DMF to dissolve the amino acid protected by FMOC and adopts the HCTU/DIEA scheme of FastMoc chemistry to activate, and each amino acid reacts for 1-2 hours; thereafter, fmoc at the N-terminus was removed using a 20% piperidine/DMF solution, repeated twice for 5min each; then, TFA/H is used 2 O/TIS/anisole (90/3/4/3, v/v/v/v) and treating the polypeptide at room temperature for 3 hours while removing the side chain protection and cleaving the polypeptide from the resin; then using nitrogen to blow dry the system, using methyl tertiary butyl ether to wash twice, centrifuging to obtain the precipitatePrecipitating;
(2) Coupling amino acids one by one to Rink Amide MBHA resin according to a preset amino acid sequence, wherein HCTU and Fmoc protected amino acids are dissolved in DMF containing 0.4mol/L DIPEA during coupling, and each coupling time>1h; and then carrying out a deprotection flow: removing Fmoc groups by using a DMF solution containing 20% piperidine, wherein each deprotection time is 5min, and repeating the steps twice; then the reaction is carried out for 2 to 3 hours under the action of strong acid to remove the side chain protecting group, wherein the strong acid comprises TFA90 to 95 percent and H by mass 2 O 2-5％、TIS 2-5％、EDT 2％-5％。
In a third aspect, the invention also provides a molecular probe coupled to a CXCR4 targeting polypeptide as hereinbefore described by a fluorescent label or radionuclide.
Further, the fluorescent label is one or more of IRDye800, cy5, cy7, ICG, rhodamine, FITC and other fluorescent dyes.
Still further, the fluorescent label may be labeled by click chemistry such as NHS, EDC, MAL.
Further, the radionuclide may be 18 F， 68 Ga， 64 Cu may also be 99m Tc， 90 Y may also be 111 In， 125 I, 131 I or 177 Lu。
Further, the chelating agent of the radionuclide is HYNIC, DOTA, NOTA or DTPA and derivatives thereof, and the radionuclide ligand is tris (hydroxymethyl) methylglycine (Tricine) or sodium-trisulphonate triphenylphosphine (TPPTS).
In a fourth aspect, the invention also provides the use of a CXCR4 targeting peptide as described hereinbefore, a molecular probe as described hereinbefore, in the manufacture of a reagent for the diagnosis or treatment of cancer.
Further, the reagent is a fluorescent imaging or radioactive imaging reagent.
Still further, the imaging agent is one or more of a radionuclide, a radionuclide label, a magnetic resonance contrast agent, or a molecular imaging agent.
Further, the agent also includes a pharmaceutically acceptable carrier thereof. Such as PLGA polymers, dendrimer dendrimers, hydrogels, micelles, liposomes or inorganic nanoparticles, etc.
Further, the cancer is all tumors over-expressing CXCR4, such as one or more of melanoma, lymphoma, breast cancer, cervical cancer, lung cancer, colorectal cancer, glioma, prostate cancer, pancreatic cancer.
Further, the cancer is one or more of melanoma, breast cancer, cervical cancer, lung cancer and prostate cancer
The invention also protects the use of the aforementioned agents for the preparation of a medicament for the diagnosis of CXCR4 positive tumor imaging or the detection of surgical navigation precise excision.
The positive tumor is all tumors over-expressed by CXCR4, such as one or more of melanoma, lymphoma, breast cancer, cervical cancer, lung cancer, colorectal cancer, glioma, prostate cancer and pancreatic cancer.
Advantageous effects
The CXCR4 targeting polypeptide provided by the invention can specifically target CXCR4, and is coupled with fluorescent markers or radionuclides, so that high-sensitivity in vivo imaging of tumors is realized.
The CXCR4 targeting polypeptide can be used for screening and early diagnosis of tumors and can also realize treatment monitoring of tumors.
The polypeptide provided by the invention has good biocompatibility and safety.
The polypeptide of the invention is reported for the first time, and is convenient to obtain and popularize.
Drawings
The structure of the polypeptide YQ-X-1 in FIG. 1.
FIG. 2 shows the structure of MPA-YQ-X-1 in example 2.
FIG. 3A flow affinity assay of probe MPA-YQ-X-9 on B16 cells.
FIG. 4 fluorescent imaging of probe MPA-YQ-X-1 on A549 tumor-bearing mice.
FIG. 5 HYNIC-YQ-X-1.
FIG. 6 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-1 in melanoma cell B16 tumor-bearing mice.
FIG. 7 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-1 in lung cancer cell A549 tumor-bearing mice.
FIG. 8 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-2 in lung cancer cell A549 tumor-bearing mice.
FIG. 9 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-3 in melanoma cell B16 tumor-bearing mice.
FIG. 10 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-4 in melanoma cell B16 tumor-bearing mice.
FIG. 11 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-5 in prostate cancer cells PC3 tumor-bearing mice.
FIG. 12 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-6 in melanoma cell B16 tumor-bearing mice.
FIG. 13 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-6 in cervical cancer cell Hela tumor-bearing mice.
FIG. 14 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-7 in melanoma cell B16 tumor-bearing mice.
FIG. 15 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-7 in cervical cancer cell Hela tumor-bearing mice.
FIG. 16 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-8 in melanoma cell B16 tumor-bearing mice.
FIG. 17 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-9 in melanoma cell B16 tumor-bearing mice.
FIG. 18 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-9 in breast cancer cell MCF7 tumor-bearing mice.
FIG. 19 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-9 in cervical cancer cell Hela tumor-bearing mice.
FIG. 20 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-10 in melanoma cell B16 tumor-bearing mice.
Detailed Description
The preparation of the compounds of the present invention described in the following examples is illustrative only and is not intended to limit the scope of the invention. The reagents or instrumentation used are not manufacturer specific and are considered to be commercially available conventional products.
N, N-Diisopropylethylamine (DIPEA), piperidine, trifluoroacetic acid (TFA), dichloromethane (DCM), N, N-Dimethylformamide (DMF), methanol, phenol, ninhydrin, anhydrous diethyl ether, resin, triisopropylsilane (TIS), 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU), 6-chlorobenzotriazole-1, 3-tetramethylurea Hexafluorophosphate (HCTU), dimethyl sulfoxide (DMSO), various Fmoc protected amino acids, polypeptide synthesis tubes, shaking tables, vacuum water pumps and other instruments are all commercially available.
Example 1
The present example provides a method for preparing polypeptide YQ-X-n (n=1 to 10), the polypeptide is synthesized by FMOC solid phase synthesis, the specific synthesis method is as follows:
1. 400mg Rink Amide MBHA resin was weighed into a solid phase synthesis tube, after which 6mL DCM (dichloromethane) was added and shaken on a shaker for 20min in order to swell the resin; 2. the reaction tube was then drained, washed twice with DMF and drained after shaking, 5mL of piperidine/DMF (2/8,v/v) solution was added and the reaction was repeated twice for 5 min. The synthesis tube was washed five times with 5mL DMF and drained, after which 1mmol of FMOC amino acid, 0.4mL DIEA, 400mg HCTU, 5mL DMF were added and the mixture was placed in a shaker for reaction for 1 hour; 3. repeating the step 2, adding FMOC amino acid according to the amino terminal to the carboxyl terminal according to the amino acid sequence of the designed polypeptide each time, and keeping the rest components unchanged until all the amino acids of the polypeptide sequence are added; 4. after removal of the FMOC protection of the last amino acid with piperidine/DMF (2/8,v/v) solution, the system was washed twice each with DMF, DCM, meOH and pumped dry, 5mL (cleavage solution TFA90-95%, H) was added 2 O2-5%, TIS 2-5% and EDT 2-5%) are treated for 3 hours at room temperature, the volume of the cutting fluid is volatilized to be unchanged rapidly by utilizing nitrogen flow, 5mL of methyl tertiary butyl ether is added, and the precipitated peptide is obtained after shaking and centrifugation.
The precipitated peptide was diluted with water and purification of the peptide was accomplished using high performance liquid chromatography techniques using a reversed phase C18 column with a chromatography packing of 10 μm eluting with a mobile phase system of 0.1% TFA/water (V/V) and 0.1% TFA/acetonitrile (V/V) gradient under 220nm UV detection. And after the final product is further determined by mass spectrometry, the collected eluent is put into a freeze dryer for concentration, and is freeze-dried into white powder.
YQ-X-n (n=1 to 10) was confirmed by mass spectrometry, and YQ-X-1 was taken as an example, and its structure was as shown in FIG. 1, and [ M+H ]] + cal.:1071.59,[M+H] + 1071.67; YQ-X-2 its [ M+H ]] + cal.:1001.53,[M+H] + 1001.64; YQ-X-3 its [ M+H ]] + cal.:1005.56,[M+H] + 1005.79; YQ-X-4 its [ M+H ]] + cal.:988.54,[M+H] + 988.98; YQ-X-5 its [ M+H ]] + cal.:1111.62,[M+H] + 1111.98; YQ-X-6 its [ M+H ]] + cal.:1143.62,[M+H] + 1143.88; YQ-X-7 its [ M+H ]] + cal.:1211.65,[M+H] + 1211.98; YQ-X-8 its [ M+H ]] + cal.:1200.65,[M+H] + 1200.86; YQ-X-9 its [ M+H ]] + cal.:1154.62,[M+H] + 1154.90; YQ-X-10 its [ M+H ]] + cal.:1154.62,[M+H] + obs.:1154.32。
Example 2
This example provides a process for the preparation of YQ-X-n (n=1 to 10) coupled MPA, wherein MPA is a near infrared fluorescent dye from the invention patent (CN 101440282) filed by the subject group, comprising the following synthetic steps:
2.0mg of YQ-X-n (n=1 to 10) polypeptide obtained in example 1, 3.0mg of HATU and 3.0mg of fluorescent dye MPA (structure shown in the following diagram) were dissolved in 200. Mu.L of DMSO solution, then 1.0. Mu.L of DIPEA was added thereto and reacted in a metal bath at 50℃for 2 hours. Then, the high-efficiency preparation liquid phase is adopted for separation and purification, and the preparation liquid phase conditions are as follows: a Agilent 1220 Infinicity II series HPLC system was used equipped with a Agilent ZORBAX SB-C18 semi-preparative column (9.4X105 mm,5 um) gradient elution for 60 minutes at a flow rate of 2mL/min with mobile phase A of 0.01% TFA/ultra pure water (v/v) and B of 0.01% TFA/acetonitrile (v/v). The elution gradient was set as: the final product collected at 0-5 min 90% A and 10% B,20-25 min 70% A and 30% B,30 min 65% A and 35% B,40 min 50% A and 50% B,50-60 min 10% A and 90% B was MPA-YQ-X-n (n=1-10), the structure of which is shown in FIG. 2.
Example 3
The affinity of MPA-YQ-X-9 in CXCR4 high expression cell line B16 is detected by flow cytometry in this example, and the specific procedure is as follows:
b16 cells were 1×10 per well 6 Cell density was seeded in six well plates. After 24h incubation, 1mL of fresh medium containing 10. Mu.M/L of MPA-YQ-X-9 and 100. Mu.M/L of AMD3100, 1mL of fresh medium containing 10. Mu.M/L of MPA-YQ-X-9 and 200. Mu.M/L of AMD3100, 1mL of fresh medium containing 10. Mu.M/L of MPA, 1mL of fresh blank medium were added to each well in this order, and incubated at 37℃for 2h. Cells were then washed 3 times per well with PBS (pH 7.4) to remove unbound drug. Cells were then trypsinized, centrifuged at 1000rpm for 3min, resuspended in 300 μl PBS and kept in the dark in a flow tube, and loaded by flow cytometry on its own with near infrared FL 4 channels, and finally the raw data analysis was expressed as mean fluorescence intensity (mean fluorescence intensity, MFI) using FlowJo 7.0 software and GraphPad Prism 8.0 software.
The results are shown in FIG. 3, which illustrates that the polypeptide MPA-YQ-X-9 has strong affinity to CXCR4 high expression cell line and the CXCR4 inhibitor AMD3100 has blocking effect on the CXCR4 high expression cell line, and the polypeptide probe can specifically target CXCR4.
Example 4
In this example, the probe MPA-YQ-X-1 prepared in example 2 was used for in vivo fluorescence imaging in tumor-bearing mice, and the specific procedure is as follows:
a549 cells were inoculated subcutaneously in 6-8 week old female mice and used for imaging experiments after tumor formation. One group of rat tails was intravenously injected with 15. Mu.g of probe MPA-YQ-X-1, and the other group of rat tails was intravenously injected with 15. Mu.g of pure dye MPA and fluorescence detection was performed on the in vivo imaging system of small animals at 1h, 2h, 4h, 6h, 9h, 12h and 24h post-administration. As shown in FIG. 4, the probe MPA-YQ-X-1 has stronger fluorescent signal in 1h tumor, and compared with pure dye, the fluorescent intensity of the mouse tumor injected with the probe is stronger, which proves that the probe of the invention has CXCR4 targeting and can realize high-sensitivity in vivo imaging of tumor. The probe has good biocompatibility and safety through the distribution condition of main organs.
Example 5
This example provides for the preparation of HYNIC-YQ-X-n (n=1 to 10)
(1) Synthesis of bifunctional chelating agent HYNIC-NHS
5.0g of 6-chloronicotinic acid is weighed, 18mL of 80% hydrazine hydrate is added, water is used for dissolution, and the mixture is heated and refluxed for reaction for 6 hours. And then, through a decompression rotary evaporation concentration system, adjusting the PH value to be 5.5 by using concentrated hydrochloric acid, separating out solid, and carrying out suction filtration and drying to obtain yellow solid which is 2-hydrazinopyridine-4-formic acid.
4.0g of 2-hydrazinopyridine-4-carboxylic acid and 3.8g of p-dimethylaminobenzoic acid were weighed, dissolved in 25mL of DMF and stirred at room temperature for 3h. Then adding 4.5g of NHS, 9.0g of EDCI and 15mL of DMF, stirring at room temperature overnight, precipitating yellow solid with water, filtering, washing with methanol, pumping, boiling in ethyl acetate, filtering while hot, and pumping to obtain bright yellow solid which is HYNIC-NHS.
(2) Synthesis of HYNIC-YQ-X-n (n=1 to 10)
Purified 2mg of polypeptide YQ-X-n (X=1 to 10) and 2mg of HYNIC-NHS were dissolved in 0.2mL of DMSO, 1. Mu.L of DIPEA was added to react at 50℃for 3 hours, and after completion of the reaction, separation and purification were performed by preparing a liquid phase to obtain a product, the structure of which is shown in FIG. 5 as an example.
Example 6
The present embodiment provides 99m Preparation of Tc-HYNIC-YQ-X-n (n=1-10)
Preparing 100.0mg/mL TPPTS solution, 130.0mg/mL Tricine (trimethylglycine) and 102.4mg/mL succinic acid-sodium succinate buffer (77.0 mg succinic acid, 25.4mg succinic acid), mixing 10.0uL TPPTS solution, 10.0uL Tricine solution, 10.0uL succinic acid-sodium succinate buffer and 10.0uL (1.0 mg/mL) of HYNIC-YQ-X-n (n=1-10) described in example 5 in a penicillin bottle, respectively, adding 10 mCiNa 99m TcO4 is heated in a metal bath at 100 ℃ for 20 minutes and cooled to the temperature after the reaction is finishedRoom temperature, the polypeptide radiopharmaceuticals are prepared 99m Tc-HYNIC-YQ-X-n (n=1-10), and the product is analyzed and identified by Agilent ZORBAX SB-Aq analytical column. The HPLC method used was an Agilent 1220 Infinicity II series HPLC system equipped with a radioactive online detector (Flow-RAM) and a Agilent ZORBAX SB-Aq analytical column (4.6X105 mm,5 um). Gradient elution was carried out for 45 minutes at a flow rate of 1mL/min, wherein mobile phase A was ultrapure water (0.01% TFA) and B was acetonitrile (0.01% TFA). The elution gradient was set as: 95% A and 5% B at 0-5 min, 70% A and 30% B at 15 min, 65% A and 35% B at 20min, 45% A and 55% B at 25 min, 5%A and 95% B at 45 min. 99m The purity of Tc-HYNIC-YQ-X-n (n=1-10)>95％。
Example 7
Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-1 in tumor-bearing mice
The probe prepared in example 6 99m Tc-HYNIC-YQ-X-1 is prepared into physiological saline solution, and is injected into 500 μCi through tail vein, 2 tumor-bearing nude tail veins (B16, A549) are respectively injected, and at least three tumor-bearing mice are each injected. And SPECT signal acquisition is carried out at 0.5h, 1h, 2h, 3h and 4h after administration. The imaging results are shown in FIG. 6 and FIG. 7, and the probe 99m Tc-HYNIC-YQ-X-1 has obvious uptake in tumor site, and shows that the probe has excellent targeting property.
Example 8
Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-2 in tumor-bearing mice
The probe prepared in example 6 99m Tc-HYNIC-YQ-X-2 is prepared into physiological saline solution, and at least three of the tumor-bearing nude tail veins of lung cancer cells A549 are injected through tail vein injection of 500 mu Ci. And SPECT signal acquisition is carried out at 0.5h, 1h, 2h, 3h and 4h after administration. The imaging results are shown in FIG. 8, probe 99m Tc-HYNIC-YQ-X-2 has obvious uptake in tumor site, and shows that the probe has excellent targeting property.
Example 9
Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-3 in tumor-bearing mice
The probe prepared in example 6 99m Tc-HYNIC-YQ-X-3 is prepared into physiological saline solution, and is injected into 500 mu Ci through tail vein, and at least three of melanoma cell B16 tumor-bearing nude tail veins are injected. And SPECT signal acquisition is carried out at 0.5h, 1h, 2h, 3h and 4h after administration. The imaging results are shown in FIG. 9, probe 99m Tc-HYNIC-YQ-X-3 has obvious uptake in tumor site, and shows that the probe has excellent targeting property.
Example 10
Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-4 in tumor-bearing mice
The probe prepared in example 6 99m Tc-HYNIC-YQ-X-4 is prepared into physiological saline solution, and is injected into 500 mu Ci through tail vein, and at least three of melanoma cell B16 tumor-bearing nude tail veins are injected. And SPECT signal acquisition is carried out at 0.5h, 1h, 2h, 3h and 4h after administration. The imaging results are shown in FIG. 10, probe 99m Tc-HYNIC-YQ-X-4 has obvious uptake in tumor site, and shows that the probe has excellent targeting property.
Example 11
Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-5 in tumor-bearing mice
The probe prepared in example 6 99m Tc-HYNIC-YQ-X-5 is prepared into physiological saline solution, and is injected into 500 mu Ci through tail vein, and at least three human prostate cancer cells PC3 are injected into tumor-bearing tail veins of the nude tail. And SPECT signal acquisition is carried out at 0.5h, 1h, 2h, 3h and 4h after administration. The imaging results are shown in FIG. 11, probe 99m Tc-HYNIC-YQ-X-5 has obvious uptake in tumor site, and shows that the probe has excellent targeting property.
Example 12
Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-6 in tumor-bearing mice
The probe prepared in example 6 99m Tc-HYNIC-YQ-X-6 is prepared into physiological saline solution, and 500 μCi is injected into tail vein, and melanoma cell B16 and human cervical cancer cell Hela tumor-bearing nude tail vein are injected into at least three of each. And SPECT signal acquisition is carried out at 0.5h, 1h, 2h, 3h and 4h after administration. The imaging result is shown in FIG. 12 and FIG. 13, probe 99m Tc-HYNIC-YQ-X-6 has obvious uptake in tumor site, and shows that the probe has excellent targeting property.
Example 13
Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-7 in tumor-bearing mice
The probe prepared in example 6 99m Tc-HYNIC-YQ-X-7 is prepared into physiological saline solution, and 500 μCi is injected into tail vein, and melanoma cell B16 and human cervical cancer cell Hela tumor-bearing nude tail vein are injected into at least three of each. And SPECT signal acquisition is carried out at 0.5h, 1h, 2h, 3h and 4h after administration. The imaging results are shown in FIGS. 14 and 15, and the probe 99m Tc-HYNIC-YQ-X-7 has obvious uptake in tumor site, and shows that the probe has excellent targeting property.
Example 14
Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-8 in tumor-bearing mice
The probe prepared in example 6 99m Tc-HYNIC-YQ-X-8 is prepared into physiological saline solution, and is injected into 500 mu Ci through tail vein, and at least three of melanoma cell B16 tumor-bearing nude tail veins are injected. And SPECT signal acquisition is carried out at 0.5h, 1h, 2h, 3h and 4h after administration. The imaging results are shown in FIG. 16, probe 99m Tc-HYNIC-YQ-X-8 has obvious uptake in tumor site, and shows that the probe has excellent targeting property.
Example 15
Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-9 in tumor-bearing mice
The probe prepared in example 6 99m Tc-HYNIC-YQ-X-9 is prepared into physiological saline solution, and at least three of the physiological saline solution are injected into a tail vein by 500 mu Ci, and melanoma cells B16, human breast cancer cells MCF-7 and human cervical cancer cells Hela tumor-bearing nude tail veins are injected. And SPECT signal acquisition is carried out at 0.5h, 1h, 2h, 3h and 4h after administration. The imaging results are shown in FIG. 17, FIG. 18 and FIG. 19, and the probe 99m Tc-HYNIC-YQ-X-9 has obvious uptake in tumor site, and shows that the probe has excellent targeting property.
Example 16
Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-10 in tumor-bearing mice
The probe prepared in example 6 99m Tc-HYNIC-YQ-X-10 is prepared into physiological saline solution, and 500 μCi is injected into tail vein, and at least three of melanoma cell B16 tumor-bearing nude tail veins are injected. And SPECT signal acquisition is carried out at 0.5h, 1h, 2h, 3h and 4h after administration. The imaging results are shown in FIG. 20, probe 99m Tc-HYNIC-YQ-X-10 has obvious uptake in tumor site, and shows that the probe has excellent targeting property.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that would occur to one skilled in the art are included in the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims.
Sequence listing
| Name of the name | Polypeptide sequence |
| YQ-X-1 | GGPYRRGRGP |
| YQ-X-2 | GGpAFX 1 SRGp |
| YQ-X-3 | GGGHRRGRGp |
| YQ-X-4 | PYRX 1 SRGp |
| YQ-X-5 | GGPyRRpRGP |
| YQ-X-6 | GGPyRvGRWP |
| YQ-X-7 | GGPYrRGRX 2 P |
| YQ-X-8 | GGPYrRGRWP |
| YQ-X-9 | GGPyRvGRX 2 P |
| YQ-X-10 | GGPyRvGRX 3 P |
Wherein in the above sequences the lowercase letters denote D-amino acids, X 1 Represents L-citrulline, X 2 Represents D-2-naphthylalanine, X 3 Represents L-2-naphthylalanine.
Claims (10)
1. A CXCR4 targeting polypeptide selected from any one of the following amino acid sequences:
YQ-X-1: G-G-P-Y-R-R-G-R-G-P, as shown in SEQ ID NO. 1;
YQ-X-2: G-G-P-A-F-X-S-R-G-P, as shown in SEQ ID NO. 2;
YQ-X-3: G-G-G-H-R-R-G-R-G-P, as shown in SEQ ID NO. 3;
YQ-X-4: P-Y-R-X-S-R-G-P is shown as SEQ ID NO. 4;
YQ-X-5: G-G-P-Y-R-R-P-R-G-P, as shown in SEQ ID NO. 5;
YQ-X-6: G-G-P-Y-R-V-G-R-W-P, as shown in SEQ ID NO. 6;
YQ-X-7: G-G-P-Y-R-R-G-R-X-P, as shown in SEQ ID NO. 7;
YQ-X-8: G-G-P-Y-R-R-G-R-W-P, as shown in SEQ ID NO. 8;
YQ-X-9: G-G-P-Y-R-V-G-R-X-P, as shown in SEQ ID NO. 9;
YQ-X-10: G-G-P-Y-R-V-G-R-X-P, as shown in SEQ ID NO. 10.
Wherein, X in the polypeptides YQ-X-2 and YQ-X-4 is citrulline, X in the polypeptides YQ-X-7 and YQ-X-9 is D-2-naphthylalanine, and X in the polypeptides YQ-X-10 is L-2-naphthylalanine.
2. The CXCR4 targeting polypeptide of claim 1, wherein said CXCR4 targeting polypeptide is selected from any one of the amino acid sequences:
YQ-X-1：G-G-P-Y-R-R-G-R-G-P；
YQ-X-2：G-G-p-A-F-X 1 -S-R-G-p；
YQ-X-3：G-G-G-H-R-R-G-R-G-p；
YQ-X-4：P-Y-R-X 1 -S-R-G-p；
YQ-X-5：G-G-P-y-R-R-p-R-G-P；
YQ-X-6：G-G-P-y-R-v-G-R-W-P；
YQ-X-7：G-G-P-Y-r-R-G-R-X 2 -P；
YQ-X-8：G-G-P-Y-r-R-G-R-W-P；
YQ-X-9：G-G-P-y-R-v-G-R-X 2 -P；
YQ-X-10：G-G-P-y-R-v-G-R-X 3 -P；
wherein X is 1 Represents citrulline, X 2 Represents D-2-naphthylalanine, X 3 Represents L-2-naphthylalanine;
lower case letters in the sequences indicate amino acids of type D.
3. The CXCR4 targeting polypeptide of claim 1 or 2, wherein the proline is replaced with hydroxyproline and the arginine is replaced with homoarginine or citrulline.
4. A method of preparing a CXCR4 targeting polypeptide according to any one of claims 1 to 3, wherein said CXCR4 targeting polypeptide is prepared by an Fmoc solid phase polypeptide synthesis method.
5. The preparation method according to claim 4, wherein the method comprises the following specific steps:
(1) According to the designed polypeptide sequence, the amino acid is European to Rink Amide MBHA resin one by one, in the reaction process, the solvent adopts DMF to dissolve the amino acid protected by FMOC and adopts the HCTU/DIEA scheme of FastMoc chemistry to activate, and each amino acid reacts for 1-2 hours; thereafter, fmoc at the N-terminus was removed using a 20% piperidine/DMF solution, repeated twice for 5min each; then, TFA/H is used 2 O/TIS/anisole (90/3/4/3, v/v/v/v) and treating the polypeptide at room temperature for 3 hours while removing the side chain protection and cleaving the polypeptide from the resin; then, using nitrogen to blow the system dry, using methyl tertiary butyl ether to wash twice, and centrifuging to obtain a precipitate;
(2) Coupling amino acids one by one to Rink Amide MBHA resin according to a preset amino acid sequence, wherein HCTU and Fmoc protected amino acids are dissolved in DMF containing 0.4mol/L DIPEA during coupling, and each coupling time>1h; and then carrying out a deprotection flow: removing Fmoc groups by using a DMF solution containing 20% piperidine, wherein each deprotection time is 5min, and repeating the steps twice; then the reaction is carried out for 2 to 3 hours under the action of strong acid to remove the side chain protecting group, wherein the strong acid comprises TFA90 to 95 percent and H by mass 2 O 2-5％、TIS 2-5％、EDT 2％-5％。
6. A molecular probe consisting of a fluorescent label or radionuclide conjugated CXCR4 targeting polypeptide according to any of claims 1 to 3.
7. The molecular probe of claim 6, wherein the fluorescent label is one or more of IRDye800, cy5, cy7, ICG, rhodamine, FITC; preferably, the fluorescent label is labelled by NHS, EDC, MAL.
8. The molecular probe of claim 6, wherein the radionuclide is selected from the group consisting of 18 F， 68 Ga， 64 Cu, or 99m Tc， 90 Y, or, alternatively, 111 In， 125 I, 131 i or 177 Lu; preferably, the chelating agent of the radionuclide is HYNIC, DOTA, NOTA or DTPA and derivatives thereof, and the radionuclide ligand is tris (hydroxymethyl) methylglycine or sodium m-sulfonate triphenylphosphine.
9. Use of a CXCR4 targeting polypeptide of any one of claims 1-3, a molecular probe of any one of claims 6-8 in the preparation of a reagent for the diagnosis or treatment of cancer;
preferably, the agent further comprises a pharmaceutically acceptable carrier thereof;
more preferably, the carrier is a PLGA polymer, a Dendrimer, a hydrogel, a micelle, a liposome, or an inorganic nanoparticle;
preferably, the cancer is one or more of melanoma, lymphoma, breast cancer, cervical cancer, lung cancer, colorectal cancer, glioma, prostate cancer, pancreatic cancer.
10. The use according to claim 9, wherein the agent is a fluorescent imaging or radiological imaging agent;
preferably, the imaging agent is one or more of a radionuclide, a radionuclide label, a magnetic resonance contrast agent, or a molecular imaging agent.
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| CN116813704A (en) * | 2023-05-26 | 2023-09-29 | 豫章师范学院 | Tumor targeting fluorescent molecular probe and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116813704A (en) * | 2023-05-26 | 2023-09-29 | 豫章师范学院 | Tumor targeting fluorescent molecular probe and application thereof |
| CN116813704B (en) * | 2023-05-26 | 2024-01-23 | 豫章师范学院 | Tumor targeting fluorescent molecular probe and application thereof |
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