VEGFR3 receptor specific binding peptide and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a VEGFR3 receptor specific binding peptide and application thereof, and the amino acid sequence of the VEGFR3 receptor specific binding peptide is as shown in SEQ ID NO.1. The VEGFR3 receptor specific binding peptide can be specifically bound with a VEGFR3 receptor on a cell membrane, so that a downstream signal channel is activated, migration and proliferation of lymphatic endothelial cells are stimulated, a new lymphatic network is formed, the permeability of lymphatic vessels is increased, convenience is provided for flowing of lymph fluid, and the function of the lymphatic vessels is further promoted. The VEGFR3 receptor specific binding peptide not only has obvious effects of promoting 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 promote proliferation and migration of human umbilical vein endothelial cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a VEGFR3 receptor-specific binding peptide and its application. This 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 equivalent function of vascular endothelial cells (HUVECs). Background Technology

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

[0003] The extracellular domains of VEGFRs consist of seven Ig homologous domains, of which domains 1-3 (D1-3) are responsible for ligand binding. The crystal structure of VEGF-C is an antiparallel homodimer with an antiparallel four-stranded β-chain structure, three linker loops (L1-L3), and an extended N-terminal α-helix. The binding sites of VEGF-C and VEGFR3 exhibit high structural specificity, with the α-helix forming the most stable structure at the binding interface.

[0004] For a long time, the research and development of drugs that promote lymphogenesis has lagged behind. The number of existing clinical drugs, such as 9-cis-retinoic acid (9-cisRA) and simvastatin, is limited, while the preparation cost of recombinant VEGF-C protein is high. It is worth noting that current therapeutic agents of this type generally suffer from common problems such as low pharmacoeconomic efficiency, poor target selectivity, and a high incidence of adverse reactions.

[0005] Against this backdrop, if a peptide can specifically target and bind to the VEGFR3 receptor, activating the downstream VEGF signaling pathway to promote lymphangiogenesis, it is expected to show development potential as a novel non-natural peptide-like drug. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, the first objective of this invention is to provide a novel, structurally stable, and highly affinity-targeting VEGFR3 receptor-specific binding peptide. This peptide can specifically bind to the VEGFR3 receptor on the cell membrane, thereby activating downstream signaling pathways, stimulating the migration and proliferation of lymphatic endothelial cells, forming new lymphatic vessel networks, and increasing lymphatic vessel permeability to facilitate lymph flow, further promoting lymphatic vessel function. This VEGFR3 receptor-specific binding peptide not only significantly promotes the proliferation and migration of human lymphatic endothelial cells but also exhibits good biocompatibility and bioactivity. Furthermore, this 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 this invention has broad prospects for promoting vascularization in tissue engineering and regenerative medicine, contributing not only to basic scientific research but also providing new ideas and methods for clinical treatment.

[0007] The second objective of this invention is to apply the aforementioned VEGFR3 receptor-specific binding peptide in the preparation of drugs that promote lymphangiogenesis, angiogenesis, and / or tissue repair. This innovative strategy not only provides new ideas for the innovation of lymphangiogenesis-promoting treatment strategies, but also serves as an effective supplement to existing therapies, providing optimized solutions for clinical intervention of diseases related to imbalances in lymphangiogenesis regulation.

[0008] The third objective of this invention is to apply the above-mentioned VEGFR3 receptor-specific binding peptide in the preparation of molecular probes targeting lymphatic endothelial cells.

[0009] To achieve the above objectives, the present invention employs a technical solution consisting of the following technical measures.

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

[0011] It should be noted that in existing technical literature, the known synthetic binding peptide sequences for the VEGFR3 receptor are typically over 200 units in length, exhibiting significant structural redundancy. Furthermore, the academic community lacks a unified definition and systematic research on the precise active core domain in the interaction between VEGF-C and VEGFR3. Although the amino acid sequence shown in SEQ ID NO.1 of this invention exists within the aforementioned synthetic binding peptide sequence fragment, it is important to emphasize that this invention is the first to provide a precise functional analysis of the VEGF-C-VEGFR3 interaction interface, clearly defining the α-helix region that bears the core biological function and depends on a specific spatial conformation. Simulation calculations revealed that this natural helical structure cannot exist stably independently. The design of the amino acid sequence shown in SEQ ID NO.1 optimizes and reconstructs the structure of this core functional domain, ensuring high stability during independent peptide formation while fully preserving its active conformation, and successfully synthesizing a stable powdered polypeptide. In vitro and in vivo functional experiments confirm that its biological effects are comparable to or better than those of VEGF-C at the same concentration. Therefore, this invention not only overcomes the limitations of existing knowledge, but also achieves substantial innovation in key functional domains, significantly reducing the difficulty of synthesis and production costs while ensuring optimal activity.

[0012] In this document, the pharmaceutically acceptable salt is a pharmaceutically acceptable salt of a polypeptide according to the amino acid sequence shown in SEQ ID NO.1, including, but not limited to, salt formation by acidification and salt formation by alkaliification. The term "pharmaceutically acceptable salt" as used in this invention refers to a salt that retains the VEGFR3 binding activity of the polypeptide according to the amino acid sequence shown in SEQ ID NO.1 and is suitable for use in humans or animals. Methods for preparing salts of peptides with the amino acid sequence shown in SEQ ID NO.1 are known in the art and generally involve mixing the peptide with a pharmaceutically acceptable acid or base. Examples of pharmaceutically acceptable acids and bases include organic and inorganic acids that form ammonium salts with the free amino group of the peptide, 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 thiocyanate; and bases that form carboxyl salts with the free carboxyl group of the peptide, such as ethylamine, methylamine, dimethylamine, triethylamine, isopropylamine, diisopropylamine, and other monoalkylamines, dialkylamines, trialkylamines, and arylamines.

[0013] In one of the technical solutions, the present invention 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 invention provides a composition containing the above-described VEGFR3 receptor-specific binding peptide or a pharmaceutically acceptable salt thereof, or the above-described fusion protein. The composition comprises a load composition wherein the VEGFR3 receptor-specific binding peptide and the load composition are covalently or non-covalently associated with each other. Optionally, the load composition comprises a therapeutic agent, a detectable agent, a carrier, a surface molecule, or a combination thereof. Preferably, the therapeutic agent is an antibacterial agent, an anti-inflammatory agent, an immunostimulant, an immunosuppressant, or an angiogenic agent. Preferably, the detectable agent is a labeling agent, a contrast agent, or an imaging agent. Optionally, the VEGFR3 receptor-specific binding peptide is conjugated to the load composition. Preferably, one or more of the conjugated peptides are indirectly conjugated to the load composition via a linker. Optionally, the load composition further comprises multiple linkers. Preferably, at least one of the linkers comprises polyethylene glycol or maleimide conjugation, amino-functionalized dextran chemistry conjugation, and components can be directly or indirectly conjugated to the surface molecule or covalently associated with each other via any functional group, such as amine, carbonyl, carboxyl, aldehyde, or alcohol.

[0015] In one preferred embodiment, a composition is formed by conjugating the aforementioned VEGFR3 receptor-specific binding peptide or a pharmaceutically acceptable salt thereof with a labeling agent, wherein the labeling agent includes, but is not limited to, fluorescent groups (e.g., FITC, Cy5), radionuclides (e.g.,... 99m Tc, 68 Ga).

[0016] In one preferred embodiment, the present invention is a combination of the aforementioned VEGFR3 receptor-specific binding peptide or a pharmaceutically acceptable salt thereof with a carrier, wherein the carrier is preferably a hydrogel, a spray, or a biological dressing.

[0017] In another aspect, the present invention provides a drug delivery system containing the above-mentioned VEGFR3 receptor-specific binding peptide or a pharmaceutically acceptable salt thereof, or the above-mentioned fusion protein; the drug delivery system further includes one or more of the following: 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, and exosomes.

[0018] In another aspect, the present invention also relates to compositions of the above-mentioned VEGFR3 receptor-specific binding peptide and / or the above-mentioned fusion protein, comprising a linker, said linker comprising one or more of the following: protein, polypeptide, fatty acid and its derivatives, antibody, antibody fragment, cyclodextrin and its derivatives, small molecule drug, PEG and its derivatives, fatty acid, mesoporous inorganic material, linear polymer, crosslinked polymer, or branched copolymer.

[0019] In another aspect, the present invention also relates to the above-mentioned VEGFR3 receptor-specific binding peptide and its pharmaceutically acceptable salt, the above-mentioned fusion protein, providing its use in the preparation of medicaments that promote lymphangiogenesis, angiogenesis, and / or tissue repair. Meanwhile, in another aspect, the present invention also relates to compositions comprising the above-mentioned VEGFR3 receptor-specific binding peptide or its pharmaceutically acceptable salt, or the above-mentioned fusion protein, for use in the preparation of therapeutic agents, targeting agents, preventative agents, diagnostic agents, or imaging agents, or for use in the preparation of medicaments for diagnosis and / or treatment, image-guided surgery, drug delivery, and targeted delivery of imaging agents that promote lymphangiogenesis, angiogenesis, and / or tissue repair. The composition is preferably used in the preparation of medicaments that promote lymphangiogenesis, angiogenesis, and / or tissue repair.

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

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

[0022] 2. The VEGFR3 receptor-specific binding peptide provided by this invention, as confirmed by SPR analysis, has a binding constant KD value of 89.9 nM with VEGFR3, indicating good affinity.

[0023] 3. The VEGFR3 receptor-specific binding peptide provided by this invention has been demonstrated in vitro to not only specifically promote the proliferation, migration, and tube formation of lymphatic endothelial cells (LECs), but also effectively promote the equivalent function of vascular endothelial cells (HUVECs). This indicates that it can simultaneously stimulate angiogenesis and lymphangiogenesis, providing a more comprehensive regenerative microenvironment for tissue repair.

[0024] 4. The VEGFR3 receptor-specific binding peptide provided by this invention has been shown in a mouse dorsal skin defect model to significantly accelerate wound closure when applied topically. Histological (H&E staining) results show that it can improve granulation tissue formation and epithelial regeneration.

[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 9The figures show the in vitro cell CCK-8 experimental results of the polypeptide obtained in Example 1 of this invention. Figure A shows the cell model using human lymphatic endothelial cells (HLEC), and Figure B shows the cell model using human umbilical vein endothelial cells (HUVEC).

[0035] Figure 10 This is a summary graph of the in vitro cell scratch assay and Transwell assay results of the polypeptide obtained in Example 1 of this invention. Figures A and D use human lymphatic endothelial cells (HLECs) as the cell model, and respectively summarize the results of the scratch assay and Transwell assay. Figures E and H use human umbilical vein endothelial cells (HUVECs) as the cell model, and respectively summarize the results of the scratch assay and Transwell assay.

[0036] Figure 11 This is a summary figure of the in vitro cell tube formation experiment results of the polypeptide obtained in Example 1 of the present invention. Among them, Figure A shows the cell model using human lymphatic endothelial cells (HLEC), and Figure B shows the cell model using human umbilical vein endothelial cells (HUVEC).

[0037] Figure 12 This is an in vitro colocalization verification diagram of the polypeptide obtained in Example 1 of the present invention after fluorescent labeling.

[0038] Figure 13 The figures show the results of a mouse dorsal skin defect healing experiment using the polypeptide obtained in Example 1 of this invention. Figures A and B show comparisons of wound photographs and healing diagrams at different time points; Figure C shows a statistical analysis of the wound healing rate; and Figure D shows H&E staining images of the wound tissue on days 7 and 12.

[0039] Figure 14 This is a summary image of the immunofluorescence staining results of the polypeptide obtained in Example 1 of this invention on the blood vessels and lymphatic vessels of mouse skin tissue. The top image shows a representative fluorescence image of blood vessels stained with immunofluorescence on day 12 of the healed skin wound on the back of a mouse, and the bottom image shows a representative fluorescence image of lymphatic vessels stained with immunofluorescence on day 12 of the healed skin wound on the back of a mouse. Detailed Implementation

[0040] To further understand the present invention, preferred embodiments are described below with reference to examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims. Those skilled in the art can refer to the content of this document to appropriately improve the process parameters. In particular, it should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention. Although it is believed that those skilled in the art will fully understand the following terms, the following definitions are set forth to help illustrate the subject matter disclosed in the present invention.

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

[0042] It should be noted that in existing technical literature, the known synthetic binding peptide sequences for the VEGFR3 receptor are typically over 200 units in length, exhibiting significant structural redundancy. Furthermore, the academic community lacks a unified definition and systematic research on the precise active core domain in the interaction between VEGF-C and VEGFR3. Although the amino acid sequence shown in SEQ ID NO.1 of this invention exists within the aforementioned synthetic binding peptide sequence fragment, it is important to emphasize that this invention is the first to provide a precise functional analysis of the VEGF-C-VEGFR3 interaction interface, clearly defining the α-helix region that bears the core biological function and depends on a specific spatial conformation. Simulation calculations revealed that this natural helical structure cannot exist stably independently. The design of the amino acid sequence shown in SEQ ID NO.1 optimizes and reconstructs the structure of this core functional domain, ensuring high stability during independent peptide formation while fully preserving its active conformation, and successfully synthesizing a stable powdered polypeptide. In vitro and in vivo functional experiments confirm that its biological effects are comparable to or better than those of VEGF-C at the same concentration. Therefore, this invention not only overcomes the limitations of existing knowledge, but also achieves substantial innovation in key functional domains, significantly reducing the difficulty of synthesis and production costs while ensuring optimal activity.

[0043] In this document, the pharmaceutically acceptable salt is a pharmaceutically acceptable salt of a polypeptide according to the amino acid sequence shown in SEQ ID NO.1, including, but not limited to, salt formation by acidification and salt formation by alkaliification. The term "pharmaceutically acceptable salt" as used in this invention refers to a salt that retains the VEGFR3 binding activity of the polypeptide according to the amino acid sequence shown in SEQ ID NO.1 and is suitable for use in humans or animals. Methods for preparing salts of the polypeptide with the amino acid sequence shown in 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 that form ammonium salts with the free amino group 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 thiocyanate in one embodiment; and bases that form carboxyl salts with the free carboxyl group of the peptide, such as ethylamine, methylamine, dimethylamine, triethylamine, isopropylamine, diisopropylamine, and other monoalkylamines, dialkylamines, trialkylamines, and arylamines.

[0044] In one embodiment, the present invention provides a fusion protein containing the above-mentioned VEGFR3 receptor-specific binding peptide or a pharmaceutically acceptable salt thereof.

[0045] In another aspect, the present invention provides a composition containing the above-described VEGFR3 receptor-specific binding peptide or a pharmaceutically acceptable salt thereof, or the above-described fusion protein. In one embodiment, the composition comprises a load composition wherein the VEGFR3 receptor-specific binding peptide and the load composition are covalently or non-covalently associated with each other. Optionally, the load composition comprises a therapeutic agent, a detectable agent, a carrier, a surface molecule, or a combination thereof. Preferably, the therapeutic agent is an antibacterial agent, an anti-inflammatory agent, an immunostimulant, an immunosuppressant, or an angiogenic agent. Preferably, the detectable agent is a labeling agent, a contrast agent, or an imaging agent. Optionally, the VEGFR3 receptor-specific binding peptide is conjugated to the load composition. Preferably, one or more of the conjugated peptides are indirectly conjugated to the load composition via a linker. Optionally, the load composition further comprises multiple linkers. Preferably, at least one of the linkers comprises polyethylene glycol or maleimide conjugation, amino-functionalized dextran chemistry for conjugation, and components can be directly or indirectly conjugated to the surface molecule or covalently associated with each other via any functional group, such as amine, carbonyl, carboxyl, aldehyde, or alcohol.

[0046] In one preferred embodiment, a composition is formed by conjugating the above-mentioned VEGFR3 receptor-specific binding peptide or a pharmaceutically acceptable salt thereof with a labeling agent, wherein the labeling agent includes, but is not limited to, fluorescent groups (e.g., FITC, Cy5), radionuclides (e.g., ... 99m Tc, 68 Ga).

[0047] In one preferred embodiment, it is a combination of the above-mentioned VEGFR3 receptor-specific binding peptide or a pharmaceutically acceptable salt thereof with a carrier, wherein the carrier is preferably a hydrogel, spray or bio-dressing.

[0048] In another aspect, the present invention provides a drug delivery system containing the above-mentioned VEGFR3 receptor-specific binding peptide or a pharmaceutically acceptable salt thereof, or the above-mentioned fusion protein; in one embodiment, the drug delivery system further includes one or more of the following: 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, and exosomes.

[0049] In another aspect, the present invention also relates to compositions of the above-mentioned VEGFR3 receptor-specific binding peptide and / or the above-mentioned fusion protein, comprising a linker, wherein in one embodiment, the linker comprises one or more of the following: protein, polypeptide, fatty acid and its derivatives, antibody, antibody fragment, cyclodextrin and its derivatives, small molecule drug, PEG and its derivatives, fatty acid, mesoporous inorganic material, linear polymer, crosslinked polymer, or branched copolymer.

[0050] In another aspect, the present invention also relates to the above-mentioned VEGFR3 receptor-specific binding peptide and its pharmaceutically acceptable salt, the above-mentioned fusion protein, providing its use in the preparation of medicaments that promote lymphangiogenesis, angiogenesis, and / or tissue repair. Meanwhile, in another aspect, the present invention also relates to compositions comprising the above-mentioned VEGFR3 receptor-specific binding peptide or its pharmaceutically acceptable salt, or the above-mentioned fusion protein, for use in the preparation of therapeutic agents, targeting agents, preventative agents, diagnostic agents, or imaging agents, or for use in the preparation of medicaments for diagnosis and / or treatment, image-guided surgery, drug delivery, and targeted delivery of imaging agents that promote lymphangiogenesis, angiogenesis, and / or tissue repair. The composition is preferably used in the preparation of medicaments that promote lymphangiogenesis, angiogenesis, and / or tissue repair.

[0051] The present application will be further explained in detail below with reference to embodiments. However, those skilled in the art should understand that these embodiments 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, the secondary structure analysis results of the peptides in Example 1 and Comparative Examples 1-3 show that the secondary structures of Peptide_1, Peptide_2 and Peptide_3 remained consistent during the simulation process, mainly consisting of α-helical structures. Among them, Peptide_2 had the largest proportion of α-helices in its secondary structure and was more stable.

[0064] like Figure 4 As shown, based on the docking of peptide Peptide_2 with the original receptor molecule in Example 1 and the analysis of the results, the interaction of the complex after docking of peptide Peptide_2 with the original receptor molecule was analyzed. It was found that the structure of the docked complex differed significantly from the conformation of the original protein, and its interaction mode with the original protein also changed considerably. Therefore, it can be inferred that the peptide's protein-protein binding mechanism is inconsistent, but the peptide can also bind well to protein Rec. The C / N terminus of the peptide binds to the protein via hydrogen bonds, while the middle peptide binds to the protein via hydrophobic interactions, thus forming a relatively stable peptide-protein complex. Therefore, peptide Peptide_2 was ultimately determined to be the preferred choice.

[0065] like Figure 5 As shown, mass spectrometry analysis of peptide Peptide_2 from Example 1 and high performance liquid chromatography showed that the molecular weight of the synthesized product was consistent with the theoretical value (2077.34).

[0066] like Figure 6 As shown, circular dichroism analysis was performed on peptide Peptide_2 from Example 1. Scanning in the far ultraviolet region showed that the peptide had a negative peak at 190-210 nm, indicating that it has an α-helical secondary structure in aqueous solution.

[0067] like Figure 7 As shown, nuclear magnetic resonance analysis was performed on peptide Peptide_2 from Example 1. The NMR spectrum showed good signal dispersion and no impurity peaks, indicating that the synthesized product has high purity and can be further used for solution structure analysis.

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

[0069] ① The VEGFR3-Fc fusion protein was immobilized on a CM5 chip using a Biacore series SPR instrument. Different concentrations of peptide 2 were then passed through the chip surface.

[0070] ②Result: such as Figure 8As shown, the sensor spectrum displays a typical concentration-dependent binding curve. Fitting with a 1:1 Langmuir binding model yielded binding constants KD = 89.9 nM, kon = 4547 / Ms, and koff = 0.0409 / s. This data demonstrates that the peptide of this invention has a high affinity for VEGFR3.

[0071] Based on the experiment of the dual promoting effect of peptide_2 on the function of vascular and lymphatic endothelial cells in Example 1, the cell models used were human lymphatic endothelial cells (HLEC) and human umbilical vein endothelial cells (HUVEC):

[0072] (1) CCK-8 experiment:

[0073] ① Take cells in good growth condition, digest them, centrifuge them, resuspend them in ECM medium, and dilute them to 7.5 × 10⁻⁶. 4 Cells / mL, 100 μL per well was seeded into a 96-well cell culture plate and cultured at 37 ℃, 5% CO2 for 24 h.

[0074] ② Prepare concentration gradient solutions of peptide_2.

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

[0076] The blank control group was replenished with complete culture medium, the peptide treatment group was replenished with complete culture medium containing different concentrations of peptide_2 solution (the concentration of peptide_2 solution used in the human umbilical vein endothelial cell experiment was 20 ng / mL), and the positive control group was replenished with complete culture medium containing VEGF-C (the concentration of VEGF-C solution was 20 ng / mL). The culture was carried out at 37 ℃ and 5% CO2 for 72 h.

[0077] ④ The absorbance of each well was measured at 24h, 48h and 72h after drug addition. The complete culture medium was discarded, and 100 μL of the detection reagent (CCK-8 reagent: culture medium = 1:9) was added to each well. The wells were incubated for 2 h at 37 ℃ and 5% CO2. The OD value at 450 nm was measured using a microplate reader.

[0078] like Figure 9 As shown, compared with the control group, the peptide of the present invention can significantly and in a concentration-dependent manner promote the proliferation of HLEC and HUVEC (p<0.05), which indicates that it has an activating effect on both VEGFR3 and VEGFR2 signaling pathways.

[0079] (2) Scratch test:

[0080] ① Cell Culture: Draw two longitudinal lines on the back of each well of a 6-well plate as markers. Set up a control group and a peptide treatment group. Take cells in good growth condition, digest, centrifuge, resuspend in complete culture medium, and dilute to 5×10⁻⁶. 6 Cells / mL, 2 mL of cell suspension per well, to ensure that the cell density of each group is the same, and that 90-100% confluence can be achieved after 48 h of culture.

[0081] ② Scratch assay: After 48 h of cell culture, a 200 μL pipette tip was used to vertically and closely scrape across the cell layer to form a scratch. The cells were washed three times with PBS buffer to remove the scratched cells. 2 mL of low serum (HLEC) or serum-free (HUVEC) medium was added to each of the six wells. The peptide treatment group was given complete medium containing different concentrations (10 ng / mL, 20 ng / mL, 30 ng / mL) of peptides. The blank control group was given complete medium, and the positive control group was given complete medium containing VEGF-C.

[0082] ③ Incubate in an incubator at 37 ℃ and 5% CO2, and take microscopic photographs at fixed positions after 0 h, 6 h, and 12 h.

[0083] ④ Measure the scratch area using ImageJ: Process the scratch images (0 h, 6 h, 12 h) using software to obtain the cell migration rate, such as... Figure 10 As shown in AB and EF.

[0084] (3) Transwell experiment:

[0085] ① Cell preparation: Take cells in good growth condition, digest and centrifuge them, resuspend them in serum-free medium, and adjust the cell density to 1×10⁶. 6 per mL.

[0086] ② Cell plate formation: 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 peptides to the lower chamber. The blank control group is added with complete culture medium, and the positive control group is added with complete culture medium containing VEGF-C.

[0087] ③ Cell incubation: Place the culture plate in a 37 ℃, 5% CO2 incubator for 24 hours to allow the cells to migrate.

[0088] ④ Fixation and staining: Remove the chamber and gently wipe away any unmigrated cells from 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: Take photos of multiple randomly selected fields of view under a microscope, and count the cells that have penetrated the membrane, such as... Figure 10 As shown in CD and GH.

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

[0091] (4) Tube forming experiment:

[0092] ①Preparation of Matrigel matrix: Remove Matrigel from -20 ℃ and thaw overnight in a 4 ℃ freezer. Pre-cool the 24-well container, pipettes, and 200 μL pipette tips in a 4 ℃ freezer.

[0093] ② Plating: Spread Matrigel evenly at a rate of 20 μL / well on the bottom of the culture plate, ensuring it does not stick to the sidewalls. Place the culture plate with Matrigel in a 37 ℃ incubator and let it stand for 30-60 minutes to allow the Matrigel to gel.

[0094] ③ Take cells in good growth condition, digest them, centrifuge them, resuspend them in complete culture medium, and dilute them to 1×10⁻⁶. 6 Cells / mL: Add 200 μL of cell suspension to Matrigel plates and gently shake the plate to distribute the cells evenly. The peptide treatment group received complete culture medium containing 20 ng / mL peptides; the blank control group received complete culture medium; and the positive control group received complete culture medium containing 20 ng / mL VEGF-C.

[0095] ④ Incubate in an incubator at 37℃ and 5% CO2, and take microscopic pictures after 8 hours.

[0096] ⑤ Use ImageJ to quantitatively analyze the length, number of branch points, and number of grids of the tubular structure: process the images of the tubular structure using the software.

[0097] like Figure 11 As shown, on Matrigel, the HLECs and HUVECs of the peptide-treated group formed a more complete and branched tubular network structure.

[0098] In vitro targeting validation of fluorescently labeled peptides based on peptide Peptide_2 from Example 1:

[0099] ① Preparation of fluorescent probe: The peptide was chemically coupled with 5-TAMRA and purified by high performance liquid chromatography to obtain a purified 5-TAMRA-short peptide probe.

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

[0101] ③ Probe 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 in the dark for 20 minutes.

[0102] ④ Fluorescence microscopy observation: After incubation, the cells were gently washed three times with pre-cooled PBS buffer to remove unbound probes. Subsequently, the cells were observed and images were acquired under a fluorescence microscope.

[0103] like Figure 12 As shown, the red fluorescence signal of 5-TAMRA on HLECs highly overlaps with the cell membrane of HLECs, proving that the peptide binds precisely to VEGFR3 on the cell surface, further demonstrating the peptide's specific targeting ability to VEGFR3.

[0104] Based on the healing-promoting effect of peptide Peptide_2 in a mouse skin defect model (Example 1):

[0105] Animal model: C57 mouse, full-thickness skin defect created on the back (diameter: 8mm).

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

[0107] like Figure 13 As shown, a healing assessment was performed:

[0108] Macroscopic observation: Photos were taken on days 0, 3, 6, and 12. ImageJ software was used to analyze the wound area. Results showed that the healing rate of the peptide-treated group was significantly higher than that of the blank control group at all time points, and superior to that of the positive control group. Figure 13 AC (Chinese)

[0109] Histological analysis: Samples were taken on days 7 and 12 for paraffin embedding and H&E staining. Results showed that the peptide-treated group had thicker, denser granulation tissue, more abundant new capillaries and fibroblasts, less inflammatory cell infiltration, and more complete epithelial regeneration. Figure 13 (D).

[0110] Immunofluorescence staining of blood vessels and lymphatic vessels in mouse skin tissue based on peptide_2 from Example 1:

[0111] ① Sample preparation: Skin tissue from the wound healing site on the back of mice on day 12 was taken, fixed with 4% PFA for 4 hours, dehydrated with CPT for 24 hours, embedded in embedding solution, and then frozen at -20 ℃.

[0112] ② Sectioning: After the sample is thawed, it is frozen into sections with a thickness of 100 μm.

[0113] ③ Staining: After the sections were warmed to room temperature, they were hydrated with PBS buffer, permeabilized with 0.3% Triton X-100 for 10 minutes, and then blocked in 5% donkey serum for 30 minutes.

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

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

[0116] ⑥ Image acquisition and analysis: The slices were observed under a confocal microscope and images were acquired and analyzed.

[0117] like Figure 14 As shown, the tissues of the polypeptide-treated group showed clear CD31, α-SMA, and Emcn positive vascular networks and LYVE1, Prox1, and CD31 positive lymphatic vessel structures, with more blood vessels and lymphatic vessels than the blank control group and the VEGF-C positive control group.

[0118] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

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

3. A composition, characterized in that: The composition contains the VEGFR3 receptor-specific binding peptide of claim 1 or a pharmaceutically acceptable salt thereof, or the fusion protein of claim 2.

4. The composition according to claim 3, characterized in that: It is a composition 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 includes at least one of a fluorescent group and a radionuclide.

5. 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.

6. The use of the VEGFR3 receptor-specific binding peptide of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for promoting lymphangiogenesis, angiogenesis and / or tissue repair.

7. The use of the fusion protein as described in claim 2 in the preparation of a medicament that promotes lymphangiogenesis, angiogenesis and / or tissue repair.

8. Use of the composition of claim 3 in the preparation of therapeutic agents, targeting agents, preventive agents, diagnostic agents or imaging agents.

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