Cascade three-section type self-assembly composite polypeptide nano-particles for targeted repair of endothelial barrier and regulation and control of immune recruitment as well as preparation method and application of nano-particles

Abstract

The invention discloses a three-section self-assembly composite polypeptide nanoparticle for targeted repair of endothelial barrier and regulation and control of immune recruitment cascade and a preparation method and application thereof, and the preparation method comprises the following steps: (1) preparation of a hydrophilic section head multifunctional peptide solution, (2) preparation of a middle section connector ROS response thioketal solution, and (3) preparation of a three-section self-assembly composite polypeptide nanoparticle for targeted repair of endothelial barrier and regulation and control of immune recruitment cascade. (5) preparing a three-section type compound of a hydrophilic section, the hydrophobic section and the middle section, and (6) mixing the connection compound solutions of the three types of polypeptide of the hydrophobic section, the middle section and the hydrophilic section, and dialyzing to remove DMSO (Dimethylsulfoxide), so that the compound solution is changed from an organic phase to a water phase, and the three-section type compound of the hydrophobic section, the middle section and the hydrophilic section is obtained. And promoting amphiphilic self-assembly of the compound and co-assembly of the three polypeptide compounds, and freeze-drying to obtain the three-section type self-assembly composite polypeptide nanoparticles. According to the invention, inflammatory endothelial cells are targeted, local high ROS microenvironment is responded, endothelial barrier repair peptide is released to repair endothelial barrier, complement receptor antagonistic peptide is released to regulate complement cascade reaction so as to recover immune microenvironment, and body inflammation and immune disorder are cooperatively regulated.

Description

Technical Field

[0001] The present invention relates to the technical field of polymer medical materials, and specifically to a three-segment self-assembling composite polypeptide nanoparticle that can repair endothelial barriers and regulate immune recruitment cascades, as well as a preparation method and application thereof. Background Art

[0002] In recent years, there has been an increasing number of studies on the use of nanoparticles to regulate the balance of the body's immune microenvironment. These biomaterials can respond to and adapt to the tissue microenvironment, especially the immune response when implanted in the body. The type and duration of the immune response and the multiple types of cells involved can affect the results of normal or abnormal repair. Injured or damaged tissues quickly release a large number of inflammatory factors, which in turn recruit various immune cells such as granulocytes, monocytes, macrophages and T cells to eliminate inflammation and repair trauma. Nanobiomaterials are targeted to the damaged site of the body, respond intelligently and release therapeutic drugs in real time to regulate the balance of the immune microenvironment. Self-assembled nanoscale structured polypeptide polymers and polypeptide dendrimers show broad prospects in biomedical applications due to their versatility and availability. In the past decade, dendrite / dendritic molecular structures and self-assembled nanostructures have provided researchers with countless possibilities for studying their wide range of applications in biomedicine, such as drug carriers, gene delivery carriers and immunodiagnosis. In addition, self-assembled peptide dendron / dendritic molecular nanostructures have attracted great interest in the development of targeted drugs and peptide drugs due to their molecular multivalency, efficient bioactivity and biocompatibility. They can effectively enhance the ability to resist protein hydrolysis and digestion and prolong the half-life of drugs in the body. Hydrophobic alkyl chains based on C16 motifs are a new type of biocompatible polymer material, which is widely used in drug delivery, gene therapy and biomedical engineering. For example, the ether bond in the C16Ether-PEG-SH molecular chain can improve the swelling and biocompatibility of the molecule in water, making the material have good dispersibility and stability in the body; the long-chain alkyl group can increase the hydrophobicity of the molecule in water, which helps to form a stable nano drug carrier; and the thiol group can covalently bind to a variety of biological molecules (such as proteins, DNA, etc.), thereby achieving efficient drug delivery and effective gene transfection. Since the molecular chain with C16 as the motif has excellent biocompatibility and modifiability, it can be used as an important component of drug delivery systems and gene therapy carriers. In terms of drug delivery, this material can be used to prepare nanomedicines with high drug loading, long circulation time and excellent targeting, thereby effectively improving the efficacy of drugs and reducing side effects.

[0003] Endothelial cells rely on mechanical and humoral factors to maintain endothelial barrier function and avoid inappropriate loss of fluid and solutes. They are an important barrier to maintain the local tissue physiological microenvironment. However, under pathological conditions such as inflammation, endothelial signaling cascades are activated, and endothelial barrier destruction leads to increased vascular permeability and immune cell extravasation. Immune cells mainly cross the endothelial barrier in a paracellular manner through cell-cell junctions. These cell-cell junctions are formed by two major protein complexes: adhesion junctions (AJ) and tight junctions (TJ) of endothelial cells. Endothelial barrier destruction leads to the migration of a large number of immune cells across the endothelial barrier, further aggravating the inflammatory response and forming a vicious cycle between the inflammatory cascade and the destruction of the air-blood barrier. Therefore, the endothelial barrier is crucial to controlling inflammation. Activated endothelial cells highly express relevant adhesion molecules (VCAM-1, ICAM-1), and cell adhesion molecules are very attractive targets in the treatment of inflammatory diseases. Vascular cell adhesion molecule-1 (VCAM-1) is the receptor of integrin VLA 4 (α4β1) on endothelial cells. It is not expressed in endothelial cells under normal conditions. Activated endothelial cells highly express VCAM-1 after inflammatory stimulation to respond to cytokines (such as TNF-α and IL-1β) to mediate the adhesion of leukocytes including lymphocytes and monocytes. Therefore, the use of VCAM-1 binding peptides to target VCAM-1 on endothelial cells (ECs) can achieve the purpose of targeting inflammatory endothelial cells. Various targeting peptides targeting VCAM-1, ICAM-1, E and P-selectin or platelet endothelial cell adhesion molecule (PECAM-1) are described in the literature. The cLABL peptide has previously been shown to have a high affinity for the ICAM-1 receptor on the cell surface. The VCAM-1 affinity peptide described by Fuior et al. successfully targeted liposomes to activated endothelial cells in atherosclerosis and cancer.

[0004] The inflammatory cascade is also closely related to the complement system. Inflammatory endothelial cells highly express related adhesion molecules. The ligands on immune cells bind to the receptors on the endothelial surface and migrate and aggregate to the inflammatory site under the guidance of chemokines. The over-activated effector fragments of the complement system are one of the main initiating factors of inflammatory response damage. The number of complement fragments increases under inflammatory conditions. The C4a, C3a, and C5a fragments produced during complement activation are called complement peptides (anaphylatoxin), among which C5a has the strongest ability and has strong chemotactic activity for neutrophils. C5a factor is the strongest factor that appears in the early stage of acute infection and tissue damage inflammation, and is recognized as a broad-spectrum inflammatory amplifier. Activated complement fragments activate endothelial inflammatory response and immune cell recruitment cascade by binding to receptors on the surface of endothelial cells and immune cells, increasing the expression of adhesion molecules, increasing endothelial permeability, and promoting the inflammatory process. A series of complement peptides bind to complement receptors on immune cells to promote the release of chemokines, and the combination of the two promotes the release of proinflammatory factors and various mediators by immune cells such as neutrophils. This process can be reversed by inhibiting the interaction between complement fragments and their receptors. Complement antagonist peptides bind to complement receptors on the surface of endothelial and immune cells, and C5a fragments in the environment cannot bind to them, preventing the activation of endothelial cells, inhibiting the release of chemokines, and inhibiting proinflammatory factors and proinflammatory mediators. From the perspective of the complement activation pathway, inhibiting the downstream terminal key point of the cascade reaction seems to be a good method, because it can cut off the progress of three channels at the same time. PMX-53 (3D53) is a synthetic peptide and an effective, orally active complement C5a receptor (CD88) antagonist. After binding to the C5a receptor, the complement fragment will not be able to bind, so the receptor antagonist plays a competitive binding role. Summary of the invention

[0005] In order to solve the above technical problems, the first purpose of the present invention is to provide a method for preparing a three-stage self-assembling composite polypeptide nanoparticle that targets and repairs the endothelial barrier and regulates the immune recruitment cascade, the second purpose is to provide the three-stage self-assembling composite polypeptide nanoparticle that targets and repairs the endothelial barrier and regulates the immune recruitment cascade, and the third purpose is to provide its application. By targeting inflammatory endothelial cells, responding to the local high ROS microenvironment, releasing endothelial barrier repair peptides to repair the endothelial barrier, and at the same time releasing complement receptor antagonist peptides to regulate the complement cascade reaction to restore the immune microenvironment, synergistically regulating inflammation and immune disorders in the body.

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: a method for preparing three-stage self-assembling composite polypeptide nanoparticles that target repair of endothelial barrier and regulate immune recruitment cascade, characterized in that the preparation is carried out according to the following steps:

[0007] (1) Preparation of a hydrophilic segment head multifunctional peptide solution, wherein the peptides are C-terminal cysteine-modified adhesion molecule targeting peptide, endothelial barrier repair peptide and complement antagonist peptide, wherein the adhesion molecule targeting peptide sequence is VHPKQHRGGSKGC, the endothelial barrier repair peptide sequence is MPPILRFDHPFLFIIFEEHTQSPIFVGKVVDPTHKC, and the complement antagonist peptide sequence is Ac-Cys-Phe-Orn-Pro-D-Cha-Trp-Arg-OH, and the above-mentioned peptides are added with DMSO to prepare solutions;

[0008] (2) Preparation of the intermediate linker ROS-responsive thioketal solution, using a thioketal (TK) linker with a PEG group and modified with an N-hydroxysuccinimide group (NHS) and a maleimide group (MAL) at both ends, respectively, and dissolving the ester-polyethylene glycol-thioketal-maleimide (NHS-PEG1000-TK-MAL) in a DMSO solution to obtain an intermediate linker ROS-responsive thioketal solution;

[0009] (3) Preparation of a hydrophobic end tail alkyl chain solution, dissolving a hexadecyl chain (C16-GGGKK) with two lysine residues at the end in DMSO to obtain a hydrophobic end tail alkyl chain solution;

[0010] (4) preparing a connection complex between the hydrophobic segment and the middle segment, mixing a solution of an alkyl chain at the tail of the hydrophobic segment with a solution of a ROS-responsive thioketal connector in the middle segment to obtain a connection complex solution between the hydrophobic segment and the middle segment;

[0011] (5) preparing a three-segment complex of a hydrophilic segment, a hydrophobic segment and an intermediate segment, reacting the connection complex solution of the hydrophobic segment and the intermediate segment obtained in step (4) with the prepared three hydrophilic segment head multifunctional peptide solutions respectively to obtain connection complex solutions of three hydrophobic segment, intermediate segment and hydrophilic segment polypeptides;

[0012] (6) The connection complex solutions of the three hydrophobic segment, the middle segment, and the hydrophilic segment polypeptides are mixed, and DMSO is removed by dialysis to allow the complex solution to change from an organic phase to an aqueous phase, thereby promoting the amphiphilic self-assembly of the complex and the co-assembly of the three polypeptide complexes, and freeze-drying to obtain three-segment self-assembled composite polypeptide nanoparticles.

[0013] Adhesion molecule targeting peptide (VC), endothelial barrier repair peptide (MC), and complement antagonist peptide (PMX-53) are all existing peptides purchased from Nanjing Peptide Valley Biotechnology Co., Ltd.

[0014] NHS-PEG1000-TK-MAL, active ester polyethylene glycol thioketal maleimide, also known as active ester-polyethylene glycol-thioketal-maleimide, was purchased from Xi'an Ruixi Biotechnology Co., Ltd.

[0015] C16-GGGKK was purchased from Nanjing Peptide Valley Biotechnology Co., Ltd.

[0016] The PB buffer of the present invention is prepared as follows: 1.20 g of anhydrous sodium dihydrogen phosphate (NaH2PO4, MW 119.98) and 1.42 g of anhydrous sodium dihydrogen phosphate (Na2HPO4, MW 141.96) are weighed and dissolved in ultrapure water, and the ultrapure water is diluted to 1 L to obtain a 10 mM PB buffer, which is filtered through a 0.45 nm filter.

[0017] In the above scheme: the concentration of the multifunctional peptide solution at the head of the hydrophilic segment is 10 mg / ml, the concentration of the ROS-responsive thioketal solution of the middle segment connector is 5 mg / ml, and the concentration of the alkyl chain solution at the hydrophobic end is 1 mg / ml.

[0018] In the above scheme: in step (4), after the alkyl chain solution of the tail of the hydrophobic segment and the ROS-responsive thioacetal solution of the middle segment connector are mixed in equal volumes, the pH is adjusted to 7.2 with triethylamine, and the reaction is carried out on a shaking table at room temperature and in the dark to promote the reaction between the amino group at the tail of the hydrophobic segment and the N-hydroxysuccinimide group of the middle segment connector to obtain a connection complex solution of the hydrophobic segment and the middle segment.

[0019] In the above scheme: in step (5), the volume ratio of the connection complex solution of the hydrophobic segment and the middle segment to the three hydrophilic segment head multifunctional peptide solutions is 2:1. After mixing, the pH is adjusted to 7.2 with triethylamine, and the reaction is carried out on a shaking table at room temperature and in the dark to promote the reaction between the maleimide group of the middle segment connector and the thiol group at the end of the hydrophilic segment head polypeptide.

[0020] In the above scheme: in step (6), the connection complex solutions of the three hydrophobic segment, middle segment and hydrophilic segment polypeptides are mixed, loaded into a dialysis bag with a pore size of 1 kDa, and dialyzed in PB buffer for 3 days to remove excess DMSO reaction solution, allowing the complex solution to change from organic phase to aqueous phase, thereby promoting the amphiphilic self-assembly of the complex and the co-assembly of the three polypeptide complexes.

[0021] In the above scheme: the dialyzed complex is precooled at -20°C for 2 hours, prefrozen at -80°C for 12 hours, and placed in a -80°C freeze dryer to dry, thereby obtaining three-stage self-assembled composite polypeptide nanoparticles.

[0022] A method for preparing three-segment self-assembling composite polypeptide nanoparticles that target endothelial barrier repair and regulate immune recruitment cascades. The three-segment self-assembling composite polypeptide nanoparticles that target endothelial barrier repair and regulate immune recruitment cascades are prepared.

[0023] The three-stage self-assembling composite polypeptide nanoparticles provide a reference template for the self-assembly strategy of polypeptide drugs, effectively enhancing the ability of oligopeptide drugs to resist protein hydrolysis and digestion, and prolonging the half-life of drugs in the body. At the same time, the use of co-assembly combines specific targeting, intelligent response and multi-level treatment, reflecting the concept of precision medicine in modern medicine. The nanoparticles constructed by the present invention can restore the immune microenvironment by regulating the complement cascade reaction, while repairing the endothelial barrier to control inflammation and tissue damage, reflecting the advantages of precise multi-pathway combined treatment of diseases.

[0024] Immune activation, leukocyte infiltration and endothelial barrier destruction lead to increased vascular permeability, which is an important feature of inflammation. In view of the complex inflammatory microenvironment, the composite polypeptide nanoparticles first use adhesion molecule targeting peptides to accurately target activated endothelial cells in the inflammatory state, and then release free functional peptides in response to the high concentration of reactive oxygen in the microenvironment: endothelial barrier repair peptides can promote the remodeling of tight junctions between cells to restore vascular permeability; complement antagonistic peptides downregulate the mediator molecules of the granulocyte transendothelial process, and the two synergistically regulate the immune microenvironment under inflammatory conditions. The nanoparticles described in the present invention have a simple preparation process, and the products have good stability, dispersibility and uniformity. They have the advantages of good biocompatibility, precise targeting ability and synergistic regulation of the immune microenvironment, realizing the organic combination of synthetic polypeptide drugs and biological targeting, and providing a highly promising new treatment platform for the field of precision medicine.

[0025] A method for preparing a drug for treating acute inflammation-related diseases using the three-segment self-assembling composite polypeptide nanoparticles that target and repair the endothelial barrier and regulate the immune recruitment cascade. The three-segment composite intelligent polypeptide nanoparticles exhibit excellent endothelial cell targeting ability, effectively restore the damaged endothelial barrier, and can also exert synergistic anti-oxidation and anti-inflammatory therapeutic effects.

[0026] Acute inflammatory diseases are acute inflammatory diseases with endothelial barrier damage and immune imbalance as the dominant factors.

[0027] Acute inflammatory-related diseases are diseases such as severe sepsis or acute lung injury or acute respiratory distress syndrome or acute liver failure or acute kidney injury.

[0028] Beneficial effects: (1) The innermost layer of the three-segment composite intelligent polypeptide nanoparticles of the present invention is a hydrophobic end constructed by an alkyl chain, the middle layer is a thioketal (TK) bond, and the outermost layer is a hydrophilic end constructed by a functional polypeptide. The raw materials used are cheap and easy to obtain. The method utilizes the principle of amphiphilicity of polypeptides and covalent connection between molecules. The method is simple to operate, has good repeatability, and is less time-consuming. Granular materials with a diameter of nanometers can be obtained.

[0029] (2) This method enables multi-component peptide molecules to be covalently linked within the same particle, effectively addressing the complex and ever-changing inflammatory environment from multiple angles and effectively improving the efficacy of the material.

[0030] (3) The three-segment composite intelligent polypeptide nanoparticle material prepared by the present invention utilizes the pathological specificity under inflammatory conditions and targets the adhesion molecules highly expressed in endothelial cells to exert a targeting effect and precisely regulate the various functional effects of inflammatory endothelial cells.

[0031] (4) The three-segment composite intelligent polypeptide nanoparticle material prepared by the present invention can precisely regulate the different roles of endothelial cells in participating in inflammation. It can promote the formation of tight junction proteins to repair damaged endothelial barriers, play the role of antioxidants to neutralize the excessive reactive oxygen species produced in the inflammatory response, and competitively bind to complement receptors with complement fragments to regulate the excessive release of chemokines, proinflammatory factors and various mediators. It reflects the advantages of precise multi-pathway combined treatment of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the design and preparation of three-segment self-assembling composite polypeptide nanoparticles.

[0033] Figure 2 The co-assembly characterization of the prepared three-segment self-assembling composite polypeptide nanoparticles. A. Molecular dynamics simulation of the mixed system of each group of polypeptide components (1:1:1). B. Critical micelle concentration detection. C. Atomic force microscopy. DF. Co-assembly behavior of nanoparticles.

[0034] Figure 3 The ROS response and stability characterization of the prepared three-stage self-assembled composite polypeptide nanoparticles, including A. Schematic diagram of H2O2 response of composite polypeptide nanoparticles. B. TEM, C. SEM, D. DLS, E. Zata potential detection, F. FTIR detection, G. Nile red fluorescence detection of H2O2 responsiveness of composite polypeptide particles, H. Stability, I. ROS response, disintegration of polypeptide nanoparticles to release polypeptides, K. In vitro scavenging of hydroxyl radicals, I. In vitro scavenging of superoxide anions.

[0035] Figure 4 The in vitro and in vivo targeting performance of the three-segment self-assembled composite polypeptide nanoparticles. A is an in vitro immunofluorescence experiment to detect the uptake of nanoparticles by HUVEC cells. BC are in vivo small animal live imaging and main tissue immunofluorescence experiments to observe the main organ distribution of nanoparticles.

[0036] Figure 5 A is the anti-inflammatory and anti-apoptotic properties of the three-segment self-assembled composite polypeptide nanoparticles. A-BDCFH-DA fluorescence and flow cytometry were used to detect the performance of the nanoparticles in neutralizing ROS. C was used to detect the anti-apoptotic properties of the nanoparticles by flow cytometry. D was used to detect the expression of inflammatory factors by qPCR.

[0037] Figure 6 The performance of three-segment self-assembled composite polypeptide nanoparticles in repairing the endothelial barrier in vitro, where A is an endothelial cell leakage experiment, B is a Western Blotting experiment to detect the formation of tight junction proteins and its quantitative graph, and C is an immunofluorescence experiment to detect the expression of tight junction proteins.

[0038] Figure 7 The performance of the three-segment self-assembled composite polypeptide nanoparticles in repairing the endothelial barrier in vivo, where A is the result of immunohistochemistry of tight junction protein, B is the result and quantitative diagram of Western Blotting of tight junction protein, and C is the quantitative dry-wet specific gravity of lung tissue.

[0039] Figure 8 The three-section self-assembled composite polypeptide nanoparticles have anti-inflammatory properties in vivo, where A is an immunofluorescence experiment to detect the infiltration of inflammatory cells, and B is a qPCR test to detect the anti-inflammatory effect of the nanoparticles in vivo. DETAILED DESCRIPTION

[0040] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0041] Example 1

[0042] 1. Adhesion molecule targeting peptide self-assembled nanoparticles

[0043] Preparation of the multifunctional peptide solution at the head of the hydrophilic segment: 10 mg of the C-terminal cysteine-modified adhesion molecule targeting peptide (sequence: VHPKQHRGGSKGC, abbreviated as: VC) was weighed and dissolved in 1 ml of DMSO to obtain a 10 mg / ml adhesion molecule targeting peptide solution.

[0044] Preparation of the intermediate linker ROS-responsive thioketal solution: Weigh 5 mg of the thioketal (TK) linker part (NHS-PEG1000-TK-MAL) with a PEG group and modified NHS groups and MAL groups at both ends, and dissolve it in 1 ml of DMSO solution to obtain a 5 mg / ml intermediate linker thioketal solution.

[0045] Preparation of hydrophobic end tail alkyl chain solution: Weigh 1 mg of a hexadecyl chain (C16-GGGKK) with two lysine residues at the end and dissolve it in 1 ml of DMSO solution to obtain a 1 mg / ml hydrophobic end tail alkyl chain solution.

[0046] Mix 1 ml of the hydrophobic segment tail alkyl chain solution with 1 ml of the middle segment linker thioketal solution (equal volumes v / v), adjust the pH to 7.2 with triethylamine, shake in a room temperature and dark incubator for 24 h to obtain 2 ml of the hydrophobic segment and middle segment connection complex solution.

[0047] Add 1 ml of the prepared adhesion molecule targeting peptide solution, adjust the pH to 7.2 with triethylamine again, shake at room temperature and away from light for 24 hours to obtain 3 ml of a connection complex solution of the hydrophobic segment, the middle segment, and the hydrophilic segment polypeptides.

[0048] 2. Endothelial barrier repair peptide self-assembled nanoparticles

[0049] Preparation of the hydrophilic segment head multifunctional peptide solution: Weigh the C-terminal cysteine-modified endothelial barrier repair peptide (endothelial barrier repair peptide (sequence:

[0050] MPPILRFDHPFLFIIFEEHTQSPIFVGKVVDPTHKC, abbreviated as: MC) 10 mg was dissolved in 1 ml DMSO to obtain a 10 mg / ml endothelial barrier repair peptide solution.

[0051] Preparation of the intermediate linker ROS-responsive thioketal solution: Weigh 5 mg of the thioketal (TK) linker part (NHS-PEG1000-TK-MAL) with a PEG group and modified NHS groups and MAL groups at both ends, and dissolve it in 1 ml of DMSO solution to obtain a 5 mg / ml intermediate linker thioketal solution.

[0052] Preparation of hydrophobic end tail alkyl chain solution: Weigh 1 mg of a hexadecyl chain (C16-GGGKK) with two lysine residues at the end and dissolve it in 1 ml of DMSO solution to obtain a 1 mg / ml hydrophobic end tail alkyl chain solution.

[0053] Mix 1 ml of the hydrophobic segment tail alkyl chain solution with 1 ml of the middle segment linker thioketal solution (equal volumes v / v), adjust the pH to 7.2 with triethylamine, shake in a room temperature and dark incubator for 24 h to obtain 2 ml of the hydrophobic segment and middle segment connection complex solution.

[0054] Add 1 ml of the prepared endothelial barrier repair peptide solution, adjust the pH to 7.2 with triethylamine again, shake at room temperature and away from light for 24 hours to obtain 3 ml of a connection complex solution of the hydrophobic segment, the middle segment, and the hydrophilic segment polypeptides.

[0055] 3. Complement antagonist peptide self-assembled nanoparticles

[0056] Preparation of the multifunctional peptide solution at the head of the hydrophilic segment: 10 mg of a C-terminal cysteine-modified complement antagonist peptide (sequence: Ac-Cys-Phe-Orn-Pro-D-Cha-Trp-Arg-OH (Lactam bridge: Orn-2 to Arg-6, abbreviated as: PMX-53) was weighed and dissolved in 1 ml of DMSO to obtain a 10 mg / ml complement antagonist peptide solution.

[0057] Preparation of the intermediate linker ROS-responsive thioketal solution: Weigh 5 mg of the thioketal (TK) linker part (NHS-PEG1000-TK-MAL) with a PEG group and modified NHS groups and MAL groups at both ends, and dissolve it in 1 ml of DMSO solution to obtain a 5 mg / ml intermediate linker thioketal solution.

[0058] Preparation of hydrophobic end tail alkyl chain solution: Weigh 1 mg of a hexadecyl chain (C16-GGGKK) with two lysine residues at the end and dissolve it in 1 ml of DMSO solution to obtain a 1 mg / ml hydrophobic end tail alkyl chain solution.

[0059] Mix 1 ml of the hydrophobic segment tail alkyl chain solution with 1 ml of the middle segment linker thioketal solution (equal volumes v / v), adjust the pH to 7.2 with triethylamine, shake in a room temperature and dark incubator for 24 h to obtain 2 ml of the hydrophobic segment and middle segment connection complex solution.

[0060] Add 1 ml of the prepared complement antagonist peptide solution, adjust the pH to 7.2 with triethylamine again, shake at room temperature and in the dark for 24 hours to obtain 3 ml of a connection complex solution of the hydrophobic segment, the middle segment, and the hydrophilic segment polypeptides.

[0061] 4. Mix the connection complex solutions of the three hydrophobic segment, middle segment and hydrophilic segment polypeptides, put them into a dialysis bag with a pore size of 1 kDa, dialyze in PB buffer for 3 days, remove excess DMSO reaction solution, collect the dialyzed complex solution into a centrifuge tube, pre-cool at -20°C for 2h, pre-freeze at -80°C for 12h, and put it into a -80°C freeze dryer to form three-segment self-assembled composite polypeptide nanoparticles.

[0062] from Figure 2 It can be seen that the composite polypeptide nanoparticles of the three polypeptides exhibit excellent self-assembly and co-assembly performance in the self-assembly simulation, and can effectively achieve self-assembly and uniform co-assembly in PB buffer in vitro.

[0063] from Figure 3 It can be seen that the three-segment self-assembled composite polypeptide nanoparticles have a particle size of about 50nm and a positive charge on the surface. The nanoparticles respond to ROS in vitro, causing the intermediate connector of the nanoparticles to break, releasing three functional peptides, and then the particle size becomes smaller, the potential becomes smaller, and the hydroxyl radicals and superoxide anions in the in vitro environment are effectively removed during the response process.

[0064] In the present invention, the three-segment self-assembling composite polypeptide nanoparticles can exert a biological effect of synergistically regulating inflammatory diseases. Figure 4As shown, the endothelial barrier constructed by HUVEC cells was selected as an in vitro research model to detect the uptake of nanoparticles by HUVEC cells under inflammatory conditions; the IVIS imaging system and immunofluorescence experiments were used to observe the main distribution of nanoparticles after airway administration; the results all indicated that the three-segment self-assembled composite polypeptide nanoparticles can target the local inflammatory environment under the mediation of VCAM-1.

[0065] like Figure 5 As shown, the endothelial barrier constructed by HUVEC cells was selected as an in vitro research model. Using the DCFH-DA probe and flow cytometry, it was detected that the three-segment self-assembled composite polypeptide nanoparticles significantly neutralized the excessively produced ROS and exerted a good anti-apoptotic effect; through qPCR experiments, it was found that the three-segment self-assembled composite polypeptide nanoparticles can significantly down-regulate the expression of inflammatory factors, indicating that the three-segment self-assembled composite polypeptide nanoparticles of the present invention can protect cells by neutralizing ROS and anti-apoptosis. Figure 6 As shown, the endothelial barrier constructed by HUVEC cells was selected as an in vitro research model. According to the previous preliminary experiment, LPS at a concentration of 1ug / ml was selected as the inflammatory stimulus, and nanoparticles at a concentration of 20ug / ml were added to the treatment group at the same time. Using FITC-Dextran, it was detected that the three-segment self-assembled composite polypeptide nanoparticles can restore the permeability balance of the inflammatory damaged endothelial barrier; through Western Blotting and immunofluorescence experiments, it was observed that the three-segment self-assembled composite polypeptide nanoparticles can significantly increase the expression level of the main components of tight junctions in the endothelial barrier (ZO-1, Claudin-5, Occludin), indicating that the three-segment self-assembled composite polypeptide nanoparticles of the present invention can significantly upregulate the expression of tight junctions and repair the endothelial barrier damaged by inflammatory stimulation.

[0066] like Figure 7 As shown in the figure, an acute lung injury mouse model was established by airway instillation of LPS (dose: 10 mg / kg), and at the same time, the three-segment self-assembled composite polypeptide nanoparticles (15 mg / kg) were also given to the model mice by airway instillation. After 24 hours, the lung tissue was sampled for functional verification, and it was found that the three-segment self-assembled composite polypeptide nanoparticles had good endothelial barrier repair performance in vivo. Tissue immunofluorescence results are shown in the figure. Figure 8 The number of resident macrophages in the lung tissue of the model mice shown in A decreased significantly, and was replaced by explosive aggregation of neutrophils. The three-segment self-assembled composite polypeptide nanoparticles can play an immunomodulatory role and balance the infiltration of immune cells. The tissue qPCR results are shown in Figure 8 As shown in Figure B, the three-segment composite polypeptide nanoparticles can alleviate the LPS-induced cytokine storm, and the expression of inflammatory factors in the body is significantly downregulated.

[0067] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing three-stage self-assembling composite polypeptide nanoparticles that target endothelial barrier repair and regulate immune recruitment cascade, characterized in that: Prepare according to the following steps: (1) Preparation of a hydrophilic segment head multifunctional peptide solution, wherein the peptides are C-terminal cysteine-modified adhesion molecule targeting peptide, endothelial barrier repair peptide and complement antagonist peptide, wherein the adhesion molecule targeting peptide sequence is VHPKQHRGGSKGC, the endothelial barrier repair peptide sequence is MPPILRFDHPFLFIIFEEHTQSPIFVGKVVDPTHKC, and the complement antagonist peptide sequence is Ac-Cys-Phe-Orn-Pro-D-Cha-Trp-Arg-OH, and the above-mentioned peptides are added with DMSO to prepare solutions; (2) Preparation of the intermediate linker ROS-responsive thioketal solution, using a thioketal (TK) linker with a PEG group and modified with an N-hydroxysuccinimide group (NHS) and a maleimide group (MAL) at both ends, respectively, and dissolving the ester-polyethylene glycol-thioketal-maleimide (NHS-PEG1000-TK-MAL) in a DMSO solution to obtain an intermediate linker ROS-responsive thioketal solution; (3) Preparation of a hydrophobic end tail alkyl chain solution, dissolving a hexadecyl chain (C16-GGGKK) with two lysine residues at the end in DMSO to obtain a hydrophobic end tail alkyl chain solution; (4) preparing a connection complex between the hydrophobic segment and the middle segment, mixing a solution of an alkyl chain at the tail of the hydrophobic segment with a solution of a ROS-responsive thioketal connector in the middle segment to obtain a connection complex solution between the hydrophobic segment and the middle segment; (5) preparing a three-segment complex of a hydrophilic segment, a hydrophobic segment and an intermediate segment, reacting the connection complex solution of the hydrophobic segment and the intermediate segment obtained in step (4) with the prepared three hydrophilic segment head multifunctional peptide solutions respectively to obtain connection complex solutions of three hydrophobic segment, intermediate segment and hydrophilic segment polypeptides; (6) The connection complex solutions of the three hydrophobic segment, the middle segment, and the hydrophilic segment polypeptides are mixed, and DMSO is removed by dialysis to allow the complex solution to change from an organic phase to an aqueous phase, thereby promoting the amphiphilic self-assembly of the complex and the co-assembly of the three polypeptide complexes, and freeze-drying to obtain three-segment self-assembled composite polypeptide nanoparticles.

2. The method for preparing the three-stage self-assembling composite polypeptide nanoparticles for targeted repair of endothelial barrier and regulation of immune recruitment cascade according to claim 1, characterized in that: The concentration of the multifunctional peptide solution at the head of the hydrophilic segment was 10 mg / ml, the concentration of the ROS-responsive thioketal solution at the middle segment linker was 5 mg / ml, and the concentration of the alkyl chain solution at the hydrophobic end was 1 mg / ml.

3. The method for preparing the three-stage self-assembling composite polypeptide nanoparticles for targeted repair of endothelial barrier and regulation of immune recruitment cascade according to claim 2, characterized in that: In step (4), after the alkyl chain solution of the tail of the hydrophobic segment and the ROS-responsive thioacetal solution of the middle segment connector are mixed in equal volumes, the pH is adjusted to 7.2 with triethylamine, and the reaction is carried out on a shaking table at room temperature and in the dark to promote the reaction between the amino group of the tail of the hydrophobic segment and the N-hydroxysuccinimide group of the middle segment connector to obtain a connection complex solution of the hydrophobic segment and the middle segment.

4. The method for preparing the three-stage self-assembling composite polypeptide nanoparticles for targeted repair of endothelial barrier and regulation of immune recruitment cascade according to claim 3, characterized in that: In step (5), the volume ratio of the connection complex solution of the hydrophobic segment and the middle segment to the three hydrophilic segment head multifunctional peptide solutions is 2:

1. After mixing, the pH is adjusted to 7.2 with triethylamine, and the reaction is carried out on a shaker at room temperature and in the dark to promote the reaction between the maleimide group of the middle segment connector and the thiol group at the end of the hydrophilic segment head polypeptide.

5. The method for preparing the three-stage self-assembling composite polypeptide nanoparticles for targeted repair of endothelial barrier and regulation of immune recruitment cascade according to claim 4, characterized in that: In step (6), the connection complex solutions of the three hydrophobic segment, middle segment, and hydrophilic segment polypeptides are mixed, loaded into a dialysis bag with a pore size of 1 kDa, and dialyzed in PB buffer for 3 days to remove excess DMSO reaction solution and allow the complex solution to change from an organic phase to an aqueous phase, thereby promoting the amphiphilic self-assembly of the complex and the co-assembly of the three polypeptide complexes.

6. The method for preparing the three-stage self-assembling composite polypeptide nanoparticles for targeted repair of endothelial barrier and regulation of immune recruitment cascade according to claim 5, characterized in that: The dialyzed complex was precooled at -20°C for 2 hours, prefrozen at -80°C for 12 hours, and dried in a -80°C freeze dryer to obtain three-stage self-assembled composite polypeptide nanoparticles.

7. A three-segment self-assembling composite polypeptide nanoparticle targeted to repair the endothelial barrier and regulate the immune recruitment cascade prepared by the method for preparing the three-segment self-assembling composite polypeptide nanoparticle targeted to repair the endothelial barrier and regulate the immune recruitment cascade as described in any one of claims 1 to 6.

8. Use of the three-segment self-assembling composite polypeptide nanoparticles according to claim 7 that target the repair of endothelial barrier and regulate the immune recruitment cascade in the preparation of drugs for treating acute inflammatory-related diseases.

9. The use according to claim 8, characterized in that: Acute inflammatory diseases are acute inflammatory diseases with endothelial barrier damage and immune imbalance as the dominant factors.

10. The use according to claim 9, characterized in that: The acute inflammation-related disease is severe sepsis or acute lung injury or acute respiratory distress syndrome or acute liver failure or acute kidney injury.

Images