A polypeptide crossing the blood-brain barrier, a preparation method thereof, a nanostructure and a preparation method and application thereof

Abstract

The application provides a polypeptide for crossing blood brain barrier and a preparation method thereof, a nanostructure and a preparation method and application thereof, and belongs to the technical field of biological medicine, wherein the polypeptide is obtained by connecting a cyanine dye Cy5.5 and a short peptide; the short peptide is YFFG. The polypeptide provided by the application is used for crossing complete blood brain barrier, a production process adopts an Fmoc-standard solid-phase synthesis method, and has the advantages of low cost, high yield, clear structure, single component and the like. Meanwhile, the polypeptide can be self-assembled to form nanospheres by being diluted from high-concentration mother liquor of an organic solvent in a phosphate buffer solution, the nanospheres can be combined with insulin receptors expressed on the BBB, and the crossing of the BBB can be completed under the mediation of the insulin receptors.

Description

[0001] The present application is a divisional application of a patent application with the application date of September 20, 2022, the application number of 202211142764.5, and the invention name of "Polypeptide for crossing blood-brain barrier, preparation method thereof, nanostructure, preparation method thereof and application". TECHNICAL FIELD

[0002] The present application belongs to the technical field of biological medicine, and particularly relates to a polypeptide for crossing blood-brain barrier, a preparation method thereof, a nanostructure, a preparation method thereof and application. BACKGROUND

[0003] The effective crossing of the blood-brain barrier (BBB) is a major challenge for the development of therapeutic drugs for central nervous system (CNS) diseases. The blood-brain barrier, as a defense mechanism of the brain, strictly controls the entry of certain nutrients into the CNS and limits the passage of harmful biological molecules, while also preventing almost all drugs, especially biological macromolecular drugs, from entering the brain. Traditional invasive brain delivery techniques have their own drawbacks and often cause additional trauma or danger, and non-invasive brain delivery strategies have attracted increasing attention due to their safety. Non-invasive techniques mainly utilize endogenous endocytosis, such as adsorption-mediated endocytosis, carrier-mediated endocytosis and receptor-mediated endocytosis, when crossing the BBB. Among them, receptor-mediated endocytosis is the most widely studied for crossing the blood-brain barrier, such as transferrin receptor (TfR), low-density lipoprotein receptor (LDLR) family members, melanotransferrin (MTf), CD98 heavy chain (CD98hc, also known as SLC3A2).

[0004] In recent years, with the rapid development of nanotechnology, nanodelivery systems for crossing the BBB for brain disease treatment have shown great potential. The brain-targeted delivery system that has been widely studied is by surface modification of specific ligands, including polypeptides or proteins that can bind to receptors widely expressed on the BBB. These ligands can actively recognize receptors on the BBB after entering the systemic circulation, thereby achieving active targeted delivery of drugs.

[0005] The currently widely studied brain delivery system has the following problems:

[0006] Firstly, the design and synthesis steps are complex, and the system construction process at least includes three steps of nanocarrier preparation, drug loading and surface ligand modification, and it is difficult to accurately control the drug loading and surface modification process.

[0007] Secondly, during circulation, due to the complexity of the in vivo environment, especially the presence of various hydrolytic enzymes, the ligands on the surface of the carrier will have off-target side effects, resulting in a large amount of loss during circulation.

[0008] Thirdly, the component of the nano delivery system is not single, which brings new challenges for the mechanism or pharmacokinetics research of the nano delivery system crossing the BBB; if the specific ligand used for surface modification of the nano system is a protein, there are also problems such as high price, difficult preservation, immunogenicity and the like.

[0009] Finally, it is observed that the research on the nano delivery system crossing the BBB mediated by the overexpressed insulin receptor on the BBB is less, and the insulin receptor itself is a good target. SUMMARY

[0010] Therefore, the purpose of the present application is to provide a polypeptide for crossing the blood-brain barrier and a preparation method thereof, a nano structure and a preparation method and application thereof. The polypeptide provided by the present application is used for crossing the complete blood-brain barrier, and the production process adopts the Fmoc-standard solid-phase synthesis method, has the advantages of low cost, high yield, clear structure, single component and the like. At the same time, the polypeptide can be self-assembled to form nanospheres by diluting the high-concentration mother liquor of the organic solvent in the phosphate buffer solution, and the nanospheres can combine with the insulin receptor expressed on the BBB and complete the crossing of the BBB under the mediation of the insulin receptor.

[0011] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0012] The present application provides a polypeptide for crossing the blood-brain barrier, wherein the polypeptide is obtained by connecting a cyanine dye Cy5.5 and a short peptide.

[0013] The short peptide is GFFY, YFFG, GYFF, GFYF or FGFY.

[0014] The configuration of the amino acid in the GFFY is L configuration or D configuration.

[0015] The configuration of the amino acid in the YFFG is L configuration.

[0016] The configuration of the amino acid in the GYFF is L configuration.

[0017] The configuration of the amino acid in the GFYF is L configuration.

[0018] The configuration of the amino acid in the FGFY is L configuration.

[0019] The present application also provides a preparation method of the polypeptide described in the above technical solutions, wherein the polypeptide is synthesized by using the FMOC-solid-phase synthesis method.

[0020] Preferably, the method comprises the following steps:

[0021] (1) the C-terminal of the Fmoc-amino acid is combined with the resin;

[0022] (2) removal of Fmoc protecting group, washing;

[0023] (3) coupling of next Fmoc-amino acid C-terminal with N-terminal of amino acid or polypeptide on resin, washing;

[0024] (4) repeating (2)-(3) until the last amino acid is coupled, removing Fmoc protecting group, washing;

[0025] (5) removing polypeptide derivative from resin to obtain crude product;

[0026] (6) coupling of flower dye Cy5.5 with N-terminal of polypeptide;

[0027] (7) purifying crude product by using high performance liquid chromatography.

[0028] The application further provides a self-assembled polypeptide of the polypeptide.

[0029] Preferably, the polypeptide is self-assembled by diluting the polypeptide in organic solution into phosphate buffer.

[0030] The application further provides a preparation method of the self-assembled polypeptide, comprising the following steps:

[0031] 1) mixing the polypeptide with dimethyl sulfoxide to obtain mother liquor;

[0032] 2) mixing the mother liquor obtained in step 1) with phosphate buffer, sodium carbonate solution, and oscillating to obtain self-assembled polypeptide.

[0033] Preferably, the mass / volume ratio of the polypeptide to dimethyl sulfoxide in step 1) is 0.8 mg:10-20 μL.

[0034] Preferably, the volume ratio of the mother liquor to phosphate buffer to sodium carbonate solution in step 2) is 4:194:2.

[0035] The molar concentration of the sodium carbonate solution is 1M.

[0036] The application further provides use of the polypeptide in preparation of blood-brain barrier penetrating drug.

[0037] The application further provides use of the self-assembled polypeptide in preparation of blood-brain barrier penetrating drug.

[0038] The application has the following beneficial effects:

[0039] The polypeptide provided by the application is simple to synthesize, is synthesized by standard solid-phase synthesis of short peptides and liquid-phase method marking the flower qing dye, and the raw material used is the amino acid necessary for human body every day; the assembly can be combined with the insulin receptor expressed on the blood-brain barrier and cross the blood-brain barrier under the mediation of the insulin receptor; the self-assembled polypeptide crosses the blood-brain barrier in the form of endocytosis and exocytosis. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 is a high performance liquid chromatography mass spectrum of a Cy5.5-GFFY polypeptide;

[0041] Figure 2 is a high performance liquid chromatography mass spectrum of a Cy5.5-YFFG polypeptide;

[0042] Figure 3 is a high performance liquid chromatography mass spectrum of a Cy5.5-GYFF polypeptide;

[0043] Figure 4 is a high performance liquid chromatography mass spectrum of a Cy5.5-GFYF polypeptide;

[0044] Figure 5 is a high performance liquid chromatography mass spectrum of a Cy5.5-FGFY polypeptide;

[0045] Figure 6 is a mouse live imaging diagram at different time points after intravenous injection of Cy5.5-GFFY, Cy5.5-YFFG, Cy5.5-GYFF, Cy5.5-GFYF and Cy5.5-FGFY;

[0046] Figure 7 is a fluorescence content and fluorescence statistical result of a dissected brain after intravenous injection of different self-assembled polypeptides for 24 hours;

[0047] Figure 8 is a fluorescence content and statistical result of a dissected brain after intravenous injection of equal amounts of Cy5.5-GFFY and Cy5.5-G D F D F D Y for 6 hours;

[0048] Figure 9Figure 4 is a graph of the protein binding constant of the self-assembled polypeptide to insulin receptor; the binding constant of Cy5.5-GFFY to insulin receptor protein shown in the figure is 6.47 μM, indicating that the self-assembled polypeptide Cy5.5-GFFY has specific binding ability to insulin receptor; the binding constants of self-assembled polypeptides Cy5.5-YFFG, Cy5.5-GYFF, Cy5.5-FGFY and Cy5.5-GFYF to insulin receptor are 17.7 μM, 18.7 μM, 54.2 μM and 23.0 μM, respectively, indicating that the self-assembled polypeptides Cy5.5-YFFG, Cy5.5-GYFF, Cy5.5-FGFY and Cy5.5-GFYF have different degrees of binding ability to insulin receptor protein;

[0049] Figure 10 Figure 5 is a graph of the protein binding constant of the self-assembled polypeptide Cy5.5-GFFY to insulin receptor; the binding constant shown in the figure is 6.52 μM, indicating that the self-assembled polypeptide Cy5.5-GFFY also has specific binding ability to insulin receptor. D F D F D Figure 6 is a graph of the protein binding constant of the self-assembled polypeptide Cy5.5-GFFY to insulin receptor; the binding constant shown in the figure is 6.52 μM, indicating that the self-assembled polypeptide Cy5.5-GFFY also has specific binding ability to insulin receptor. D F D F D Y and insulin receptor also has specific binding ability.

[0050] Figure 11 Figure 7 is a graph of the endocytosis and exocytosis of the self-assembled polypeptide Cy5.5-GFFY by brain vascular endothelial cells (bEnd.3 cells); the operation is performed according to the experimental process shown in the schematic diagram; the results of the confocal microscope of the cells show that the bEnd.3 cells endocytose the self-assembled polypeptide Cy5.5-GFFY and then excrete it into the culture medium; the exocytosed self-assembled polypeptide can be endocytosed by blank cells again, thereby realizing the crossing of the blood-brain barrier. DETAILED DESCRIPTION

[0051] The present application provides a polypeptide for crossing the blood-brain barrier, wherein the polypeptide is obtained by connecting a cyanine dye Cy5.5 and a short peptide; the short peptide is GFFY, YFFG, GYFF, GFYF or FGFY.

[0052] The configurations of the amino acids in the GFFY are all L configuration or D configuration;

[0053] The configurations of the amino acids in the YFFG are all L configuration;

[0054] The configurations of the amino acids in the GYFF are all L configuration;

[0055] The configurations of the amino acids in the GFYF are all L configuration;

[0056] The configurations of the amino acids in the FGFY are all L configuration.

[0057] When the polypeptide is connected by cyanine dye Cy5.5 and GFFY, the structure is as follows:

[0058]

[0059] When the polypeptide is connected by cyanine dye Cy5.5 and YFFG, the structure is as follows:

[0060]

[0061] When the polypeptide is connected by cyanine dye Cy5.5 and GYFF, the structure is as follows:

[0062]

[0063] When the polypeptide is connected by cyanine dye Cy5.5 and GFYF, the structure is as follows:

[0064]

[0065] When the polypeptide is connected by cyanine dye Cy5.5 and FGFY, the structure is as follows:

[0066]

[0067] When the polypeptide is connected by cyanine dye Cy5.5 and G D F D F D Y, the structure is as follows:

[0068]

[0069] The application also provides a preparation method of the polypeptide.

[0070] In the application, the preparation method preferably comprises the following steps:

[0071] (1) the C-terminal of Fmoc-amino acid is combined with resin;

[0072] (2) the removal of Fmoc protecting group, and washing;

[0073] (3) the C-terminal of next Fmoc-amino acid is coupled with the N-terminal of amino acid or polypeptide on the resin, and washing;

[0074] (4) the steps (2) to (3) are repeated until the coupling of the last amino acid is completed, the Fmoc protecting group is removed, and washing is performed;

[0075] (5) the polypeptide derivative is cut off from the resin to obtain a crude product;

[0076] (6) coupling of the dye Cy5.5 with the N-terminus of the polypeptide;

[0077] (7) purification of the crude product by high performance liquid chromatography.

[0078] The present application also provides a self-assembled polypeptide of the polypeptide according to the technical solution described above. In the present application, the polypeptide is preferably prepared by self-assembly of organic solution diluted into phosphate buffer.

[0079] The present application also provides a preparation method of the self-assembled polypeptide according to the technical solution described above, comprising the following steps:

[0080] 1) mixing the polypeptide with dimethyl sulfoxide to obtain a mother liquor;

[0081] 2) mixing the mother liquor obtained in step 1) with phosphate buffer, sodium carbonate solution, and oscillating to obtain a self-assembled polypeptide.

[0082] In the present application, the mass / volume ratio of the polypeptide to dimethyl sulfoxide is preferably 0.8 mg: 10-20 μL. In the present application, the volume ratio of the mother liquor to phosphate buffer, sodium carbonate solution is preferably 4:194:2. In the present application, the molar concentration of the sodium carbonate solution is preferably 1M.

[0083] The technical solutions provided by the present application will be described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0084] The sources of the preparations involved in the examples of the present application are as follows:

[0085] 2-Cl-Trt resin was purchased from Tianjin Nankai Hengcheng Technology Co., Ltd., with an activity of 1.1 mmol / mL;

[0086] Amino acids were purchased from Gil Biochemical (Shanghai) Co., Ltd., with a purity of 98%;

[0087] N,N-diisopropylethylamine (DIEPA) was purchased from Adamas, with a purity of 99%;

[0088] Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (hereinafter referred to as HBTU) was purchased from Gil Biochemical (Shanghai) Co., Ltd., with a purity of 98%;

[0089] Trifluoroacetic acid (TFA) and triisopropylsilane (TIS) were purchased from Sigma-Aldrich, with a purity of 99%;

[0090] Anhydrous dichloromethane (DCM) was purchased from Tianjin Chemical Reagent Company;

[0091] N,N-dimethylformamide (DMF), Tianjin Chemical Reagent Company;

[0092] Methanol, Tianjin Concord Technology Co., Ltd.;

[0093] Piperidine, Tianjin Chemical Reagent Company;

[0094] Flower dye Cy5.5-NHS ester, APExBIO, USA;

[0095] Amino acids, purchased from Genview Biochemical (Shanghai) Co., Ltd., purity 98%;

[0096] Cell culture medium, Gibco, USA;

[0097] Fetal bovine serum, Biological Industries;

[0098] BALB / c mice, BALB / c nude mice, 6-8 weeks old, female, purchased from Vito Li Hua Biotechnology Co., Ltd.

[0099] The device involved in the embodiments of the application is:

[0100] High performance liquid chromatograph, Germany Lumtech, HPLC;

[0101] High performance liquid chromatograph-mass spectrometer, Japan Shimadzu, model LC-MS 2020;

[0102] Electronic balance, Germany arturious, model BS124S;

[0103] Liquid chromatograph-mass spectrometer, USA Agilent, Agilent 6520Q-TOFLC / MS).

[0104] Transmission electron microscope, Talos L120C G2;

[0105] Freeze dryer, Beijing Yataike Long, model LGJ-1-50;

[0106] Laser confocal microscope, Germany Zeiss, model LSM 800 with Airyscan;

[0107] Small animal live imaging system, USA, model IVIS Lumina II or Germany, model NightOWL II LB983.

[0108] Example 1

[0109] (1) Synthesis of polypeptide Cy5.5-GFFY

[0110] The specific steps are:

[0111] 1) Take 0.5 mmol 2-Cl-Trt resin in a solid phase synthesizer, add 15 mL of anhydrous dichloromethane (DCM), and place on a shaker for 15 min to allow the 2-Cl-Trt resin to swell fully;

[0112] 2) Remove the DCM from the solid phase synthesizer using a wash bottle;

[0113] 3) Dissolve 0.5 mmol of Fmoc-protected amino acid (Fmoc-Tyr(tbu)-OH) in 10 mL of anhydrous DCM, add 1 mmol of DIEA, and then transfer to the above-mentioned solid phase synthesizer, and react at room temperature on a shaker for 1 h;

[0114] 4) Blocking: remove the reaction solution from the solid phase synthesizer using a wash bottle, and then wash with 10 mL of anhydrous DCM, a total of 5 times, 1 min each time; add 20 mL of blocking solution (DCM:DIEPA:methanol = 17:1:2), and react at room temperature on a shaker for 20 min;

[0115] 5) Remove the reaction solution from the solid phase synthesizer using a wash bottle, first wash with DCM, a total of 5 times, 1 min each time, and then wash with DMF, a total of 5 times, 1 min each time; add 10 mL of DMF containing 20% by volume of piperidine, and react for 35 min to remove the Fmoc protecting group, and then wash with DMF 5 times, 1 min each time, and proceed to the next step;

[0116] 6) Add 1 mmol of the second Fmoc-protected amino acid (Fmoc-Phe-OH), 1.5 mmol of HBTU, 2 mmol of DIEA, and 10 mL of DMF, and add the dissolved amino acid solution to the above-mentioned solid phase synthesizer, and react for 2 h;

[0117] 7) Repeat the methods of steps 5) and 6) to sequentially add Fmoc-Phe-OH and Fmoc-Gly-OH; then wash with DMF 5 times, use 20% piperidine to remove the Fmoc protecting group, and then wash with DMF 5 times, dichloromethane 5 times, and proceed to the next step;

[0118] 8) Add 10 mL of a solution composed of 95% TFA, 2.5% TIS, and 2.5% H2O by volume to the above-mentioned solid phase synthesizer, and react for 30 min to cleave the product from the 2-Cl-Trt resin, vacuum concentrate, remove the solvent, and obtain a crude product, which is then separated and purified by HPLC;

[0119] 9) 1.5 mmol of the pure polypeptide GFFY in 8) was weighed into 3 mL of DMSO, and DIEA was used to adjust the pH to 9-9.5, 1 mmol of the flower dye Cy5.5-NHS ester was weighed into the reaction system, and the reaction was carried out on a dark stirring device overnight. The reaction solution was filtered, purified by high performance liquid chromatography, and the polypeptide Cy5.5-GFFY obtained in Example 1 was detected by liquid chromatography-mass spectrometry, and the amino acid configuration was L.

[0120] Example 2

[0121] Cy5.5-YFFG was synthesized according to the synthesis steps of Example 1, and the amino acid configuration was L.

[0122] Example 3

[0123] Cy5.5-GYFF was synthesized according to the synthesis steps of Example 1, and the amino acid configuration was L.

[0124] Example 4

[0125] Cy5.5-FGFY was synthesized according to the synthesis steps of Example 1, and the amino acid configuration was L.

[0126] Example 5

[0127] Cy5.5-GFYF was synthesized according to the synthesis steps of Example 1, and the amino acid configuration was L.

[0128] Example 6

[0129] Cy5.5-G D F D F D Y.

[0130] Example 7

[0131] Formation of self-assembled polypeptide nanostructures

[0132] 0.8 mg of pure polypeptide powder (prepared in Examples 1-5) was weighed and dissolved in 20 μL of dimethyl sulfoxide to obtain a high-concentration mother liquor; 4 μL of the mother liquor was diluted in phosphate buffer and 2 μL of a 1 M sodium carbonate solution was added to adjust the pH of the solution (final concentration 0.8 mg / mL) and shaken until completely dissolved to obtain.

[0133] Example 8

[0134] Micro-morphology of self-assembled nanostructures

[0135] The nanometer microstructure of the self-assembled polypeptide (prepared in Example 7) was observed using an electron transmission microscope. 10 μL of the above polypeptide self-assembly solution was dropped onto a copper mesh and left for one minute, and excess solution was removed from the edge using filter paper; 10 μL of phosphotungstic acid was dropped onto the copper mesh and left for one minute for negative staining, and excess staining solution was removed from the edge using filter paper; the copper mesh was placed in a drying box, and after complete drying, an electron transmission microscope was used to take photographs.

[0136] Example 9

[0137] The ability of the self-assembled polypeptide to cross the blood-brain barrier

[0138] The ability of the self-assembled polypeptide to cross the blood-brain barrier was investigated by a small animal live imaging system. The polypeptide self-assembly solutions of Examples 1-5 were prepared according to a dose of 5 mg / kg of the fluorochrome Cy5.5-NHS, and were injected into the tail vein with a volume of 200 μL. The time at which the injection was completed was taken as the 0th hour (0 h), and live imaging was performed at different time points (4 h, 8 h, 24 h) after the tail vein injection. The mouse brain was dissected and photographed to quantify the fluorescence content at 24 h after the imaging, and the excitation wavelength was 645 nm.

[0139] As can be seen from Figure 6 , different self-assembled polypeptides showed the characteristic of systemic distribution (0 h) after being injected into the blood circulation of mice via the tail vein. With the extension of the administration time, the fluorescence distribution range and intensity of the self-assembled polypeptides became smaller. At 8 hours after administration, the fluorescence signal of the self-assembled polypeptide in the brain of the mice of Examples 1-3, especially Example 1, and the fluorescence signal of Example 4 almost disappeared. At 24 hours after administration, Example 3 showed weak fluorescence in the brain, and Examples 4 and 5 almost had no fluorescence. The brain of the mouse of Example 1 still retained a large amount of fluorescence signal of the self-assembled polypeptide, and the brain of the mouse of Example 2 had relatively strong fluorescence but was inferior to that of Example 1, which may be related to the reverse parallel of the amino acid sequence.

[0140] The mouse at 24 hours after tail vein administration was humanely killed, and the brain was dissected and imaged as shown in Figure 7 , which was consistent with the results of the live imaging. The content of the self-assembled polypeptide in the brain of the mouse was Example 1 > Example 2 > Example 3 > Example 5 > Example 4 after 24 hours of drug metabolism. The quantitative statistics of the fluorescence in the brain showed that the fluorescence in the brain of the mouse of Example 1 was 1.09, 1.44, 1.53 and 1.60 times that of the other groups, respectively. The above results showed that the self-assembled polypeptide Cy5.5-GFFY had the best ability to cross the blood-brain barrier.

[0141] In addition, the blood-brain barrier crossing ability of Examples 1 and 6, with the same amino acid composition but different amino acid configurations, was investigated using in vivo imaging in small animals. The brains of Examples 1 and 6 (Cy 5.5-NHS 5 mg / kg) were isolated 6 hours after intravenous injection via the tail vein. Figure 8 As can be seen, the fluorescence content in the brains of the two groups of mice is relatively similar. The fluorescence intensity of Example 1 is slightly higher than that of Example 6, but there is no significant difference.

[0142] Example 10

[0143] Binding of self-assembled peptides to insulin receptor proteins

[0144] Insulin receptor protein (Human Insulin R / CD220(28-944) Protein, His Tag (MALS & SPRVerified)) was labeled using the Monolith NT Protein Labeling Kit BLUE-NHS. Cat No: INR-H52Ha. This dye reacts effectively with the primary amine of the protein to form a highly stable protein-dye conjugate. The dye was dissolved according to the kit instructions, and the dye working solution and protein solution were mixed at a 1:1 volume ratio and incubated at room temperature in the dark for 30 minutes to label the insulin receptor protein. Excess dye was completely washed away through a pre-equilibrated molecular sieve, and quantitative buffer was added to allow the solution to flow out under gravity. The fluorescently labeled protein molecules were then collected. For testing, assemblies of the peptides described in Example 1 were prepared with different concentration gradients (using initial concentrations of 1460 μM from Examples 1, 2, 3, 4, 5, and 6, which were then sequentially diluted 2-fold to obtain peptide assembly solutions of 730 μM, 365 μM, 182.5 μM, 91.25 μM, etc.; then, the assembly solutions of different concentrations were uniformly mixed with an equal volume of labeled insulin receptor protein solution and incubated at room temperature in the dark for 5 minutes). Assemblies of different concentration gradients were incubated with an equal volume of labeled insulin receptor protein at room temperature for 5 minutes. Finally, data collection and final analysis were performed using a microthermophoresis apparatus (Monolith NT.115), and the results are as follows: Figures 9-10 As shown.

[0145] from Figure 9 As can be seen, the binding constants of the assemblies in Examples 1, 2, 3, 4, and 5 to the insulin receptor are 6.47, 17.7, 18.7, 54.2, and 23.0 μM, respectively. These results indicate that the self-assembled peptides of the examples, especially Example 1, have a strong binding affinity to the insulin receptor. After entering the bloodstream, they can cross the blood-brain barrier and reach the brain by binding to the overexpressed insulin receptor on the blood-brain barrier.

[0146] From Figure 10 It can be seen that the binding constant of the embodiment 6 assembly with the insulin receptor is 6.52 μM, indicating that the polypeptide assembly still has specific binding ability with the insulin receptor when the configuration of the amino acids F, F and Y is the D configuration.

[0147] Embodiment 11

[0148] Endocytosis and exocytosis of self-assembled polypeptides by brain microvascular endothelial cells

[0149] The manner of the self-assembled polypeptide crossing the blood-brain barrier was investigated using mouse brain microvascular endothelial cells (bEnd.3 cells) and by means of laser confocal microscopy. Briefly, (1) well-grown bEnd.3 cells were seeded into a well plate, and the cells were incubated with culture medium containing the self-assembled polypeptide of embodiment 1 (100 μM) (1 st batch) for 8 hours; (2) the culture medium was discarded, the cells were washed with sterile PBS and replaced with fresh blank culture medium, and the incubation was continued for 8 hours and the culture medium was collected; (3) the collected culture medium was used to incubate blank cells (2 nd batch) for 8 hours; (4) the 1 st batch and 2 nd batch cells were observed after being incubated for 8 hours, respectively.

[0150] As Figure 11 shown, after the 1 st batch cells were incubated with the self-assembled polypeptide of embodiment 1 for 8 hours, a large amount of fluorescent signal appeared inside the cells, i.e. the self-assembled polypeptide incubated with the cells for 8 hours could be taken up by the bEnd.3 cells in a large amount; in addition, a large amount of fluorescent signal also appeared inside the 2 nd batch cells, indicating that the self-assembled polypeptide endocytosed by the bEnd.3 cells could be exocytosed to the outside of the cells and could be taken up by new blank cells again. Based on this, the self-assembled polypeptide of embodiment 1 crosses the blood-brain barrier by endocytosis and exocytosis in combination with the insulin receptor.

[0151] From the above embodiments, it can be seen that the nanospheres self-assembled from the polypeptides provided by the present application can bind to the insulin receptor protein, and when they are injected into the body through the tail vein, they can cross the blood-brain barrier to reach the brain tissue by binding to the insulin receptor overexpressed on the blood-brain barrier and being mediated by the insulin receptor.

[0152] The above only describes the preferred embodiments of the present application, and it should be noted that those of ordinary skill in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. The use of self-assembled peptides in the preparation of drugs that cross the blood-brain barrier; The method for preparing the self-assembled polypeptide includes the following steps: 1) Mix the polypeptide with dimethyl sulfoxide to obtain the mother liquor; The polypeptide was obtained by linking cyanine dye Cy5.5 with a short peptide; The short peptide is YFFG; the amino acids in YFFG are all in the L configuration. 2) Mix the mother liquor obtained in step 1) with phosphate buffer and sodium carbonate solution, and shake to obtain a self-assembled polypeptide; In step 1), the mass ratio of the polypeptide to the volume of dimethyl sulfoxide is 0.8 mg: 10–20 μL. The volume ratio of the mother liquor to the phosphate buffer and sodium carbonate solution in step 2) is 4:194:

2. The molar concentration of the sodium carbonate solution is 1M.

2. The application according to claim 1, characterized in that, The method for preparing the polypeptide includes the following steps: (1) The C-terminus of Fmoc-amino acids binds to the resin; (2) Removal of Fmoc protecting groups and washing; (3) The C-terminus of the next Fmoc-amino acid is coupled to the N-terminus of the amino acid or polypeptide on the resin, and then washed. (4) Repeat steps (2) to (3) until the last amino acid is coupled, remove the Fmoc protecting group, and wash. (5) The polypeptide derivative is removed from the resin to obtain the crude product; (6) The N-terminus of the jasmine dye Cy5.5 is coupled to the polypeptide; (7) The crude product was purified by high performance liquid chromatography.

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