CN112961215B - Polypeptide and tumor targeting peptide, tumor detection reagent, tumor surgery navigation contrast medium and tumor targeting drug thereof - Google Patents
Polypeptide and tumor targeting peptide, tumor detection reagent, tumor surgery navigation contrast medium and tumor targeting drug thereof Download PDFInfo
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Abstract
The invention relates to a polypeptide and tumor targeting peptide thereof, a tumor detection reagent, a tumor operation navigation contrast medium and a tumor targeting drug, belonging to the field of biological medicine. The polypeptide is selected from the group consisting of Va-C, Va-RA, Va-RS, Va-RK, Va-KRA, Va-RL; the amino acid sequence of Va-C is shown in SEQ ID NO. 1; the amino acid sequence of Va-RA is shown in SEQ ID NO. 2; the amino acid sequence of the Va-RS is shown in SEQ ID NO. 3; the amino acid sequence of Va-RK is shown in SEQ ID NO. 4; the amino acid sequence of the Va-KRA is shown as SEQ ID NO. 5; the amino acid sequence of the Va-RL is shown in SEQ ID NO. 6. Compared with the existing commonly used tumor targeting peptide such as P47, the polypeptide of the invention has better targeting property, and can be applied to clinical detection, surgical navigation and treatment of tumors.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a polypeptide, a tumor targeting peptide thereof, a tumor detection reagent, a tumor surgery navigation contrast medium and a tumor targeting medicine.
Background
Hepatocellular carcinoma (HCC) is one of common malignant tumors in China, and has the characteristics of high morbidity and high mortality. The liver cancer is high in malignancy degree and rapid in development, once obvious symptoms appear, about 1/3 belongs to the late stage, and the average survival time is about half a year, so that early diagnosis of the liver cancer has great influence on prognosis of patients. At present, there are many clinical examination methods for liver cancer, including traditional imaging examination methods such as ultrasound, CT, nuclear magnetic resonance, etc., which mainly rely on nonspecific imaging means to analyze different tissue physical characteristics, so as to examine the disease. Although the resolution of the images obtained is continuously improved with the development of imaging technology, the defects in sensitivity and specificity still exist, so that the optimal time for treatment is missed when the focus is detected. The molecular imaging integrating biochemistry, nanotechnology and imaging technology has great significance for tumor detection. In particular, the PET-CT technique can detect the extrahepatic metastasis of liver cancer well, but the effect of detecting primary liver cancer is not ideal. The reason for this is that because the liver itself is active in glycometabolism, 18-fluorodeoxyglucose (18F-FDG), which is a probe molecule used in PET-CT, is also absorbed in normal liver tissues, resulting in high background noise. More importantly, the liver is a detoxifying organ of a human body, and liver cells can be combined with various substances, so that the application of a plurality of molecular imaging probes in liver cancer detection is limited. Therefore, the development of a molecular probe with high specificity and high sensitivity is urgently needed for liver cancer detection.
Some patients with liver cancer can obtain better prognosis by surgical resection, but accurate positioning of tumor boundaries is a problem which always troubles clinicians and researchers. The fluorescence operation system guided by the contrast molecules with tumor affinity can display the shape and the edge of the tumor under the excitation of fluorescence, provide an operation boundary for surgeons and enable the complete excision of the tumor to be possible. However, indocyanine green (ICG), the only FDA-approved contrast agent for surgical navigation of liver cancer, is insufficient in sensitivity and specificity and is difficult to satisfy clinical needs.
Disclosure of Invention
Based on the above disadvantages and needs in the art, the present invention provides a series of polypeptides with tumor targeting properties, and a large number of experiments prove that the polypeptides have better targeting properties than the existing commonly used tumor targeting peptides such as P47, and can be applied to clinical detection, surgical navigation and treatment of tumors.
The technical scheme of the invention is as follows:
a polypeptide selected from the group consisting of Va-C, Va-RA, Va-RS, Va-RK, Va-KRA, Va-RL;
the amino acid sequence of Va-C is shown in SEQ ID NO. 1;
the amino acid sequence of the Va-RA is shown in SEQ ID NO. 2;
the amino acid sequence of the Va-RS is shown in SEQ ID NO. 3;
the amino acid sequence of Va-RK is shown in SEQ ID NO. 4;
the amino acid sequence of the Va-KRA is shown as SEQ ID NO. 5;
the amino acid sequence of Va-RL is shown in SEQ ID NO. 6.
In a particular embodiment of the invention, the polypeptide of the invention is Va-C, or Va-RA, or Va-RS, or Va-RK, or Va-KRA, or Va-RL.
The skilled person can also use any 2, any 3, any 4, any 5, any 6 of the polypeptides Va-C, Va-RA, Va-RS, Va-RK, Va-KRA and Va-RL according to the present invention to achieve the purpose.
Any act of using, synthesizing, expressing, manufacturing, producing, selling, or offering for sale a polypeptide Va-C, or Va-RA, or Va-RS, or Va-RK, or Va-KRA, or Va-RL of the present invention, any 2, any 3, any 4, any 5, any 6 acts of using, synthesizing, expressing, manufacturing, producing, selling, offering for sale a polypeptide Va-C, Va-RA, Va-RS, Va-RK, Va-KRA, or Va-RL of the present invention are within the scope of the present invention.
Preferably, the polypeptide is a tumor targeting peptide.
Preferably, the tumor is selected from: liver cancer, breast cancer, pancreatic cancer, intestinal cancer, brain tumor, cervical cancer, lung cancer, gastric cancer, bone cancer, ovarian cancer, lymphoma, renal cancer, nasopharyngeal cancer, testicular cancer, esophageal cancer, bladder cancer, prostate cancer, thyroid cancer, neuroblastoma, soft tissue sarcoma;
preferably, the polypeptide is Va-RS with an amino acid sequence shown in SEQ ID NO. 3.
A tumor targeting peptide comprising a polypeptide; wherein the polypeptide is selected from the group consisting of Va-C, Va-RA, Va-RS, Va-RK, Va-KRA, Va-RL;
the amino acid sequence of Va-C is shown in SEQ ID NO. 1;
the amino acid sequence of the Va-RA is shown in SEQ ID NO. 2;
the amino acid sequence of the Va-RS is shown in SEQ ID NO. 3;
the amino acid sequence of Va-RK is shown in SEQ ID NO. 4;
the amino acid sequence of the Va-KRA is shown as SEQ ID NO. 5;
the amino acid sequence of the Va-RL is shown in SEQ ID NO. 6.
In other embodiments of the invention, the tumor targeting peptide of the invention is Va-C, or Va-RA, or Va-RS, or Va-RK, or Va-KRA, or Va-RL.
The skilled person can also use any 2, any 3, any 4, any 5, or any 6 of the tumor targeting peptides Va-C, Va-RA, Va-RS, Va-RK, Va-KRA, Va-RL of the present invention in combination to achieve the purpose of tumor targeting according to the teaching of the present invention.
Any act of using, synthesizing, expressing, manufacturing, producing, selling, or marketing the tumor targeting peptide Va-C, or Va-RA, or Va-RS, or Va-RK, or Va-KRA, or Va-RL of the present invention, any 2, any 3, any 4, any 5, any 6 acts of using, synthesizing, expressing, manufacturing, producing, selling, or marketing the tumor targeting peptide Va-C, Va-RA, Va-RS, Va-RK, Va-KRA, or Va-RL of the present invention, fall within the scope of the present invention.
The tumor targeting peptide also comprises a conventional tumor targeting peptide; preferably, the conventional tumor targeting peptide is selected from the group consisting of: p47, a54, SP 94;
preferably, the tumor is selected from: liver cancer, breast cancer, pancreatic cancer, intestinal cancer, cerebroma, cervical cancer, lung cancer, gastric cancer, bone cancer, ovarian cancer, lymphoma, renal cancer, nasopharyngeal cancer, testicular cancer, esophageal cancer, bladder cancer, prostate cancer, thyroid cancer, neuroblastoma, and soft tissue sarcoma.
A tumor detection reagent comprising a polypeptide; wherein the polypeptide is selected from the group consisting of Va-C, Va-RA, Va-RS, Va-RK, Va-KRA, Va-RL;
the amino acid sequence of Va-C is shown in SEQ ID NO. 1;
the amino acid sequence of the Va-RA is shown in SEQ ID NO. 2;
the amino acid sequence of the Va-RS is shown in SEQ ID NO. 3;
the amino acid sequence of Va-RK is shown in SEQ ID NO. 4;
the amino acid sequence of the Va-KRA is shown as SEQ ID NO. 5;
the amino acid sequence of the Va-RL is shown in SEQ ID NO. 6.
The tumor detection reagent further comprises: color-developing agent, coloring agent;
preferably, the colour developer is selected from: ninhydrin, dimethylaminobenzaldehyde, diaminobenzidine;
preferably, the staining agent is selected from fluorescent dyes, preferably selected from: FAM, DAPI, CY5, IR800, ICG;
preferably, the tumor is selected from: liver cancer, breast cancer, pancreatic cancer, intestinal cancer, cerebroma, cervical cancer, lung cancer, gastric cancer, bone cancer, ovarian cancer, lymphoma, renal cancer, nasopharyngeal cancer, testicular cancer, esophageal cancer, bladder cancer, prostate cancer, thyroid cancer, neuroblastoma, and soft tissue sarcoma.
A tumor surgery navigation contrast agent, comprising a polypeptide; wherein the polypeptide is selected from the group consisting of Va-C, Va-RA, Va-RS, Va-RK, Va-KRA, Va-RL;
the amino acid sequence of Va-C is shown in SEQ ID NO. 1;
the amino acid sequence of the Va-RA is shown in SEQ ID NO. 2;
the amino acid sequence of the Va-RS is shown in SEQ ID NO. 3;
the amino acid sequence of Va-RK is shown in SEQ ID NO. 4;
the amino acid sequence of the Va-KRA is shown as SEQ ID NO. 5;
the amino acid sequence of the Va-RL is shown in SEQ ID NO. 6.
Preferably, the tumor operation navigation contrast agent further comprises a color developing agent and a coloring agent;
preferably, the colour developer is selected from: ninhydrin, dimethylaminobenzaldehyde, diaminobenzidine;
preferably, the staining agent is selected from fluorescent dyes, preferably selected from: FAM, DAPI, CY5, IR800, ICG;
preferably, the tumor is selected from: liver cancer, breast cancer, pancreatic cancer, intestinal cancer, cerebroma, cervical cancer, lung cancer, gastric cancer, bone cancer, ovarian cancer, lymphoma, renal cancer, nasopharyngeal cancer, testicular cancer, esophageal cancer, bladder cancer, prostate cancer, thyroid cancer, neuroblastoma, and soft tissue sarcoma.
A tumor targeting medicine comprises a medicine active component and a tumor targeting peptide; wherein the tumor targeting peptide is selected from the group consisting of Va-C, Va-RA, Va-RS, Va-RK, Va-KRA, Va-RL;
the amino acid sequence of Va-C is shown in SEQ ID NO. 1;
the amino acid sequence of Va-RA is shown in SEQ ID NO. 2;
the amino acid sequence of the Va-RS is shown in SEQ ID NO. 3;
the amino acid sequence of Va-RK is shown in SEQ ID NO. 4;
the amino acid sequence of the Va-KRA is shown as SEQ ID NO. 5;
the amino acid sequence of the Va-RL is shown in SEQ ID NO. 6.
Preferably, the pharmaceutical active ingredient is selected from antitumor antibiotics, antitumor nucleoside drugs, alkylating agents, plant alkaloids, antitumor hormone drugs, platinum compounds and antimetabolites;
the antitumor antibiotic is selected from: doxorubicin, mitomycin, bleomycin, daunorubicin;
preferably, the antineoplastic nucleoside drug is selected from: anti-tumor antisense oligonucleotides, nucleoside analogs; preferably, the anti-tumor antisense oligonucleotide is selected from: the antisense oligonucleotides Sur-AS of Survivin gene, the antisense oligonucleotide Bcl2-AS of Bcl2 gene, the antisense oligonucleotide MDM2-AS of MDM2 gene, the antisense oligonucleotide BCLXL-AS of BCLXL gene, the antisense oligonucleotide RelA-AS of RelA gene;
preferably, the nucleoside analogue is selected from: deoxyfluoroguanosine, hydroxyurea and cyclocytidine;
preferably, the alkylating agent is selected from: nimustine, cyclophosphamide, carmustine;
preferably, the plant alkaloid is selected from: vincristine, colchicine, cephalotaxine;
preferably, the anti-tumor hormone drug is selected from: tamoxifen, flutamide, leuprorelin;
preferably, the platinum-based compound is selected from: cisplatin, oxaliplatin, carboplatin, phenanthroline;
preferably, the antimetabolite is selected from the group consisting of: methotrexate, 5-fluorouracil, 6-mercaptopurine.
Preferably, the tumor targeting drug also comprises a drug carrier which can load the pharmacodynamically active component; preferably, the drug carrier is selected from nanoparticles;
preferably, the nanoparticles are selected from: exosomes, extracellular vesicles, liposomes;
more preferably, the action targets of the tumor targeting drug are located in the tumor cell nucleus and nucleolus regions.
An antitumor drug enhancer comprises tumor targeting peptide as active ingredient; wherein the tumor targeting peptide is selected from the group consisting of Va-C, Va-RA, Va-RS, Va-RK, Va-KRA, Va-RL;
the amino acid sequence of Va-C is shown in SEQ ID NO. 1;
the amino acid sequence of the Va-RA is shown in SEQ ID NO. 2;
the amino acid sequence of the Va-RS is shown in SEQ ID NO. 3;
the amino acid sequence of Va-RK is shown in SEQ ID NO. 4;
the amino acid sequence of the Va-KRA is shown as SEQ ID NO. 5;
the amino acid sequence of the Va-RL is shown in SEQ ID NO. 6.
Preferably, the antineoplastic drug is selected from antitumor antibiotics, antitumor nucleoside drugs, alkylating agents, plant alkaloids, antitumor hormones, platinum compounds and antimetabolites;
preferably, the antitumor antibiotic is selected from the group consisting of: doxorubicin, mitomycin, bleomycin, daunorubicin;
preferably, the antineoplastic nucleoside drug is selected from: anti-tumor antisense oligonucleotides, nucleoside analogs;
preferably, the anti-tumor antisense oligonucleotide is selected from: the antisense oligonucleotides of Survivin gene SuraS, Bcl2 gene antisense oligonucleotides Bcl2-AS, MDM2 gene antisense oligonucleotides MDM2-AS, BCLXL gene antisense oligonucleotides BCLXL-AS, RelA gene antisense oligonucleotides RelA-AS; these anti-tumor antisense oligonucleotides are reported in the art, and the anti-tumor antisense oligonucleotides known to those skilled in the art can find their description.
Preferably, the nucleoside analogue is selected from: deoxyfluoroguanosine, hydroxyurea and cyclocytidine;
preferably, the alkylating agent is selected from: nimustine, cyclophosphamide, carmustine;
preferably, the plant alkaloid is selected from: vincristine, colchicine, cephalotaxine;
preferably, the anti-tumor hormone drug is selected from: tamoxifen, flutamide, leuprorelin;
preferably, the platinum compound is selected from: cisplatin, oxaliplatin, carboplatin, phenanthroline;
preferably, the antimetabolite is selected from the group consisting of: methotrexate, 5-fluorouracil, 6-mercaptopurine. 10. A coupling reagent for modifying polypeptide and/or short peptide, wherein the coupled active ingredients comprise tumor targeting peptide; wherein the tumor targeting peptide is selected from the group consisting of Va-C, Va-RA, Va-RS, Va-RK, Va-KRA, Va-RL;
the amino acid sequence of Va-C is shown in SEQ ID NO. 1;
the amino acid sequence of the Va-RA is shown in SEQ ID NO. 2;
the amino acid sequence of the Va-RS is shown in SEQ ID NO. 3;
the amino acid sequence of Va-RK is shown in SEQ ID NO. 4;
the amino acid sequence of the Va-KRA is shown as SEQ ID NO. 5;
the amino acid sequence of the Va-RL is shown in SEQ ID NO. 6.
Preferably, the polypeptide and/or short peptide to be modified is selected from conventional tumor targeting peptides, and/or, tumor killing polypeptides, cell shuttle peptides;
preferably, the conventional tumor targeting peptide is selected from the group consisting of: a group consisting of RGD, NGR, RGD-NGR, HCBP1, SP94, HCl, A54, HCC 79;
more preferably, the conventional tumor targeting peptide is RGD-NGR;
preferably, the tumor killing polypeptide is selected from the group consisting of: melittin, octreotide, polymyxin, bacitracin, LTX-302, cecropin;
preferably, the cell shuttle peptide is selected from: TAT, (RXR) 4 、B;
Cell-penetrating peptides, also known as cell-penetrating peptides (CPPs), cell-penetrating peptides TAT, (RXR) are well known in the art 4 Cell shuttle peptide B is described in "A fusion peptide directions enhanced system dynamic therapy exon scraping and functional recovery in dynamic-specific mdx mice".
Preferably, the amino acid sequence of the conventional tumor targeting peptide RGD-NGR is shown in SEQ ID NO. 7; ARYCRGDCFDALNGREE
The amino acid sequence of the conventional tumor targeting peptide HCBP1 is shown in SEQ ID NO. 8; FQHPSFI
The amino acid sequence of the conventional tumor targeting peptide SP94 is shown in SEQ ID NO. 9; SFSIIHTPILPL
The amino acid sequence of the conventional tumor targeting peptide HCl is shown in SEQ ID NO. 10; RGWCRPLPKGEG
The amino acid sequence of the conventional tumor targeting peptide A54 is shown in SEQ ID NO. 11; AGKGTPSLETTP
The amino acid sequence of the conventional tumor targeting peptide HCC79 is shown in SEQ ID NO. 12. KSLSRHDHIHHH is added.
A series of novel variant peptides Va-C, Va-RA, Va-RS, Va-RK, Va-KRA and Va-RL are obtained in the invention, and a large number of experiments prove that the variant peptides (polypeptides) have better targeting property, as shown in figure 1 and figure 2. Meanwhile, the signal-to-noise ratio of the polypeptide in subcutaneous, in situ and lung metastasis mouse models is proved to be superior to that of the known targeting peptide P47, and the polypeptide can also indicate other tumors such as cervical cancer, lung cancer, intestinal cancer and the like. Compared with the known targeting peptide P47, the variant peptide has better tumor-to-tumor signal-to-noise ratio and larger clinical application range.
Drawings
FIG. 1 shows targeting comparisons of Va-C, Va-S, Va-R and P47: wherein each label in the upper panel across represents tumor tissue from a different mouse site, as specified below: b-brain, Q-quadriceps, Lu-lung, S-spleen, K-kidney, H-heart, Li-liver, Tu-subcutaneous tumor; Tumor-Liver ratio on the ordinate in the lower panel represents the ratio of Tumor fluorescence intensity to Liver fluorescence intensity (i.e., signal-to-noise ratio).
FIG. 2 shows targeting comparisons of Va-C, Va-RA, Va-RS, Va-RK, Va-KRA and Va-RL: wherein each label in the upper panel in the horizontal direction represents tumor tissue from a different mouse site, as shown in detail below: b-brain, Q-quadriceps, Lu-lung, S-spleen, K-kidney, H-heart, Li-liver, Tu-subcutaneous tumor; Tumor-Liver ratio on the ordinate in the lower panel represents the ratio of Tumor fluorescence intensity to Liver fluorescence intensity (i.e., signal-to-noise ratio).
FIG. 3 is an analysis of the uptake of Va-RS in normal and tumor cells: the horizontal label in the figure represents a specific cell line, as follows: h7702-normal liver cell line, Hepa 1-6-mouse liver cancer cell line, 97H-human liver cancer cell line, LM 3-human liver cancer cell line, Hela-cervical cancer cell line, MC 38-intestinal cancer cell line; the arrows in the figure indicate the nucleolar region, which is a dark stained region after DAPI staining because of the high RNA content and low DNA content of the nucleolar region.
FIG. 4 is a comparison of targeting of Va-RS, P47 and NC peptides in a mouse model of hepatoma subcutaneous tumor: wherein each marker in the upper panel in the horizontal direction and each marker in the lower panel in the horizontal axis represents tumor tissue derived from a different mouse site, as specified below: b-brain, Q-quadriceps, Lu-lung, S-spleen, K-kidney, H-heart, Li-liver, Tu-subcutaneous tumor; Tumor-Liver ratio on the ordinate in the lower panel represents the ratio of Tumor fluorescence intensity to Liver fluorescence intensity (i.e., signal-to-noise ratio).
FIG. 5 shows the targeting comparison of Va-RS and P47 in a mouse model of carcinoma in situ: in which the horizontal markers in the left panel represent tumor tissues originating from different mouse sites, as specified below: b-brain, Q-quadriceps femoris, Lu-lung, S-spleen, K-kidney, H-heart, Li & Tu-liver and orthotopic tumor, BF 1-bright field orthotopic tumor and liver tissue marking tumor margin, BF 2-bright field orthotopic tumor and liver tissue not marking tumor margin; Tumor-Liver ratio on the ordinate in the right panel represents the ratio of Tumor fluorescence intensity to Liver fluorescence intensity (i.e., signal-to-noise ratio).
FIG. 6 shows the targeting comparison of Va-RS and P47 in mouse model of hepatoma lung metastasis (expressing m-chery fluorescent protein): wherein the horizontal labels in the left panel represent tumor tissues derived from different mouse sites, as shown below: b-brain, Q-quadriceps femoris, Lu-lung, S-spleen, K-kidney, H-heart, Li & Tu-liver and subcutaneous tumors, peptide represents the fluorescence distribution of polypeptide RS; m-chery represents the autofluorescence emitted by tumor cells and is used for indicating the fluorescence distribution of the tumor; Tumor-Liver ratio on the ordinate in the right panel represents the ratio of Tumor fluorescence intensity to Liver fluorescence intensity (i.e., signal-to-noise ratio).
FIG. 7 shows the targeting analysis of Va-RS in mouse model of liver cancer lung metastasis tumor: the horizontal markers in the figure represent tumor tissues originating from different mouse sites, as shown in detail below: b-brain, Q-quadriceps, Lu-lung, S-spleen, K-kidney, H-heart, Li-liver, BF is bright field.
FIG. 8 is the uptake of Va-RS in mouse models of breast cancer (4T1), pancreatic cancer (panc02), liver cancer (Hepa1-6) and intestinal cancer (MC 38): the horizontal markers in the figure represent tumor tissue from different mouse sites, as specified below: b-brain, Q-quadriceps, Lu-lung, S-spleen, K-kidney, H-heart, Li-liver, 4T 1-mouse breast cancer, Panc 02-pancreatic cancer, Hepa 1-6: liver cancer, MC 38-bowel cancer; the vertical notation is: FAM-fluorescence results, BF-bright field results; Tumor-Liver ratio on the ordinate of the bar graph indicates the ratio of Tumor fluorescence intensity to Liver fluorescence intensity (i.e., signal-to-noise ratio), and each marker on the abscissa has the same meaning as the corresponding marker described above.
FIG. 9 shows the uptake of Va-RS in human hepatocellular carcinoma (LM3), breast cancer (MDA-MB-231), lung cancer (A549), cervical cancer (Hela) subcutaneous tumor models: the horizontal markers in the figure represent tumor tissues originating from different mouse sites, as shown in detail below: b-brain, Q-quadriceps, Lu-lung, S-spleen, K-kidney, H-heart, Li-liver, A549-lung cancer, MDA-MB-231: breast cancer, LM 3-human hepatocellular carcinoma, Hela-cervical carcinoma; the vertical notation is: FAM-fluorescence results, BF-bright field results; Tumor-Liver ratio on the ordinate of the bar graph indicates the ratio of Tumor fluorescence intensity to Liver fluorescence intensity (i.e., signal-to-noise ratio), and each marker on the abscissa has the same meaning as the corresponding marker described above.
FIG. 10 shows the uptake and quantitative analysis of Va-RS, P47 and NC in liver cancer, intestinal cancer and the corresponding paracarcinoma tissues of clinical patients. The A picture and the B picture are the condition of taking Va-RS by liver cancer of a clinical patient, liver cancer tissues in the pictures are gray, liver tissues are yellow, the yellow-gray junction is a tumor boundary, and the marked meanings in the pictures are listed as follows: photo-direct photograph of patient tissue, FAM-fluorescence distribution of patient tissue, Quantification-Tumor fluorescence Quantification, Tumor-Liver cancer tissue, boundary-para-carcinoma tissue, Liver-normal Liver tissue. C picture shows the intestinal cancer uptake of Va-RS, P47 and NC, and the meanings of the markers in the picture are as follows: photo-direct photograph of patient tissue, FAM-fluorescence distribution of patient tissue, Quantification-Tumor fluorescence Quantification, Tumor-intestinal cancer tissue, Colon-normal intestinal tissue. The graph D shows the quantitative analysis results of Va-RS and P47 in Liver cancer and intestinal cancer tissues, wherein Tumor-Liver ratio on the ordinate represents the ratio of Tumor fluorescence intensity to Liver fluorescence intensity (i.e. signal-to-noise ratio), Liver cancer tissue on the abscissa, and Bowel cancer tissue on the abscissa.
Fig. 11 shows distribution (a) and quantification (B) of RS-targeted modified exosome-loaded doxorubicin in hepatoma subcutaneous tumors: the horizontal marks in panel A represent tissues from various parts of a mouse model of subcutaneous hepatoma, and the specific meanings are listed as follows: quadrupileces-quadriceps, lung, spleen, kidney, heart, liver, Subcutaneous tumor; the meaning of the longitudinal mark is listed below: PBS-blank control, Free-Dox: free doxorubicin, EXO Dox Exosomes loaded with doxorubicin, RS-EXO Dox : RS target-modified and adriamycin-loaded exosomes; the meanings of the marks on the abscissa in the graph B are the same as those of the marks corresponding to the names, the meanings of the column marks with different colors in the histogram are the same as those of the marks corresponding to the names, and the ordinate Ratio represents the multiple of the intake amount of each group compared with that of the PBS group.
FIG. 12 is an evaluation of the effect of RS-targeted modified exosomes loaded with doxorubicin on treatment of hepatoma subcutaneous tumors. Graph A is a tumor growth curve; panel B shows tumor size comparison. The labeled meanings of the curves in the A diagram are as follows: PBS-blank control, Free-Dox: free doxorubicin, EXO Dox Exosomes loaded with doxorubicin, RS-EXO Dox : RS target-modified and adriamycin-loaded exosomes; the ordinate is the size of the tumor volume, and the abscissa is days; the vertical marks in the drawing B have the same meaning as the marks of the corresponding names.
FIG. 13 shows the same dose of Dox in PBS, free Dox, EXO Dox And RS-EXO Dox Pathological analysis of group tumors and comparison of ability to induce apoptosis. The meanings of the lateral labels in the figures are as follows: h&HE staining at E20X-20 fold, H&Results of HE staining at E40X-40 fold, TUNEL&DAPI-TUNEL and DAPI staining results; the meaning of the longitudinal markings is as follows: PBS-blank control, Free-Dox: free doxorubicin, EXO Dox Exosomes loaded with doxorubicin, RS-EXO Dox :RS targeting modified and doxorubicin-loaded exosomes.
FIG. 14 shows the distribution of RS-modified and free SurAS in tumor-bearing mouse brain (B), muscle (Q), lung (Lu), spleen (S), kidney (K), heart (H), liver (Li) and liver cancer (Tu) tissues, with the horizontal symbols indicating tissues from different mouse sites, as shown below: b-brain, Q-muscle, Lu-lung, S-spleen, K-kidney, H-heart, Li-liver, Tu-liver cancer; the longitudinal markers are listed below: NC-NC peptide, RS-SurAS: RS-modified SurAS, SurAS-free SurAS.
FIG. 15 shows the targeting comparison of P47, RGD-NGR-P47, Va-RS and RGD-NGR-RS in mouse model of subcutaneous tumor of liver cancer. The horizontal symbols in the figure represent tissues originating from different mouse sites, as specified below: b-brain, Q-quadriceps, Lu-lung, S-spleen, K-kidney, H-heart, Li-liver, Tu-subcutaneous tumor; R-N-P47 in the longitudinal marker indicates the result of RGD-NGR-P47, and R-N-RS indicates the result of RGD-NGR-RS; the longitudinal Tumor-Liver ratio in the bar graph shows the ratio of Tumor fluorescence intensity to Liver fluorescence intensity (i.e., signal-to-noise ratio), and the horizontal R-N-P47 and R-N-RS have the meanings as described above.
Detailed Description
The following detailed description of the present invention is provided in connection with specific embodiments and accompanying drawings, but not intended to limit the scope of the invention.
The reagents used in the examples and experimental examples of the present invention, for example, m-cherry expression vector, FAM marker, and DAPI, are commercially available.
Sources of biological material
The mice used in the experimental examples were purchased from experimental animal technology ltd, viton, beijing;
the cancer cells used in the experimental examples (7) to (9), including hepatoma cells, breast cancer (4T1), pancreatic cancer (panc02), liver cancer (Hepa1-6), intestinal cancer (MC38), human hepatoma cell (LM3), breast cancer (MDA-MB-231), lung cancer (A549), cervical cancer (Hela), and the like, were derived from the ATCC cell resource center
The tumors, normal organ controls and tumor boundary tissues of liver cancer and intestinal cancer patients used in Experimental example (10) were obtained from hepatobiliary surgery of tumor hospital, Tianjin.
The DC cells used in section (11) of the Experimental examples were obtained from ATCC cell resource center.
The tumor-bearing mice used in the experimental examples of the parts (11) and (14) are independently constructed in the laboratory of the applicant, and the specific construction method is as follows: will be 1 × 10 6 The tumor cells are inoculated to the subcutaneous part of the mouse, and after the tumor grows to the proper size, the injection of the medicament and the imaging observation of the small animal are carried out.
Group 1 example, Polypeptides of the invention
The present group of embodiments provides a polypeptide. All embodiments of this group share the following common features: the polypeptide is selected from the group consisting of Va-C, Va-RA, Va-RS, Va-RK, Va-KRA, Va-RL;
the amino acid sequence of Va-C is shown in SEQ ID NO. 1;
the amino acid sequence of the Va-RA is shown in SEQ ID NO. 2;
the amino acid sequence of the Va-RS is shown in SEQ ID NO. 3;
the amino acid sequence of Va-RK is shown in SEQ ID NO. 4;
the amino acid sequence of the Va-KRA is shown as SEQ ID NO. 5;
the amino acid sequence of the Va-RL is shown in SEQ ID NO. 6.
According to the elicitation of the present invention, a person skilled in the art can modify, change, add, delete, or modify the amino acid sequence of any of the above polypeptides of the present invention, and obtain variant peptides which have 80%, 81%, 82%, … …, 88%, 89%, 90%, 91%, … … 95%, 96%, … … 99% of amino acid sequence homology with the polypeptide of the present invention and also have tumor targeting property, and all of them fall into the protection scope of the present invention.
In a specific embodiment, the polypeptide is a tumor targeting peptide.
Preferably, the tumor is selected from: liver cancer, breast cancer, pancreatic cancer, intestinal cancer, cerebroma, cervical cancer, lung cancer, gastric cancer, bone cancer, ovarian cancer, lymphoma, renal cancer, nasopharyngeal cancer, testicular cancer, esophageal cancer, bladder cancer, prostate cancer, thyroid cancer, neuroblastoma, and soft tissue sarcoma.
In some preferred embodiments, the polypeptide refers to Va-RS having an amino acid sequence as set forth in SEQ ID NO.3
As used herein, both RS and Va-RS refer to a polypeptide Va-RS of the invention having an amino acid sequence as set forth in SEQ ID NO. 3.
Any action of detection and treatment by the polypeptide of the present invention falls within the scope of the present invention.
Any action of tumor detection (including but not limited to tumor detection, observation and diagnosis), tumor treatment (including but not limited to tumor administration treatment, tumor surgical navigation and tumor drug enhancement treatment) by using the polypeptide of the present invention falls within the protection scope of the present invention.
Any action of utilizing the polypeptide of the present invention to prepare detection reagents, therapeutic auxiliary reagents, drugs, drug enhancers falls within the scope of the present invention.
Any action of preparing a tumor detection reagent, an antitumor drug, an auxiliary reagent for tumor surgery and an antitumor drug reinforcing agent by using the polypeptide of the present invention falls within the scope of the present invention.
Such tumors include, but are not limited to: liver cancer, breast cancer, pancreatic cancer, intestinal cancer, cerebroma, cervical cancer, lung cancer, gastric cancer, bone cancer, ovarian cancer, lymphoma, renal cancer, nasopharyngeal cancer, testicular cancer, esophageal cancer, bladder cancer, prostate cancer, thyroid cancer, neuroblastoma, and soft tissue sarcoma.
The present set of embodiments provides a tumor targeting peptide. All embodiments of this group share the following common features: the tumor targeting peptide comprises a polypeptide selected from the group consisting of Va-C, Va-RA, Va-RS, Va-RK, Va-KRA, Va-RL;
the amino acid sequence of Va-C is shown in SEQ ID NO. 1;
the amino acid sequence of Va-RA is shown in SEQ ID NO. 2;
the amino acid sequence of the Va-RS is shown in SEQ ID NO. 3;
the amino acid sequence of Va-RK is shown in SEQ ID NO. 4;
the amino acid sequence of the Va-KRA is shown as SEQ ID NO. 5;
the amino acid sequence of the Va-RL is shown in SEQ ID NO. 6.
In further embodiments, the tumor targeting peptide further comprises a conventional tumor targeting peptide; preferably, the conventional tumor targeting peptide is selected from the group consisting of: p47, a54, SP 94;
preferably, the tumor is selected from: liver cancer, breast cancer, pancreatic cancer, intestinal cancer, cerebroma, cervical cancer, lung cancer, gastric cancer, bone cancer, ovarian cancer, lymphoma, renal cancer, nasopharyngeal cancer, testicular cancer, esophageal cancer, bladder cancer, prostate cancer, thyroid cancer, neuroblastoma, and soft tissue sarcoma.
According to the description of the present invention, a person skilled in the art can use the tumor targeting peptide provided by the present invention to perform tumor detection (including but not limited to tumor detection, observation and confirmation), tumor treatment (including but not limited to tumor administration treatment, tumor surgical navigation, tumor drug enhancement treatment), preparation of tumor detection reagents, anti-tumor drugs, tumor surgical auxiliary reagents, anti-tumor drug enhancers, etc., and can also use the tumor targeting peptide provided by the present invention in combination with any known tumor targeting peptide reported at present to perform the above-mentioned actions, and the person skilled in the art can also be inspired by the present invention to perform amino acid modification, alteration, addition, deletion, etc., on the tumor targeting peptide provided by the present invention, and any of the above-mentioned actions falls into the protection scope of the present invention.
Group 3 example, tumor detection reagent of the present invention
The present group of embodiments provides a tumor detection reagent. All embodiments of this group share the following common features: the tumor detection reagent comprises a polypeptide; said polypeptide is selected from the group consisting of Va-C, Va-RA, Va-RS, Va-RK, Va-KRA, Va-RL;
the amino acid sequence of Va-C is shown in SEQ ID NO. 1;
the amino acid sequence of the Va-RA is shown in SEQ ID NO. 2;
the amino acid sequence of the Va-RS is shown in SEQ ID NO. 3;
the amino acid sequence of Va-RK is shown in SEQ ID NO. 4;
the amino acid sequence of the Va-KRA is shown as SEQ ID NO. 5;
the amino acid sequence of Va-RL is shown in SEQ ID NO. 6.
In still further embodiments, the tumor detection reagent further comprises: color-developing agent, coloring agent;
preferably, the colour developer is selected from: ninhydrin, dimethylaminobenzaldehyde, diaminobenzidine;
preferably, the staining agent is selected from fluorescent dyes, preferably selected from: FAM, DAPI, CY5, IR800, ICG;
preferably, the tumor is selected from: liver cancer, breast cancer, pancreatic cancer, intestinal cancer, cerebroma, cervical cancer, lung cancer, gastric cancer, bone cancer, ovarian cancer, lymphoma, renal cancer, nasopharyngeal cancer, testicular cancer, esophageal cancer, bladder cancer, prostate cancer, thyroid cancer, neuroblastoma, and soft tissue sarcoma.
The present group of embodiments provides a tumor surgery navigation contrast agent. All embodiments of this group share the following common features: the tumor surgery navigation contrast agent comprises polypeptide; said polypeptide is selected from the group consisting of Va-C, Va-RA, Va-RS, Va-RK, Va-KRA, Va-RL;
the amino acid sequence of Va-C is shown in SEQ ID NO. 1;
the amino acid sequence of the Va-RA is shown in SEQ ID NO. 2;
the amino acid sequence of the Va-RS is shown in SEQ ID NO. 3;
the amino acid sequence of Va-RK is shown in SEQ ID NO. 4;
the amino acid sequence of the Va-KRA is shown as SEQ ID NO. 5;
the amino acid sequence of the Va-RL is shown in SEQ ID NO. 6.
Preferably, the tumor operation navigation contrast agent further comprises a color developing agent and a coloring agent;
preferably, the colour developer is selected from: ninhydrin, dimethylaminobenzaldehyde, diaminobenzidine;
preferably, the staining agent is selected from fluorescent dyes, preferably selected from: FAM, DAPI, CY5, IR800, ICG;
preferably, the tumor is selected from: liver cancer, breast cancer, pancreatic cancer, intestinal cancer, cerebroma, cervical cancer, lung cancer, gastric cancer, bone cancer, ovarian cancer, lymphoma, renal cancer, nasopharyngeal cancer, testicular cancer, esophageal cancer, bladder cancer, prostate cancer, thyroid cancer, neuroblastoma, and soft tissue sarcoma.
The embodiment of the group provides a tumor targeting drug. All embodiments of this group share the following common features: the tumor targeting drug comprises a pharmaceutical active component and a tumor targeting peptide; wherein the tumor targeting peptide is selected from the group consisting of Va-C, Va-RA, Va-RS, Va-RK, Va-KRA, Va-RL;
the amino acid sequence of Va-C is shown in SEQ ID NO. 1;
the amino acid sequence of the Va-RA is shown in SEQ ID NO. 2;
the amino acid sequence of the Va-RS is shown in SEQ ID NO. 3;
the amino acid sequence of Va-RK is shown in SEQ ID NO. 4;
the amino acid sequence of Va-KRA is shown in SEQ ID NO. 5;
the amino acid sequence of the Va-RL is shown in SEQ ID NO. 6.
Preferably, the active pharmaceutical ingredient is selected from antitumor antibiotics, antitumor nucleoside drugs, alkylating agents, plant alkaloids, antitumor hormone drugs, platinum compounds and antimetabolites.
In specific embodiments, the anti-tumor antibiotic is selected from the group consisting of: doxorubicin, mitomycin, bleomycin, daunorubicin;
preferably, the antineoplastic nucleoside drug is selected from: anti-tumor antisense oligonucleotides, nucleoside analogs; preferably, the anti-tumor antisense oligonucleotide is selected from: the antisense oligonucleotides of Survivin gene, Sur-AS, Bcl2 gene, Bcl2-AS, MDM2 gene, MDM2-AS, BCLXL gene, BCLXL-AS, RelA gene, and RelA gene, respectively;
preferably, the nucleoside analogue is selected from: deoxyfluoroguanosine, hydroxyurea and cyclocytidine;
preferably, the alkylating agent is selected from: nimustine, cyclophosphamide, carmustine;
preferably, the plant alkaloid is selected from: vincristine, colchicine, cephalotaxine;
preferably, the anti-tumor hormone drug is selected from: tamoxifen, flutamide, leuprorelin;
preferably, the platinum compound is selected from: cisplatin, oxaliplatin, carboplatin, phenanthroline;
preferably, the antimetabolite is selected from the group consisting of: methotrexate, 5-fluorouracil, 6-mercaptopurine.
Preferably, the tumor targeting drug also comprises a drug carrier which can load the pharmacodynamically active component; preferably, the drug carrier is selected from nanoparticles;
preferably, the nanoparticles are selected from: exosomes, extracellular vesicles, liposomes;
more preferably, the action targets of the tumor targeting drug are located in the tumor cell nucleus and nucleolus regions.
The present group of embodiments provides an antitumor drug potentiator. All embodiments of this group share the following common features: the active component of the anti-tumor drug enhancer comprises tumor targeting peptide; wherein the tumor targeting peptide is selected from the group consisting of Va-C, Va-RA, Va-RS, Va-RK, Va-KRA, Va-RL;
the amino acid sequence of Va-C is shown in SEQ ID NO. 1;
the amino acid sequence of the Va-RA is shown in SEQ ID NO. 2;
the amino acid sequence of the Va-RS is shown in SEQ ID NO. 3;
the amino acid sequence of Va-RK is shown in SEQ ID NO. 4;
the amino acid sequence of the Va-KRA is shown as SEQ ID NO. 5;
the amino acid sequence of the Va-RL is shown in SEQ ID NO. 6.
Preferably, the antineoplastic drug is selected from antitumor antibiotics, antitumor nucleoside drugs, alkylating agents, plant alkaloids, antitumor hormones, platinum compounds and antimetabolites;
preferably, the antitumor antibiotic is selected from the group consisting of: doxorubicin, mitomycin, bleomycin, daunorubicin;
preferably, the antineoplastic nucleoside drug is selected from: anti-tumor antisense oligonucleotides, nucleoside analogs;
preferably, the anti-tumor antisense oligonucleotide is selected from: the antisense oligonucleotides of Survivin gene SuraS, Bcl2 gene antisense oligonucleotides Bcl2-AS, MDM2 gene antisense oligonucleotides MDM2-AS, BCLXL gene antisense oligonucleotides BCLXL-AS, RelA gene antisense oligonucleotides RelA-AS;
preferably, the nucleoside analogue is selected from: deoxyfluoroguanosine, hydroxyurea and cyclocytidine;
preferably, the alkylating agent is selected from: nimustine, cyclophosphamide, carmustine;
preferably, the plant alkaloid is selected from: vincristine, colchicine, cephalotaxine;
preferably, the anti-tumor hormone drug is selected from: tamoxifen, flutamide, leuprorelin;
preferably, the platinum-based compound is selected from: cisplatin, oxaliplatin, carboplatin, phenanthroline;
preferably, the antimetabolite is selected from the group consisting of: methotrexate, 5-fluorouracil, 6-mercaptopurine.
Example 7 coupling reagents of the invention
The present set of embodiments provides a coupling reagent for modifying polypeptides and/or short peptides. The present group of embodiments has the following common features: the active ingredient for coupling of the coupling reagent for modifying the polypeptide and/or the short peptide comprises a tumor targeting peptide; the tumor targeting peptide is selected from the group consisting of Va-C, Va-RA, Va-RS, Va-RK, Va-KRA and Va-RL;
the amino acid sequence of Va-C is shown in SEQ ID NO. 1;
the amino acid sequence of the Va-RA is shown in SEQ ID NO. 2;
the amino acid sequence of the Va-RS is shown in SEQ ID NO. 3;
the amino acid sequence of Va-RK is shown in SEQ ID NO. 4;
the amino acid sequence of the Va-KRA is shown as SEQ ID NO. 5;
the amino acid sequence of the Va-RL is shown in SEQ ID NO. 6.
Preferably, the polypeptide and/or short peptide to be modified is selected from conventional tumor targeting peptides, and/or, tumor killing polypeptides, cell shuttle peptides;
preferably, the conventional tumor targeting peptide is selected from the group consisting of: a group consisting of RGD, NGR, RGD-NGR, HCBP1, SP94, HCl, A54, HCC 79;
more preferably, the conventional tumor targeting peptide is RGD-NGR;
preferably, the tumor killing polypeptide is selected from the group consisting of: melittin, octreotide, polymyxin, bacitracin, LTX-302, cecropin;
preferably, the cell shuttle peptide is selected from: TAT, (RXR) 4 、B;
Preferably, the amino acid sequence of the conventional tumor targeting peptide RGD-NGR is shown in SEQ ID NO. 7; ARYCRGDCFDALNGREE
The amino acid sequence of the conventional tumor targeting peptide HCBP1 is shown in SEQ ID NO. 8; FQHPSFI
The amino acid sequence of the conventional tumor targeting peptide SP94 is shown as SEQ ID NO. 9; SFSIIHTPILPL
The amino acid sequence of the conventional tumor targeting peptide HCl is shown in SEQ ID NO. 10; RGWCRPLPKGEG
The amino acid sequence of the conventional tumor targeting peptide A54 is shown in SEQ ID NO. 11; AGKGTPSLETTP
The amino acid sequence of the conventional tumor targeting peptide HCC79 is shown in SEQ ID NO. 12. KSLSRHDHIHHH are provided.
Experimental examples, the polypeptide of the present invention and functional verification thereof
Synthesis of polypeptide
Peptide sequence: CGRCKCCNGERS (Va-RS, SEQ ID NO.3)
1 polypeptide synthesis: from the C end to the N end of the sequence, the steps are as follows:
a. weighing n equivalents of resin, putting the resin into a reactor, adding DCM (dichloromethane) to swell for half an hour, then pumping out DCM, adding 2n equivalents of the first amino acid in the sequence, adding 2n equivalents of DIEA, an appropriate amount of DMF (dimethyl formamide), DCM (the appropriate amount is that the resin can be fully stirred), DIEA (diisopropylethylamine), DMF (dimethyl formamide), DCM, and nitrogen for bubbling reaction for 60 min. Then adding about 5n equivalent of methanol, reacting for half an hour, pumping out reaction liquid, and washing with DMF and MEOH;
b. the second amino acid in the sequence (also 2N equivalents), 2N equivalents HBTU (1-hydroxy, benzo, trichloroazol tetramethyl hexafluorophosphate) and DIEA, N2 were bubbled through the reactor for half an hour, washed off the liquid, assayed for ninhydrin, and then capped with pyridine and acetic anhydride. Finally, cleaning, adding a proper amount of decapping liquid to remove the Fmoc (9-fluorenylmethyloxycarbonyl) protecting group, cleaning, and detecting ninhydrin;
c. c, sequentially adding different amino acids in the sequence according to the mode of the step b and carrying out various modifications;
d. blowing the resin to dry with nitrogen, taking the resin out of the reaction column, pouring the resin into a flask, adding a certain amount of cutting fluid (the composition is 95% TFA, 2% ethanedithiol, 2% triisopropylsilane and 1% water) (the ratio of the cutting fluid to the resin is about 10 ml/g) into the flask, shaking and filtering the resin;
e. obtaining filtrate, then adding a large amount of ether into the filtrate to separate out a crude product, then centrifuging and cleaning to obtain a crude product of the sequence;
2, polypeptide purification:
and purifying the crude product to the required purity by adopting high performance liquid chromatography.
3, polypeptide freeze-drying:
and (4) putting the purified liquid into a freeze dryer for concentration, and freeze-drying to obtain white powder.
Functional verification of polypeptide
(1) The invention designs a series of variant peptides Va-C, Va-S and Va-R, and verifies the targeting property of the variant peptides to a conventional targeting peptide P47, and the specific experimental process is as follows: each mouse (tumor-bearing mice from the laboratory of the Applicant independently constructed, i.e., 1X 10 of the total mice were cultured in the laboratory 6 The tumor cells are inoculated to the subcutaneous part of the mouse, and after the tumor grows to the proper size, the injection of the medicament and the imaging observation of the small animal are carried out. ) Injecting Va-C, Va-S, Va-R and P47 polypeptide labeled by 0.5mg carboxyfluorescein (FAM) into tail vein, wherein the carboxyfluorescein (FAM) is labeled at the N end of the polypeptide; taking out brain (B), quadriceps femoris (Q), lung (Lu), spleen (S), kidney (K), heart (H), liver (Li) and subcutaneous tumor (Tu) for small animal imaging observation after two hours of injection; the test result is shown in figure 1, wherein the Va-C has optimal targeting, and the amino acid sequence of the Va-C is shown in SEQ ID NO. 1.
(2) Based on the variant peptides Va-C, the invention further designs a series of second-generation variant peptides Va-RA, Va-RS, Va-RK, Va-KRA and Va-RL, wherein the respective amino acids of the second-generation variant peptides are respectively shown in SEQ ID NO.2-6, and the targeting property of the second-generation variant peptides Va-RA, Va-RS, Va-RK, Va-KRA and Va-RL is verified at the same time, and the experimental operation and the experimental process in the part (1) are similar, in particular as follows:
injecting 0.5mg of carboxyfluorescein (FAM) labeled Va-C (CQRCRCWNGTRS, SEQ ID NO.1), Va-RA (CGCCKCCNGERA, SEQ ID NO.2), Va-RS (CGRCKCCNGERS, SEQ ID NO.3), Va-RK (CGCCKCCNGERK, SEQ ID NO.4), Va-KRA (CKCCKCCNGERA, SEQ ID NO.5) and Va-RL (CGCCKCCNGERL, SEQ ID NO.6) polypeptide into each mouse tail, wherein the carboxyfluorescein (FAM) is labeled at the N end of the polypeptide; two hours after injection, the brain (B), quadriceps femoris (Q), lung (Lu), spleen (S), kidney (K), heart (H), liver (Li) and subcutaneous tumor (Tu) were taken for small animal imaging observation; the observations are shown in FIG. 2, where targeting of Va-RS is optimal.
(3) The invention detects the enrichment condition of Va-RS peptide with optimal targeting in a normal liver cell line (H7702), a mouse liver cancer cell line (Hepa1-6), a human liver cancer cell line (97H and LM3), a cervical cancer cell line (Hela) and an intestinal cancer cell line (MC38), and the experimental process is as follows: the polypeptide was labeled with carboxyfluorescein (FAM) at the N-terminus, the FAM-labeled Va-RS peptide was incubated with a normal liver cell line (H7702), a mouse liver cancer cell line (Hepa1-6), a human liver cancer cell line (97H and LM3), a cervical cancer cell line (Hela) and an intestinal cancer cell line (MC38) at a concentration of 5uM for 6 hours, followed by staining the cell nuclei for 10min using DAPI, rinsing to remove free dye, and then photographed under a fluorescent microscope. The experimental results are shown in fig. 3: the targeting peptide RS is more localized to the nucleolar region of the tumor cell nucleus and is not taken up by normal liver cells (7702).
(4) The targeting comparison of Va-RS, P47 and NC peptides in a mouse model of liver cancer subcutaneous tumor is verified in the section. The specific experimental operations were as follows: injecting 0.5mg of carboxyfluorescein (FAM) labeled P47 and Va-RS and a control polypeptide NC polypeptide (DQDIEAKNGVIS, SEQ ID NO.13) which is reported not to have tumor targeting property into the tail of each mouse, labeling the carboxyfluorescein (FAM) at the N end of the polypeptide, taking out the brain (B), the quadriceps femoris (Q), the lung (Lu), the spleen (S), the kidney (K), the heart (H), the liver (Li) and the subcutaneous tumor (Tu) after injecting for two hours, and carrying out imaging observation on the small animals; the observation results are shown in fig. 4: in the subcutaneous tumor model, targeting of Va-RS (tumor-liver ratio 23.4) was significantly stronger than that of P47(tumor-liver ratio 5.2).
(5) The invention compares the intake conditions of Va-RS and P47 on a liver cancer orthotopic tumor model, and the specific experimental process is as follows: injecting 0.5mg of carboxyfluorescein (FAM) -labeled P47, Va-RS and NC polypeptide into the tail of each mouse, wherein the carboxyfluorescein (FAM) is labeled at the N end of the polypeptide; taking brain (B), quadriceps femoris (Q), lung (Lu), spleen (S), kidney (K), heart (H), liver and carcinoma in situ (Li & Tu), bright field carcinoma in situ and liver tissue (BF1) marked at the edge of the tumor, and bright field carcinoma in situ and liver tissue (BF2) unmarked at the edge of the tumor for small animal imaging observation after two hours of injection; Tumor-Liver ratio shows the ratio of Tumor fluorescence intensity to Liver fluorescence intensity (i.e., signal-to-noise ratio), and the results show that, as shown in FIG. 5, the ratio of Tumor fluorescence intensity to Liver fluorescence intensity (i.e., signal-to-noise ratio) of Va-RS on hepatoma carcinoma-in-situ (medium-sized) is 18.2, and the ratio of P47 on hepatoma carcinoma-in-situ is 5.1.
(6) The tail vein of the invention is inoculated with cells expressing m-cherry fluorescent protein, and a liver cancer lung metastasis model is obtained after 3 weeks, and the specific operation is as follows: and (3) injecting the tail vein of the liver cancer cell expressing m-cherry fluorescence into a mouse to obtain a liver cancer lung metastasis mouse. The invention injects FAM labeled variant peptide RS and targeting peptide P47 into a model mouse, and the specific operation is as follows: tail vein injecting P47 and Va-RS polypeptide labeled by 0.5mg carboxyfluorescein (FAM) which is labeled at the N end of the polypeptide; two hours after injection, brain (B), quadriceps femoris (Q), lung (Lu), spleen (S), kidney (K), heart (H) and liver (Li) were taken for small animal imaging observation and analysis, and the results showed that as shown in FIG. 6, Va-RS and P47 all indicated the location of liver cancer lung metastasis, and the fluorescence distribution of polypeptide Va-RS completely coincided with that of tumor, while the tumor-liver ratio of Va-RS (9.4) was significantly better than that of P47 (2.6).
(7) The invention detects the detection capability of Va-RS to lung metastasis focus, and the specific operation is as follows: injecting tail vein of liver cancer cell into mouse to obtain liver cancer lung transfer mouse. The brain (B), quadriceps femoris (Q), lung (Lu), spleen (S), kidney (K), heart (H) and liver (Li) were then taken two hours after tail vein injection of 0.5mg carboxyfluorescein (FAM) -labeled Va-RS polypeptide, and the results of small animal imaging observations showed that the Va-RS polypeptide could indicate lung metastasis with a diameter of only 0.3mm, as shown in FIG. 7.
(8) In order to detect the indication effect of the variant peptide Va-RS on different tumors, the invention establishes a model mouse loaded with subcutaneous tumors of breast cancer (4T1), pancreatic cancer (panc02), liver cancer (Hepa1-6) and intestinal cancer (MC38), the establishment method can refer to the acquisition of a 'liver cancer lung metastasis mouse' of part (7), and then Va-RS is injected, and the specific operation can refer to the corresponding part of part (7). The results showed that Va-RS was taken up in 4 different tumor sources (tumor-liver ratio was above 20) (FIG. 8).
(9) The invention also establishes a subcutaneous tumor model mouse simultaneously loading human liver cell cancer (LM3), breast cancer (MDA-MB-231), lung cancer (A549) and cervical cancer (Hela), and then injecting Va-RS, and similarly, the specific operation can refer to the part (7). The results show that Va-RS is taken up in 4 different tumor sources. Tumor tissues except breast cancer, each tumor had a tumor ratio of 20 or more (FIG. 9).
(10) In order to detect the clinical availability of the variant peptide RS, the invention soaks the tumor, normal organ contrast and tumor boundary tissue of liver cancer and intestinal cancer patients in FAM fluorescence labeled variant peptide Va-RS, P47 and NC solution respectively, and carries out fluorescence imaging detection after rinsing. The specific operation is as follows: liver cancer, intestinal cancer and tissues beside the cancer of clinical patients are soaked in 10uM FAM marked Va-RS, P47 and NC solution for 30 minutes, and then the residual FAM marked polypeptide is removed by PBS rinsing. The patient tissue is then photographed using a small animal imager. The results show that the variant peptides Va-RS can accurately indicate the borders of liver cancer, as shown in figure 10, and are not ingested in the heterogeneous liver disease context of patients. In liver cancer, the tumor-liver ratio of the variant peptide RS can reach 21.6, and in intestinal cancer can reach 10.7.
(11) In order to test the capacity of the variant peptide RS carrying the drug to target the tumor, the invention connects the targeting peptide RS with an exosome loaded with adriamycin, and tests the distribution of the exosome after targeted modification in a tumor-bearing mouse. The specific operation is as follows: incubating DC cells and adriamycin (Dox), simultaneously stimulating the DC cells to secrete an exosome containing adriamycin by ultraviolet irradiation, collecting the exosome carrying the adriamycin by ultracentrifugation, and then using an exosome anchoring peptide CP05 described in a patent 201510520565.7 with independent intellectual property rights in the laboratory to load a targeting peptide RS on the surface of the exosome, thereby obtaining the exosome carrying the adriamycin and modified by the RS targeting. Injecting free adriamycin, an adriamycin-loaded exosome and an RS targeting modified adriamycin-loaded exosome into a mouse body respectively through tail veins, and observing the distribution of adriamycin fluorescence in each tissue of the mouse by using animal imaging.
The advantages of using exosomes to load doxorubicin in this section are: the exosome is a natural biological nano material and has small immunogenicity. ② the medicine is slow-released, and the duration of the medicine effect is long. And thirdly, the exosome can directly send the medicine into the cell through the endocytosis fusion effect, so that the adverse reaction is reduced by using high-concentration free medicine. Fourthly, the capillary wall of the normal tissue is complete, most of the exosomes cannot permeate, the permeability of the capillary vessel of the tumor growth part is increased, the aggregation amount of the exosome adriamycin is increased, and the exosome adriamycin is directly applied to the tumor part, so that the treatment effect is improved.
The results show (fig. 11) that uptake of the loaded drug in the tumor is significantly increased after RS-targeted modification. (12) To evaluate RS-EXO Dox The invention injects free Dox and EXO in a liver cancer subcutaneous tumor model according to a dose of Dox (adriamycin) of 1.5mg/kg (HPLC quantitative) every other day system Dox And RS-EXO Dox PBS was injected as a blank control for two consecutive weeks of treatment. The specific operation is as follows: free Dox and EXO were injected every other day at a dose of 1.5mg/kg Dox (HPLC quantitation) in the hepatoma model Dox And RS-EXO Dox The treatment is continued for two weeks, the tumor growth curve is recorded during the treatment process, and the tumor is picked up and photographed after one week of treatment. The results show that: 1 week after the end of treatment, RS-EXO Dox The treated mice still survived and were in good condition, and the volume of the hepatoma subcutaneous tumor was significantly smaller than that of free Dox and EXO Dox Treatment group (fig. 12).
(13) This section continues the experimental procedure of section (12) above, and one week after the end of the treatment the tumors were harvested and paraffin-embedded, sectioned and HE-stained. And analyzing the apoptosis condition of the tumor cells by using a TUNEL apoptosis staining kit, wherein the green area is an apoptosis staining area. The results are shown in FIG. 13, and the pathological analysis of the tumor tissues in each group showed that RS-EXO Dox The tumor cells become round and small, the cell nucleus shrinks and the cell gap increases. And PBS, free Dox and EXO Dox The group of tumor cells still substantially maintained a relatively normal growth state. TUNEL apoptosis staining results show RS-EXO Dox The group tumors showed more apoptotic regions (green fluorescence signal) with loosely arranged cells, while other various tumor tissues showed less apoptotic regions.
(14) In order to test the ability of the variant peptide RS carrying nucleic acid drug to target tumor, the invention covalently connects the targeting peptide RS and the antisense oligonucleotide (SuraS) of the Survivin gene to obtain RS-SuraS, and injects the NC peptide marked by FAM, RS-SuraS and SuraS into the tumor-bearing mouse body by the mode of intravenous injection, and tests the distribution of the RS-SuraS after targeted modification in the tumor-bearing mouse. The imaging results of the small animals show that after the coupling of the liver cancer targeting peptide RS, the aggregation of the fluorescent-labeled SurAS in the tumor tissues is obviously enhanced (FIG. 14).
(15) In order to test whether RS peptide can be modified by other polypeptide to further improve the targeting effect, the invention leads RS and P47 to be embedded with polypeptide RGD-NGR (with the sequence of ARYCRGDCFDALNGREE, SEQ ID NO.7), 0.5mg of carboxyl Fluorescein (FAM) labeled P47(SQDIRTWNGTRS, SEQ ID NO.14), RGD-NGR-P47(ARYCRGDCFDALNGREESQDIRTWNGTRS, SEQ ID NO.15), Va-RS (CGRCKCCNGERS, SEQ ID NO.3) and RGD-NGR-RS (ARYCRGDCFDALNGREECGRCKCCNGERS, SEQ ID NO.16) are injected into the tail vein of each mouse, carboxyl Fluorescein (FAM) is labeled at the N end of the polypeptide, and brain (B), quadriceps (Q), lung (Lu), spleen (S), kidney (K), heart (H), liver (Li) and subcutaneous tumor (Tu) are taken for small animal imaging observation after two hours of injection; Tumor-Liver ratio shows the ratio of Tumor fluorescence intensity to Liver fluorescence intensity (i.e. signal-to-noise ratio), and the results show that the targeting ability of RS and P47 is improved after the RGD-NGR is embedded, and the improvement range is about two times. The final tumor-liver ratio of RGD-NGR-RS can reach 55.5 (FIG. 15).
The solvent of the polypeptide injection in all the above experimental examples (1) to (15) of the present invention is PBS, and the concentration of the carboxyfluorescein (FAM) -labeled polypeptide in the solvent is 5 mg/ml.
SEQUENCE LISTING
<110> Tianjin medical university
<120> polypeptide and tumor targeting peptide, tumor detection reagent, tumor surgery navigation contrast medium and tumor targeting drug thereof
<130> P200953/TJY
<160> 16
<170> PatentIn version 3.5
<210> 1
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Va-C
<400> 1
Cys Gln Arg Cys Arg Cys Trp Asn Gly Thr Arg Ser
1 5 10
<210> 2
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Va-RA
<400> 2
Cys Gly Cys Cys Lys Cys Cys Asn Gly Glu Arg Ala
1 5 10
<210> 3
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Va-RS
<400> 3
Cys Gly Arg Cys Lys Cys Cys Asn Gly Glu Arg Ser
1 5 10
<210> 4
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Va-RK
<400> 4
Cys Gly Cys Cys Lys Cys Cys Asn Gly Glu Arg Lys
1 5 10
<210> 5
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Va-KRA
<400> 5
Cys Lys Cys Cys Lys Cys Cys Asn Gly Glu Arg Ala
1 5 10
<210> 6
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Va-RL
<400> 6
Cys Gly Cys Cys Lys Cys Cys Asn Gly Glu Arg Leu
1 5 10
<210> 7
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> conventional tumor targeting peptide RGD-NGR
<400> 7
Ala Arg Tyr Cys Arg Gly Asp Cys Phe Asp Ala Leu Asn Gly Arg Glu
1 5 10 15
Glu
<210> 8
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> conventional tumor targeting peptide HCBP1
<400> 8
Phe Gln His Pro Ser Phe Ile
1 5
<210> 9
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> conventional tumor targeting peptide SP94
<400> 9
Ser Phe Ser Ile Ile His Thr Pro Ile Leu Pro Leu
1 5 10
<210> 10
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> conventional tumor targeting peptide HCl
<400> 10
Arg Gly Trp Cys Arg Pro Leu Pro Lys Gly Glu Gly
1 5 10
<210> 11
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> conventional tumor targeting peptide A54
<400> 11
Ala Gly Lys Gly Thr Pro Ser Leu Glu Thr Thr Pro
1 5 10
<210> 12
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> conventional tumor targeting peptide HCC79
<400> 12
Lys Ser Leu Ser Arg His Asp His Ile His His His
1 5 10
<210> 13
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> NC Polypeptides
<400> 13
Asp Gln Asp Ile Glu Ala Lys Asn Gly Val Ile Ser
1 5 10
<210> 14
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> P47
<400> 14
Ser Gln Asp Ile Arg Thr Trp Asn Gly Thr Arg Ser
1 5 10
<210> 15
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> RGD-NGR-P47
<400> 15
Ala Arg Tyr Cys Arg Gly Asp Cys Phe Asp Ala Leu Asn Gly Arg Glu
1 5 10 15
Glu Ser Gln Asp Ile Arg Thr Trp Asn Gly Thr Arg Ser
20 25
<210> 16
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> RGD-NGR-RS
<400> 16
Ala Arg Tyr Cys Arg Gly Asp Cys Phe Asp Ala Leu Asn Gly Arg Glu
1 5 10 15
Glu Cys Gly Arg Cys Lys Cys Cys Asn Gly Glu Arg Ser
20 25
Claims (47)
1. A polypeptide selected from the group consisting of Va-C, Va-RA, Va-RS, Va-RK, Va-KRA, Va-RL;
the amino acid sequence of Va-C is shown in SEQ ID NO. 1;
the amino acid sequence of the Va-RA is shown in SEQ ID NO. 2;
the amino acid sequence of the Va-RS is shown in SEQ ID NO. 3;
the amino acid sequence of Va-RK is shown in SEQ ID NO. 4;
the amino acid sequence of the Va-KRA is shown as SEQ ID NO. 5;
the amino acid sequence of the Va-RL is shown in SEQ ID NO. 6.
2. A polypeptide according to claim 1,
the polypeptide is Va-RS with an amino acid sequence shown in SEQ ID NO. 3.
3. A tumor detection reagent comprising a polypeptide; wherein the polypeptide is selected from the group consisting of Va-C, Va-RA, Va-RS, Va-RK, Va-KRA, Va-RL;
the amino acid sequence of Va-C is shown in SEQ ID NO. 1;
the amino acid sequence of the Va-RA is shown in SEQ ID NO. 2;
the amino acid sequence of the Va-RS is shown in SEQ ID NO. 3;
the amino acid sequence of Va-RK is shown in SEQ ID NO. 4;
the amino acid sequence of the Va-KRA is shown as SEQ ID NO. 5;
the amino acid sequence of Va-RL is shown in SEQ ID NO. 6.
4. The reagent for detecting tumor according to claim 3, further comprising: color developing agent and coloring agent.
5. The reagent of claim 4, wherein the color-developing agent is selected from the group consisting of: ninhydrin, dimethylaminobenzaldehyde, and diaminobenzidine.
6. The reagent of claim 4, wherein the staining agent is selected from the group consisting of fluorescent dyes.
7. The reagent of claim 6, wherein the fluorescent dye is selected from the group consisting of: FAM, DAPI, CY5, IR800, ICG.
8. The reagent for detecting tumor according to any one of claims 3 to 7, wherein the tumor is selected from the group consisting of: liver cancer, breast cancer, pancreatic cancer, intestinal cancer, brain tumor, cervical cancer, lung cancer, gastric cancer, bone cancer, ovarian cancer, lymphoma, renal cancer, nasopharyngeal carcinoma, testicular cancer, esophageal cancer, bladder cancer, prostate cancer, thyroid cancer, neuroblastoma, soft tissue sarcoma.
9. A tumor surgery navigation contrast agent, comprising a polypeptide; wherein the polypeptide is selected from the group consisting of Va-C, Va-RA, Va-RS, Va-RK, Va-KRA, Va-RL;
the amino acid sequence of Va-C is shown in SEQ ID NO. 1;
the amino acid sequence of the Va-RA is shown in SEQ ID NO. 2;
the amino acid sequence of the Va-RS is shown in SEQ ID NO. 3;
the amino acid sequence of Va-RK is shown in SEQ ID NO. 4;
the amino acid sequence of the Va-KRA is shown as SEQ ID NO. 5;
the amino acid sequence of the Va-RL is shown in SEQ ID NO. 6.
10. The tumor surgery navigation contrast medium according to claim 9, further comprising a color developing agent and a coloring agent.
11. The tumor surgery navigation contrast agent according to claim 10, wherein the color-developing agent is selected from the group consisting of: ninhydrin, dimethylaminobenzaldehyde, and diaminobenzidine.
12. The tumor surgical navigation contrast agent according to claim 10, wherein the staining agent is selected from fluorescent dyes.
13. The tumor surgical navigation contrast agent according to claim 12, wherein the fluorescent dye is selected from the group consisting of: FAM, DAPI, CY5, IR800, ICG.
14. The tumor surgery navigation contrast agent according to claim 9, wherein the tumor is selected from the group consisting of: liver cancer, breast cancer, pancreatic cancer, intestinal cancer, cerebroma, cervical cancer, lung cancer, gastric cancer, bone cancer, ovarian cancer, lymphoma, renal cancer, nasopharyngeal cancer, testicular cancer, esophageal cancer, bladder cancer, prostate cancer, thyroid cancer, neuroblastoma, and soft tissue sarcoma.
15. A tumor targeting medicine comprises a medicine active component and a tumor targeting peptide; wherein the tumor targeting peptide is selected from the group consisting of Va-C, Va-RA, Va-RS, Va-RK, Va-KRA, Va-RL;
the amino acid sequence of Va-C is shown in SEQ ID NO. 1;
the amino acid sequence of the Va-RA is shown in SEQ ID NO. 2;
the amino acid sequence of the Va-RS is shown in SEQ ID NO. 3;
the amino acid sequence of Va-RK is shown in SEQ ID NO. 4;
the amino acid sequence of the Va-KRA is shown as SEQ ID NO. 5;
the amino acid sequence of the Va-RL is shown in SEQ ID NO. 6.
16. The tumor-targeting agent according to claim 15, wherein said pharmaceutically active ingredient is selected from the group consisting of antitumor antibiotics, antitumor nucleosides, alkylating agents, plant alkaloids, antitumor hormones, platinum compounds, and antimetabolites.
17. The tumor targeting agent according to claim 16, wherein said antitumor antibiotic is selected from the group consisting of: adriamycin, mitomycin, bleomycin and daunorubicin.
18. The tumor targeting agent according to claim 16, wherein said antineoplastic nucleoside drug is selected from the group consisting of: antitumor antisense oligonucleotide, nucleoside analogue.
19. The tumor targeting agent according to claim 18, wherein said anti-tumor antisense oligonucleotide is selected from the group consisting of: the antisense oligonucleotide of Survivin gene, Sur-AS, Bcl2 gene, Bcl2-AS, MDM2 gene, MDM2-AS, BCLXL gene, BCLXL-AS, RelA gene, are antisense oligonucleotides.
20. The tumor targeting drug of claim 18, wherein said nucleoside analog is selected from the group consisting of: deoxyfluoroguanosine, hydroxyurea and cyclocytidine.
21. The tumor targeting drug of claim 16, wherein said alkylating agent is selected from the group consisting of: nimustine, cyclophosphamide, carmustine.
22. The tumor targeting agent according to claim 16, wherein said plant alkaloid is selected from the group consisting of: vincristine, colchicine, and cephalotaxine.
23. The tumor targeting agent according to claim 16, wherein said anti-tumor hormone is selected from the group consisting of: tamoxifen, flutamide, leuprorelin.
24. The tumor targeting drug according to claim 16, wherein said platinum group compound is selected from the group consisting of: cisplatin, oxaliplatin, carboplatin, phenanthrplatinum.
25. The tumor targeting agent according to claim 16, wherein said antimetabolite is selected from the group consisting of: methotrexate, 5-fluorouracil, 6-mercaptopurine.
26. The tumor-targeted drug according to claim 15, further comprising a drug carrier capable of carrying the pharmaceutically active ingredient.
27. The tumor targeting drug according to claim 26, wherein said drug carrier is selected from the group consisting of nanoparticles.
28. The tumor targeting agent according to claim 27, wherein said nanoparticles are selected from the group consisting of: exosomes, extracellular vesicles, liposomes.
29. The tumor-targeting agent of claim 15, wherein the target of action of said tumor-targeting agent is located in the nuclear and nucleolar region of a tumor cell.
30. An anti-tumor drug enhancer, the active component of which comprises tumor targeting peptide, wherein the tumor targeting peptide is selected from the group consisting of Va-C, Va-RA, Va-RS, Va-RK, Va-KRA and Va-RL;
the amino acid sequence of Va-C is shown in SEQ ID NO. 1;
the amino acid sequence of Va-RA is shown in SEQ ID NO. 2;
the amino acid sequence of the Va-RS is shown in SEQ ID NO. 3;
the amino acid sequence of Va-RK is shown in SEQ ID NO. 4;
the amino acid sequence of the Va-KRA is shown as SEQ ID NO. 5;
the amino acid sequence of the Va-RL is shown in SEQ ID NO. 6.
31. The enhancer of claim 30, wherein the anti-tumor drug is selected from the group consisting of anti-tumor antibiotics, anti-tumor nucleosides, alkylating agents, plant alkaloids, anti-tumor hormones, platinum compounds, and anti-metabolites.
32. The enhancer of claim 31, wherein said antitumor antibiotic is selected from the group consisting of: adriamycin, mitomycin, bleomycin and daunorubicin.
33. An enhancer for antitumor drugs according to claim 31, wherein said antitumor nucleoside drugs are selected from the group consisting of: antitumor antisense oligonucleotide, nucleoside analogue.
34. An enhancer for antitumor drugs according to claim 33, wherein said antitumor antisense oligonucleotide is selected from the group consisting of: the antisense oligonucleotides of Survivin gene SuraS, Bcl2 gene Bcl2-AS, MDM2 gene MDM2-AS, BCLXL gene BCLXL-AS, RelA gene RelA-AS.
35. An enhancer for an antitumor drug according to claim 31, wherein said nucleoside analog is selected from the group consisting of: deoxyfluoroguanosine, hydroxyurea and cyclocytidine.
36. An enhancer for antitumor drugs according to claim 31, wherein said alkylating agent is selected from the group consisting of: nimustine, cyclophosphamide, carmustine.
37. The enhancer of claim 31, wherein said plant alkaloid is selected from the group consisting of: vincristine, colchicine, and cephalotaxine.
38. The enhancer of claim 31, wherein said anti-tumor hormone is selected from the group consisting of: tamoxifen, flutamide, leuprorelin.
39. The enhancer for antitumor drugs according to claim 31, wherein said platinum compound is selected from the group consisting of: cisplatin, oxaliplatin, carboplatin, phenanthrplatinum.
40. An enhancer for an antitumor drug according to claim 31, wherein said antimetabolite is selected from the group consisting of: methotrexate, 5-fluorouracil, 6-mercaptopurine.
41. A coupling reagent for modifying polypeptide and/or short peptide, wherein the coupled active ingredients comprise tumor targeting peptide; wherein the tumor targeting peptide is selected from the group consisting of Va-C, Va-RA, Va-RS, Va-RK, Va-KRA, Va-RL;
the amino acid sequence of Va-C is shown in SEQ ID NO. 1;
the amino acid sequence of the Va-RA is shown in SEQ ID NO. 2;
the amino acid sequence of the Va-RS is shown in SEQ ID NO. 3;
the amino acid sequence of Va-RK is shown in SEQ ID NO. 4;
the amino acid sequence of the Va-KRA is shown as SEQ ID NO. 5;
the amino acid sequence of the Va-RL is shown in SEQ ID NO. 6.
42. A coupling reagent for modifying polypeptides and/or short peptides according to claim 41, wherein the polypeptide and/or short peptide to be modified is selected from the group consisting of conventional tumor targeting peptides, and/or tumor killing polypeptides, cell shuttle peptides.
43. A coupling reagent for modifying polypeptides and/or short peptides according to claim 42, wherein said conventional tumor targeting peptide is selected from the group consisting of: RGD, NGR, RGD-NGR, HCBP1, SP94, HCl, A54, HCC 79.
44. A coupling reagent for modifying polypeptides and/or short peptides according to claim 43, wherein said conventional tumor targeting peptide is RGD-NGR.
45. A coupling reagent for modifying polypeptides and/or short peptides according to claim 42, wherein said tumor killing polypeptide is selected from the group consisting of: consisting of melittin, octreotide, polymyxin, bacitracin, LTX-302, cecropin.
46. A coupling reagent for the modification of polypeptides and/or short peptides according to claim 42, wherein said cell shuttle peptide is selected from the group consisting of: TAT, (RXR) 4 、B。
47. The coupling reagent for modifying polypeptide and/or short peptide according to claim 43, wherein the amino acid sequence of the conventional tumor targeting peptide RGD-NGR is shown in SEQ ID NO. 7;
the amino acid sequence of the conventional tumor targeting peptide HCBP1 is shown as SEQ ID NO. 8;
the amino acid sequence of the conventional tumor targeting peptide SP94 is shown in SEQ ID NO. 9;
the amino acid sequence of the conventional tumor targeting peptide HCl is shown in SEQ ID NO. 10;
the amino acid sequence of the conventional tumor targeting peptide A54 is shown in SEQ ID NO. 11;
the amino acid sequence of the conventional tumor targeting peptide HCC79 is shown in SEQ ID NO. 12.
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