A polypeptide that crosses vascular endothelium and selectively anchors HSCs and its application

Abstract

The present invention relates to the fields of medicine and pharmaceutical technology, disclosing a polypeptide that crosses the vascular endothelium and selectively anchors to hepatic stellate cells (HSCs), and its use. The polypeptide obtained by the invention selectively binds to hepatic stellate cells (HSCs), promoting the recruitment of NK cells to HSCs, which are associated with liver fibrosis. This enhances NK cell immune cytotoxicity while inhibiting the activation and abnormal proliferation of HSCs, thereby alleviating the symptoms of liver fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of medicine and pharmaceutical technology, and in particular to a polypeptide that crosses vascular endothelium and selectively anchors HSCs and applications thereof. Background Art

[0002] The interactions between different cell types, chemokines, and signaling molecules form a vast information network that collectively regulates and influences the progression of liver fibrosis. With the deepening understanding of the immune system, supramolecular therapeutic strategies based on intercellular interactions are gradually emerging in precision medicine. Classic intercellular interactions occur through two pathways: direct contact and signaling, both of which are closely related to the development and progression of various diseases. Modulating intercellular interactions to influence and reverse disease progression has become a new strategy in nanomedicine development. Intercellular interactions in the body are highly dynamic. Along with cell proliferation, differentiation, and migration, intercellular interactions and the extracellular microenvironment also influence cell fate and function. Immune checkpoint blockade, widely used in tumor immunotherapy, such as the well-known immune checkpoint TIGIT-PVR, is a new immune checkpoint following the T cell-based PD-1 / PD-L1.

[0003] Previous studies have shown that preventing the activation of HSCs (hepatic stellate cells) or inducing apoptosis of activated stellate cells has become a new target for the treatment of liver fibrosis. In particular, many HSCs survival signals are obtained from the extracellular matrix, while active pro-apoptotic signals are provided by immune cells, especially natural killer (NK) cells. Therefore, as a mediator between the key immune cells NK and effector cells in the development of liver fibrosis, significantly slowing and inhibiting the progression of liver fibrosis by affecting intercellular interactions in both direct contact and signal transduction is a new strategy for the development of liver fibrosis-specific drugs. To date, there have been no studies on immune drugs that use small molecules to affect intercellular interactions in liver fibrosis. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a polypeptide that crosses the vascular endothelium and selectively anchors HSCs and its application. The polypeptide obtained by the present invention can selectively connect with hepatic stellate cells (HSCs) and facilitate NK cells to approach hepatic stellate cells (HSCs) associated with liver fibrosis, thereby enhancing NK immune killing activity while inhibiting the activation and abnormal proliferation of HSCs, thereby alleviating the symptoms of liver fibrosis.

[0005] The present invention provides a polypeptide that spans the vascular endothelium and selectively anchors HSCs, and the structural formula of the polypeptide is:

[0006] .

[0007] Furthermore, the polypeptide is composed of D-configuration D It consists of GFFY amino acid sequence, cysteine, and L-configuration VHPKQHR sequence.

[0008] Furthermore, the D configuration D The amino acid sequence of GFFY is D-configuration glycine-D-configuration phenylalanine-D-configuration phenylalanine-D-configuration tyrosine.

[0009] Furthermore, the thiol group of the cysteine ​​is also connected to a near-infrared fluorescent group Cy5.5.

[0010] Furthermore, the L-configuration VHPKQHR sequence is L-configuration valine-L-configuration histidine-L-configuration proline-L-configuration lysine-L-configuration glutamic acid-L-configuration histidine-L-configuration arginine.

[0011] Furthermore, the present invention also provides the use of the polypeptide that crosses the vascular endothelium and selectively anchors HSCs in the preparation of a drug for alleviating liver fibrosis.

[0012] The present invention also provides a polypeptide that enhances NK cell immune killing and inhibits HSCs activation, the structural formula of the polypeptide is:

[0013] .

[0014] Furthermore, the polypeptide is composed of D-configuration D GFFY amino acid sequence, D configuration D TBP-3 targeting sequence, L-configuration competitive antagonistic tetrapeptide LSKL, and capping composition.

[0015] In the present invention, the main chain of the above polypeptide is composed of end-capped, D-configured D GFFY amino acid sequence, D configuration D The TBP-3 targeting sequence is composed of a terminal, D-configuration D GFFY amino acid sequence and D-configuration D The GFFY amino acid sequences are linked sequentially.

[0016] Furthermore, the D configuration D The amino acid sequence of GFFY is D-configuration glycine-D-configuration phenylalanine-D-configuration phenylalanine-D-configuration tyrosine.

[0017] Furthermore, the end capping is 2-naphthylacetic acid (Nap).

[0018] Furthermore, the D configuration DThe TBP-3 targeting sequence is D-configuration tyrosine-D-configuration cysteine-D-configuration phenylalanine-D-configuration histidine-D-configuration tryptophan-D-configuration histidine-D-configuration arginine-D-configuration leucine-D-configuration aspartic acid-D-configuration proline.

[0019] Furthermore, the L-configuration competitive antagonistic tetrapeptide LSKL is L-configuration histidine-L-configuration serine-L-configuration lysine-L-configuration histidine.

[0020] The present invention also provides the use of the polypeptide for enhancing NK cell immune killing and inhibiting HSCs activation in the preparation of a drug for alleviating liver fibrosis.

[0021] The present invention also provides a method for preparing a bioactive solution, the method comprising: and Disperse in PBS buffer, adjust the pH value of the solution, heat, and cool to obtain a bioactive solution.

[0022] Furthermore, the pH value of the PBS buffer (phosphate buffer) is 5-9.

[0023] Further, in PBS buffer, the and The molar concentration ratio is 4:1.

[0024] Furthermore, the The mass concentration in PBS buffer is 3mg / 1.2mL-3mg / 0.8mL.

[0025] Furthermore, the pH value of the solution is adjusted to 6-8.

[0026] Furthermore, the solution is heated to a temperature of 90°C-100°C.

[0027] Those skilled in the art should understand that heating is to promote the dissolution of the polypeptide in the solution, and the heating time is until the polypeptide is completely dissolved.

[0028] Furthermore, the cooling is to room temperature.

[0029] Furthermore, the room temperature is 26°C-28°C.

[0030] The present invention also provides application of the bioactive solution in preparing a medicine for alleviating liver fibrosis.

[0031] The embodiments of the present invention have the following technical effects:

[0032] 1. First, the polypeptide of the present invention can selectively connect with hepatic stellate cells (HSCs) and promote the proximity of NK cells to hepatic stellate cells (HSCs) related to liver fibrosis, thereby enhancing the NK immune killing activity while inhibiting the activation and abnormal proliferation of HSCs, thereby alleviating the symptoms of liver fibrosis; in addition, the design of the polypeptide structure itself in the present invention is conducive to enabling the polypeptide to cross the vascular endothelium under inflammatory conditions and enter the liver parenchyma without changing the polypeptide structure. The design of the polypeptide structure is the key technical point that enables the polypeptide of the present invention to alleviate the symptoms of liver fibrosis.

[0033] 2. The polypeptide of the present invention has high biocompatibility and basically has no other effects on normal cells and the body.

[0034] 3. The polypeptide of the present invention can effectively slow down the progression of liver fibrosis, maintain and reverse fibrosis-related phenotypes. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0036] Figure 1 This is the high performance liquid chromatogram of the polypeptide PepA-TT in Example 1.

[0037] Figure 2 This is the mass spectrum of the polypeptide PepA-TT in Example 1.

[0038] Figure 3 For the polypeptide in Example 1 Cy5.5 HPLC chromatogram of PepB-V.

[0039] Figure 4 For the polypeptide in Example 1 Cy5.5 Mass spectrum of PepB-V.

[0040] Figure 5 The microscopic morphology of the two polypeptides with different molar concentrations in the biologically active solution after co-assembly in the examples and comparative examples is shown in FIG. Figure 5 Figure a is a microscopic morphology diagram of Example 1. Figure 5 Middle b is the microscopic morphology of Example 2, Figure 5 Middle c is the microscopic morphology of Example 3, Figure 5 Middle d is the microscopic morphology of Example 1.

[0041] Figure 6 The polypeptide PepA-TT and polypeptide in Example 1 Cy5.5 Microscopic morphology of PepB-V after self-assembly, where Figure 6 Figure a is the microscopic morphology of the self-assembled polypeptide PepA-TT. Figure 6 b is a polypeptide Cy5.5 Microscopic morphology of PepB-V after self-assembly.

[0042] Figure 7 The polypeptide PepA-TT and polypeptide in Example 1 Cy5.5 The concentration of PepB-V used in the cell-level experiments was selected (PepA-TT concentration was 50 μM, PepA-TT molar concentration: Cy5.5 Microscopic morphology after co-assembly at PepB-V molar concentration = 4:1, where Figure 7 Middle a is the microscopic morphology after co-assembly, Figure 7 b is Figure 7 A partial enlarged view of a.

[0043] Figure 8 The bioactive solutions of the examples and comparative examples were used to test the endothelial transendothelial ability of the polypeptides. Figure 8 Middle a is the microscopic picture of Example 4, Figure 8 Middle b is a microscopic image of endocytosis in Example 1, Figure 8 Figure c is a microscopic image of exocytosis in Example 1. Figure 8 D is Figure 8 A partial enlarged view of a in the middle. Figure 8 The middle e is Figure 8 A partial enlarged view of b in the middle. Figure 8 f is Figure 8 A partial enlarged view of center c.

[0044] Figure 9 The binding constants of the peptides to the target proteins TIGIT and VCAM-1 were measured by microthermophoresis. Figure 9 Figure a is the binding constant diagram of the target protein TIGIT and the peptide PepA-TT. Figure 9 b is the target protein TIGIT and peptide Cy5.5 Binding constant diagram of PepB-V, Figure 9 Figure c is the binding constant diagram of the target protein TIGIT and the biologically active solution of Example 1. Figure 9 The figure d in the middle is the binding constant diagram of the target protein VCAM-1 and the peptide PepA-TT. Figure 9 The target protein VCAM-1 and peptide Cy5.5 Binding constant diagram of PepB-V, Figure 9Figure f is a graph of the binding constants between the target protein VCAM-1 and the biologically active solution of Example 1.

[0045] Figure 10 The number and fluorescence intensity of the red fluorescent bright spots of the indicator polypeptide in the cytoplasm after incubation of HUVEC cells for different time periods in Example 1 are as the incubation time elapses, wherein Figure 10 In the middle, a is the fluorescent point of the fluorescent group Cy5.5 at 0h during endocytosis. Figure 10 Middle b is the fluorescent spot of the cell nuclear dye DAPI at 0h during endocytosis. Figure 10 The middle c is the fluorescence point of all fluorescence channels at 0h during endocytosis. Figure 10 The d in the middle is the fluorescence point of the fluorescent group Cy5.5 at 0h during exocytosis. Figure 10 Middle e is the fluorescence point of the nuclear dye DAPI at 0h during exocytosis. Figure 10 The middle f is the fluorescence point of all fluorescence channels at 0h during exocytosis. Figure 10 The middle g is the fluorescent point of the fluorescent group Cy5.5 at 4h during endocytosis. Figure 10 The middle h is the fluorescence point of the nuclear dye DAPI at 4h during endocytosis. Figure 10 The i in the middle is the fluorescence point of all fluorescence channels at 4 hours during endocytosis. Figure 10 The middle j is the fluorescent point of the fluorescent group Cy5.5 at 4h during exocytosis. Figure 10 The k in the middle is the fluorescence point of the nuclear dye DAPI at 4 hours during exocytosis. Figure 10 The middle l is the fluorescence point of all fluorescence channels at 4 hours during exocytosis. Figure 10 The m in the middle is the fluorescent point of the fluorescent group Cy5.5 at 8h during endocytosis. Figure 10 The n in the middle is the fluorescence point of the nuclear dye DAPI at 8h during endocytosis. Figure 10 The o in the middle is the fluorescence point of all fluorescence channels at 8 hours during endocytosis. Figure 10 p in the middle is the fluorescence point of the fluorescent group Cy5.5 at 8h during exocytosis. Figure 10 The middle q is the fluorescence point of the nuclear dye DAPI at 8 hours during exocytosis. Figure 10 The middle r is the fluorescence point of all fluorescence channels at 8h during exocytosis. Figure 10 c1 is Figure 10 A partial enlarged view of c in the middle. Figure 10 f1 is Figure 10 The local enlarged view of f in the middle, Figure 10 i1 is Figure 10 A partial enlarged view of the i in the figure, Figure 10 l1 is Figure 10 A partial enlarged view of the middle l, Figure 10 o1 is Figure 10 A partial enlarged view of o in the middle, Figure 10 r1 is Figure 10 A partial enlarged view of r in the middle.

[0046] Figure 11 The NK cells and HSCs cells were treated with the bioactive solutions prepared in Example 1 and Comparative Example 4, and the fluorescence points at different culture times were as follows: Figure 11 a in the middle is the fluorescent spot of the fluorescent group Cy5.5 when cultured for 1 hour in Example 1, Figure 11 Figure b is the fluorescent spot of the cell nucleus dye DAPI when cultured for 1 hour in Example 1. Figure 11 Figure c in the middle is the fluorescent spot of CFSE, a live cell dye pre-stained with NK, after culturing for 1 hour in Example 1. Figure 11 The d in the middle is the fluorescence point of all fluorescence channels when cultured for 1 hour in Example 1. Figure 11 e in the middle is the fluorescent spot of the fluorescent group Cy5.5 when cultured for 2 hours in Example 1, Figure 11 Figure f in the middle is the fluorescent spot of the cell nucleus dye DAPI when cultured for 2 hours in Example 1. Figure 11 Figure g in the middle is the fluorescent spot of NK live cell dye CFSE pre-stained after culturing for 2 hours in Example 1. Figure 11 h in the middle is the fluorescence point of all fluorescence channels when cultured for 2 hours in Example 1, Figure 11 Where i is the fluorescent spot of the fluorescent group Cy5.5 when cultured for 4 hours in Example 1, Figure 11 In the middle, j is the fluorescent spot of the cell nucleus dye DAPI when cultured for 4 hours in Example 1. Figure 11 k in the figure is the fluorescence point of NK live cell dye CFSE pre-stained after culturing for 4 hours in Example 1. Figure 11 Figure 1 is the fluorescence points of all fluorescence channels when cultured for 4 hours in Example 1. Figure 11 The middle m is the fluorescent point of the fluorescent group Cy5.5 when cultured for 4 hours in comparative example 4, Figure 11 The n in the middle is the fluorescence point of the nuclear dye DAPI when cultured for 4 hours in Example 4. Figure 11 The o in the middle is the fluorescent spot of NK live cell dye CFSE when cultured for 4 hours in Example 4. Figure 11 In the middle, p is the fluorescence point of all fluorescence channels when the comparative example 4 was cultured for 4 hours.

[0047] Figure 12 It is the test result of Western blot test, where "+" represents addition and "-" represents non-addition.

[0048] Figure 13 is the effect of the embodiments and comparative examples on liver indexes, wherein Figure 13 a is the alanine aminotransferase content, Figure 13 b in the middle is the aspartate aminotransferase content, Figure 13 c is the blood urea nitrogen content, Figure 13 Where d is the total bilirubin content. DETAILED DESCRIPTION

[0049] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0050] In a first aspect, some embodiments of the present invention further provide a polypeptide that spans the vascular endothelium and selectively anchors HSCs, the polypeptide having an amino acid sequence of SEQ ID NO. 2;

[0051] The structural formula of the polypeptide having the amino acid sequence of SEQ ID NO.2 is:

[0052] .

[0053] In the present invention, the polypeptide having the amino acid sequence of SEQ ID NO.2 can selectively connect to hepatic stellate cells (HSCs), and facilitate NK cells to approach hepatic stellate cells (HSCs) associated with liver fibrosis, thereby enhancing NK immune killing activity while inhibiting the activation and abnormal proliferation of HSCs, thereby alleviating the symptoms of liver fibrosis.

[0054] In some embodiments, the polypeptide having the amino acid sequence of SEQ ID NO. 2 is composed of D- D It consists of GFFY amino acid sequence, cysteine, and L-configuration VHPKQHR sequence.

[0055] In the present invention, D-configuration D The GFFY amino acid sequence and the L-configuration VHPKQHR sequence are linked via cysteine.

[0056] In some embodiments, the D-configuration D The amino acid sequence of GFFY is D-configuration glycine-D-configuration phenylalanine-D-configuration phenylalanine-D-configuration tyrosine.

[0057] In some embodiments, the thiol group of the cysteine ​​is further linked to a near-infrared fluorescent group Cy5.5.

[0058] In some embodiments, the L-configuration VHPKQHR sequence is L-configuration valine-L-configuration histidine-L-configuration proline-L-configuration lysine-L-configuration glutamic acid-L-configuration histidine-L-configuration arginine.

[0059] In a second aspect, some embodiments of the present invention further provide use of the polypeptide that spans the vascular endothelium and selectively anchors to HSCs in the preparation of a drug for alleviating liver fibrosis.

[0060] In a third aspect, some embodiments of the present invention further provide a polypeptide for enhancing NK cell immune killing and inhibiting HSCs activation, wherein the polypeptide has an amino acid sequence of SEQ ID NO.1;

[0061] The structural formula of the polypeptide having the amino acid sequence of SEQ ID NO.1 is:

[0062] .

[0063] In the present invention, the polypeptide having the amino acid sequence of SEQ ID NO.1 induces NK cells to approach hepatic stellate cells (HSCs) associated with liver fibrosis, thereby enhancing NK immune killing activity and inhibiting the activation and abnormal proliferation of HSCs, thereby alleviating the symptoms of liver fibrosis.

[0064] In some embodiments, the polypeptide having the amino acid sequence of SEQ ID NO.1 is composed of D- D GFFY amino acid sequence, D configuration D TBP-3 targeting sequence, L-configuration competitive antagonistic tetrapeptide LSKL, and capping composition.

[0065] In some embodiments, the D-configuration D The amino acid sequence of GFFY is D-configuration glycine-D-configuration phenylalanine-D-configuration phenylalanine-D-configuration tyrosine.

[0066] In some embodiments, the end-capping agent is 2-naphthylacetic acid (Nap).

[0067] In some embodiments, the D-configuration D The TBP-3 targeting sequence is D-configuration tyrosine-D-configuration cysteine-D-configuration phenylalanine-D-configuration histidine-D-configuration tryptophan-D-configuration histidine-D-configuration arginine-D-configuration leucine-D-configuration aspartic acid-D-configuration proline.

[0068] In some embodiments, the L-configuration competitive antagonist tetrapeptide LSKL is L-configuration histidine-L-configuration serine-L-configuration lysine-L-configuration histidine.

[0069] In a fourth aspect, some embodiments of the present invention further provide the use of the polypeptide that enhances NK cell immune killing and inhibits HSCs activation in the preparation of a drug for alleviating liver fibrosis.

[0070] In a fifth aspect, some embodiments of the present invention also provide a method for preparing a bioactive solution, which comprises dispersing a polypeptide having an amino acid sequence of SEQ ID NO.1 and a polypeptide having an amino acid sequence of SEQ ID NO.2 in a PBS buffer solution, adjusting the pH value of the solution, heating, and cooling to obtain a bioactive solution.

[0071] In the bioactive solution of the present invention, the co-assembly system of the two polypeptides can completely cross the vascular endothelium, further inducing NK cells and hepatic stellate cells HSCs related to liver fibrosis to approach each other, enhancing the NK immune killing activity while inhibiting the activation and abnormal proliferation of HSCs, thereby alleviating the symptoms of liver fibrosis.

[0072] In some embodiments, the pH value of the PBS buffer (phosphate buffered saline) is 5-9.

[0073] In some embodiments, in PBS buffer, the molar concentration ratio of the polypeptide having the amino acid sequence of SEQ ID NO. 1 to the polypeptide having the amino acid sequence of SEQ ID NO. 2 is 4:1.

[0074] In some embodiments, the mass concentration of the polypeptide having the amino acid sequence of SEQ ID NO. 1 in PBS buffer is 3 mg / 1.2 mL-3 mg / 0.8 mL.

[0075] In some embodiments, the pH value of the adjusted solution is 6-8.

[0076] In some embodiments, the solution is heated to a temperature of 90°C-100°C.

[0077] In some embodiments, the cooling is to room temperature.

[0078] In some embodiments, the room temperature is 26°C-28°C.

[0079] In a sixth aspect, some embodiments of the present invention further provide use of the bioactive solution in the preparation of a drug for alleviating liver fibrosis.

[0080] The following is elaborated with reference to specific embodiments:

[0081] Example 1:

[0082] Preparation method of polypeptide having amino acid sequence of SEQ ID NO.1: Nap-G D F D F D YK(LSKL)GG D Y D T D FD H D W D H D R D L D N D Synthesis of P

[0083] The Fmoc-short peptide solid phase synthesis method was used for synthesis. The specific steps are:

[0084] (1) Weigh 0.5 mmol of 2-Cl-Trt resin into a solid phase synthesizer, add 10 mL of anhydrous dichloromethane (DCM), and shake on a shaker for 10 min to allow the 2-Cl-Trt resin to fully swell;

[0085] (2) Use an ear bulb to remove DCM from the solid phase synthesizer containing 2-Cl-Trt resin;

[0086] (3) Dissolve 0.5 mmol of Fmoc-protected amino acid (Fmoc-D-Phe-OH) in 10 mL of anhydrous DCM, add 1 mmol of DIEPA (N,N-diisopropylethylamine), and then transfer to the above-mentioned solid phase synthesizer and react at room temperature for 1 h;

[0087] (4) Blocking: Use an ear bulb to remove the reaction solution in the solid phase synthesizer, then wash with 10 mL of anhydrous DCM for 1 min each time, for a total of 5 washes, add 20 mL of a solution with a volume ratio of anhydrous DCM: DIEPA: methanol of 17:1:2, and react at room temperature for 20 min;

[0088] (5) Remove the reaction solution from the solid phase synthesizer with an ear bulb, first wash with anhydrous DCM, 10 mL of DCM each time, 1 min of washing time, 5 times in total, then wash with DMF (dimethylformamide), 10 mL of DMF each time, 1 min of washing time, 5 times in total, add 10 mL of a mixed solution of piperidine and DMF, wherein the volume ratio of piperidine to DMF is 2:8, react for 25 min, then wash with DMF, 10 mL of DMF each time, 1 min of washing time, 5 times in total, and proceed to the next step of reaction;

[0089] (6) Add 1 mmol of the second Fmoc-protected amino acid (Fmoc-D-His(Boc)-OH), 1.5 mmol of HBTU (benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate), 2 mmol of DIEPA, and 10 mL of DMF. Add the prepared solution to the above solid phase synthesizer and react for 2 h.

[0090] (7) Repeat steps (5) and (6) to sequentially add L-configuration and D-configuration amino acids and a blocking group (2-naphthylacetic acid) according to the amino acid sequence of SEQ ID NO. 1; then wash with DMF 5 times and dichloromethane 5 times before proceeding to the next step;

[0091] (8) 10 mL of a solution consisting of 95% TFA (trifluoroacetic acid), 2.5% TIS (triisopropylsilane), and 2.5% H2O (water) by volume was added to the solid phase synthesizer and reacted for 0.5 h. The product was cut from the 2-Cl-Trt resin, concentrated in vacuo, and the solvent was removed to obtain a crude product, which was then separated and purified by HPLC. A person skilled in the art can also prepare a TFA solution with a TFA volume percentage concentration of 1% by volume by mixing TFA and DCM in a volume ratio of 1:99, and add 3 mL of the TFA solution to the solid phase synthesizer ten times, with each reaction time of 1 min.

[0092] (9) The purified tetrapeptide LSKL was connected to the amino group of lysine in the main chain by liquid phase synthesis. 1mmol of tetrapeptide LSKL, 1.1mmol NHS (N-hydroxysuccinimide), and 1.2mmol DCC (1,3-dicyclohexylcarbodiimide) were dissolved in a small amount of DMF until clear, and the exposed carboxyl group of LSKL was activated. The reaction was carried out for more than 4 hours, and the reaction liquid was filtered. 1mmol of the main chain pure product and 1mmol of DIEPA were added to the system, and the pH value was adjusted to 8-10. After the reaction was allowed to proceed overnight, the product was separated and purified by HPLC. A polypeptide having an amino acid sequence of SEQ ID NO.1 was obtained, which was designated as PepA-TT. The amino acid sequence of the obtained PepA-TT is shown in SEQ ID NO.1. The result was detected by high performance liquid chromatography-mass spectrometry. Figure 1-Figure 2 ;

[0093] Preparation method of the polypeptide having the amino acid sequence of SEQ ID NO.2: The Fmoc-short peptide solid phase synthesis method of the polypeptide having the amino acid sequence of SEQ ID NO.1 can also be applied to the synthesis of the amino acid sequence of SEQ ID NO.2, which is recorded as Cy5.5 PepB-V.

[0094] (1) Weigh 0.5 mmol of 2-Cl-Trt resin into a solid phase synthesizer, add 10 mL of anhydrous dichloromethane (DCM), and shake on a shaker for 10 min to allow the 2-Cl-Trt resin to fully swell;

[0095] (2) Use an ear bulb to remove DCM from the solid phase synthesizer containing 2-Cl-Trt resin;

[0096] (3) Dissolve 0.5 mmol of Fmoc-protected amino acid (Fmoc-D-Phe-OH) in 10 mL of anhydrous DCM, add 1 mmol of DIEPA (N,N-diisopropylethylamine), and then transfer to the above-mentioned solid phase synthesizer and react at room temperature for 1 h;

[0097] (4) Blocking: Use an ear bulb to remove the reaction solution in the solid phase synthesizer, then wash with 10 mL of anhydrous DCM for 1 min each time, for a total of 5 washes, add 20 mL of a solution with a volume ratio of anhydrous DCM: DIEPA: methanol of 17:1:2, and react at room temperature for 20 min;

[0098] (5) Remove the reaction solution from the solid phase synthesizer with an ear bulb, first wash with anhydrous DCM, 10 mL of DCM each time, 1 min of washing time, 5 times in total, then wash with DMF (dimethylformamide), 10 mL of DMF each time, 1 min of washing time, 5 times in total, add 10 mL of a mixed solution of piperidine and DMF, wherein the volume ratio of piperidine to DMF is 2:8, react for 25 min, then wash with DMF, 10 mL of DMF each time, 1 min of washing time, 5 times in total, and proceed to the next step of reaction;

[0099] (6) Add 1 mmol of the second Fmoc-protected amino acid (Fmoc-D-His(Boc)-OH), 1.5 mmol of HBTU (benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate), 2 mmol of DIEPA, and 10 mL of DMF. Add the prepared solution to the above solid phase synthesizer and react for 2 h.

[0100] (7) Repeat steps (5) and (6) to sequentially add L-configuration and D-configuration amino acids and a blocking group (2-naphthylacetic acid) according to the main chain amino acid sequence of SEQ ID NO. 2; then wash with DMF 5 times and dichloromethane 5 times, and proceed to the next step;

[0101] (8) 10 mL of a solution consisting of 95% TFA (trifluoroacetic acid), 2.5% TIS (triisopropylsilane), and 2.5% H2O (water) by volume was added to the solid phase synthesizer and reacted for 0.5 h. The product was cut from the 2-Cl-Trt resin, concentrated in vacuo, and the solvent was removed to obtain a crude product, which was then separated and purified by HPLC. A person skilled in the art can also prepare a TFA solution with a TFA volume percentage concentration of 1% by volume by mixing TFA and DCM in a volume ratio of 1:99, and add 3 mL of the TFA solution to the solid phase synthesizer ten times, with each reaction time of 1 min.

[0102] (9) Then, the backbone pure product and Cy5.5-MAL (Cy5.5-maleimide) were added to the system at a mass ratio of 1:1 and vortexed for 30 seconds. The pH was adjusted to 7.5 using sodium carbonate (0.01 M) and placed at 4°C overnight. Finally, the peptide was obtained by freeze drying. Cy5.5 PepB-V. The polypeptide having the amino acid sequence of SEQ ID NO.2 was detected by high performance liquid chromatography-mass spectrometry. Figure 3-Figure 4 ;

[0103] Add 3mg of PepA-TT peptide, Cy5.5 0.75 mg of PepB-V peptide was added to the system, and 1 mL of PBS buffer solution (pH = 7.4) was added to the system. In the PBS buffer solution, PepA-TT peptide and Cy5.5 The molar concentration ratio of the PepB-V peptides was 4:1. The pH was adjusted to 7.4 using a sodium carbonate solution and completely dissolved by Vortex for 3-5 seconds to obtain the pre-assembly bioactive solution. A heating-cooling reaction was then performed, heating the system to boiling and then allowing it to cool to room temperature for 3-5 seconds to obtain the post-co-assembly bioactive solution.

[0104] Comparative Example 2:

[0105] The preparation method is the same as that of Example 1, but PepA-TT polypeptide and Cy5.5 The added mass of PepB-V peptide was different. In PBS buffer, PepA-TT peptide and Cy5.5 The molar concentration ratio of PepB-V polypeptide was 2:1.

[0106] Comparative Example 3:

[0107] The preparation method is the same as that of Example 1, but PepA-TT polypeptide and Cy5.5 The added mass of PepB-V peptide was different. In PBS buffer, PepA-TT peptide and Cy5.5 The molar concentration ratio of PepB-V polypeptide was 3:1.

[0108] Comparative Example 4:

[0109] Weigh 8g NaCl (sodium chloride), 0.2g KCl (potassium chloride), 1.44g Na2HPO4 (disodium hydrogen phosphate), and 0.24g KH2PO4 (potassium hydrogen phosphate) and dissolve in 800mL of distilled water. Adjust the pH of the solution to 7.4 with HCl (hydrochloric acid). Finally, add distilled water to 1L to obtain a PBS solution. Sterilize in an autoclave and store in a refrigerator at 4°C.

[0110] The bioactive solutions obtained in the examples and comparative examples were tested experimentally:

[0111] Results and Analysis:

[0112] Weigh 3 mg of the polypeptide PepA-TT prepared in Example 1 and the polypeptides with different molar concentration ratios thereof. Cy5.5 PepB-V, add 1 mL of 1×PBS buffer solution (pH=7.4) to the system, adjust its pH to 7.4 with sodium carbonate solution, and Vortex for 3-5 seconds to completely dissolve it to obtain the bioactive solution before assembly. Then, a heating-cooling reaction is performed, heating the system to boiling, and then cooling it to room temperature for 3-5 seconds to obtain the bioactive solution after co-assembly. The bioactive solutions obtained in Example 1 and Comparative Examples 1-3 were observed using a transmission electron microscope for their micromorphology after assembly. The results are shown in FIG. Figure 5 . Figure 5 It shows that the peptide PepA-TT and peptide Cy5.5 PepB-V will present a completely different microstructure after co-assembly. As the molar concentration of the PepA-TT polypeptide in the system gradually increases, the nanostructure presented after co-assembly gradually transforms from the relatively independent existence of nanofibers and nanoparticles to a more ordered higher-level structure. Cy5.5 When the molar concentration ratio of PepB-V is 4:1, the co-assembly presents a nanofiber network shaped like a vascular network and loaded with fine nanoparticles, indicating that the system has reached a more stable assembly state at this ratio. Therefore, it is further preferred that the concentration of the polypeptide PepA-TT in PBS buffer is 4:1. Cy5.5 The molar concentration ratio of PepB-V in PBS buffer was 4:1.

[0113] Weigh 3 mg of the polypeptide PepA-TT prepared in Example 1 and 3 mg of the polypeptide prepared in Example 1 respectively. Cy5.5 PepB-V 0.6 mg, each was added with 1 mL of 1× PBS buffer (pH = 7.4), and the pH value was adjusted to 7.4 with sodium carbonate solution. After sonication, the solution was completely dissolved to obtain a PepA-TT solution with a concentration of 1096 μM and a PepA-TT solution with a concentration of 274 μM. Cy5.5 The PepB-V solution was prepared to meet the concentration required for biological transmission electron microscopy experiments, and then a heating-cooling reaction was performed. The system was heated to boiling and then cooled at room temperature after 3-5 seconds to obtain the assembled PepA-TT bioactive solution and the assembled peptide. Cy5.5 PepB-V biological active solution. The assembled peptides PepA-TT and peptides were observed using transmission electron microscopy. Cy5.5 The microscopic morphology of the PepB-V bioactive solution was shown in Figure 2. Figure 6 shown. Figure 6 The results showed that the nanostructure of the assembled polypeptide PepA-TT was a network of tangled long nanofibers. Cy5.5 PepB-V forms nanoparticles with different diameters.

[0114] Next, 0.7 mg of the polypeptide PepA-TT prepared in Example 1 and 0.7 mg of the polypeptide Cy5.5 PepB-V 0.15 mg, 1× PBS buffer (pH = 7.4) 5 mL was added to the system, the pH value was adjusted to 7.4 with sodium carbonate solution, and ultrasonication was used to dissolve it completely to obtain a solution with a peptide PepA-TT concentration of 50 μM (PepA-TT molar concentration: Cy5.5 The PepB-V molar concentration was 4:1), and the bioactive solution of the co-assembled system was obtained after heating-cooling reaction. The micromorphology of the bioactive solution after the co-assembly was observed by transmission electron microscopy. The results are as follows Figure 7 shown. Figure 7 It shows that the bioactive solution at a concentration of 50 μM (PepA-TT molar concentration: Cy5.5 The nanostructure presented by the PepB-V molar concentration = 4:1 is still a vascular nanofiber network, with single fibers arranged longitudinally and aggregated into bundles. It should be understood by those skilled in the art that when the polypeptide concentration in the cell-level experiment is changed, the polypeptide in the prior art will undergo structural changes due to poor structural stability. However, in the polypeptide structure of the present invention, in the cell-level experiment, the polypeptide in the biologically active solution at the selected drug concentration still has a highly ordered nanostructure, which shows that the polypeptide structure of the present invention is stable and can be seen from the Figure 7 Verified in.

[0115] In order to explore the ability of the polypeptide of the present invention to cross the vascular endothelium, we conducted a biological transmission electron microscopy experiment. 6 Human umbilical vein endothelial cells (HUVECs) were seeded in a 10 cm diameter culture dish at a density of 10 cells / dish. After the cells attached to the dish, the culture medium was removed. The co-assembly system of Example 1 was added to the system, i.e., the peptide PepA-TT concentration was 50 μM (PepA-TT molar concentration: Cy5.5A bioactive solution containing PepB-V (molar concentration = 4:1) was prepared, with the PBS buffer of Comparative Example 4 serving as a blank control. After 8 hours of incubation, the culture medium in the dish was replaced with fresh drug-free culture medium and incubated for a further 6 hours. HUVEC cells were collected after 8 hours of incubation and after replacing the culture medium and incubating for a further 6 hours into 1.5 mL EP tubes. Cell pellets were obtained by centrifugation and gently scratched with a needle to form tiny cell clusters. Fixative (2.5% glutaraldehyde solution) was slowly added along the tube wall and fixed overnight at 4°C. The fixative in the tube was removed by centrifugation again, and the sample was rinsed three times with pH 7.0 PBS buffer for 15 minutes each time. The sample was then fixed with 1% osmium acid solution for 1-2 hours. Subsequently, the osmium acid waste solution was carefully removed and the sample was rinsed three times with pH 7.0 PBS buffer for 15 minutes each time. The samples were dehydrated with ethanol solutions of gradient concentrations (including 30%, 50%, 70%, 80%, 90% and 95%), treated for 15 minutes at each concentration, then treated with 100% ethanol for 20 minutes, and finally transitioned to pure acetone for 20 minutes. The samples were treated with a mixture of Spurr embedding agent and acetone (V / V=1 / 1) for 1 hour, a mixture of Spurr embedding agent and acetone (V / V=3 / 1) for 3 hours, and pure embedding agent for overnight. The infiltrated samples were embedded and heated at 70°C overnight to obtain embedded samples. The samples were sliced ​​in an ultrathin microtome to obtain 70nm-90nm slices. The slices were stained with lead citrate solution, uranyl acetate and 50% ethanol saturated solution for 5-10 minutes each, and then observed in a Hitachi-7800 transmission electron microscope. The results are shown in Figure 2. Figure 8 shown.

[0116] Figure 8 The results indicate that after endocytosis, HUVEC cells exhibited a large number of nanofiber structures in the cytoplasm, while after exocytosis, secretory vesicles formed on the cell membrane and the number of small fibers in the cytoplasm decreased and was significantly reduced. These results demonstrate that endocytosis and exocytosis of vascular endothelial cells do not affect the morphology of the co-assembly system of Example 1, achieving complete endothelial bridging.

[0117] According to the Monolith NT™ Protein Labeling Kit BLUE - NHS protein labeling kit instructions, 5 μL of "Dye NT-495 NHS" (DMSO (dimethyl sulfoxide)) was diluted 20-fold in PBS. This was then mixed with 100 μL of 10 μM rhTIGIT and rhVCAM-1 proteins (buffer: 1× PBS pH 7.4, Tween-20 (0.05%)) by pipetting until uniform. The mixture was then allowed to react in the dark for 30 minutes. After the reaction, the excess dye was completely washed through molecular sieves, and quantitative buffer was added to allow the solution to drain by gravity, after which the fluorescently labeled protein was collected. According to the method for preparing a bioactive solution in Example 1, the two peptides prepared in Example 1 and the co-assembly system (PepA-TT molar concentration: Cy5.5 Bioactive solutions of varying concentrations were prepared using a PepB-V molar concentration of 4:1. The peptide bioactive solutions of varying concentration gradients were incubated with equal volumes of fluorescently labeled rhTIGIT and rhVCAM-1 proteins at room temperature for 5 minutes. The solutions were then aspirated via capillary tubes and tested on a microfluidic system. Finally, the binding ability of the different peptides to the PD-L1 protein was determined using a microcalorimetry instrument (Monolith NT.115). The results are shown in Figure 2. Figure 9 shown.

[0118] Figure 9 Different peptides have different binding constants for TIGIT protein and VCAM-1 protein. The peptides PepA-TT and peptides Cy5.5 PepB-V and bioactive solution (PepA-TT molar concentration: Cy5.5 PepB-V (molar concentration = 4:1) was mixed with TIGIT protein and VCAM-1 protein respectively, and the protein binding capacity was determined by microthermophoresis. D The smaller the value, the stronger its affinity. Regarding the binding ability to TIGIT protein, the polypeptide PepA-TT in the present invention has a stronger ability due to the presence of TIGIT affinity sequence. The K D The value was 2.19 μM, which was comparable to the peptide lacking the TIGIT affinity sequence. Cy5.5 K of PepB-V D The affinity of the polypeptide of the present invention to TIGIT protein was increased by 19.5 times compared with the value of 44.8 μM. Cy5.5 PepB-V has a strong ability, and the peptide PepA-TT K DThe value reached 8.72 μM, which is 5.8 times that of the polypeptide PepA-TT prepared in Example 1. In addition, the bioactive solution of the present invention has good binding ability for both TIGIT protein and VCAM-1 protein. It can be seen that the co-assembly system in Example 1 (PepA-TT molar concentration: Cy5.5 PepB-V (molar concentration = 4:1) has good binding ability to both target proteins, laying the foundation for its cell selectivity and functional specificity.

[0119] According to 5×10 4 Human umbilical vein endothelial cells (HUVECs) were seeded onto a 24-well plate covered with a circular cell slide at a density of 10 cells / well. After the cells attached to the surface of the cell slide, the culture medium in the wells was removed. The bioactive solution prepared according to Example 1 was added to each well to prepare a peptide PepA-TT concentration of 50 μM (PepA-TT molar concentration: Cy5.5 The bioactive solution of PepB-V molar concentration = 4:1 was used, and the PBS solution of comparative example 1 was used as a blank control. Incubate at different time points to observe the endocytosis of HUVEC, and then replace the culture medium in the wells with fresh drug-free culture medium and continue to incubate at different time points to explore the exocytosis of HUVEC to verify the vascular endothelial crossing ability of the polypeptide of the present invention. After the above steps are completed, the solution in the wells is aspirated, and the cells are washed 3 times with PBS buffer for 1 minute each time, and then stained with 0.5 μg / mL DAPI (4', 6-diamidino-2-phenylindole) at room temperature for 4 minutes. All the above operations must be protected from light. After staining, images were taken at the same voltage using a laser scanning confocal microscope (Carl Zeiss LSM 900) to detect the vascular endothelial crossing of the polypeptide, as shown Figure 10 shown.

[0120] from Figure 10 It can be seen that after incubating HUVEC cells for different periods of time with the bioactive solution prepared in Example 1, the number and fluorescence intensity of the red fluorescent bright spots of the indicator polypeptide in the cytoplasm gradually increased with the passage of incubation time, and were closely distributed around the cell nucleus. The above results indicate that HUVEC cells expressing the adhesion receptor VCAM-1 are able to endocytose the co-assembly system of Experimental Example 1 into the cytoplasm through the receptor-mediated endocytosis pathway. However, after replacing the drug-containing culture medium in the well with fresh culture medium, the red fluorescent bright spots in the cytoplasm gradually decreased as exocytosis proceeded, and the small amount of fluorescent bright spots remaining in the cytoplasm were also distributed away from the cell nucleus. The above results indicate that after the polypeptide in Example 1 enters the vascular endothelial cells by endocytosis, it is further transported to the outside of the cell by receptor-mediated exocytosis, creating conditions for further enhancing the interaction between effector cells.

[0121] Next, follow the 5×104 Human hepatic stellate cells LX-2 were seeded onto 24-well plates at a density of 10 cells / well. After the cells adhered, the culture medium in the wells was removed. The bioactive solution prepared according to Example 1 was added to each well to obtain a 50 μM concentration of the peptide PepA-TT (PepA-TT molar concentration: Cy5.5 At the same time, human NK cells NK92-MI were added to the wells at a certain cell number ratio to explore the effect of the polypeptide of the present invention on the interaction between the two effector cells and whether it can enhance the adhesion of NK cells to HSCs. 2.5×10 5 NK cells were co-cultured. After incubating the cells for different time points, the solution in the wells was aspirated and the cells were washed 3 times with PBS buffer for 1 minute each time. The cells attached to the coverslips were fixed with 4% paraformaldehyde in PBS buffer for 10 minutes at room temperature. Then, the solution in the wells was aspirated and the cells were washed 3 times with PBS buffer for 1 minute each time, and then stained with 0.5μg / mL DAPI for 4 minutes at room temperature. All operations must be protected from light. CFSE (hydroxyfluorescein diacetate succinimidyl ester) is a live cell dye. After staining, images were taken at the same voltage using a laser scanning confocal microscope (Carl Zeiss LSM 900) to detect the effect of the polypeptide on the adhesion ability of NK cells, such as Figure 11 shown.

[0122] from Figure 11 As can be seen, the number of green-fluorescent NK cells attached to the LX-2 cell surface gradually increased at different time points during the co-culture of NK cells and HSCs treated with the bioactive solution prepared in Example 1. Using the PBS buffer group in Comparative Example 1 as a blank control, after the same 4-hour incubation, Example 1 significantly enhanced the adhesion of NK cells to HSCs.

[0123] The effect of the polypeptide of the present invention on the liver fibrosis-related phenotype of HSCs was detected by immunoblotting. LX-2 cells were seeded on a cell culture dish with a diameter of 10 mm and cultured for 24 hours until the cells attached to the wall and grew to a density of about 80%. PepA-TT, prepared by the method of the polypeptide bioactive solution prepared according to Example 1, Cy5.5 PepB-V and co-assembled polypeptide bioactive solutions, with the PBS solution described in Comparative Example 1 as a blank control, the PepA-TT concentration was 50 μM, Cy5.5The concentration of PepB-V was 12.5 μM. The cells were incubated for 24 hours and the culture medium was removed. The cells were then washed three times with PBS buffer. The cells were collected and lysed in cell lysis buffer containing protease inhibitors. After quantitative analysis with BCA, equal amounts (30 μg) of samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% BSA (bovine serum albumin) to eliminate nonspecific interference. The membranes were incubated with the corresponding primary antibodies at 4°C overnight and then incubated with ImmunoWay's HRP (horseradish peroxidase)-labeled secondary antibodies for 1.5 hours. Finally, Tanon 5500 was used to detect the membrane using Immobilon Western HRP substrate to detect the ability of the peptide to reverse the TGF-β1-induced LX-2 liver fibrosis-related phenotype. GAPDH was used as the internal reference protein, and Collegen III, Desmin, and α-SMA were used as fibrosis-related indicators. The results are shown in Figure 2. Figure 12 as shown (where "+" represents addition and "-" represents non-addition).

[0124] from Figure 12 It can be seen that, with the PBS group as the blank control group, under the condition that the internal reference protein bands of each group were basically consistent, the single peptide PepA-TT, peptide Cy5.5 After 24 hours of incubation with PepB-V and Example 1, the fibrosis-related phenotypes of LX-2 cells were significantly reduced, and the degree of fibrosis induced by TGF-β1 was effectively reversed, indicating that the peptide PepA-TT, peptide Cy5.5 PepB-V and Example 1 have significant in vitro inhibitory effects on LX-2 cell fibrosis, providing a basis for further in vivo experiments.

[0125] We used CCL4 (carbon tetrachloride) to induce a mouse liver fibrosis model to explore the application of the polypeptide of the present invention in anti-liver fibrosis in vivo. CCL4 was dissolved in mineral oil to prepare an injection solution with a final concentration of 25%, and intraperitoneal injection was adopted at a dose of 2mL / kg, twice a week for four weeks. The mice in the control group were intraperitoneally injected with equal amounts of mineral oil and fed with normal feed. The drug was administered by tail vein injection, and the dosage concentration was selected as 10mg / kg. The drug was administered once on the 1st, 4th and 7th day, for a total of three times. The serum of mice in each group was collected 24 hours after the last administration for blood biochemical analysis of liver damage-related indicators, such as Figure 13 shown.

[0126] The main test indicators include aspartate aminotransferase (AST) and alanine aminotransferase (ALT) which represent liver parenchymal damage, total bilirubin (TBIL) which is an important indicator of liver function, and blood urea nitrogen (BUN) which is an indicator for evaluating kidney performance. Figure 13 It can be seen that after three doses, the peptide PepA-TT, Cy5.5 The liver damage-related indicators of mice treated with PepB-V and the co-assembled system of Example 1 via tail vein were reversed to varying degrees compared with those of the model group mice.

[0127] Therefore, the polypeptide provided by the present invention can cross the vascular endothelium and enhance intercellular interactions, inhibiting the proliferation and activation of HSCs from both direct contact and immune killing by enhancing the adhesion and killing of NK cells, thereby slowing down the progression of liver fibrosis.

[0128] It should be noted that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the scope of this application. As shown in the present specification, unless the context clearly indicates an exception, the words "one", "a", "a kind of" and / or "the" do not specifically refer to the singular and may also include the plural. The terms "comprise", "include" or any other variants thereof are intended to cover non-exclusive inclusion, so that the process, method or device comprising a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method or device. In the absence of further restrictions, the elements defined by the sentence "comprise a..." do not exclude the presence of other identical elements in the process, method or device comprising the elements.

[0129] It should also be noted that the terms "center", "up", "down", "left", "right", "vertical", "horizontal", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, they cannot be understood as limitations on the present invention. Unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", etc. should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or an indirect connection through an intermediate medium, or it can be a communication between the internal parts of two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0130] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the technical solutions of the embodiments of the present invention.

Claims

1. A polypeptide that crosses the vascular endothelium and selectively anchors HSCs, characterized in that: The structural formula of the polypeptide is: 。 2. Use of the polypeptide that spans the vascular endothelium and selectively anchors HSCs according to claim 1 in the preparation of a drug for alleviating liver fibrosis.

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