Peptides specific for vegfr3 receptor and uses thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a VEGFR3 receptor specific binding peptide and application thereof, wherein the amino acid sequence of the VEGFR3 receptor specific binding peptide is shown as SEQ ID NO. 1. The VEGFR3 receptor specific binding peptide can specifically bind with the VEGFR3 receptor on the cell membrane, thereby activating a downstream signal path, stimulating the migration and proliferation of lymphatic endothelial cells, forming a new lymphatic network, and increasing the permeability of the lymphatic vessel, thereby providing convenience for the flow of lymph fluid and further promoting the function of the lymphatic vessel. The VEGFR3 receptor specific binding peptide not only has a significant effect of promoting the proliferation and migration of human lymphatic endothelial cells, but also shows good biocompatibility and biological activity. In addition, the VEGFR3 receptor specific binding peptide can also promote the proliferation and migration of human umbilical vein endothelial cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a VEGFR3 receptor specific binding peptide and application thereof, the VEGFR3 receptor specific binding peptide can specifically promote the proliferation, migration and tube formation of lymphatic endothelial cells (LECs), and can also effectively promote the same functions of vascular endothelial cells (HUVECs). BACKGROUND

[0002] Vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) are key drivers of blood and lymphatic vessel formation in normal development and some pathological processes. VEGF-C signals promote lymphangiogenesis through VEGFR3, and lymphangiogenesis is a clinically relevant target for treating lymphatic insufficiency and blocking tumor angiogenesis and metastasis.

[0003] The extracellular domain of VEGFRs is composed of 7 Ig homology domains, of which domains 1-3 (D1-3) are responsible for ligand binding. The crystal structure of VEGF-C is an anti-parallel homodimer with an anti-parallel four-stranded beta sheet structure, three connecting loops (L1-L3) and an extended N-terminal alpha-helix. The binding site of VEGF-C and VEGFR3 has high structural specificity, in which the alpha-helix constitutes the most stable structure in the binding interface.

[0004] For a long time, the development of drugs for promoting lymphangiogenesis has been relatively lagging behind, and the existing clinical drugs such as 9-cis retinoic acid (9-cisRA) and simvastatin are limited in variety, and the preparation cost of recombinant VEGF-C protein is high. It is worth noting that the current such therapeutic preparations generally have the common problems of low pharmacoeconomic benefit, poor target selectivity and high incidence of adverse reactions.

[0005] Under this background, if a polypeptide can specifically target and bind to VEGFR3 receptor and activate the downstream VEGF signaling pathway to promote lymphangiogenesis, it is expected to show the development potential as a new type of non-natural peptidomimetic drug. SUMMARY

[0006] In view of the deficiencies in the prior art, a first object of the present application is to provide a novel VEGFR3 receptor specific binding peptide with stable structure and high affinity target binding characteristics, which can specifically bind to the VEGFR3 receptor on the cell membrane, thereby activating the downstream signaling pathway, stimulating the migration and proliferation of lymphatic endothelial cells, forming a new lymphatic network, and increasing the permeability of the lymphatic vessels, providing convenience for the flow of lymph fluid, and further promoting the function of the lymphatic vessels. The VEGFR3 receptor specific binding peptide not only has a significant effect of promoting the proliferation and migration of human lymphatic endothelial cells, but also exhibits good biocompatibility and biological activity. In addition, the VEGFR3 receptor specific binding peptide can also promote the proliferation and migration of human umbilical vein endothelial cells. Therefore, the VEGFR3 receptor specific binding peptide provided by the present application has a broad prospect in the application of promoting vascularization in the field of tissue engineering and regenerative medicine, which not only helps basic scientific research, but also provides a new idea and method for clinical treatment.

[0007] A second object of the present application is to use the VEGFR3 receptor specific binding peptide described above in the preparation of a drug for promoting lymphangiogenesis, angiogenesis and / or tissue repair. This innovative strategy not only provides a new idea for the innovation of lymphangiogenesis promotion therapy, but also can be used as an effective supplement to existing therapies, and provides an optimized scheme for clinical intervention of diseases related to imbalance of lymphangiogenesis regulation.

[0008] A third object of the present application is to use the VEGFR3 receptor specific binding peptide described above in the preparation of a molecular probe targeting lymphatic endothelial cells.

[0009] To achieve the above object, the present application is realized by the following technical measures.

[0010] A VEGFR3 receptor specific binding peptide or a pharmaceutically acceptable salt thereof, the amino acid sequence of the VEGFR3 receptor specific binding peptide is shown in SEQ ID NO. 1.

[0011] It should be noted that in the prior art documents, the length of the known artificial synthetic binding peptide sequence for VEGFR3 receptor is usually more than 200, there is obvious structural redundancy, and the exact active core domain in the interaction between VEGF-C and VEGFR3 has not been uniformly defined and systematically studied in the academic field. Although the amino acid sequence shown in SEQ ID NO. 1 exists in the above-mentioned artificial synthetic binding peptide sequence fragment, it should be emphasized that the present application first precisely analyzes the function of the binding interface between VEGF-C and VEGFR3, and clearly defines the alpha helix region that bears the core biological function and must rely on a specific spatial conformation. It is found through simulation calculation that the natural helical structure cannot exist independently and stably. The design of the amino acid sequence shown in SEQ ID NO. 1 optimizes and restructures the core functional domain, which can maintain high stability when it is synthesized into a peptide independently on the premise of completely retaining its active conformation, and successfully synthesizes a stable powdery polypeptide. In vivo and in vitro functional experiments confirm that its biological effect is equivalent to or better than that of VEGF-C at the same concentration. Therefore, the present application not only overcomes the limitations of the existing knowledge, but also realizes substantial innovation of the key functional domain, significantly reduces the synthesis difficulty and production cost while ensuring the best activity.

[0012] In this text, the pharmaceutically acceptable salt is the pharmaceutically acceptable salt of the polypeptide according to the amino acid sequence shown in SEQ ID NO. 1, and such salt includes but is not limited to acid salt and base salt. The "pharmaceutically acceptable salt" mentioned in the present application refers to a salt that retains the binding activity of VEGFR3 of the polypeptide according to the amino acid sequence shown in SEQ ID NO. 1 and is suitable for use in humans or animals. The method for preparing the salt of the polypeptide according to the amino acid sequence shown in SEQ ID NO. 1 is known in the technical field, and generally involves mixing the polypeptide with a pharmaceutically acceptable acid or base. Examples of the pharmaceutically acceptable acid and base include organic acids and inorganic acids that form ammonium salts with free amino groups of the polypeptide, such as formic acid, acetic acid, propionic acid, lactic acid, glycolic acid, oxalic acid, pyruvic acid, succinic acid, maleic acid, malonic acid, trifluoroacetic acid, cinnamic acid, sulfuric acid, hydrochloric acid, hydrobromic acid, nitric acid, perchloric acid, phosphoric acid and thiocyanic acid; and bases that form carboxylate salts with free carboxyl groups of the polypeptide, such as ethylamine, methylamine, dimethylamine, triethylamine, isopropylamine, diisopropylamine and other monoalkyl amines, dialkyl amines and trialkyl amines, and aryl amines.

[0013] In one of the technical solutions, the present application provides a fusion protein containing the above-mentioned VEGFR3 receptor specific binding peptide or a pharmaceutically acceptable salt thereof.

[0014] In another aspect, the present application provides a composition comprising the VEGFR3 receptor specific binding peptide or a pharmaceutically acceptable salt thereof described above, or the fusion protein described above. The composition comprises a payload composition, wherein the VEGFR3 receptor specific binding peptide and the payload composition are covalently coupled to each other or non-covalently associated, optionally, wherein the payload composition comprises a therapeutic agent, a detectable agent, a carrier, a surface molecule, or a combination thereof, preferably, wherein the therapeutic agent is an anti-bacterial agent, an anti-inflammatory agent, an immune stimulatory agent, an immune suppressive agent, or a pro-angiogenic agent, preferably, wherein the detectable agent is a labeling agent, a contrast agent, an imaging agent, optionally, wherein the VEGFR3 receptor specific binding peptide is conjugated to the payload composition, preferably, wherein one or more of the conjugated peptides are indirectly conjugated to the payload composition via a linker, optionally, wherein the payload composition further comprises a plurality of linkers, preferably, wherein at least one of the linkers comprises polyethylene glycol or maleimide coupling, amino-functionalized dextran chemistry to couple, and components can be covalently bound to surface molecules or to each other directly or indirectly through any functional group, for example, amine, carbonyl, carboxyl, aldehyde, alcohol.

[0015] In one preferred embodiment, the present application provides a composition comprising the VEGFR3 receptor specific binding peptide or a pharmaceutically acceptable salt thereof described above conjugated to a labeling agent, including but not limited to a fluorescent group (e.g., such as FITC, Cy5), a radionuclide (e.g., such as Tc, 99m Tc, 68 Ga).

[0016] In one preferred embodiment, the present application provides a composition comprising the VEGFR3 receptor specific binding peptide or a pharmaceutically acceptable salt thereof described above conjugated to a carrier, preferably a hydrogel, a spray, or a biological dressing.

[0017] In another aspect, the present application provides a drug delivery system comprising the VEGFR3 receptor specific binding peptide or a pharmaceutically acceptable salt thereof described above, or the fusion protein described above; the drug delivery system further comprises one or more of a micelle, a liposome, a nanoparticle, a nanoemulsion, a microemulsion, a nanocapsule, a phospholipid complex, a polymer complex, a carbon nanotube, a quantum dot, a microcapsule, a hydrogel, a microsphere, a bacteriophage, a bacteriophage capsid, a bacteriophage particle, a virus, a virus capsid, a virus particle, an extracellular vesicle, an exosome.

[0018] In another aspect, the present application also relates to a composition of the above-mentioned VEGFR3 receptor specific binding peptide and / or the above-mentioned fusion protein, which comprises a linker, the linker comprising one or more of a protein, a polypeptide, a fatty acid and its derivative, an antibody, an antibody fragment, a cyclodextrin and its derivative, a small molecule drug, PEG and its derivative, a fatty acid, a mesoporous inorganic material, a linear polymer, a cross-linked polymer, or a branched copolymer.

[0019] In another aspect, the present application also relates to the above-mentioned VEGFR3 receptor specific binding peptide and its pharmaceutically acceptable salt, the above-mentioned fusion protein, and provides the use thereof in the preparation of a medicament for promoting lymphangiogenesis, angiogenesis and / or tissue repair. Meanwhile, in another aspect, the present application also relates to a composition comprising the above-mentioned VEGFR3 receptor specific binding peptide or its pharmaceutically acceptable salt, or the above-mentioned fusion protein, and the use of the composition in the preparation of a therapeutic agent, a targeting agent, a prophylactic agent, a diagnostic agent or an imaging agent, or the use of the composition in the preparation of a medicament for promoting lymphangiogenesis, angiogenesis and / or tissue repair, diagnosis and / or treatment, image-guided surgery, drug delivery, targeted delivery of an imaging agent. The composition is preferably used in the preparation of a medicament for promoting lymphangiogenesis, angiogenesis and / or tissue repair.

[0020] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0021] 1. The VEGFR3 receptor specific binding peptide provided by the present application can form a stable spatial conformation and tightly bind to the pocket of the VEGFR3 receptor, as shown by molecular dynamics simulation.

[0022] 2. The VEGFR3 receptor specific binding peptide provided by the present application has a binding constant KD value of 89.9 nM to VEGFR3, as confirmed by SPR analysis, and has good affinity.

[0023] 3. The VEGFR3 receptor specific binding peptide provided by the present application can not only specifically promote the proliferation, migration and tube formation of lymphatic endothelial cells (LECs), but also effectively promote the same functions of vascular endothelial cells (HUVECs), as confirmed by in vitro experiments. This indicates that it can simultaneously stimulate angiogenesis and lymphangiogenesis, and provide a more comprehensive regenerative microenvironment for tissue repair.

[0024] 4. The VEGFR3 receptor specific binding peptide provided by the present application can significantly accelerate wound closure when applied locally, as confirmed by a mouse dorsal skin defect model, and can improve granulation tissue formation and epithelial regeneration, as shown by histology (H&E staining).

[0025] 5. The VEGFR3 receptor-specific binding peptide provided by this invention exhibits specific co-localization with lymphatic endothelial cells (LECs) in vitro after fluorescent labeling, indicating that it is an ideal carrier for developing lymphatic vessel-specific molecular probes and provides a core tool for "therapeutic integration". Attached Figure Description

[0026] Figure 1 This is a summary chart of the molecular models and root mean square fluctuation (RMSF) calculation results of the polypeptides obtained in Example 1 and Comparative Examples 1-3 of the present invention. Among them, Figures (A), (B), (C), and (D) correspond to the polypeptides obtained in Example 1, Example 1, Comparative Example 2, and Comparative Example 3, respectively.

[0027] Figure 2 This is a summary diagram of the Gibbs free energy conformational analysis results of the polypeptides obtained in Example 1 and Comparative Examples 1-3 of the present invention. Among them, Figures (A), (B), (C), and (D) correspond to the polypeptides obtained in Example 1, Example 1, Comparative Example 2, and Comparative Example 3, respectively.

[0028] Figure 3 This is a summary diagram of the secondary structure analysis results of the polypeptides obtained in Example 1 and Comparative Examples 1-3 of the present invention, based on molecular dynamics simulations. Figures (A), (B), (C), and (D) correspond to the polypeptides obtained in Example 1, Comparative Example 2, and Comparative Example 3, respectively.

[0029] Figure 4 Figure 1 is a summary diagram of the docking of the polypeptide obtained in Example 1 of this invention with the original receptor molecule and the results thereof. Specifically, Figure (A) is a schematic diagram of the stick-like structure of the polypeptide and the original receptor molecule complex in Example 1; Figure (B) is a three-dimensional schematic diagram of the polypeptide and the original receptor molecule complex in Example 1; Figure (C) is a schematic diagram of the interaction between the polypeptide and the original receptor molecule in Example 1; and Figure (D) is a 2D schematic diagram of the interaction between the polypeptide and the original receptor molecule in Example 1.

[0030] Figure 5 This is the mass spectrometry spectrum of the polypeptide obtained in Example 1 of the present invention.

[0031] Figure 6 This is a circular dichroism chromatogram of the polypeptide obtained in Example 1 of the present invention.

[0032] Figure 7 The NMR spectrum of the polypeptide obtained in Example 1 of this invention is shown.

[0033] Figure 8 This is the SPR sensor spectrum of the polypeptide obtained in Example 1 of the present invention.

[0034] Figure 9Figure for in vitro cell CCK-8 experiment results of polypeptide obtained in Example 1 of the present application. Wherein, Figure A is human lymphatic endothelial cells (HLEC) as a cell model, Figure B is human umbilical vein endothelial cells (HUVEC) as a cell model.

[0035] Figure 10 Figure for in vitro cell scratch test and Transwell experiment results of polypeptide obtained in Example 1 of the present application. Wherein, Figure A-D is human lymphatic endothelial cells (HLEC) as a cell model, respectively corresponding to the results of scratch test and Transwell experiment summary figure, Figure E-H is human umbilical vein endothelial cells (HUVEC) as a cell model, respectively corresponding to the results of scratch test and Transwell experiment summary figure.

[0036] Figure 11 Figure for in vitro cell tube formation experiment results of polypeptide obtained in Example 1 of the present application. Wherein, Figure A is human lymphatic endothelial cells (HLEC) as a cell model, Figure B is human umbilical vein endothelial cells (HUVEC) as a cell model.

[0037] Figure 12 Figure for in vitro co-localization verification of polypeptide obtained in Example 1 of the present application.

[0038] Figure 13 Figure for mouse back skin defect healing experiment results of polypeptide obtained in Example 1 of the present application. Wherein, Figure A-B is a comparison of wound photos at different time points and a healing schematic diagram; Figure C is a statistical analysis diagram of wound healing rate; Figure D is a H&E staining diagram of wound tissue at 7th day and 12th day.

[0039] Figure 14 Figure for mouse skin tissue blood vessel and lymphatic vessel immunofluorescence staining results of polypeptide obtained in Example 1 of the present application. Wherein, the upper figure is a representative fluorescence image of immunofluorescence staining blood vessels of mouse back skin wound healing tissue at 12th day, and the lower figure is a representative fluorescence image of immunofluorescence staining lymphatic vessels of mouse back skin wound healing tissue at 12th day. DETAILED DESCRIPTION

[0040] For a further understanding of the present application, preferred embodiments thereof will be described in conjunction with examples, it being understood, however, that this description is made only by way of further illustration and is not intended to limit the scope of the claimed application. Those skilled in the art can readily improve the process parameters using the contents described herein. It is particularly pointed out that all such further modifications and variations that fall within the scope of the present application are considered to be included herein, as are expressly set forth in the following claims. The methods and applications of the present application have been described by way of preferred embodiments, and it will be obvious to those skilled in the art that the methods and applications described herein can be modified or appropriately changed and combined without departing from the content, spirit and scope of the present application, to realize and apply the present application. Although the following terms are believed to be well understood by those of ordinary skill in the art, the following definitions are set forth to facilitate the understanding of the presently disclosed subject matter.

[0041] A VEGFR3 receptor specific binding peptide or a pharmaceutically acceptable salt thereof, the amino acid sequence of the VEGFR3 receptor specific binding peptide is shown as SEQ ID NO. 1.

[0042] It should be noted that in the prior art documents, the length of the known artificially synthesized binding peptide sequence for VEGFR3 receptor is usually more than 200, there is obvious structural redundancy, and the academic community lacks unified definition and systematic study on the exact active core domain in the interaction between VEGF-C and VEGFR3. Although the amino acid sequence shown in SEQ ID NO. 1 of the present application exists in the above-mentioned artificially synthesized binding peptide sequence fragment, it should be emphasized that the present application first accurately analyzes the function of the binding interface between VEGF-C and VEGFR3, and clearly defines the alpha helix region that bears the core biological function and must rely on a specific spatial conformation. Through simulation calculation, it is found that the natural helical structure cannot exist independently and stably. The design of the amino acid sequence shown in SEQ ID NO. 1 optimizes and restructures the core functional domain, which can maintain high stability when synthesized into a peptide independently on the premise of completely retaining its active conformation, and successfully synthesizes a stable powdered polypeptide. In vivo and in vitro functional experiments confirm that its biological effect is equivalent or superior to that of VEGF-C at the same concentration. Therefore, the present application not only overcomes the limitations of existing knowledge, but also realizes substantial innovation of the key functional domain, while ensuring optimal activity, significantly reducing the difficulty of synthesis and production cost.

[0043] In the present context, the pharmaceutically acceptable salt is a pharmaceutically acceptable salt of a polypeptide according to the amino acid sequence of SEQ ID NO. 1, such salts include but are not limited to acid addition salts and base salts. The "pharmaceutically acceptable salt" mentioned in the present application means a salt of a polypeptide according to the amino acid sequence of SEQ ID NO. 1 which retains the binding activity of VEGFR3 and is suitable for use in humans or animals. Methods for preparing salts of polypeptides according to the amino acid sequence of SEQ ID NO. 1 are known in the art and generally involve mixing the polypeptide with a pharmaceutically acceptable acid or base, examples of pharmaceutically acceptable acids and bases include organic and inorganic acids which form ammonium salts with the free amino groups of the polypeptide, in one embodiment, for example, formic acid, acetic acid, propionic acid, lactic acid, glycolic acid, oxalic acid, pyruvic acid, succinic acid, maleic acid, malonic acid, trifluoroacetic acid, cinnamic acid, sulfuric acid, hydrochloric acid, hydrobromic acid, nitric acid, perchloric acid, phosphoric acid and thiocyanic acid; and bases which form carboxylate salts with the free carboxyl groups of the polypeptide, for example, ethylamine, methylamine, dimethylamine, triethylamine, isopropylamine, diisopropylamine and other mono-, di- and trialkylamines, and arylamines.

[0044] In one embodiment, the present application provides a fusion protein comprising the VEGFR3 receptor specific binding peptide described above or a pharmaceutically acceptable salt thereof.

[0045] In another aspect, the present application provides a composition comprising the VEGFR3 receptor specific binding peptide described above or a pharmaceutically acceptable salt thereof, or the fusion protein described above. In one embodiment, the composition comprises a payload composition, wherein the VEGFR3 receptor specific binding peptide and the payload composition are covalently coupled or non-covalently associated with each other, optionally, wherein the payload composition comprises a therapeutic agent, a detectable agent, a carrier, a surface molecule or a combination thereof, preferably, wherein the therapeutic agent is an antibacterial agent, an anti-inflammatory agent, an immune stimulant, an immune suppressant or a pro-angiogenic agent, preferably, wherein the detectable agent is a labeling agent, a contrast agent, an imaging agent, optionally, wherein the VEGFR3 receptor specific binding peptide is conjugated to the payload composition, preferably, wherein one or more of the conjugated peptides are indirectly conjugated to the payload composition via a linker, optionally, wherein the payload composition further comprises a plurality of linkers, preferably, wherein at least one of the linkers comprises polyethylene glycol or maleimide coupling, amino-functionalized dextran chemistry for coupling, and components can be covalently bound to surface molecules or to each other directly or indirectly through any functional group, for example, amine, carbonyl, carboxyl, aldehyde, alcohol.

[0046] In one preferred embodiment, the VEGFR3 receptor specific binding peptide or its pharmaceutically acceptable salt is coupled with a labeling agent to form a composition, which includes but is not limited to a fluorescent group (e.g. FITC, Cy5), a radionuclide (e.g. Tc, 99m 68 Ga).

[0047] In one preferred embodiment, the VEGFR3 receptor specific binding peptide or its pharmaceutically acceptable salt is combined with a carrier, which is preferably a hydrogel, a spray or a biological dressing.

[0048] In another aspect, the present application provides a drug delivery system containing the VEGFR3 receptor specific binding peptide or its pharmaceutically acceptable salt, or the fusion protein. In one embodiment, the drug delivery system further comprises one or more of micelles, liposomes, nanoparticles, nanoemulsions, microemulsions, nanocapsules, phospholipid complexes, polymer complexes, carbon nanotubes, quantum dots, microcapsules, hydrogels, microspheres, bacteriophages, bacteriophage capsids, bacteriophage particles, viruses, viral capsids, viral particles, extracellular vesicles, exosomes.

[0049] In another aspect, the present application also relates to a composition of the VEGFR3 receptor specific binding peptide and / or the fusion protein, which comprises a linker. In one embodiment, the linker comprises one or more of proteins, polypeptides, fatty acids and their derivatives, antibodies, antibody fragments, cyclodextrins and their derivatives, small molecule drugs, PEG and its derivatives, fatty acids, mesoporous inorganic materials, linear polymers, cross-linked polymers, or branched copolymers.

[0050] In another aspect, the present application also relates to the VEGFR3 receptor specific binding peptide and its pharmaceutically acceptable salt, and the fusion protein, and provides the use thereof in the preparation of a medicament for promoting lymphangiogenesis, angiogenesis and / or tissue repair. Meanwhile, in another aspect, the present application also relates to a composition comprising the VEGFR3 receptor specific binding peptide or its pharmaceutically acceptable salt, or the fusion protein, and provides the use of the composition in the preparation of a therapeutic agent, a targeting agent, a prophylactic agent, a diagnostic agent or an imaging agent, or the use of the composition in the preparation of a medicament for promoting lymphangiogenesis, angiogenesis and / or tissue repair, image-guided surgery, drug delivery, targeted delivery of imaging agents in diagnosis and / or treatment. Preferably, the composition is used in the preparation of a medicament for promoting lymphangiogenesis, angiogenesis and / or tissue repair.

[0051] The present application will be further explained in detail with reference to the following examples. However, those skilled in the art will understand that the examples are provided for illustrative purposes only, and are not intended to limit the present application.​

[0052] Example

[0053] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be construed as limiting the scope of this application. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all commercially available conventional products. This application should not be construed as being limited to the specific embodiments described.

[0054] Example 1, Comparative Examples 1-3

[0055] Example 1 and Comparative Examples 1-3: Target peptides were synthesized using microwave-assisted solid-phase peptide synthesis.

[0056] Example 1: The amino acid sequence of the polypeptide is shown in SEQ ID NO.1, and is denoted as Peptide_2;

[0057] The amino acid sequence of the polypeptide in Comparative Example 1 is shown in SEQ ID NO.2 and is denoted as Peptide_1;

[0058] The amino acid sequence of the polypeptide in Comparative Example 2 is shown in SEQ ID NO.3, and is denoted as Peptide_3;

[0059] The amino acid sequence of the polypeptide in Comparative Example 3 is shown in SEQ ID NO.4, and is denoted as Peptide_4;

[0060] The synthesized product was purified by preparative high-performance liquid chromatography.

[0061] Based on Example 1 and Comparative Examples 1-3, the amino acid sequences were modeled using GROMACS 2019.6 and molecular dynamics simulations were performed from a kinetic and thermodynamic perspective.

[0062] like Figure 1 , 2 As shown, the root mean square fluctuation (RMSF) values ​​of the peptides in Example 1 and Comparative Examples 1-3 were calculated, and the Gibbs free energy conformation of the peptides was analyzed. Throughout the simulation process, the four peptides remained in a stable state, suggesting that no significant structural changes or refolding occurred during the simulation. Based on the comparison of the total atomic RMSF values ​​of the four peptides (…), Figure 1 Among them, Peptide_2 is more stable than the other three peptide systems, and the region with the lowest relative free energy conformation accounts for the largest proportion. Figure 2 ).

[0063] like Figure 3As shown in the secondary structure analysis results of the polypeptides of Example 1, Comparative Examples 1-3, the secondary structures of Peptide_1, Peptide_2 and Peptide_3 are consistent during the simulation process, mainly in the form of α-helix structure, and the proportion of α-helix in the secondary structure of Peptide_2 is the largest, and the structure is more stable.

[0064] As shown in the secondary structure analysis results of the polypeptides of Example 1, Comparative Examples 1-3, the secondary structures of Peptide_1, Peptide_2 and Peptide_3 are consistent during the simulation process, mainly in the form of α-helix structure, and the proportion of α-helix in the secondary structure of Peptide_2 is the largest, and the structure is more stable. Figure 4 As shown in the secondary structure analysis results of the polypeptides of Example 1, Comparative Examples 1-3, the secondary structures of Peptide_1, Peptide_2 and Peptide_3 are consistent during the simulation process, mainly in the form of α-helix structure, and the proportion of α-helix in the secondary structure of Peptide_2 is the largest, and the structure is more stable.

[0065] As shown in the secondary structure analysis results of the polypeptides of Example 1, Comparative Examples 1-3, the secondary structures of Peptide_1, Peptide_2 and Peptide_3 are consistent during the simulation process, mainly in the form of α-helix structure, and the proportion of α-helix in the secondary structure of Peptide_2 is the largest, and the structure is more stable. Figure 5 As shown in the secondary structure analysis results of the polypeptides of Example 1, Comparative Examples 1-3, the secondary structures of Peptide_1, Peptide_2 and Peptide_3 are consistent during the simulation process, mainly in the form of α-helix structure, and the proportion of α-helix in the secondary structure of Peptide_2 is the largest, and the structure is more stable.

[0066] As shown in the secondary structure analysis results of the polypeptides of Example 1, Comparative Examples 1-3, the secondary structures of Peptide_1, Peptide_2 and Peptide_3 are consistent during the simulation process, mainly in the form of α-helix structure, and the proportion of α-helix in the secondary structure of Peptide_2 is the largest, and the structure is more stable. Figure 6 As shown in the secondary structure analysis results of the polypeptides of Example 1, Comparative Examples 1-3, the secondary structures of Peptide_1, Peptide_2 and Peptide_3 are consistent during the simulation process, mainly in the form of α-helix structure, and the proportion of α-helix in the secondary structure of Peptide_2 is the largest, and the structure is more stable.

[0067] As shown in the secondary structure analysis results of the polypeptides of Example 1, Comparative Examples 1-3, the secondary structures of Peptide_1, Peptide_2 and Peptide_3 are consistent during the simulation process, mainly in the form of α-helix structure, and the proportion of α-helix in the secondary structure of Peptide_2 is the largest, and the structure is more stable. Figure 7 As shown in the secondary structure analysis results of the polypeptides of Example 1, Comparative Examples 1-3, the secondary structures of Peptide_1, Peptide_2 and Peptide_3 are consistent during the simulation process, mainly in the form of α-helix structure, and the proportion of α-helix in the secondary structure of Peptide_2 is the largest, and the structure is more stable.

[0068] Based on the affinity and specificity analysis (SPR) of the polypeptide Peptide_2 of Example 1 and VEGFR3:

[0069] ①Using a Biacore series SPR instrument, VEGFR3-Fc fusion protein was immobilized on a CM5 chip. Different concentrations of polypeptide Peptide_2 were flowed through the chip surface.

[0070] ②Results: As shown in the secondary structure analysis results of the polypeptides of Example 1, Comparative Examples 1-3, the secondary structures of Peptide_1, Peptide_2 and Peptide_3 are consistent during the simulation process, mainly in the form of α-helix structure, and the proportion of α-helix in the secondary structure of Peptide_2 is the largest, and the structure is more stable. Figure 8As shown, the sensorgram showed a typical concentration-dependent binding curve. By fitting with a 1:1 Langmuir binding model, the binding constant KD=89.9 nM, kon=4547 / Ms, koff=0.0409 / s were obtained. The data proved that the polypeptide of the application has high affinity with VEGFR3.

[0071] Based on the experiment of dual promotion of polypeptide Peptide_2 of Example 1 to the function of vascular and lymphatic endothelial cells, the cell model respectively takes human lymphatic endothelial cells (HLEC) and human umbilical vein endothelial cells (HUVEC) as the model:

[0072] (1) CCK-8 experiment:

[0073] ① Take the cells to be tested in good growth state, digest, centrifuge, resuspend with ECM medium, dilute to 7.5×10 4 / mL, inoculate 100 μL per well into a 96-well cell culture plate, 37 ℃, 5 % CO2, and culture for 24 h.

[0074] ② Prepare polypeptide Peptide_2 solution with concentration gradient.

[0075] ③ Based on the cells cultured in step ①, discard the old culture medium;

[0076] add complete culture medium to the blank control group, add complete culture medium containing different concentrations of polypeptide Peptide_2 solution to the polypeptide treatment group (20 ng / mL of polypeptide Peptide_2 solution is used in the experiment of human umbilical vein endothelial cells), and add complete culture medium containing VEGF-C to the positive control group (the concentration of VEGF-C solution is 20 ng / mL), 37 ℃, 5 % CO2, and culture for 72 h.

[0077] ④ Measure the absorbance of each well at 24 h, 48 h and 72 h after adding the drug. Discard the complete culture medium, add 100 μL of detection agent (CCK-8 reagent: culture medium = 1:9) to each well, incubate in the incubator for 2 h, 37 ℃, 5 % CO2, and detect the OD value at 450 nm with an enzyme-labeled instrument.

[0078] As Figure 9 shown, compared with the control group, the polypeptide of the application can significantly and concentration-dependently promote the proliferation of HLEC and HUVEC (p<0.05), which indicates that it has activation effect on VEGFR3 and VEGFR2 signaling pathways.

[0079] (2) Scratch test:

[0080] 6 Cell culture: Draw two lines in the back of each hole of the 6-hole plate as a marker. Set up the control group and the polypeptide treatment group. Take the well-grown cells to be tested, digest, centrifuge, resuspend in complete culture medium, dilute to 5 x 10 6

[0081] Scratch test: After the cells are cultured for 48 h, use a 200 μL gun head to vertically and closely scratch the cell layer to form a scratch mark. Rinse the cells with PBS buffer for 3 times to remove the scratched cells. Add 2 mL of low serum (HLEC) or serum-free (HUVEC) culture medium to the 6 holes, respectively. The polypeptide treatment group is complete culture medium containing different concentrations (10 ng / mL, 20 ng / mL, 30 ng / mL) of polypeptides. The blank control group is added with complete culture medium. The positive control group is added with complete culture medium containing VEGF-C.

[0082] Cultivate in a 37 ℃, 5 % CO2 incubator, and take microphotographs at fixed positions at 0 h, 6 h, and 12 h.

[0083] Image J is used to measure the scratch area: the scratch pictures (0 h, 6 h, and 12 h) are processed by using the software, and finally the cell migration rate is obtained, as shown in A-B, E-F in Figure 10

[0084] (3) Transwell test:

[0085] Cell preparation: take well-grown cells to be tested, digest, centrifuge, resuspend in serum-free culture medium, and adjust the cell density to 1 x 10 6

[0086] Chamber plating: add 200 μL of cell suspension to the upper chamber of the Transwell, and add 500 μL of complete culture medium containing different concentrations (10 ng / mL, 20 ng / mL, 30 ng / mL) of polypeptides to the lower chamber, respectively. The blank control group is added with complete culture medium. The positive control group is added with complete culture medium containing VEGF-C.

[0087] Cell incubation: place the culture plate in a 37 ℃, 5 % CO2 incubator for 24 hours to allow the cells to complete migration.

[0088] Fixation and staining: take out the chamber, and gently wipe off the non-migrated cells in the upper chamber with a cotton swab. Then, fix the chamber in 4 % paraformaldehyde for 15 minutes, and stain with 0.1 % crystal violet for 2 hours.

[0089] ​​⑤ Observation and counting: randomly select multiple fields under a microscope for photography, and count the cells penetrating the membrane, such as Figure 10 C-D, G-H in the middle.

[0090] As shown in Figure 10 , based on the results of (2) scratch test and (3) Transwell experiment, the scratch healing rate and the number of cells penetrating the membrane of the polypeptide treatment group HLEC were significantly higher than those of the control group (p<0.05), indicating that it can promote the migration of two kinds of endothelial cells at the same time.

[0091] (4) Tube formation experiment:

[0092] ① Preparation of Matrigel matrix: take Matrigel out from -20 ℃, and place it in 4 ℃ refrigerator overnight for thawing. Put 24-well, pipette and 200 uL gun head into 4 ℃ refrigerator for pre-cooling.

[0093] ② Plating: evenly spread Matrigel at 20 μL / well on the bottom of the culture plate, and make sure it does not stick to the side wall. Place the culture plate with Matrigel on it in a 37 ℃ incubator for 30-60 minutes to gel the Matrigel.

[0094] ③ Take the cells to be tested in good growth state, digest, centrifuge, resuspend with complete culture medium, and dilute to 1×10 6 / mL. Add the cell suspension to Matrigel at 200 μL / well, and gently shake the culture plate to make the cells evenly distributed. The polypeptide treatment group is complete culture medium containing 20 ng / mL concentration of polypeptide, the blank control group is added with complete culture medium, and the positive control group is added with complete culture medium containing 20 ng / mL concentration of VEGF-C.

[0095] ④ Incubate in a 37 ℃, 5 % CO2 incubator, and take microphotographs after 8 h.

[0096] ⑤ Use Image J to quantitatively analyze the length of the tubular structure, the number of branch points, the number of grids, etc.: use the software to process the tube formation pictures.

[0097] As shown in Figure 11 , on Matrigel, HLEC and HUVEC of the polypeptide treatment group formed more complete and branched tubular network structures.

[0098] In vitro targeting verification of fluorescently labeled polypeptide based on polypeptide Peptide_2 of Example 1:

[0099] ① Preparation of fluorescent probe: couple polypeptide with 5-TAMRA by chemical method, and purify by high performance liquid chromatography to obtain purified 5-TAMRA-short peptide probe.

[0100] ②Cell preparation: Take human lymphatic endothelial cells (HLEC) in good growth condition, digest, centrifuge and resuspend in complete culture medium, inoculate in confocal culture dish at appropriate density, and incubate in 37 ℃, 5% CO2 incubator until the cell density reaches 70-80%.

[0101] ③Probe incubation: discard the old culture medium, add fresh complete culture medium containing 5-TAMRA-short peptide probe (working concentration: 20 ng / mL), and incubate at room temperature for 20 minutes.

[0102] ④Fluorescence microscope observation: after incubation, gently wash the cells with pre-cooled PBS buffer for 3 times to remove unbound probes. Then, observe under fluorescence microscope and collect images.

[0103] As shown in Figure 12 , the red fluorescence signal of 5-TAMRA highly overlaps with the cell membrane of HLEC on HLEC, proving that the polypeptide precisely binds to VEGFR3 on the cell surface, which helps to prove the specific targeting ability of the polypeptide to VEGFR3.

[0104] Promoting effect of polypeptide Peptide_2 in example 1 on wound healing in a mouse skin defect model:

[0105] Animal model: C57 mice, full-thickness skin defects (diameter: 8 mm) were made on the back.

[0106] Grouping and administration: randomly divided into polypeptide treatment group (polypeptide-loaded hydrogel), blank control group (only blank hydrogel) and positive control group (VEGF-C-loaded hydrogel with the same concentration), 10 mice in each group.

[0107] As shown in Figure 13 , the healing evaluation was carried out:

[0108] Macroscopic observation: take photos on days 0, 3, 6 and 12. ImageJ software was used to analyze the wound area. The results showed that the healing rate of the polypeptide treatment group was significantly higher than that of the blank control group at each time point, and better than that of the positive control group (Fig. Figure 13 A-C).

[0109] Histological analysis: take samples on days 7 and 12, paraffin-embedded and H&E stained. The results showed that the granulation tissue of the polypeptide treatment group was thicker and denser, the new capillary blood vessels and fibroblasts were more abundant, the inflammatory cell infiltration was less, and the epithelial regeneration was more complete (Fig. Figure 13 D).

[0110] Based on the polypeptide Peptide_2 in example 1, mouse skin tissue blood vessels and lymphatic vessels were immunofluorescently stained:

[0111] ① Sample preparation: Take the 12th day mouse dorsal wound healing skin tissue, fixed with 4% PFA for 4 hours, CPT dehydrated for 24 hours, then embedded with embedding medium, and then frozen at-20℃.

[0112] ② Section: After thawing the sample, freeze sectioning was performed with a thickness of 100 μm.

[0113] ③ Staining: After the section was warmed at room temperature, it was hydrated with PBS buffer, permeabilized with 0.3% Triton X-100 for 10 minutes, and blocked in 5% donkey serum for 30 minutes.

[0114] ④ Primary antibody incubation: Add primary antibody diluent (anti-CD31, LYVE1, etc.) and incubate at room temperature for 4 hours. Wash with PBS buffer for 7-8 times.

[0115] ⑤ Secondary antibody incubation and mounting: Add fluorescent secondary antibody and DAPI and incubate at room temperature in the dark for 90 minutes. After washing with PBS buffer for 7-8 times, mount with antifade mounting medium.

[0116] ⑥ Image acquisition and analysis: Observe the section under a confocal microscope, collect images and analyze.

[0117] As shown in Figure 14 , clear CD31, α-SMA, Emcn positive vascular network and LYVE1, Prox1, CD31 positive lymphatic vessel structure can be seen in the polypeptide treatment group, and the number of blood vessels and lymphatic vessels is more than that of the blank control group and the VEGF-C positive control group.

[0118] The above specific embodiments further illustrate the purpose, technical solutions and beneficial effects of the present application. It should be understood that the above description is only a specific embodiment of the present application and is not intended to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A VEGFR3 receptor-specific binding peptide or a pharmaceutically acceptable salt thereof, characterized in that: The amino acid sequence of the VEGFR3 receptor-specific binding peptide is shown in SEQ ID NO.

1.

2. A coupling agent, characterized in that: The conjugate is formed by conjugating the VEGFR3 receptor-specific binding peptide of claim 1 or a pharmaceutically acceptable salt thereof with a labeling agent, wherein the labeling agent is a fluorescent group.

3. A composition, characterized in that: The composition contains the VEGFR3 receptor-specific binding peptide of claim 1 or a pharmaceutically acceptable salt thereof.

4. The composition according to claim 3, characterized in that: It is a composition of the VEGFR3 receptor-specific binding peptide of claim 1 or a pharmaceutically acceptable salt thereof with a carrier, wherein the carrier comprises any one of hydrogels, sprays, and biological dressings.

5. The application of the conjugate as described in claim 2 in the preparation of VEGFR3 targeting agents and VEGFR3 imaging agents.

6. The use of the composition of claim 3 in the preparation of VEGFR3 targeting agents and VEGFR3 imaging agents.

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