Self-assembled polypeptide for inhibiting angiogenesis as well as preparation method and application of self-assembled polypeptide

Abstract

The invention relates to a self-assembled polypeptide for inhibiting angiogenesis as well as a preparation method and application of the self-assembled polypeptide. The self-assembled polypeptide comprises a hydrophobic unit, an assembly unit, a polypeptide specifically targeting a Tie2 receptor and a polypeptide specifically targeting a VEGFR2 (vascular endothelial growth factor receptor 2) receptor. The self-assembled polypeptide provided by the invention simultaneously targets a Tie2 receptor and a VEGFR2 receptor, specifically targets vascular endothelial cells, forms nano-particles through self-assembly under hydrophilic and hydrophobic effects, and after interaction of the receptor and the ligand, the nano-particles are converted into nano-fibers to prevent VEGF from being combined with VEGFR2 and prevent Ang2 from being combined with Tie2, so that rapid enrichment in a neovascularization focus area is realized, and the self-assembled polypeptide has a good application prospect. And endothelial cell migration is inhibited, so that blood vessel generation is inhibited.

Description

Technical Field

[0001] The present invention relates to the field of medicine and biology, and in particular to a self-assembling polypeptide for inhibiting angiogenesis, and a preparation method and application thereof. Background Art

[0002] When cancer develops to the late stage, there are abundant blood vessels in the tumor tissue. These blood vessels play an important role in the life cycle of the tumor, providing oxygen and nutrients to the tumor, thereby promoting the proliferation of tumor cells and becoming a channel for tumor cell metastasis. Selecting anti-tumor angiogenesis therapy can not only inhibit the formation of new blood vessels, but also normalize the existing blood vessels of the tumor, so that the efficacy of chemotherapy drugs is enhanced or the immunosuppressive microenvironment is relieved, and the infiltration of immune killer cells is enhanced, thereby achieving the purpose of treating tumors. New blood vessels play an important role in the development of fundus diseases, such as choroidal neovascularization (CNV) and oxygen-induced retinopathy (OIR), leading to visual impairment. New blood vessels are fragile, prone to bleeding and leakage, causing retinal edema and fibrosis, and ultimately leading to vision loss. Anti-angiogenesis therapy can reduce the formation and leakage of new blood vessels, effectively slow down the progression of the disease, and improve vision, which is of great significance for the treatment of fundus neovascular diseases.

[0003] Self-assembly refers to the process by which molecules spontaneously form an ordered and stable structure under non-covalent interactions. As one of the common molecular self-assembly phenomena in nature, the study of self-assembly behavior is of great significance for understanding the molecular interactions between biomolecules and preparing new functional materials. As a molecule with biological homology, peptides are ideal self-assembly components with superior biological and chemical properties. Through reasonable molecular design and diversified peptide synthesis methods, a variety of peptides with different structures and functions can be generated, providing sufficient sequence analogs for screening and optimizing peptides. Self-assembling peptides can be widely used in the field of biomedicine, such as drug delivery, tumor treatment, antibacterial treatment, etc.

[0004] CN114099701A discloses a self-assembling composite polypeptide and a preparation method and medicine thereof, wherein the self-assembling composite polypeptide comprises a targeting recognition unit, a self-assembling unit and a functional unit connected in sequence. The self-assembling unit is a polypeptide fragment including a phenylalanine dipeptide, and the phenylalanine polypeptide contains multiple hydrophobic benzene ring structures, which can be β-folded to form supramolecular nano-micelles through π-π superposition and intermolecular hydrogen bonding.

[0005] In summary, providing a novel self-assembling polypeptide and applying it in the field of biomedicine has become one of the problems to be solved urgently in this field. Summary of the invention

[0006] To solve the above technical problems, the present invention provides a self-assembling polypeptide for inhibiting angiogenesis, and a preparation method and application thereof. The self-assembling polypeptide can target VEGFR2 and Tie2 related to angiogenesis, inhibit ligand receptor binding, and achieve the inhibition of tumor and fundus angiogenesis.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a self-assembling polypeptide for inhibiting angiogenesis, wherein the self-assembling polypeptide comprises a hydrophobic unit, an assembly unit, a polypeptide specifically targeting Tie2 (angiopoietin) receptor, and a polypeptide specifically targeting VEGFR2 (vascular endothelial growth factor receptor 2) receptor.

[0009] The self-assembling polypeptide provided by the present invention simultaneously targets Tie2 receptors and VEGFR2 receptors, specifically targets vascular endothelial cells, self-assembles to form nanoparticles under the hydrophilic and hydrophobic effects, and after the ligand-receptor interaction, the nanoparticles are transformed into nanofibers, thereby hindering VEGF from binding to VEGFR2 and Ang2 from binding to Tie2, achieving rapid enrichment in the area of ​​new blood vessel lesions, inhibiting endothelial cell migration, and further inhibiting angiogenesis.

[0010] Preferably, the hydrophobic unit comprises any one of saturated fatty acids having 8 to 22 carbon atoms (an integer from 8 to 22, such as 8, 10, 12, 14, 16, 18, 20 or 22).

[0011] Preferably, the hydrophobic unit is palmitic acid.

[0012] Preferably, the amino acid sequence of the assembly unit includes the sequence shown in SEQ ID NO.1.

[0013] SEQ ID NO.1: KLVFF.

[0014] In the present invention, the role of the assembly unit is that when the polypeptide targets the target protein, the hydrogen bonds between the FF amino acid molecules in the structure drive the self-assembly to form nanofibers. Therefore, the connection mode of the amino acids in the assembly unit does not affect the self-assembly effect of the polypeptide. The assembly unit can be KLVFF from the N-terminus to the C-terminus, or the assembly unit can be inverted as a whole, and FFVLK from the N-terminus to the C-terminus.

[0015] Preferably, the leucine in the assembly unit is D-leucine or L-leucine.

[0016] Preferably, the amino acid sequence of the polypeptide specifically targeting Tie2 receptor includes the sequence shown in SEQ ID NO.2.

[0017] SEQ ID NO.2: SAAMLLN.

[0018] Preferably, the amino acid sequence of the polypeptide specifically targeting the VEGFR2 receptor includes the sequence shown in SEQ ID NO.3.

[0019] SEQ ID NO.3: ATWLPPR.

[0020] Preferably, the leucine in the polypeptide specifically targeting the VEGFR2 receptor is D-leucine or L-leucine.

[0021] Preferably, the structure of the self-assembling polypeptide is as shown in Formula I:

[0022]

[0023] In the self-assembling polypeptide of the present invention as shown in formula I, the hydrophobic unit is palmitic acid, the amino acid sequence of the assembly unit is shown in SEQ ID NO.1, the amino acid sequence of the polypeptide specifically targeting the Tie2 receptor is shown in SEQ ID NO.2, and the amino acid sequence of the polypeptide specifically targeting the VEGFR2 receptor is shown in SEQ ID NO.3.

[0024] Preferably, in the self-assembling polypeptide, the 5th position of the N-terminus of the polypeptide specifically targeting the Tie2 receptor is D-leucine, and the 4th position of the N-terminus of the polypeptide specifically targeting the VEGFR2 receptor is D-leucine. The self-assembling polypeptide is hereinafter referred to as D PAK(N)A.

[0025] Preferably, in the self-assembling polypeptide, all leucines are L-leucines. Hereinafter, this self-assembling polypeptide is abbreviated as PAK(N)A.

[0026] Preferably, the preparation method of the self-assembling polypeptide is a polypeptide solid phase synthesis method.

[0027] In a second aspect, the present invention provides a polypeptide self-assembling nanoparticle, wherein the raw materials for preparing the polypeptide self-assembling nanoparticle include the self-assembling polypeptide described in the first aspect.

[0028] Preferably, the method for preparing the polypeptide self-assembling nanoparticles comprises: mixing the self-assembling polypeptide and a DMSO solution.

[0029] Preferably, the concentration of the self-assembling polypeptide after mixing is 3×10 -5 ~5×10 -5 M (for example, it can be 3×10 - 5 M, 3.5×10 -5 M, 3.8×10 -5 M, 4×10-5 M, 4.2×10 -5 M, 4.5×10 -5 M or 5×10 -5 M, etc.).

[0030] Preferably, the volume ratio of DMSO to water in the DMSO solution is 1:(90-110).

[0031] The specific point values ​​of the above (90-110) can be selected from 90, 95, 98, 99, 100, 101, 102, 105 or 110, etc.

[0032] In a third aspect, the present invention provides a polypeptide self-assembling nanofiber, wherein the raw materials for preparing the polypeptide self-assembling nanoparticles include the self-assembling polypeptide described in the first aspect and a targeting protein of the self-assembling polypeptide.

[0033] The self-assembling polypeptide provided by the present invention can specifically bind to VEGFR2 protein or Tie2 protein, accelerate the transformation of nanoparticles into nanofibers, thereby inhibiting the binding of VEGF and Ang2 to their respective receptors, and further inhibiting endothelial cell migration and angiogenesis.

[0034] Preferably, the targeting protein of the self-assembling polypeptide includes VEGFR2 protein or Tie2 protein.

[0035] Preferably, the method for preparing the polypeptide self-assembling nanofibers comprises: mixing the self-assembling polypeptide, the targeting protein and a DMSO solution.

[0036] Preferably, the concentration of the self-assembling polypeptide after mixing is 3×10 -5 ~5×10 -5 M (for example, it can be 3×10 - 5 M, 3.5×10 -5 M, 3.8×10 -5 M, 4×10 -5 M, 4.2×10 -5 M, 4.5×10 -5 M or 5×10 -5 M, etc.).

[0037] Preferably, the concentration of the target protein after mixing is 3×10 -8 ~5×10 -8 M (for example, it can be 3×10 -8 M, 3.5×10 -8 M, 3.8×10 -8 M, 4×10 -8 M, 4.2×10 -8 M, 4.5×10 -8M or 5×10 -8 M, etc.).

[0038] Preferably, the volume ratio of DMSO to water in the DMSO solution is 1:(90-110).

[0039] The specific point values ​​of the above (90-110) can be selected from 90, 95, 98, 99, 100, 101, 102, 105 or 110, etc.

[0040] In a fourth aspect, the present invention provides the use of any one or a combination of at least two of the self-assembling polypeptide for inhibiting angiogenesis as described in the first aspect, the polypeptide self-assembling nanoparticles as described in the second aspect, or the polypeptide self-assembling nanofibers as described in the third aspect in the preparation of drugs for inhibiting endothelial cell migration, inhibiting vascular leakage and inhibiting angiogenesis.

[0041] In the fifth aspect, the present invention provides the use of any one or a combination of at least two of the self-assembling polypeptide for inhibiting angiogenesis as described in the first aspect, the polypeptide self-assembling nanoparticles as described in the second aspect, or the polypeptide self-assembling nanofibers as described in the third aspect in the preparation of drugs for inhibiting tumor proliferation and treating fundus neovascularization diseases.

[0042] Preferably, the treatment of fundus neovascularization diseases includes choroidal neovascularization and / or oxygen-induced retinopathy.

[0043] The self-assembling polypeptide provided by the present invention has biological activity in mice, and can be used for the treatment of tumors by inhibiting the formation of tumor angiogenesis and thus inhibiting the proliferation of tumors. D PAK(N) and PAK(N)A were prepared into eye drops and applied to CNV and OIR mice, showing the effects of inhibiting angiogenesis and vascular leakage. They can also be used to treat fundus neovascularization diseases.

[0044] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] Anti-tumor angiogenesis has become an important field in the treatment of cancer. In addition, anti-angiogenesis therapy is also a first-line treatment strategy for fundus diseases such as CNV and OIR. By inhibiting the formation of new blood vessels, cancer metastasis can be reduced and the survival rate of patients can be improved or the vision of patients with eye diseases can be improved. With the continuous development and progress of science, people's research and treatment methods in the field of anti-angiogenesis are also constantly advancing. The anti-angiogenesis self-assembling polypeptide strategy proposed in the present invention will provide new ideas for future anti-tumor angiogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Schematic diagram of the structures of four self-assembling polypeptides in the present invention.

[0048] Figure 2 Schematic diagram of the self-assembly process of four self-assembling polypeptides in the present invention.

[0049] Figure 3 Spectra of the four self-assembling polypeptides in the present invention, wherein a is D CD spectrum of PAK(N)A without protein induction, Figure b is D The CD spectrum of PAK(N)A induced by Tie2 protein, Figure c is D Figure 1 is the CD spectrum of PAK(N)A under the induction of VEGFR2 protein, Figure 2 is the CD spectrum of PAK(N)A under the induction of Tie2 protein, Figure 3 is the CD spectrum of PAK(N)A under the induction of VEGFR2 protein, Figure 4 is the CD spectrum of PAK(N)A under the induction of Tie2 protein, Figure 5 is the CD spectrum of PAK(N)A under the induction of VEGFR2 protein, Figure 6 is the CD spectrum of PAFN under the induction of Tie2 protein, Figure 7 is the CD spectrum of PAFA under the induction of VEGFR2 protein, Figure 8 is the CD spectrum of PAFA under the induction of VEGFR2 protein.

[0050] Figure 4 The transmission electron microscopy images of the morphological transformation of the four self-assembling polypeptides in the present invention are shown in Figure a. D a is the transmission electron microscopy image of PAK(N)A, b is the transmission electron microscopy image of PAK(N)A, c is the transmission electron microscopy image of PAFN, and d is the transmission electron microscopy image of PAFA.

[0051] Figure 5 This is a fluorescence confocal microscope image of the four self-assembling polypeptides of the present invention after being labeled with the fluorescent molecule Cy5 and interacting with endothelial cells.

[0052] Figure 6 Figure 4 is a graph showing the results of four self-assembling polypeptides inhibiting endothelial cell migration in the present invention, wherein Figure a is a microscopic graph showing the scratch experiment for inhibiting endothelial cell migration, Figure b is a quantitative analysis result graph showing the scratch healing rate in Figure a, Figure c is a microscopic graph showing the Transwell chamber experiment for inhibiting endothelial cell migration, and Figure d is a quantitative analysis result graph showing the cell migration in Figure c.

[0053] Figure 7Figure 4 is a diagram showing the tumor inhibition effect of the self-assembling polypeptide of the present invention after treatment in mice, wherein Figure a is a schematic diagram of the construction of a mouse liver cancer model and the treatment process, Figure b is a curve diagram showing the change of tumor volume over time during the treatment process of mice, Figure c is a curve diagram showing the change of body weight during the treatment process of mice, Figure d is an immunofluorescence image of tumor tissue after anti-tumor angiogenesis treatment, the blue signal is DAPI, and the red signal is CD31, and Figure e is a quantitative analysis result of the number of new blood vessels in Figure d.

[0054] Figure 8 Figure 3 is a diagram showing the enrichment of the self-assembling polypeptide in the present invention in mice and in major organs and tumor tissues, wherein Figure a is a distribution diagram of Cy5-labeled polypeptides and Cy5-labeled bevacizumab in mice, Figure b is a quantitative analysis result diagram of the fluorescence intensity in tumor tissues in Figure a, Figure c is a distribution diagram of Cy5-labeled polypeptides and Cy5-labeled bevacizumab in various organs of mice, and Figure d is a quantitative analysis result diagram of the fluorescence intensity of tumor tissues and organs in Figure c.

[0055] Fig. 9 This is a biological projection electron micrograph of the self-assembling polypeptide of the present invention at a tumor tissue section.

[0056] Fig.10 HE staining sections of the heart, liver, spleen, lung and kidney tissues of mice after treatment with the self-assembling polypeptide of the present invention and bevacizumab, with a scale bar of 50 μm.

[0057] Fig.11 Figure 1 is a diagram showing the treatment of OIR mice by the self-assembling polypeptide eye drops of the present invention, wherein Figure a is a diagram showing the therapeutic effect of the self-assembling polypeptide on OIR, Figure b is a diagram showing the quantitative analysis results of the area of ​​the OIR neovascularization region in Figure a, and Figure c is a diagram showing the quantitative analysis results of the neovascularization cure rate in Figure a.

[0058] Fig.12 Figure 1 is a diagram showing the treatment of CNV mice by the self-assembling polypeptide eye drops of the present invention, wherein Figure a is a diagram showing the therapeutic effect of the self-assembling polypeptide on CNV, and Figure b is a diagram showing the quantitative analysis results of the area of ​​the new blood vessel region in Figure a.

[0059] Fig.13 Figure 1 is a diagram of vascular leakage after treatment with the self-assembling polypeptide eye drops of the present invention, wherein Figure a is a fundus fluorescein angiography of the CNV area after drug treatment, Figure b is a quantitative analysis result diagram of the area of ​​the vascular leakage region in Figure a, and Figure c is a quantitative analysis result diagram of the reduction ratio of the area of ​​the vascular leakage region in Figure a. DETAILED DESCRIPTION

[0060] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0061] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0062] Example 1

[0063] This embodiment provides a method for preparing a self-assembling polypeptide, which is prepared and synthesized using a standard polypeptide solid phase synthesis method (SPSS), comprising the following steps:

[0064] (1) Swelling resin: Weigh 400 mg of Fmoc-Arg-Wang resin into a peptide synthesis tube, add 8 mL of anhydrous DMF and shake for 6 h to swell the resin.

[0065] (2) Deprotection: Remove DMF, add DMF / DCM solvent to rinse the resin alternately three times, then add deprotection agent (DMF:DBU=98:2, v / v, add 5% anhydrous piperazine to completely dissolve it) to remove the Fmoc protecting group to ensure that the amino group is completely exposed, the time is 15 min.

[0066] (3) Detection: Remove the deprotection agent, rinse the resin three times with DMF / DCM solvents alternately, take a small amount of resin in ninhydrin detection reagent, heat it in a boiling water bath for 1 min and observe whether the resin changes color. If it turns blue-purple, it means that the deprotection is complete. If there is no color change, it means that the deprotection is unsuccessful and the second step needs to be repeated.

[0067] (4) Coupling: Weigh 10 times the amount of proline with Fmoc protection group and HBTU catalyst relative to the resin loading amount of amino acids, add 8 mL of coupling agent (DMF: N-methylmorpholine = 19:1, v / v), mix well, add to the peptide synthesis tube and shake for reaction for 1 h.

[0068] (5) Detection: Remove the coupling agent, add DMF / DCM solvent to rinse the resin three times alternately, take a small amount of resin and test it with ninhydrin detection reagent, heat it in a boiling water bath for 1 min to observe whether the resin changes color. If there is no color change, it means that the coupling is successful. If there is a color change, it means that the coupling is incomplete and the coupling needs to be repeated.

[0069] (6) Side chain synthesis: After the palmitic acid coupling is successful, add 8 mL of hydrazine hydrate deprotection agent (DMF: hydrazine hydrate = 50:1, v / v) to the peptide synthesis tube and react for 15 minutes to remove the Dde protecting group of the lysine side chain. After the time is up, rinse with DMF / DCM solvents alternately three times, and then use ninhydrin detection reagent to detect whether the resin changes color. If it changes color, the amino group on the lysine side chain is successfully exposed. If it does not change color, the deprotection operation needs to be repeated. This operation step is only for D PAK(N) and PAK(N)A.

[0070] (7) Contraction: When all amino acids in the peptide sequence are successfully coupled, remove the Fmoc protecting group with a deprotecting agent, and then rinse with DMF / DCM solvents alternately three times. After the resin turns blue with the ninhydrin detection reagent, add 8 mL of methanol solution to the peptide synthesis tube and soak for 10 minutes. After removing the methanol, transfer the resin to a clean serum bottle.

[0071] (8) Lysis: Add the prepared lysate (TFA:H2O:TIS=95:2.5:2.5, v / v) to the serum bottle containing the resin, stir for 2.5 h in an ice bath, filter, collect the lysate, and concentrate the lysate under a nitrogen atmosphere. Finally, add icy ether to precipitate the polypeptide, transfer the suspension to a centrifuge tube, centrifuge at 8000 r / min for 3 min, discard the supernatant, and repeat the above operation for two more washes. Dry the polypeptide in a vacuum oven at 37°C to obtain a white polypeptide solid powder.

[0072] Example 2

[0073] In this example, four self-assembling polypeptides were prepared by referring to the method provided in Example 1. Figure 1 As shown, the green part is the hydrophobic unit palmitic acid, the orange part is the assembly unit, the amino acid sequence is shown in SEQ ID NO.1, the red part is a polypeptide specifically targeting the Tie2 receptor, the amino acid sequence is shown in SEQ ID NO.2, and the blue part is a polypeptide specifically targeting the VEGFR2 receptor, the amino acid sequence is shown in SEQ ID NO.3. D PAK(N) and PAK(N)A target both Tie2 and VEGFR2 receptors, PAFN only targets Tie2 receptors, and PAFA only targets VEGFR2 receptors.

[0074] The obtained peptides were dissolved in DMSO solvent to prepare 4×10 -3 M mother solution, take 10 μL of the mother solution into 990 μL of ultrapure water, and mix thoroughly to obtain a solution concentration of 4×10 -5M, nanoparticles were obtained. Targeting proteins VEGFR2 or Tie2 were added to the mixed solution, and the final concentration of VEGFR2 or Tie2 was 4×10 -8 M, to obtain nanofibers. The self-assembling polypeptide provided by the present invention D The self-assembly process of PAK(N), PAK(N)A, PAFN and PAFA is shown in Figure 2 Figure 2 shown.

[0075] The circular dichroism spectra of the above four self-assembling peptides under the conditions of protein induction and without protein induction are as follows Figure 3 As shown, the transmission electron microscope images of the morphological transformation at 0h and 24h are shown in Figure 4 As shown. Figure 3 and Figure 4 It can be seen that in the initial state, D PAK(N), PAK(N)A, PAFN and PAFA are in the form of nanoparticles under electron microscope; the self-assembling polypeptide provided by the present invention D PAK(N), PAK(N)A, PAFN and PAFA have targeting ability to VEGFR2 or Tie2. After incubation with the corresponding targeted proteins for 24 hours, it was observed that the microscopic morphology of each group of polypeptides was transformed into nanofibers. At the same time, the circular dichroism spectrum showed a peak at about 195nm and a valley at about 215nm, showing a standard β-folded secondary structure.

[0076] Example 3

[0077] In this example, four peptides were labeled with Cy5 fluorescent molecules, and incubated with HUVEC cells for 4 hours to conduct fluorescence co-localization experiments on the cell membrane surface. The single-photon laser confocal microscopy images are shown in Figure 2. Figure 5 As shown, the four polypeptides provided in the present invention are D PAK(N), PAK(N)A, PAFN and PAFA can all bind to HUVEC cells. The distribution of fluorescence signals showed that the polypeptide molecules were mainly distributed on the surface of HUVEC cells.

[0078] Example 4

[0079] The present invention provides D PAK(N), PAK(N)A, PAFN and PAFA polypeptides can form nanoparticle structures in aqueous solutions. Once bound to the corresponding targeting proteins, the nanoparticles can be transformed into nanofibers under the action of the assembly unit. Based on this, the polypeptides provided in the present invention are applied to inhibit endothelial cell migration, and the targeting and self-assembly process is completed on the surface of endothelial cells. The scratch test results of inhibiting endothelial cell migration are as follows: Figure 6As shown in Figures a and b. HUVEC cells were starved and added to the upper chamber of the Transwell chamber together with the medium containing the polypeptide molecule. The medium containing 15% FBS was added to the lower chamber. FBS was used as a chemotactic factor to induce the cells to pass through the polycarbonate membrane from the upper chamber to the bottom of the chamber, completing the cell migration process. The number of cells on the lower chamber membrane was counted using an optical microscope to evaluate the cell migration ability. The results of the Transwell chamber experiment that inhibited endothelial cell migration are shown in Figure 1. Figure 6 As shown in Figures c and d. D PAK(N) and PAK(N)A can significantly inhibit the migration of endothelial cells. PAFN and PAFA can also inhibit the migration of endothelial cells, but their inhibitory abilities are weaker than those of D PAK(N) and PAK(N)A are different.

[0080] Example 5

[0081] In this example, Hepa 1-6 cells in the logarithmic growth phase were inoculated subcutaneously on the right posterior back of female C57BL / 6 mice to construct a liver cancer model. On the eighth day after cell implantation, when the tumor volume reached 100 mm 3 The experiment was started at 5 mg / kg. The mice were divided into 4 groups: blank group (PBS group), 5 mg / kg D PAK(N) group, 5mg / kg PAK(N)A group and Bevacizumab group (Bvz, bevacizumab, can inhibit vascular endothelial growth factor). Tail vein injection, administration every other day, a total of six times. After administration, the size of the mouse tumor was measured and the weight of the mouse was weighed to determine the therapeutic effect of the self-assembling polypeptide, and finally the tumor tissue was processed by immunofluorescence sectioning to determine the effect of the self-assembling polypeptide in inhibiting tumor angiogenesis.

[0082] The results are as follows Figure 7 As shown in the figure, except for the blank group, the tumor volume of mice decreased over time, and the weight of mice did not change significantly. D The tumor progression of mice treated with PAK(N), PAK(N)A, and bevacizumab was inhibited to varying degrees. D PAK(N)>PAK(N)A>bevacizumab, and during the treatment, the mice did not show abnormal physiological conditions as shown by weight changes, and D The number of new blood vessels in the PAK(N) and PAK(N)A groups was significantly reduced, and the effect was better than that in the Bevacizumab group. Figure 8 As shown, D The distribution and enrichment of PAK(N), PAK(N)A and bevacizumab in mice were studied, proving that DPAK(N) and PAK(N)A can be rapidly enriched in the tumor area, and the enrichment rate D PAK(N)>PAK(N)A>bevacizumab. In in vitro tissue and organ fluorescence imaging, D Weak fluorescence signals were still visible in the tumor tissues of mice in the PAK(N) and PAK(N)A groups. Fig. 9 As shown, D Transmission electron microscopy was performed on the tumor tissue sections of mice in the PAK(N), PAK(N)A, bevacizumab, and PBS groups. D Nanofiber structures were observed in the tissues of both PAK(N) and PAK(N)A groups. Fig.10 As shown, after D No lesions occurred in the major organs of mice treated with PAK(N), PAK(N)A and bevacizumab.

[0083] Example 6

[0084] In this example, an OIR animal model was established. Newly born C57BL / 6J mice were placed in a normal air environment for 6 days. On the 7th day, the pups and foster mother mice (no more than 8 pups per cage) were exposed to a 73%±2% oxygen environment for 5 days, and were transferred to a normoxic environment again on the 12th day. Drug treatment began on the 13th day. Mice treated with aflibercept were injected intravitreally with aflibercept (1 μL, 40 μg) once on the 13th day, and mice treated with polypeptides were dripped with 1 μL of 40 μg twice a day. D PAK(N) and PAK(N)A eye drops (5 μL, 2 mM) were administered until the 17th day to observe neovascularization-related indicators.

[0085] like Fig.11 As shown in the figure, after treatment, the number of new blood vessel clusters in the mouse retinal flat mounts was significantly reduced, and the therapeutic effect D PAK(N)>PAK(N)A=Aflibercept.

[0086] Example 7

[0087] In this embodiment, a CNV animal model was established. An 8-week-old adult male mouse was used. One drop of tropicamide was dripped to dilate the pupil. Then, 2% tribromoethanol (Sigma-Aldrich, T48402) was used to anesthetize the mouse in a volume of 0.15 mL / 10 g body weight. The mouse cornea was coated with diclofenac eye gel. The mouse was positioned on its stand so that it was placed horizontally, perpendicular to the slit lamp beam, and the mouse was gently turned so that its head was close to and at an angle of about 170° to the laser operator until the optic nerve was seen. The optic nerve is generally yellow, and multiple blood vessels radiate from it. The laser beam of the Nd:YAG ophthalmic laser therapy instrument Ophthalmic therapy laser (Carl Zeiss, Visulas YAG III, Germany) was focused on the retina around the optic nerve, and laser photocoagulation was applied around the optic nerve. The laser parameters were: 100 μm spot size, 532 nm wavelength, 150 mW power, 200 ms duration, and the appearance of retinal photocoagulation bubbles after laser emission indicated that the laser penetrated Bruch's membrane.

[0088] In the CNV mouse model, aflibercept (1 μL, 40 μg) was injected intravitreally once on day 1 and then instilled twice daily. D PAK(N) and PAK(N)A eye drops (5 μL, 2 mM) were administered until day 6 as the treatment period.

[0089] GSL I-B4 staining was performed on the 7th day after choroidal laser modeling to observe the area of ​​CNV neovascularization. Mice were anesthetized with 2% tribromoethanol (Sigma-Aldrich, T48402) at a volume of 0.2 mL / 10 g body weight, the eyeballs were removed, fixed with 4% paraformaldehyde at room temperature for 2 h, the connective tissue and muscle around the eyeballs were removed, the cornea, lens and retina were peeled off, and four radial incisions were made in the remaining posterior half of the eye cup to cut into a petal-like appearance. Placed in 1:100 diluted GSL I-B4, Fluorescein (Vector Laboratories, FL-1201-5), incubated overnight at 4 ° C. Images were taken by an upright fluorescence microscope (Nikon Ts2, Japan), and the CNV formation area was quantified using the Image Pro Plus image analysis software program.

[0090] like Fig.12 As shown, in accepting D In CNV mice treated with PAK(N), PAK(N)A, and aflibercept, the fluorescent area of ​​the microvascular region was significantly reduced, and the therapeutic effect was D PAK(N)>PAK(N)A>Aflibercept.

[0091] FFA (fluorescein sodium angiography) was performed on the 2nd and 6th days after choroidal laser modeling to observe the severity of CNV leakage. Mice were anesthetized with 2% tribromoethanol (Sigma-Aldrich, T48402) at a volume of 0.15 mL / 10 g body weight. At the same time, 0.05 mL 10% fluorescein sodium (Alcon, Japan) was mixed with the anesthetic for intraperitoneal injection. Three minutes later, fundus angiography photographs were taken using an optical coherence tomography (Optical Coherence Tomography, OCT) (Heidelberg, Germany). Using Image J software, contours were drawn around each CNV area, and the average fluorescence was measured and then read.

[0092] like Fig.13 As shown, the eyes of the CNV mice and the PBS group mice were subjected to FFA imaging. D The vascular leakage of mice in the PAK(N) and PAK(N)A groups was significantly improved, and the therapeutic effect D PAK(N)>PAK(N)A>Aflibercept.

[0093] In summary, the present invention provides a self-assembling polypeptide for inhibiting angiogenesis. The self-assembling polypeptide can target VEGFR2 and Tie2 related to angiogenesis, inhibit ligand receptor binding, and achieve inhibition of tumor and fundus angiogenesis.

[0094] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A self-assembling polypeptide for inhibiting angiogenesis, characterized in that: The self-assembling polypeptide comprises a hydrophobic unit, an assembly unit, a polypeptide specifically targeting Tie2 receptor and a polypeptide specifically targeting VEGFR2 receptor.

2. The self-assembling polypeptide according to claim 1, characterized in that The hydrophobic unit includes any one of saturated fatty acids having 8 to 22 carbon atoms; Preferably, the hydrophobic unit is palmitic acid; Preferably, the amino acid sequence of the assembly unit includes the sequence shown in SEQ ID NO.1; Preferably, the leucine in the assembly unit is D-leucine or L-leucine; Preferably, the amino acid sequence of the polypeptide specifically targeting Tie2 receptor includes the sequence shown in SEQ ID NO.2; Preferably, the amino acid sequence of the polypeptide specifically targeting VEGFR2 receptor includes the sequence shown in SEQ ID NO.3; Preferably, the leucine in the polypeptide specifically targeting the VEGFR2 receptor is D-leucine or L-leucine.

3. The self-assembling polypeptide according to claim 1 or 2, characterized in that The structure of the self-assembling polypeptide is shown in Formula I:

4. The self-assembling polypeptide according to any one of claims 1 to 3, characterized in that The preparation method of the self-assembling polypeptide is a polypeptide solid phase synthesis method.

5. A polypeptide self-assembled nanoparticle, characterized in that: The raw materials for preparing the polypeptide self-assembling nanoparticles include the self-assembling polypeptide according to any one of claims 1 to 4.

6. The polypeptide self-assembled nanoparticle according to claim 5, characterized in that: The preparation method of the polypeptide self-assembling nanoparticles comprises: mixing the self-assembling polypeptide and a DMSO solution; Preferably, the concentration of the self-assembling polypeptide after mixing is 3×10 -5 ~5×10 -5 M; Preferably, the volume ratio of DMSO to water in the DMSO solution is 1:(90-110).

7. A polypeptide self-assembled nanofiber, characterized in that: The raw materials for preparing the polypeptide self-assembling nanoparticles include the self-assembling polypeptide according to any one of claims 1 to 4 and a targeting protein of the self-assembling polypeptide.

8. The polypeptide self-assembled nanofiber according to claim 7, characterized in that: The targeting protein of the self-assembling polypeptide includes VEGFR2 protein or Tie2 protein; Preferably, the method for preparing the polypeptide self-assembling nanofiber comprises: mixing the self-assembling polypeptide, the targeting protein and a DMSO solution; Preferably, the concentration of the self-assembling polypeptide after mixing is 3×10 -5 ~5×10 -5 M; Preferably, the concentration of the target protein after mixing is 3×10 -8 ~5×10 -8 M; Preferably, the volume ratio of DMSO to water in the DMSO solution is 1:(90-110).

9. Use of any one of the self-assembling polypeptide for inhibiting angiogenesis as described in any one of claims 1 to 4, the polypeptide self-assembling nanoparticles as described in claim 5 or 6, or the polypeptide self-assembling nanofibers as described in claim 7 or 8, or a combination of at least two of them in the preparation of drugs for inhibiting endothelial cell migration, inhibiting vascular leakage and inhibiting angiogenesis.

10. Use of any one of the self-assembling polypeptide for inhibiting angiogenesis according to any one of claims 1 to 4, the polypeptide self-assembling nanoparticles according to claim 5 or 6, or the polypeptide self-assembling nanofibers according to claim 7 or 8, or a combination of at least two thereof, in the preparation of a drug for inhibiting tumor proliferation and treating fundus neovascularization diseases; Preferably, the treatment of fundus neovascularization diseases includes choroidal neovascularization and / or oxygen-induced retinopathy.

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