Protein degradation agent based on endosome-phagosome connection assembly and application thereof

Abstract

The invention discloses a protein degradation agent based on an endosome-phagosome connection assembly and application of the protein degradation agent, and belongs to the technical field of protein degradation and nano biological medicine. According to the endosome-phagosome connecting assembly, by inserting an endosome membrane and marking endosome degradation protein, a special degradation technology strategy enables the endosome-phagosome connecting assembly to expand an autophagy degradation target spectrum in traditional cytoplasm to various target proteins outside a cell membrane / cell, and the current development situation of a current targeted protein degradation tool can be thoroughly changed possibly. The preparation method is simple, raw materials are easy to obtain, the method is suitable for research and application transformation, the traditional degradation thought is broken through by utilizing an endosome-phagosome connecting assembly, most target proteins can be degraded through an endosome-autophagy-lysosome approach, the design is novel, the application range is wide, the cost is low, the efficiency is high, a very good degradation effect can be achieved, and the method is suitable for popularization and application. The method can be applied to the field of various complex diseases and has huge potential development and transformation prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein degradation and nano-biomedicine, and in particular relates to a protein degradation agent based on an endosome-phagosome junction assembly and an application thereof. Background Art

[0002] In eukaryotic cells, damaged proteins or organelles can be cleared by proteasomes or lysosomes. Proteasomes eliminate short-lived proteins and soluble misfolded proteins through the ubiquitin-proteasome system (UPS). In contrast, lysosomes are responsible for degrading long-lived proteins, insoluble protein aggregates, and even entire organelles, macromolecular compounds, and intracellular parasites (such as certain bacteria) through endocytosis, phagocytosis, or autophagy. Targeted protein degradation (TPD) through the proteasome and lysosome pathways is a new tool for exploring cellular pathways and a promising therapeutic approach. TPD strategies that rely on the UPS, such as protein degradation targeting chimeras (PROTACs), molecular glues, degradation tags (dTAGs), etc., mainly degrade intracellular proteins; TPD strategies that rely on lysosomes, such as lysosome targeting chimeras (LYTACs), use specific lysosomal transport proteins to degrade membrane proteins, extracellular proteins and protein aggregates; at the same time, degradation technologies based on the traditional autophagy-lysosome pathway, such as autophagosome tethering complexes (ATTECs) and autophagy targeting chimeras (AUTACs), anchor targets and recruit autophagic vesicles to deliver specific targets to autophagolysosomes for degradation, mainly targeting cytoplasmic components (proteins or non-proteins).

[0003] However, degradation strategies focused on membrane / extracellular specific target proteins face a series of problems and challenges. First, the construction of LYTAC molecules with lysosomal targeting functions usually requires complex design and synthesis. Second, the degradation effect is highly dependent on the expression level and tissue distribution of lysosomal targeted transport receptors (such as the cation-independent mannose-6-phosphate receptor CI-M6PR and the insulin-like growth factor 2 receptor IGF2R). Third, degradation through bifunctional chimeras may be interfered with by potential "hook" effects. Therefore, a new technology paradigm with simple design and universality is urgently needed to upgrade the technology platform for targeted protein degradation of membrane / extracellular specific target proteins. Summary of the Invention

[0004] To solve the above technical problems, the present invention has developed a new protein degradation tool, which is an endosome-phagosome connection assembly. The degradation strategy is to degrade cell membranes and / or extracellular proteins by inserting into the endosomal membrane, marking endosomes, recruiting phagosomes and the "endosome-phagosome-lysosome" pathway.

[0005] The first object of the present invention is to provide a protein degradation agent, which comprises an assembly, wherein monomers forming the assembly contain the following parts connected in a connected manner: Target protein (protein to be degraded) specific ligand; phagosome-targeting peptides; The endosomal membrane inserting polypeptide is located between the target protein-specific ligand and the phagosome targeting polypeptide, and the endosomal membrane inserting polypeptide is a C-terminal membrane inserting polypeptide (i.e., after insertion into the endosomal membrane, the C-terminus is located on the cytoplasmic side and the N-terminus is located on the endosomal cavity side).

[0006] Furthermore, the target protein ligand is located at the C-terminus of the endosomal membrane-inserting polypeptide.

[0007] Furthermore, at least one of the following is included: (1) The target protein-specific ligand is chemically linked to the endosomal membrane-inserting polypeptide; (2) the phagosome targeting polypeptide is chemically linked to the endosomal membrane inserting polypeptide; (3) The monomer contains a hydrophilic modification; (4) The target protein is an extracellular protein or a cell membrane protein; (5) The target protein-specific ligand includes a target protein-specific polypeptide ligand.

[0008] Furthermore, at least one of the following is included: (1) The target protein-specific ligand is covalently linked to the endosomal membrane-inserting polypeptide; The target protein-specific ligand and the endosomal membrane inserting polypeptide can be linked in any manner. For example, when the target protein-specific ligand is a polypeptide ligand, they can be directly linked. Preferably, the target protein-specific ligand and the endosomal membrane inserting polypeptide are linked via a covalent bond formed by an amino group and a thiol group (that is, the target protein-specific ligand is modified with a thiol group or an amino group, and the endosomal membrane inserting polypeptide is modified with a thiol group or an amino group, and the modifications on the two groups are different); Most preferably, the endosomal membrane inserting polypeptide is modified with maleimide, and the target protein specific ligand is modified with a thiol-containing amino acid (such as cysteine), and the connection is achieved by an addition reaction in the presence of an organic solvent; the organic solvent is a solvent that can dissolve the polypeptide, including but not limited to N , N -Dimethylformamide, dimethyl sulfoxide, etc.; (2) the phagosome targeting polypeptide is covalently linked to the endosomal membrane inserting polypeptide; The phagosome targeting polypeptide and the endosomal membrane inserting polypeptide can be linked in any manner, such as by reaction with a modifying group. Preferably, the phagosome targeting polypeptide and the endosomal membrane inserting polypeptide are linked via a peptide bond. (3) The monomer is modified with a hydrophilic group; Preferably, the source of the hydrophilic group includes but is not limited to hydrophilic amino acids; Preferably, the hydrophilic group includes but is not limited to hydroxyl, carboxyl, amino, and amide groups; Preferably, the hydrophilic group is modified on the target protein-specific ligand; more preferably, the hydrophilic group is modified on one end of the target protein-specific ligand close to the endosomal membrane-inserting polypeptide.

[0009] Furthermore, at least one of the following is included: (1) The amino acid sequence of the endosomal membrane inserting polypeptide is shown in SEQ ID NO.5; (2) The amino acid sequence of the phagosome targeting polypeptide is shown in SEQ ID NO. 4; (3) The target protein includes but is not limited to one or more of programmed death receptor-ligand 1, beta-amyloid protein, advanced glycation end product ligand, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), prostate-specific membrane antigen (PAMA), and mitochondrial p32 molecular chaperone protein (p32); Preferably, when the target protein includes programmed death receptor-ligand 1, the target protein-specific ligand includes a programmed death receptor-ligand 1-specific polypeptide ligand, and the amino acid sequence of the programmed death receptor-ligand 1-specific polypeptide ligand is shown in SEQ ID NO.1; Preferably, when the target protein includes β-amyloid protein, the target protein-specific ligand includes a β-amyloid protein-specific polypeptide ligand, and the amino acid sequence of the β-amyloid protein-specific polypeptide ligand is shown in SEQ ID NO.2; Preferably, when the target protein includes an advanced glycation ligand end product, the target protein-specific ligand includes an advanced glycation ligand end product-specific polypeptide ligand, and the amino acid sequence of the advanced glycation ligand end product-specific polypeptide ligand is shown in SEQ ID NO.3.

[0010] The present invention discloses an endosome-phagosome junction assembly, which achieves targeted degradation of extracellular proteins by binding to target proteins, triggering endocytosis, inserting into endosomal membranes, labeling endosomes, recruiting phagosomes, and utilizing the "endosome-phagosome-lysosome" pathway. One end of a polypeptide that can be inserted into a lipid membrane in an acidic environment is functionalized with maleimide, and the other end is functionalized with a phagosome targeting peptide to construct an endosomal anchored phagosome targeting peptide. The specific ligand of the target protein is functionalized with histidine to increase hydrophilicity, and is also functionalized with cysteine. The endosomal anchored phagosome targeting peptide and the ligand of the target protein are connected through the addition reaction of maleimide and thiol and self-assembled to form an endosomal-phagosome junction assembly. The endosome-phagosome junction assembly specifically recognizes and binds to the target protein, entering the cell's endosomes through the target protein's constitutive endocytosis or receptor-mediated propagation into the cell. In the acidic environment of the endosomal body, the endosome-phagosome junction assembly inserts its membrane-inserting peptide into the endosomal lipid membrane, anchoring the terminal phagosome targeting peptide to the endosomal surface, recruiting phagosomes in the cytoplasm to engulf the endosome and degrading it using the autophagy-lysosome pathway. By degrading PD-L1 and the receptor for advanced glycation end products RAGE on the cell membrane, as well as extracellular amyloid protein Aβ, the endosome-phagosome junction assembly proposed for the first time in this invention has been effectively demonstrated to have the ability to efficiently utilize the "endosome-autophagy-lysosome" pathway to target and degrade cell membrane proteins, especially extracellular proteins.

[0011] The second object of the present invention is to provide a method for preparing the protein degradation agent, which includes the preparation of an assembly, and the preparation of the assembly includes the following steps: dispersing the monomers in an aqueous solution at a set concentration, and the monomers self-assembly to form the assembly.

[0012] Furthermore, the set concentration is not lower than the critical concentration for micelle formation. For example, when the protein degrader is a degrader of programmed death receptor-ligand 1, the critical concentration for micelle formation is 1.3-4.2 nM. Of course, other options can be determined based on existing technology to determine the corresponding "critical concentration for micelle formation."

[0013] Furthermore, a phagosome targeting polypeptide and an endosomal membrane-inserting polypeptide (including modifications) are first prepared in a connected manner to obtain an endosomal-anchored phagosome targeting peptide, and a target protein-specific ligand (including modifications) is prepared; when preparing a monomer, the endosomal-anchored phagosome targeting peptide and the target protein-specific ligand are mixed and reacted in the presence of an organic solvent.

[0014] Furthermore, the molar ratio of the endosomal-anchored phagosome targeting peptide to the target protein-specific ligand is 1:(1-20).

[0015] Furthermore, after the self-assembly is completed, a purification step is also included, such as dialysis to obtain a purified assembly.

[0016] The third object of the present invention is to provide the use of the protein degrading agent or the protein degrading agent obtained by the preparation method in the preparation of preventive or therapeutic drugs.

[0017] Furthermore, the medical uses of the preventive or therapeutic drugs include at least one of the following: prevention or treatment of solid tumors (such as breast cancer, lung cancer, liver cancer, gastric cancer, kidney cancer, colon cancer, colorectal cancer, etc.), prevention or treatment of neurological diseases (such as Alzheimer's disease), prevention or treatment of inflammatory diseases (such as diabetes and its vascular complications, cardiovascular diseases, etc.).

[0018] Furthermore, the prevention or treatment is achieved by degrading abnormal proteins, and the degradation is achieved through the endosomal-autophagy-lysosomal pathway.

[0019] The fourth object of the present invention is to provide a pharmaceutical composition comprising the protein degrader or the protein degrader obtained by the preparation method.

[0020] Furthermore, the pharmaceutical composition is an anti-tumor pharmaceutical composition.

[0021] By means of the above solution, the present invention has at least the following advantages: The present invention discloses an endosome-phagosome ligation assembly, which achieves targeted degradation of cell membrane proteins, especially extracellular proteins, by binding to target proteins, triggering endocytosis, inserting into the endosomal membrane, labeling endosomes, recruiting phagosomes, and utilizing the "endosome-phagosome-lysosome" pathway. By degrading PD-L1 and the receptor for advanced glycation end products RAGE on the cell membrane and the extracellular amyloid protein Aβ, it is effectively demonstrated that the endosome-phagosome ligation assembly proposed for the first time in the present invention has the ability to efficiently utilize the "endosome-autophagy-lysosome" pathway to target and degrade extracellular proteins. This degradation technology strategy based on the endosome-phagosome ligation assembly can expand the target spectrum of autophagic degradation to a variety of target proteins on the cell membrane / extracellularly, which may completely change the current development status of targeted protein degradation tools; the preparation method of the assembly is simple, which may have a profound impact on biomedical research and clinical treatment, and has potential huge development and transformation prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings.

[0023] Figure 1 This is a schematic diagram of the construction of the endosome-phagosome junction assembly and its technical tools for inserting into the endosomal membrane and labeling endosomes to degrade cell membrane / extracellular proteins, as described in the present invention, and its intracellular behavior.

[0024] Figure 2Quantitative graphs of the hemolytic efficiency of different endosomally anchored phagosome-targeting peptides with a maleimide at one end and a phagosome-targeting peptide at the other end, as well as the effect of different end insertions of acidic membrane-inserted peptides on degradation.

[0025] Figure 3 The particle size data of different assemblies were characterized by dynamic light scattering.

[0026] Figure 4 The polydispersity index data of different assemblies were characterized by dynamic light scattering.

[0027] Figure 5 The zeta potential data of different assemblies were characterized by dynamic light scattering.

[0028] Figure 6 The graphs showing the changes in the electrokinetic potential of different assemblies within one week after preparation were characterized by dynamic light scattering.

[0029] Figure 7 Quantitative graph of the critical micelle concentration of different assemblies determined by pyrene fluorescence.

[0030] Figure 8 The whole protein distribution diagram of the protein corona formed by different assemblies and mouse plasma was characterized by SDS-PAGE.

[0031] Figure 9 This is a quantitative graph of the protein concentration of the protein corona formed by different assemblies and mouse plasma measured by a full-wavelength microplate reader.

[0032] Figure 10 Quantitative graph of the zeta potential of the protein corona formed by different assemblies with mouse plasma measured by dynamic light scattering.

[0033] Figure 11 Confocal microscopy images and quantitative graphs of the co-localization of PD-L1 and phagosomes in HEK293T cells after treatment with different assemblies.

[0034] Figure 12 Confocal microscopy images and quantitative graphs of the co-localization of PD-L1 and lysosomes in HEK293T cells after treatment with different assemblies.

[0035] Figure 13 Confocal microscopy images and quantitative graphs of the co-localization of early endosomes and phagosomes in HEK293T cells after treatment with different assemblies.

[0036] Figure 14 Confocal microscopy images and quantitative graphs of the co-localization of early endosomes and lysosomes in HEK293T cells after treatment with different assemblies.

[0037] Figure 15 Confocal microscopy images and quantitative graphs of the localization of PD-L1 and phagosomes at different times in HEK293T cells after treatment with the PD-L1 and phagosome dual-targeting assembly.

[0038] Figure 16 Confocal microscopy images and quantitative graphs of the localization of PD-L1 and lysosomes at different times in HEK293T cells after treatment with the PD-L1 and phagosome dual-targeting assembly.

[0039] Figure 17 Confocal microscopy images and quantitative graphs of the localization of early endosomes and phagosomes at different times in HEK293T cells after treatment with PD-L1 and phagosome dual-targeting assembly.

[0040] Figure 18 Confocal microscopy images and quantitative graphs of the localization of early endosomes and lysosomes at different times in HEK293T cells after treatment with PD-L1 and phagosome dual-targeting assembly.

[0041] Figure 19 Western blot analysis was used to characterize the expression of PD-L1 in 4T1 cells after treatment with different concentrations of PD-L1 and phagosome dual-targeting assembly.

[0042] Figure 20 The expression of PD-L1 in 4T1 cells after treatment with different concentrations of PD-L1 single-targeting assembly was characterized by western blot.

[0043] Figure 21 The expression of PD-L1 in 4T1 cells after treatment with PD-L1 and phagosome dual-targeting assembly for different time periods was characterized by western blot.

[0044] Figure 22 The expression of PD-L1 in 4T1 cells after treatment with PD-L1 single-targeting assembly for different time periods was characterized by western blot.

[0045] Figure 23 The expression of PD-L1 in 4T1 cells after treatment with different assemblies was characterized by western blot at a concentration of 0.5 μM for 12 h.

[0046] Figure 24 The figure is a quantitative graph of PD-L1 expression after 4T1 cells were treated with different inhibitors and PD-L1 and phagosome dual-targeting assembly by western blot.

[0047] Figure 25Confocal microscopy images of calcein leakage after cells were treated with PD-L1-associated assemblies.

[0048] Figure 26 Characterization of RAGE upregulation in mouse brain microvascular endothelial cells.

[0049] Figure 27 The figure shows the quantitative expression of RAGE protein after treating mouse brain endothelial cells with RAGE-related assemblies.

[0050] Figure 28 Characterization of RAGE upregulation in BV-2 cells.

[0051] Figure 29 Quantitative graph of Aβ uptake by microglia after treatment with different concentrations of Aβ single-targeting assembly.

[0052] Figure 30 Quantitative graph of Aβ uptake by microglia after treating cells with Aβ single-targeting assembly for different time periods.

[0053] Figure 31 The figure shows the quantitative expression of Aβ and RAGE proteins in microglia treated with Aβ-related assemblies.

[0054] Figure 32 Confocal microscopy images of the localization of FITC-labeled Aβ and phagosomes in microglial cells treated with an Aβ and phagosome dual-targeting assembly.

[0055] Figure 33 Confocal microscopy images of the localization of FITC-labeled Aβ and lysosomes in microglia treated with an Aβ and phagosome dual-targeting assembly.

[0056] Figure 34 Confocal microscopy images of the localization of early endosomes and phagosomes in microglia treated with an Aβ and phagosome dual-targeting assembly.

[0057] Figure 35 Confocal microscopy images of the localization of early endosomes and lysosomes in microglia treated with Aβ and phagosome dual-targeting assemblies.

[0058] Figure 36 Schematic diagram of the treatment scheme for 4T1 breast cancer model mice using the PD-L1 and phagosome dual-targeting assembly designed for the present invention.

[0059] Figure 37Schematic diagram for quantitatively characterizing the changes in mouse tumor volume after intravenous injection of normal saline, PD-L1 single-targeting nanoparticles, PD-L1 and phagosome dual-targeting assembly, PD-L1 single-targeting assembly, phagosome targeting peptide control group assembly, phagosome single-targeting assembly and intraperitoneal injection of PD-L1 antibody.

[0060] Figure 38 Schematic diagram for quantitative characterization of mouse tumor weight after intravenous injection of normal saline, PD-L1 single-targeting nanoparticles, PD-L1 and phagosome dual-targeting assembly, PD-L1 single-targeting assembly, phagosome targeting peptide control group assembly, phagosome single-targeting assembly and intraperitoneal injection of PD-L1 antibody.

[0061] Figure 39 Schematic diagram for quantitative characterization of the effects of intravenous injection of normal saline, PD-L1 single-targeting nanoparticles, PD-L1 and phagosome dual-targeting assembly, PD-L1 single-targeting assembly, phagosome-targeting peptide control group assembly, phagosome single-targeting assembly and intraperitoneal injection of PD-L1 antibody on mouse lung metastatic nodules.

[0062] Figure 40 Schematic diagram for quantitatively characterizing the degradation effects of PD-L1 protein in mouse tumor tissues after intravenous injection of normal saline, PD-L1 single-targeting nanoparticles, PD-L1 and phagosome dual-targeting assembly, PD-L1 single-targeting assembly, phagosome targeting peptide control group assembly, phagosome single-targeting assembly and intraperitoneal injection of PD-L1 antibody.

[0063] Figure 41 To quantitatively characterize the effects of intravenous injection of saline, PD-L1 single-targeting nanoparticles, PD-L1 and phagosome dual-targeting assembly, PD-L1 single-targeting assembly, phagosome targeting peptide control assembly, phagosome single-targeting assembly, and intraperitoneal injection of PD-L1 antibody on the expression of CD4 + T cells and CD8 + Schematic diagram of quantitative improvement of T cell stimulation. DETAILED DESCRIPTION

[0064] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0065] The scheme that the present invention relates to is as follows: The present invention transforms a polypeptide that can be inserted into the lipid membrane in an acidic environment to construct a technical tool based on an endosome-phagosome connection assembly and its ability to insert into the endosomal membrane and label the endosome to degrade cell membrane / extracellular proteins. Specifically, the present invention provides an endosome-phagosome junction assembly and its application of inserting into the endosomal membrane and marking the endosome to degrade cell membrane / extracellular proteins. The endosome-phagosome junction assembly constructs a polypeptide with endosomal anchored phagosome targeting function by functionally modifying a polypeptide that can insert into the lipid membrane in an acidic environment; the cell membrane or extracellular protein-specific ligand is functionally modified for subsequent reaction; the endosomal anchored phagosome targeting peptide and the target protein ligand are chemically reacted and connected together, and then self-assembled to form an endosome-phagosome junction assembly; the endosome-phagosome junction assembly specifically recognizes and binds to the cell membrane or extracellular target protein, and enters the endosome of the cell through the constitutive endocytosis of the target protein or the receptor-mediated propagation into the cell mechanism; the endosome-phagosome junction assembly inserts its membrane-inserting peptide into the endosomal lipid membrane in the endosomal acidic environment to anchor the terminal phagosome targeting peptide on the endosome surface, recruiting phagosomes to engulf the endosome and degrade it using the autophagy-lysosome pathway.

[0066] An endosomal-phagosome junction assembly and a preparation strategy for a technical tool thereof by inserting into the endosomal membrane and marking the endosomal degradation of cell membrane / extracellular proteins, comprising: Step (1) functionally modifying the various parts of the endosomal anchored phagosome targeting peptide: functionally modifying one end of a polypeptide that can insert into the lipid membrane under acidic conditions (i.e., an "endosomal inserting polypeptide") with maleimide and the other end with a phagosome targeting peptide to construct an endosomal anchored phagosome targeting peptide. The specific ligand of the cell membrane / extracellular protein is functionalized with histidine to increase hydrophilicity and is also functionalized with cysteine.

[0067] Step (2), preparation of a target protein and phagosome dual-targeting assembly: the endosomal-anchored phagosome targeting peptide and the target protein-specific ligand are connected by utilizing the sulfhydryl addition reaction of maleimide and cysteine ​​to form an endosomal-phagosome connection assembly with dual targeting of the target protein and the phagosome.

[0068] Step (3), the process of endosome-phagosome junction assembly entering the cell: the constructed endosome-phagosome junction assembly can specifically recognize and bind to the target protein on the cell membrane or outside the cell, and enter the endosome of the cell through the constitutive endocytosis of the target protein or the receptor-mediated propagation mechanism.

[0069] The prepared endosome-phagosome junction assembly and its technical tools for inserting into the endosomal membrane and labeling the endosomal degradation of cell membrane / extracellular proteins can be found in Figure 1 . Figure 1This is a schematic diagram of the construction of the technical tool for degrading cell membrane / extracellular proteins based on the endosomal-phagosome junction assembly described in the present invention, as well as its degradation function after entering the cell: first, by screening and modifying polypeptides that can insert into the lipid membrane under an acidic environment, a polypeptide that can insert into the endosomal membrane under the acidic environment of the endosome is obtained, the target protein ligand is functionalized with cysteine, and the endosomal anchored phagosome targeting peptide is constructed by the addition reaction of maleimide and cysteine. This polypeptide binds to the target protein, triggers endocytosis, inserts into the endosomal membrane, marks the endosome, recruits the phagosome, and utilizes the "endosome-phagosome-lysosome" pathway to achieve targeted degradation of cell membrane / extracellular proteins.

[0070] In one embodiment of the present invention, in step (1), the design method for performing maleimide functional modification on one end of a polypeptide that inserts into a lipid membrane under an acidic environment is targeted at all polypeptides that insert into a lipid membrane under an acidic environment.

[0071] In one embodiment of the present invention, in step (1), the sequence of the phagosome targeting peptide used is: GIYVIWGPI.

[0072] In one embodiment of the present invention, in step (1), the endosomal anchored phagosome targeting peptide is a combination of a maleimide-modified polypeptide that inserts into the lipid membrane under an acidic environment and a phagosome targeting peptide.

[0073] In one embodiment of the present invention, in step (1), the C-terminus of the polypeptide inserted into the lipid membrane under an acidic environment is modified with maleimide functionalization, and the N-terminus is modified and connected to the phagosome targeting peptide. The final sequence of the resulting polypeptide is preferably Mal-acid membrane-inserted peptide 5-phagosome targeting peptide, and the sequence is: GIYVIWGPI-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT-maleimide modification.

[0074] In one embodiment of the present invention, in step (1), the design method of performing histidine functional modification and cysteine ​​functional modification on the cell membrane / extracellular target protein ligand is targeted at all protein targeting ligands.

[0075] In one embodiment of the present invention, in step (1), the PD-L1 protein targeting peptide: Cysteine-CVRARTR (containing a hydrophilic modification sequence: Cysteine-H 15 CVRARTR); Advanced glycation end product (RAGE) protein targeting peptide: Cysteine-ELKVLMEKEL; Extracellular amyloid protein Aβ targeting peptide: Cysteine-RGTFEGKF.

[0076] In one embodiment of the present invention, in step (2), the endosome-phagosome junction assembly is generated by utilizing the maleimide of the endosomal anchored phagosome targeting peptide to undergo an addition reaction with the sulfhydryl group on the cysteine ​​of the target protein-specific ligand and then self-assemble. This method is applicable to all chemical connection methods that can produce this reaction.

[0077] In one embodiment of the present invention, in step (2), the endosomal anchored phagosome targeting peptide is weighed and dissolved in an appropriate amount. N , N -dimethylformamide.

[0078] In one embodiment of the present invention, in step (2), the PD-L1 protein targeting peptide is weighed to 5 times the amount of the endosomal anchored phagosome targeting peptide, the advanced glycosylation ligand end product RAGE protein targeting peptide is weighed to 2 times the amount of the endosomal anchored phagosome targeting peptide, and the extracellular amyloid protein Aβ targeting peptide is weighed to 2 times the amount of the endosomal anchored phagosome targeting peptide, and dissolved in N , N -dimethylformamide.

[0079] In one embodiment of the present invention, in step (2), the endosomal anchored phagosome targeting peptide and the PD-L1 protein targeting peptide are stirred at room temperature for 1-10 hours; the endosomal anchored phagosome targeting peptide and the advanced glycation end product RAGE protein targeting peptide / extracellular amyloid protein Aβ targeting peptide are stirred at room temperature for 20-30 hours.

[0080] In one embodiment of the present invention, in step (2), the mixture of the endosomal anchored phagosome targeting peptide and the cell membrane / extracellular protein targeting peptide is injected into pure water and stirred at room temperature for 0.1-3 hours.

[0081] In one embodiment of the present invention, in step (2), the assembly formed by the endosomal anchored phagosome targeting peptide and the cell membrane / extracellular protein targeting peptide is dialyzed in water for 24-72 h using a dialysis bag.

[0082] In one embodiment of the present invention, in step (3), the endosomal-phagosome junction assembly enters the endosome of the cell through the constitutive endocytosis of the target protein or the receptor-mediated propagation endocytosis mechanism, which utilizes the endocytosis of the target protein itself on the cell membrane and the indirect propagation endocytosis mechanism mediated by the cell membrane endocytic target protein receptor for extracellular proteins to enter the endosome.

[0083] Example 1

[0084] This example demonstrates the preparation method of the endosomal-phagosome junction assembly and its technical tool for inserting into the endosomal membrane and labeling the endosomal degradation of cell membrane / extracellular proteins as follows: (1) Preparation of PD-L1 (programmed death receptor-ligand 1) and phagosome dual-targeting assembly 2.8 mg of endosomal anchored phagosome targeting peptide (made by linking maleimide-modified endosomal inserting peptide and phagosome targeting peptide, sequence: maleimide-modified-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT-GIYVIWGPI) and 8 mg of PD-L1 targeting peptide (PD-L1 targeting peptide is a cysteine ​​and histidine-modified peptide, sequence: cysteine-modified-HHHHHHHHHHHHHH-CVRARTR, the same below. CVRARTR is SEQ ID NO. 1) were weighed (molar ratio of 1:5) and dissolved in N , N The mixture was stirred in dimethylformamide for 4 hours, then injected into 5 mL of purified water and stirred at room temperature for another 0.5 hour to prepare a preliminary PD-L1 and phagosome dual-targeting assembly. The mixture was then dialyzed for 24 hours using a 3500 Da dialysis bag and then replaced with a 7000 Da dialysis bag for another 24 hours. Freshly prepared PD-L1 and phagosome dual-targeting assembly was obtained. (2) Preparation of PD-L1 single-target assembly 2.8 mg of maleimide functionalized peptide capable of inserting into lipid membrane in acidic environment and 9.9 mg of PD-L1 targeting peptide (molar ratio of 1:5) were dissolved in N , N The mixture was stirred in dimethylformamide for 4 hours, then injected into 5 mL of purified water and stirred at room temperature for another 0.5 hour to form an assembly. The assembly was then dialyzed for 24 hours using a 3500 Da dialysis bag and then replaced with a 7000 Da dialysis bag for another 24 hours. A freshly prepared PD-L1 single-target assembly was obtained. (3) Preparation of Aβ (β-amyloid protein) and phagosome dual-targeting assemblies 2.8 mg of endosomal anchored phagosome targeting peptide and 1.14 mg of Aβ targeting peptide (Aβ targeting peptide is a cysteine-modified peptide with the sequence: cysteine-modified-RGTFEGKF, the same below. RGTFEGKF is SEQ ID NO. 2) were weighed and dissolved in a 1:2 molar ratio. N , N -dimethylformamide and stirred for 24 h, then the mixture was injected into 5 mL of pure water and stirred for another 0.5 h at room temperature to prepare an assembly, which was then dialyzed using a 3500 Da dialysis bag for 48 h to obtain a freshly prepared Aβ and phagosome dual-targeted assembly; (4) Preparation of Aβ single-targeted assemblies 2.8 mg of maleimide-functionalized peptide capable of inserting into lipid membranes in acidic environments and 1.37 mg of Aβ-targeting peptide (molar ratio of 1:2) were dissolved in N , N -dimethylformamide and stirred for 24 h, then the mixture was injected into 5 mL of pure water and stirred for another 0.5 h at room temperature to prepare an assembly, which was then dialyzed using a 3500 Da dialysis bag for 48 h to obtain a freshly prepared Aβ single-targeted assembly; (5) Preparation of RAGE (advanced glycation end products) and phagosome dual-targeting assemblies 2.8 mg of endosomal anchored phagosome targeting peptide and 1.4 mg of RAGE targeting peptide (RAGE targeting peptide is a cysteine-modified polypeptide with the sequence: cysteine-modified-ELKVLMEKEL, the same below. ELKVLMEKEL is SEQ ID NO. 3) were weighed and dissolved in a 1:2 molar ratio. N , N -dimethylformamide and stirred for 24 h, then the mixture was injected into 5 mL of pure water and stirred for another 0.5 h at room temperature to prepare an assembly, which was then dialyzed using a 3500 Da dialysis bag for 48 h to obtain a freshly prepared RAGE and phagosome dual-targeted assembly; (6) Preparation of RAGE single-target assembly 2.8 mg of maleimide-functionalized peptide capable of inserting into lipid membranes in acidic environments and 1.7 mg of RAGE targeting peptide (molar ratio of 1:2) were dissolved in N , N -dimethylformamide for 24 h, and then the mixture was injected into 5 mL of pure water and stirred at room temperature for another 0.5 h to prepare an assembly, which was then dialyzed using a 3500 Da dialysis bag for 48 h to obtain a freshly prepared RAGE single-targeted assembly.

[0085] Example 2

[0086] (1) In order to screen polypeptides that can be inserted into lipid membranes in acidic environments and that conform to the assembly constructed by the present invention, the hemoglobin leakage rate is used to indicate the interaction between different polypeptides and endosomal membranes after they enter the cell. Figure 2 , Figure 2 This is a quantitative graph of the damage of red blood cell membrane by different peptides using full wavelength microplate reader. Figure 2As shown in the results of hemoglobin leakage, the Mal-acidic membrane inserting peptide 5-phagosome targeting peptide showed higher pH sensitivity. Furthermore, the Mal-acidic membrane inserting peptide 5-phagosome targeting peptide and its parallel control group, Mal-acidic membrane inserting peptide 5, were more consistent with the design of this protocol. These results indicate that the Mal-acidic membrane inserting peptide 5-phagosome targeting peptide was selected as the endosomal-anchored phagosome targeting peptide. Therefore, in subsequent experiments, the Mal-acidic membrane inserting peptide 5-phagosome targeting peptide was used as the endosomal-anchored phagosome targeting peptide. The phagosome targeting peptide sequence is shown in SEQ ID NO. 4 (GIYVIWGPI), and the endosomal membrane inserting peptide is shown in SEQ ID NO. 5 (AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT).

[0087] (2) Effects of different end (N-terminal or C-terminal) exposure on acidic membrane-inserting peptides: Acidic membrane-inserting peptide 4 was designed, and a PD-L1 and phagosome dual-targeting assembly was constructed according to the above method to compare the degradation effects. The results are shown in Figure 2 Acidic membrane-inserting peptide 5 is C-terminal inserted, acidic membrane-inserting peptide 3 and acidic membrane-inserting peptide 4 are homologous sequences, and the PD-L1 and phagosome dual-targeting assembly formed by the two is an analogue, but acidic membrane-inserting peptide 4 is C-terminal inserted, and acidic membrane-inserting peptide 3 is slightly N-terminal inserted. A comparison of the degradation effects found that although the PD-L1 and phagosome dual-targeting assembly prepared by acidic membrane-inserting peptide 3 can achieve hemolysis, it has no obvious effect on PD-L1 degradation, while acidic membrane-inserting peptide 4 and acidic membrane-inserting peptide 5 can achieve PD-L1 degradation, which proves that the assembly formed by N-terminal insertion cannot degrade PD-L1.

[0088] The peptides involved in the above experiments are as follows: Mal-acidic membrane-inserting peptide 1-phagosome targeting peptide: maleimide-IWLTALKFLGKHAAKHEAKQQLSKL-GIYVIWGPI; Mal-acidic membrane-inserting peptide 2-phagosome targeting peptide: maleimide-GALFLGFLGAAGSTMGAWSQPHHHHHV-GIYVIWGPI; Mal-acidic membrane-inserting peptide 3-phagosome targeting peptide: maleimide-FSGSGSGSGHQIHIWFQNHHMHWHHG-GIYVIWGPI; Mal-acidic membrane-inserting peptide 4-phagosome targeting peptide: maleimide-FSGSGSGSGRQIKIWFQAARMLWKK-GIYVIWGPI; Mal-acidic membrane-inserting peptide 5-phagosome targeting peptide: maleimide-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT-GIYVIWGPI.

[0089] Example 3

[0090] This example investigates the particle size of the assembly constructed by the present invention. Figure 3 , Figure 3 The particle size of all assemblies prepared in Example 1 was determined by dynamic light scattering. Figure 3 As shown in the figure, dynamic light scattering method showed that the particle size of the phagosome-targeted peptide control group assembly was 89 nm; the particle size of the phagosome-targeted assembly was 97 nm; the particle size of the PD-L1-targeted assembly was 176 nm; the particle size of the PD-L1 and phagosome-targeted assembly was 177 nm; the particle size of the Aβ-targeted assembly was 128 nm; the particle size of the Aβ and phagosome-targeted assembly was 116 nm; the particle size of the RAGE-targeted assembly was 134 nm; and the particle size of the RAGE and phagosome-targeted assembly was 157 nm, indicating that the constructed nanocarrier may form an assembly-like structure and the particle size of the assembly system is small.

[0091] The preparation methods of the phagosome-targeted peptide control group assembly and the phagosome-targeted single assembly are the same as those in Example 1, specifically: Preparation of phagosome-targeted single assembly: 2.8 mg of endosomal anchored phagosome targeting peptide and 5.3 mg of DSPE-PEG-NH2 (α-amino-ω-1,2-distearate-3-phosphatidylethanolamine polyethylene glycol) (molar ratio of 1:5) were dissolved in N , N The mixture was stirred in 5 mL of dimethylformamide for 4 hours, then poured into 5 mL of purified water and stirred at room temperature for another 0.5 hour to form an assembly. The assembly was then dialyzed for 24 hours using a 3500D dialysis bag and then a 7000D dialysis bag for another 24 hours. Freshly prepared phagosome-specific assemblies were obtained.

[0092] Preparation of the phagosome targeting peptide control group assembly: 2.8 mg of a maleimide-functionalized peptide capable of inserting into lipid membranes in acidic environments and 6.6 mg of DSPE-PEG-NH2 (molar ratio of 1:5) were dissolved in N , N The mixture was stirred in 5 mL of dimethylformamide for 4 hours, then poured into 5 mL of purified water and stirred at room temperature for another 0.5 hour to form an assembly. The assembly was then dialyzed for 24 hours using a 3500D dialysis bag and then a 7000D dialysis bag for another 24 hours. Freshly prepared phagosome-specific assemblies were obtained.

[0093] Example 4

[0094] This example investigates the uniformity of the nanoassemblies constructed above. Figure 4 , Figure 4 The degree of homogeneity of all assemblies was determined by dynamic light scattering. Figure 4 As shown, the dynamic light scattering method showed that the polydispersity index of the phagosome targeting peptide control group assembly was 0.146, the polydispersity index of the phagosome single targeting assembly was 0.150; the polydispersity index of the PD-L1 single targeting assembly was 0.176; the polydispersity index of the PD-L1 and phagosome dual targeting assembly was 0.167; the polydispersity index of the Aβ single targeting assembly was 0.158; the polydispersity index of the Aβ and phagosome dual targeting assembly was 0.164; the polydispersity index of the RAGE single targeting assembly was 0.196; the polydispersity index of the RAGE and phagosome dual targeting assembly was 0.196, indicating that the constructed nanocarrier may form an assembly and the assembly system is uniform.

[0095] Example 5

[0096] This example investigates the electrokinetic potential of the nanoassembly constructed above. Figure 5 , Figure 5 The zeta potential of all assemblies was determined by dynamic light scattering. Figure 5 As shown, the dynamic light scattering method showed that the zeta potential of the phagosome-targeted peptide control group assembly was -15 mV, the zeta potential of the phagosome-targeted assembly was -19 mV; the zeta potential of the PD-L1-targeted assembly was +13 mV; the zeta potential of the PD-L1 and phagosome dual-targeted assembly was +16 mV; the zeta potential of the Aβ-targeted assembly was -14 mV; the zeta potential of the Aβ-targeted assembly was -15 mV; the zeta potential of the RAGE-targeted assembly was -16 mV; and the zeta potential of the RAGE and phagosome dual-targeted assembly was -14 mV, indicating that the constructed nanocarriers may form assemblies.

[0097] Example 6

[0098] This example investigates the stability of the nanoassembly constructed above, and uses dynamic light scattering to investigate the changes in the zeta potential of the assembly prepared in this solution within one week of preparation. Figure 6 , Figure 6 Dynamic light scattering was used to measure the electrokinetic potential changes of the prepared RAGE and phagosome dual-targeting assembly, Aβ single-targeting assembly, and Aβ and phagosome dual-targeting assembly within one week of preparation. Figure 6 As shown, the structures of the three assemblies are stable, and the zeta potential does not change significantly within one week of preparation.

[0099] Example 7

[0100] This example investigates the critical micelle concentration (CMC) of the assembly used in this protocol and measures the critical micelle concentration (CMC) of the assembly using the pyrene fluorescence probe method. Figure 7 ,like Figure 7 As shown, the critical micelle concentration of Aβ single-targeted assembly was determined to be 1.25-5.8 nM using the pyrene fluorescent probe method; the critical micelle concentration of RAGE single-targeted assembly was 3.5-6.8 nM; the critical micelle concentration of PD-L1 single-targeted assembly was 1.2-4.7 nM; and the critical micelle concentration of PD-L1 and phagosome dual-targeted assembly was 1.3-4.2 nM.

[0101] Example 8

[0102] Preparation of plasma adsorption control group assembly. Take an appropriate amount of poly (lactic acid - glycolic acid) copolymer activated by succinimide and react with a certain amount of amino-modified polyethylene glycol carboxymethyl and add a small amount of N, N-diisopropylethylamine in dichloromethane for 2 hours, then use argon to evaporate the dichloromethane, and then add an appropriate amount of N , N After adding dimethylformamide, the successfully prepared control group assembly was injected into pure water. After reacting for 0.5 h, it was dialyzed with pure water for 48 h and then the freshly prepared assembly was collected as a control for protein corona formation.

[0103] 100 μL of freshly prepared assembly (0.4 mg / mL in PBS 7.4) was incubated with 100 μL of PBS 7.4 or plasma for 1 hour at 37°C and then centrifuged at 70,000 g for 20 minutes at 4°C. The pellet was then resuspended in PBS 7.4 and lysed with protein lysis buffer on ice for 30 minutes and centrifuged at 12,000 g for 15 minutes at 4°C for protein electrophoresis to assess plasma protein adsorption. Figure 8 , Figure 8 To characterize the protein corona formed by different assemblies adsorbing plasma, the whole protein map was used by SDS-PAGE, which showed that all assemblies formed a protein corona after incubation with plasma.

[0104] Example 9

[0105] The protein concentration was determined using the same sample preparation method as in Example 8. Figure 9 , Figure 9 The quantitative graph of plasma protein concentration adsorbed by different assemblies was measured by full-wavelength microplate reader, showing that all assemblies formed protein corona after incubation with plasma, and the protein concentration was higher than that of the phosphate buffer group.

[0106] Example 10

[0107] The same method as in Example 8 was used to prepare the plasma adsorption control group assembly.

[0108] 100 μL of freshly prepared assembly (0.4 mg / mL in PBS 7.4) was incubated with 100 μL of PBS 7.4 or plasma for 1 hour at 37°C and then centrifuged at 70,000 g for 20 minutes at 4°C. The pellet was then resuspended in PBS 7.4 and measured by electrokinetic potential. Figure 10 , Figure 10 The dynamic light scattering method is used to determine the quantitative electrokinetic potential of different assemblies after adsorption of plasma to form a protein corona, which shows that all assemblies will form a protein corona after incubation with plasma.

[0109] Example 11

[0110] To investigate whether the PD-L1 and phagosome dual-targeting assembly can transfer PD-L1 to the phagosome, HEK293T cells were transfected with pEGFP-N1 / PD-L1 and pmRFP-LC3 plasmids in a 48-well plate in serum-free medium for 48 h. The transfected cells were further treated with 0.5 μM of different assemblies for 12 h, and then fixed with 4% PFA (paraformaldehyde) solution for 15 min. Finally, images were obtained using a laser confocal scanning microscope. For the conclusion of this example, please refer to Figure 11 , Figure 11 Confocal microscopy images of the localization of PD-L1 and phagosomes in cells after being treated with different assemblies. Figure 11 As shown, in the blank control group, phagosome-targeted peptide control group assembly, phagosome-targeted assembly, PD-L1-targeted nanoparticles, and PD-L1-targeted assembly, PD-L1 was located on the cell membrane and would not induce PD-L1 to reach the phagosome, while PD-L1 and phagosome dual-targeted assembly group PD-L1 was highly co-localized with the phagosome, indicating that the PD-L1 and phagosome dual-targeted assembly has the ability to bind to PD-L1 and recruit phagosomes.

[0111] To prepare PD-L1-targeted nanoparticles, 100 mg of poly(lactic-co-glycolic acid)-polylysine (PLG) as a base carrier was dissolved in 2 mL of ethyl acetate. This mixture was added dropwise to 4 mL of 2.5% poly(vinyl alcohol) under vortexing and immediately sonicated at 300 W for 3 × 10 s to form an oil-in-water emulsion. This emulsion was then poured into 100 mL of 0.3% poly(vinyl alcohol) and stirred overnight to evaporate the ethyl acetate. The nanosuspension was purified from the poly(vinyl alcohol) by centrifugation at 70,000 g for 20 minutes, and the precipitate was resuspended in PBS 7.4 by water bath sonication. Subsequently, 11.2 mg of MAL-PEG-SCM (maleimide-poly(ethylene glycol)-succinimidyl carboxymethyl ester, purchased from Beijing Jiankai Technology Co., Ltd., product number A5002) was added and allowed to react at room temperature for 1 hour. The excess MAL-PEG-SCM was subsequently removed by centrifugation at 70,000 g for 20 minutes, and the nanosuspension was resuspended and further modified with excess cysteine-containing PD-L1-targeting ligand via maleimide-thiol reaction chemistry at room temperature for 1 hour. Finally, the excess unreacted peptide was removed by centrifugation at 70,000 g for 20 minutes, and the nanosuspension was resuspended in water and lyophilized to obtain PD-L1 single-targeting nanoparticles.

[0112] Example 12

[0113] To investigate whether the PD-L1 and phagosome dual-targeting assembly can transfer PD-L1 to lysosomes, HEK293T cells were transfected with pEGFP-N1 / PD-L1 and mTagBFP2-Lysosomes-20 plasmids in 48-well plates in serum-free medium for 48 h. The transfected cells were further treated with 0.5 μM of different assemblies for 12 h, and then fixed with 4% PFA solution for 15 min. Finally, images were obtained using a laser confocal scanning microscope. For the conclusion of this example, please refer to Figure 12 , Figure 12 Confocal microscopy images of the localization of PD-L1 and lysosomes in cells after being treated with different assemblies. Figure 12 As shown, in the blank control group, phagosome-targeted peptide control group assembly, phagosome-targeted assembly, PD-L1-targeted nanoparticles, and PD-L1-targeted assembly, PD-L1 was located on the cell membrane and did not induce PD-L1 to reach the lysosome, while PD-L1 and phagosome dual-targeted assembly group PD-L1 was highly co-localized with lysosomes. Figure 11 This indicates that the PD-L1 and phagosome dual-targeting assembly has the ability to bind to PD-L1 and recruit phagosomes, as well as degrade through lysosomes.

[0114] Example 13 To investigate whether the PD-L1 and phagosome dual-targeting assembly can transfer endosomes containing PD-L1 to phagosomes, HEK293T cells were transfected with GFP-Rab5B and pmRFP-LC3 plasmids in 48-well plates in serum-free medium for 48 h. The transfected cells were further treated with 0.5 μM of different assemblies for 12 h, and then fixed with 4% PFA solution for 15 min. Finally, images were obtained using a laser confocal scanning microscope. For the conclusion of this example, please refer to Figure 13 , Figure 13 This is a confocal microscopy image of the localization of early endosomes and phagosomes in cells after being processed by different assemblies. Figure 13 As shown, in the blank control group, phagosome-targeted peptide control group assembly, phagosome-targeted assembly, PD-L1-targeted nanoparticles, and PD-L1-targeted assembly, the early endosomes and phagosomes were clearly separated, while the early endosomes and phagosomes in the PD-L1 and phagosome dual-targeted assembly group were highly co-localized, indicating that the PD-L1 and phagosome dual-targeted assembly can shorten the spatial distance between the early endosomes and the phagosome, which is beneficial to pulling the proteins contained in the endosomes into the phagosome.

[0115] Example 14

[0116] To investigate whether the PD-L1 and phagosome dual-targeting assembly can ultimately transfer PD-L1-containing endosomes to lysosomes, HEK293T cells were transfected with GFP-Rab5B and mTagBFP2-Lysosomes-20 plasmids in a 48-well plate in serum-free medium for 48 h. The transfected cells were further treated with 0.5 μM of different assemblies for 12 h, and then fixed with 4% PFA solution for 15 min. Finally, images were taken using a laser confocal scanning microscope. For the conclusion of this example, please refer to Figure 14 , Figure 14 This is a confocal microscopy image of the localization of early endosomes and lysosomes in cells after being processed by different assemblies. Figure 14 As shown, in the blank control group, phagosome-targeted peptide control group assembly, phagosome-targeted assembly, PD-L1-targeted nanoparticles, and PD-L1-targeted assembly, the early endosomes and lysosomes were clearly separated, while the early endosomes and lysosomes of the PD-L1 and phagosome dual-targeted assembly group were highly co-localized. Figure 13 This indicates that the PD-L1 and phagosome dual-targeting assembly can shorten the spatial distance between the early endosome and the phagosome, pull the proteins contained in the early endosome into the phagosome and degrade them through the lysosome.

[0117] Example 15

[0118] To investigate the time it takes for the PD-L1 and phagosome dual-targeting assembly to transfer PD-L1 to the phagosome, HEK293T cells were transfected with pEGFP-N1 / PD-L1 and pmRFP-LC3 plasmids in a 48-well plate in serum-free medium for 48 h. The transfected cells were treated with the PD-L1 and phagosome dual-targeting assembly for 0-12 h, then fixed with 4% PFA solution for 15 minutes and imaged using a laser confocal scanning microscope. For the conclusion of this example, please refer to Figure 15 , Figure 15 The confocal microscopy images show the localization of PD-L1 and phagosomes in cells after treatment with the PD-L1 and phagosome dual-targeting assembly for 0-12 h. Figure 15 As shown, increasing the amount of PD-L1 and phagosome dual-targeting assembly over time can gradually bring PD-L1 and phagosome closer in space.

[0119] Example 16

[0120] To investigate the time it takes for the PD-L1 and phagosome dual-targeting assembly to transfer PD-L1 to lysosomes, HEK293T cells were transfected with pEGFP-N1 / PD-L1 and mTagBFP2-Lysosomes-20 plasmids in a 48-well plate in serum-free medium for 48 h. The transfected cells were treated with the PD-L1 and phagosome dual-targeting assembly for 0-12 h, then fixed with 4% PFA solution for 15 minutes and imaged using a laser confocal scanning microscope. For the conclusion of this example, please refer to Figure 16 , Figure 16 The confocal microscopy images show the localization of PD-L1 and lysosomes in cells after treatment with the PD-L1 and phagosome dual-targeting assembly for 0-12 h. Figure 16 As shown, increasing the amount of PD-L1 and phagosome dual-targeting assembly over time can gradually bring PD-L1 and lysosome closer in space.

[0121] Example 17

[0122] To investigate the time it takes for the PD-L1 and phagosome dual-targeting assembly to transfer PD-L1-containing early endosomes to phagosomes, HEK293T cells were transfected with GFP-Rab5B and pmRFP-LC3 plasmids in a 48-well plate in serum-free medium for 48 hours. The transfected cells were treated with the PD-L1 and phagosome dual-targeting assembly for 0-12 hours, then fixed with 4% PFA solution for 15 minutes and imaged using a laser confocal scanning microscope. For the conclusion of this example, please refer to Figure 17 , Figure 17 The confocal microscopy images show the localization of early endosomes and phagosomes containing PD-L1 in cells after treatment with the PD-L1 and phagosome dual-targeting assembly for 0-12 h. Figure 17 As shown, as time goes by, the co-localization of early endosomes and phagosomes in the PD-L1 and phagosome dual-targeting assembly group increases, which proves that the dual-targeting assembly group has the ability to recruit phagosomes, spatially shorten the distance between phagosomes and early endosomes anchored with PD-L1, and use phagosomes to engulf early endosomes anchored with PD-L1.

[0123] Example 18

[0124] To investigate the efficiency of the PD-L1 and phagosome dual-targeting assembly in transferring PD-L1-containing early endosomes to lysosomes, HEK293T cells were transfected with GFP-Rab5B and mTagBFP2-Lysosomes-20 plasmids in a 48-well plate in serum-free medium for 48 h. The transfected cells were treated with the PD-L1 and phagosome dual-targeting assembly for 0-12 h, then fixed with 4% PFA solution for 15 minutes and imaged using a laser confocal scanning microscope. For the conclusion of this example, please refer to Figure 18 , Figure 18 The confocal microscopy images show the localization of early endosomes and lysosomes containing PD-L1 in cells after treatment with the PD-L1 and phagosome dual-targeting assembly for 0-12 h. Figure 18 As shown in the figure, as time goes by, the co-localization of early endosomes and lysosomes in the PD-L1 and phagosome dual-targeting assembly group increases, which proves that the dual-targeting assembly group can eventually bring the early endosomes anchored with PD-L1 into the lysosome for degradation.

[0125] Example 19

[0126] To investigate the effect of the concentration of the PD-L1 and phagosome dual-targeting assembly on PD-L1 degradation, 4T1 mouse breast cancer cells in a 6-well plate were treated with different concentrations of the PD-L1 and phagosome dual-targeting assembly for 12 hours. Western blot was used to investigate the degradation of PD-L1 in 4T1 mouse breast cancer cells by the PD-L1 and phagosome dual-targeting assembly. For the conclusion of this example, please refer to Figure 19 , Figure 19 This is a quantitative graph of the effects of different concentrations of PD-L1 and phagosome dual-targeting assembly on PD-L1 expression in 4T1 mouse breast cancer cells by western blot. Figure 19 As shown in the figure, as the concentration of the PD-L1 and phagosome dual-targeting assembly gradually increases, the efficiency of the PD-L1 and phagosome dual-targeting assembly in degrading PD-L1 becomes higher and higher.

[0127] Example 20

[0128] To investigate the necessity of the phagosome-targeting ligand in the PD-L1 and phagosome dual-targeting assembly for the degradation of PD-L1, 4T1 mouse breast cancer cells in 6-well plates were treated with different concentrations of the PD-L1 single-targeting assembly for 12 hours. Western blot was used to investigate the degradation of PD-L1 in 4T1 mouse breast cancer cells by different concentrations of the PD-L1 single-targeting assembly. For the conclusion of this example, please refer to Figure 20 , Figure 20 This is a quantitative graph of the effects of different concentrations of PD-L1 single-target assembly on 4T1 mouse breast cancer cells by western blot. Figure 20 As shown, although the concentration of PD-L1 single-targeting assembly is gradually increasing, the PD-L1 single-targeting assembly does not have a degradation effect on cellular PD-L1.

[0129] Example 21

[0130] In order to investigate the effect of treatment time of PD-L1 and phagosome dual-targeting assembly on PD-L1 degradation, 4T1 mouse breast cancer cells were incubated with PD-L1 and phagosome dual-targeting assembly for 0, 2, 4, 8, 12, and 24 h, respectively. Western blot was used to investigate the effect of PD-L1 and phagosome dual-targeting assembly on PD-L1 degradation at different incubation times. For the conclusion of this example, please refer to Figure 21 , Figure 21 In order to investigate the effect of PD-L1 and phagosome dual-targeting assembly on cellular PD-L1 protein at different times by western blot, Figure 21 As shown, as the time of treating cells with the PD-L1 and phagosome dual-targeting assembly gradually increased, the efficiency of the PD-L1 and phagosome dual-targeting assembly in degrading PD-L1 became higher and higher.

[0131] Example 22

[0132] In order to investigate the necessity of the phagosome-targeted degradation ligand in the PD-L1 and phagosome dual-targeting assembly for the degradation of PD-L1, 4T1 mouse breast cancer cells in 6-well plates were treated with the PD-L1 single-targeting assembly for 0-24 hours. Western blot technology was used to investigate the degradation of PD-L1 in 4T1 mouse breast cancer cells by the PD-L1 single-targeting assembly at different times. For the conclusion of this example, please refer to Figure 22 , Figure 22 This is a quantitative graph of the PD-L1 expression of 4T1 mouse breast cancer cells by western blot at different times. Figure 22As shown, although the treatment time of cells with PD-L1 single-targeting assembly is gradually increasing, PD-L1 single-targeting assembly does not have a degradation effect on cellular PD-L1.

[0133] Example 23

[0134] In order to investigate the necessity of inserting the lipid membrane peptide and the phagosome targeting peptide into the PD-L1 targeting ligand under acidic environment for the degradation of PD-L1, please refer to the conclusion of this example. Figure 23 , Figure 23 The phagosome-targeted peptide control group assembly, phagosome-targeted assembly, PD-L1-targeted nanoparticles, PD-L1-targeted assembly, and PD-L1 and phagosome-targeted assembly were treated with 0.5 μM concentration for 12 h to quantitatively investigate the effects of different assemblies on PD-L1 expression in 4T1 cells by western blot. Figure 23 As shown, PD-L1 degradation in 4T1 cells can only be achieved when PD-L1 targeting ligand, pH-sensitive membrane-inserting peptide and phagosome-targeting peptide are all present.

[0135] Example 24

[0136] To investigate the potential mechanism of PD-L1 degradation mediated by the PD-L1 and phagosome dual-targeting assembly, please refer to the conclusion of this example. Figure 24 , Figure 24 4T1 cells were pretreated with bafilomycin A1 (lysosomal acidification inhibitor, 100 nM), chloroquine (autophagic flux inhibitor, 20 μM), NH4Cl (autophagic inhibitor, 10 mM), MG132 (proteasome inhibitor, 5 μM), or rapamycin (phagosome formation enhancer, 10 μM) for 24 hours, and then treated with 0.5 μM of a mixture of PD-L1 and phagosome dual-targeting assembly and the above functional molecules for further 12 hours. The protein level of PD-L1 in the treated cells was then quantitatively examined by western blot, as shown in Figure 5. Figure 24 Bafilomycin A1, chloroquine, and NH4Cl inhibited the degradation of PD-L1, rapamycin enhanced PD-L1 degradation, and MG132 showed no effect, demonstrating that the PD-L1 and phagosome dual-targeting assembly degrades PD-L1 through the autophagic degradation mechanism.

[0137] Example 25

[0138] To explore whether the degradation process of the PD-L1 and phagosome dual-targeting assembly will destroy the endosome, the conclusion of this example can be found in Figure 25 , Figure 25HEK293T cells were treated simultaneously with different assemblies and 0.1 mg / mL calcein, because membrane-impermeable calcein is usually sequestered in endosomes after cellular uptake. Figure 25 As shown, cells co-incubated with PD-L1 single-targeting nanoparticles showed distinct calcein spots. However, cells treated with other groups of assemblies containing pH-sensitive peptides showed calcein released from endosomes into the cytoplasm and evenly distributed within the cells, indicating that the PD-L1 and phagosome dual-targeting assembly disrupts endosomes during PD-L1 degradation.

[0139] Example 26

[0140] Preparation of Aβ oligomers. Human amyloid-β monomer 1-42 (Aβ1-42) powder was dissolved in cold hexafluoroisopropanol to 1 mM, sealed and incubated at room temperature for 60 min to completely dissolve, aliquoted into centrifuge tubes on ice, and the hexafluoroisopropanol was evaporated overnight at room temperature. Finally, a dry transparent film was obtained as an Aβ monomer. 44 μL of dimethyl sulfoxide was added per 1 mg of Aβ monomer to dissolve the peptide film and form a 5 mM solution. Reduced serum medium was then added to dilute to a concentration of 100 μM. The solution was further incubated at 4°C for 24 hours and filtered through a 0.22 μm membrane to obtain uniform Aβ oligomers, and RAGE-upregulated mouse brain microvascular endothelial cells ( Figure 26 showed that RAGE was upregulated in mouse brain microvascular endothelial cells after 24 h of Aβ incubation).

[0141] Preparation of RAGE-targeted nanoparticles. First, 100 mg of poly(lactic-co-glycolic acid)-polylysine (PLG) as a base carrier was dissolved in 2 mL of ethyl acetate. This mixture was added dropwise to 4 mL of 2.5% poly(vinyl alcohol) under vortexing and immediately sonicated at 300 W for 3 × 10 s to form an oil-in-water emulsion. This emulsion was immediately poured into 100 mL of 0.3% poly(vinyl alcohol) and stirred overnight to evaporate the ethyl acetate. The nanosuspension was purified from the poly(vinyl alcohol) by centrifugation at 70,000 g for 20 minutes, and the precipitate was resuspended in PBS 7.4 using waterbath sonication. Subsequently, 11.2 mg of maleimide-poly(ethylene glycol) was added and reacted at room temperature for 1 hour. Excess maleimide-poly(ethylene glycol) was then removed by centrifugation at 70,000 g for 20 minutes, and the nanosuspension was resuspended and further modified with excess cysteine-targeting RAGE ligand via maleimide-thiol chemistry at room temperature for 1 hour. Finally, excess unreacted peptide was removed by centrifugation at 70,000 g for 20 min, and the nanosuspension was resuspended in water and lyophilized to obtain RAGE single-targeted nanoparticles.

[0142] To explore the degradation of RAGE upregulated in mouse brain endothelial cells by the dual-targeting assembly of RAGE and phagosome, please refer to the conclusion of this example. Figure 27 , Figure 27 The figure shows the quantitative expression of RAGE on mouse brain endothelial cells by western blot after treating them with blank culture medium, phagosome-targeted peptide control group assembly, phagosome-targeted assembly, RAGE-targeted nanoparticles, RAGE-targeted assembly, and RAGE and phagosome-targeted assembly at a concentration of 0.5 μM for 12 h. Figure 27 As shown, the RAGE and phagosome dual-targeting assembly produced a significant effect on the degradation of RAGE.

[0143] Example 27

[0144] Aβ-targeted nanoparticles were prepared. First, 100 mg of poly(lactic-co-glycolic acid-polylysine) as a base carrier was dissolved in 2 mL of ethyl acetate. This mixture was added dropwise to 4 mL of 2.5% poly(vinyl alcohol) under vortexing and immediately sonicated at 300 W for 3 × 10 s to form an oil-in-water emulsion. This emulsion was immediately poured into 100 mL of 0.3% poly(vinyl alcohol) and stirred overnight to evaporate the ethyl acetate. The nanosuspension was purified from the poly(vinyl alcohol) by centrifugation at 70,000 g for 20 minutes, and the precipitate was resuspended in PBS 7.4% by waterbath sonication. Subsequently, 11.2 mg of maleimide-linked polyethylene glycol was added and reacted at room temperature for 1 hour. Excess maleimide-linked polyethylene glycol was then removed by centrifugation at 70,000 g for 20 minutes, and the nanosuspension was resuspended and further modified with excess cysteine-containing Aβ-targeting ligand via maleimide-thiol chemistry at room temperature for 1 hour. Finally, excess unreacted peptide was removed by centrifugation at 70,000 g for 20 min, and the nanosuspension was resuspended in water and lyophilized to obtain Aβ single-targeted nanoparticles.

[0145] Considering that Aβ oligomers can be internalized through RAGE-mediated endocytosis and spread between brain parenchymal cells, promoting the progression of Alzheimer's disease, the Aβ and phagosome dual-targeting assembly is expected to form a ternary complex with Aβ oligomers and RAGE for cellular uptake of Aβ and RAGE.

[0146] In order to investigate the effect of the Aβ targeting assembly on the uptake of Aβ by RAGE-upregulated microglia, the conclusion of this example can be found in Figure 29 , Figure 29 To establish RAGE upregulation in BV-2 microglia in 6-well plates (treated in the same manner as above, BV-2 cells with RAGE upregulation were incubated with 10 μM Aβ oligomers for 24 h, and the results are shown in Figure 28) were incubated with a mixture of 10 μM Aβ oligomers and different concentrations of Aβ single-targeted assemblies for 12 hours, and the protein expression of RAGE-upregulated microglia in Aβ uptake was quantified by western blot. Figure 29 As shown, as the concentration of Aβ single-targeting assembly treated cells increased, the uptake of Aβ by RAGE-upregulated microglia gradually increased.

[0147] Example 28

[0148] In order to investigate the effect of the Aβ targeting assembly on the Aβ uptake time of RAGE upregulated microglia, the conclusion of this example can be found in Figure 30 , Figure 30 To set different incubation times, namely 0, 2, 4, 6, 8, and 12 h, BV-2 microglia cells with upregulated RAGE were incubated with a mixture of 10 μM Aβ oligomers and Aβ single-targeted assemblies, and the uptake of Aβ protein by cells was measured by western blot technology. Figure 30 As shown, as the time of treating cells with Aβ single-targeting assembly increased, the uptake of Aβ by RAGE-upregulated microglia gradually increased.

[0149] Example 29

[0150] In order to investigate the effect of the dual-targeting assembly of Aβ and phagosome on the degradation of both Aβ and RAGE proteins in BV-2 cells with upregulated RAGE, the conclusion of this example can be found in Figure 31 , Figure 31 BV-2 microglia with upregulated RAGE were incubated with 10 μM Aβ oligomers and mixtures of different assemblies for 12 h, and the protein expression of Aβ and RAGE in RAGE-upregulated microglia was quantitatively examined by western blot. Figure 31 As shown, both the nanoparticles and assemblies with single Aβ targeting enhanced the cellular uptake of Aβ, while the Aβ and phagosome dual targeting assembly simultaneously degraded both Aβ and RAGE proteins in RAGE-upregulated BV-2 cells, indicating that the Aβ and phagosome dual targeting assembly can simultaneously bind to the endocytic receptor RAGE on the cell membrane and the extracellular Aβ protein, and degrade them through endosome-phagosome-lysosome.

[0151] Example 30

[0152] In order to investigate the co-localization of Aβ and phagosomes after treating cells with the dual-targeting assembly of Aβ and phagosomes, please refer to the conclusion of this example. Figure 32 , Figure 32Confocal microscopy images of BV-2 cells transfected with pmRFP-LC3 and then treated with blank medium, phagosome-targeting peptide control assembly, phagosome-targeting assembly, Aβ-targeting assembly, and Aβ- and phagosome-targeting assembly together with FITC-labeled Aβ oligomers for 12 h. The administration concentration of the assembly was 10 μM. Figure 32 As shown, only in cells treated with the Aβ and phagosome dual-targeting assembly, internalized Aβ and phagosomes were highly co-localized, indicating that the Aβ and phagosome dual-targeting assembly has the ability to recruit phagosomes.

[0153] Example 31

[0154] In order to investigate the co-localization of Aβ and lysosomes after treating cells with the dual-targeting assembly of Aβ and phagosomes, please refer to the conclusion of this example. Figure 33 , Figure 33 Confocal microscopy images of BV-2 cells transfected with mTagBFP2-Lysosomes-20 and then treated with blank medium, phagosome-targeting peptide control assembly, phagosome-targeting assembly, Aβ-targeting assembly, and Aβ- and phagosome-targeting assembly together with FITC-labeled Aβ oligomers for 12 h. The administration concentration of the assembly was 10 μM. Figure 33 As shown, only in cells treated with the Aβ and phagosome dual-targeting assembly, internalized Aβ and lysosomes were highly co-localized, indicating that the Aβ and phagosome dual-targeting assembly enabled the internalized Aβ to be engulfed by phagosomes and then taken into lysosomes for degradation.

[0155] Example 32

[0156] In order to investigate the co-localization of Aβ-containing endosomes and phagosomes after treating cells with the dual-targeting assembly of Aβ and phagosomes, the conclusion of this example can be found in Figure 34 , Figure 34 Confocal microscopy images of BV-2 cells transfected with pmRFP-LC3 and then treated with blank medium, phagosome-targeting peptide control assembly, phagosome-targeting assembly, Aβ-targeting assembly, and Aβ and phagosome-targeting assembly together with 10 μM Aβ oligomers for 12 h to show RAGE upregulation. The administration concentration of the assembly was 10 μM. Figure 34 As shown, only in cells treated with the Aβ and phagosome dual-targeting assembly were Aβ-containing endosomes and phagosomes highly co-localized, indicating that the Aβ and phagosome dual-targeting assembly enabled the transfer of Aβ-containing endosomes to phagosomes.

[0157] Example 33

[0158] To investigate the co-localization of Aβ-containing endosomes and lysosomes after treatment of cells with the dual-targeting assembly of Aβ and phagosomes, please refer to the conclusion of this example. Figure 35 , Figure 35 Confocal microscopy images of BV-2 cells transfected with mTagBFP2-Lysosomes-20 and then treated with blank medium, phagosome-targeting peptide control assembly, phagosome-targeting assembly, Aβ-targeting assembly, and Aβ and phagosome-targeting assembly together with 10 μM Aβ oligomers for 12 h to show RAGE upregulation. The administration concentration of the assembly was 10 μM. Figure 35 As shown, only in cells treated with the Aβ and phagosome dual-targeting assembly were Aβ-containing endosomes and lysosomes highly co-localized, indicating that the Aβ and phagosome dual-targeting assembly enabled the transfer of Aβ-containing endosomes to phagosomes and subsequent degradation by lysosomes.

[0159] Example 34

[0160] To investigate the therapeutic effects of the PD-L1 and phagosome dual-targeting assembly, different assemblies were injected intravenously and PD-L1 antibodies were injected intraperitoneally (PD-L1 antibody dose: 100 μg intraperitoneally every two days; other groups were injected intravenously every other day at 1.8 μmol / kg, the same below). Figure 36 , Figure 36 Schematic diagram of the PD-L1 and phagosome dual-targeting assembly therapy designed in this invention. Specifically, a breast cancer model was established by subcutaneous injection of 4T1 breast cancer cells. Dosing was performed in groups starting on day 7 after tumor inoculation. The therapeutic efficacy was subsequently assessed by measuring tumor volume, tumor weight, PD-L1 degradation efficiency, and T cell enhancement.

[0161] The treatment effects are as follows: Treatment was performed by intravenous injection of normal saline (blank control group), PD-L1 single-targeting nanoparticles, PD-L1 and phagosome dual-targeting assembly, PD-L1 single-targeting assembly, phagosome-targeting peptide control assembly, phagosome single-targeting assembly, and intraperitoneal injection of PD-L1 antibody. Figure 37 , Figure 37 To quantitatively characterize the changes in tumor volume, it can be seen that the tumor volume of the group receiving intravenous injection of the PD-L1 and phagosome dual-targeting assembly was significantly inhibited compared with the other groups, with a significant difference, demonstrating the significant therapeutic significance of the PD-L1 and phagosome dual-targeting assembly. Figure 38 To quantitatively characterize the tumor weight statistics, it can be seen that the group injected intravenously with the PD-L1 and phagosome dual-targeting assembly significantly inhibited the tumor weight compared with other groups, with significant differences, reflecting the great significance of the PD-L1 and phagosome dual-targeting assembly for treatment.

[0162] Example 35

[0163] To investigate the therapeutic effects of the PD-L1 and phagosome dual-targeting assembly, patients were treated with intravenous injection of normal saline, PD-L1 single-targeting nanoparticles, PD-L1 and phagosome dual-targeting assembly, PD-L1 single-targeting assembly, phagosome-targeting peptide control assembly, phagosome single-targeting assembly, and intraperitoneal injection of PD-L1 antibody. Figure 39 , Figure 39 To quantitatively characterize the PD-L1 degradation effects of different groups on mouse tumor tissue, normal saline, PD-L1 single-targeted nanoparticles, PD-L1 and phagosome dual-targeted assembly, PD-L1 single-targeted assembly, phagosome-targeted peptide control assembly, phagosome single-targeted assembly, and PD-L1 antibody were injected intravenously. Figure 39 As shown, the PD-L1 protein on the tissues of the group injected intravenously with the dual-targeting assembly of PD-L1 and phagosome was significantly degraded compared with that in other groups, reflecting the high degradation efficiency of the dual-targeting assembly of PD-L1 and phagosome.

[0164] Example 36

[0165] To investigate the side effects of the PD-L1 and phagosome dual-targeting assembly, patients were treated with intravenous injection of normal saline, PD-L1 single-targeting nanoparticles, PD-L1 and phagosome dual-targeting assembly, PD-L1 single-targeting assembly, phagosome-targeting peptide control assembly, phagosome single-targeting assembly, and intraperitoneal injection of PD-L1 antibody. Figure 40 , Figure 40 To quantitatively characterize the number of lung nodules produced in mice by different groups, the results showed that the group receiving intravenous injection of the PD-L1 and phagosome dual-targeting assembly significantly reduced the number of lung nodules compared to the other groups, demonstrating the low toxicity and high safety of the PD-L1 and phagosome dual-targeting assembly.

[0166] Example 37

[0167] To investigate the therapeutic effects of the PD-L1 and phagosome dual-targeting assembly, patients were treated with intravenous injection of normal saline, PD-L1 single-targeting nanoparticles, PD-L1 and phagosome dual-targeting assembly, PD-L1 single-targeting assembly, phagosome-targeting peptide control assembly, phagosome single-targeting assembly, and intraperitoneal injection of PD-L1 antibody. Figure 41 , Figure 41 To quantitatively characterize the CD4 + T cells and CD8 + Schematic diagram of T cell stimulation improvement. The results show that the group of intravenous injection of PD-L1 and phagosome dual-targeting assembly has a higher number of CD4 on tumor tissue than other groups.+ T cells and CD8 + The significant increase in T cells indicates that after the PD-L1 and phagosome dual-targeting assembly degrades PD-L1, tumor cells lose key immunosuppressive molecules, relieve T cell inhibition, and restore anti-tumor immune response.

[0168] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A protein degradation agent, characterized in that The protein degradation agent includes an assembly, wherein the monomers forming the assembly contain the following parts in a linked arrangement: Target protein-specific ligand; phagosome-targeting peptides; The endosomal membrane inserting polypeptide is located between the target protein-specific ligand and the phagosome targeting polypeptide, and the endosomal membrane inserting polypeptide is a C-terminal membrane inserting polypeptide.

2. The protein degradation agent according to claim 1, characterized in that Contains at least one of the following characteristics: (1) The target protein-specific ligand is chemically linked to the endosomal membrane-inserting polypeptide; (2) the phagosome targeting polypeptide is chemically linked to the endosomal membrane inserting polypeptide; (3) The monomer contains a hydrophilic modification; (4) The target protein is an extracellular protein or a cell membrane protein; (5) The target protein-specific ligand includes a target protein-specific polypeptide ligand.

3. The protein degradation agent according to claim 2, characterized in that Contains at least one of the following characteristics: (1) The target protein-specific ligand is covalently linked to the endosomal membrane-inserting polypeptide; The target protein-specific ligand and the endosomal membrane-inserting polypeptide are connected by a covalent bond formed by an amino group and a sulfhydryl group or by a peptide bond; (2) The phagosome targeting polypeptide is covalently linked to the endosomal membrane inserting polypeptide; The connection between the phagosome targeting polypeptide and the endosomal membrane inserting polypeptide includes connection via a peptide bond or connection via a chemical reaction of a modifying group; (3) The monomer is modified with hydrophilic groups.

4. The protein degradation agent according to claim 3, characterized in that Contains at least one of the following characteristics: (1) The hydrophilic group includes at least one of a hydroxyl group, a carboxyl group, an amino group, and an amide group; (2) The sources of hydrophilic groups include hydrophilic amino acids; (3) Modifying the target protein-specific ligand with a hydrophilic group; the modification includes modifying the target protein-specific ligand with a hydrophilic group at one end close to the endosomal membrane inserting polypeptide.

5. The protein degradation agent according to claim 1, characterized in that Contains at least one of the following characteristics: (1) The amino acid sequence of the endosomal membrane inserting polypeptide is shown in SEQ ID NO.5; (2) The amino acid sequence of the phagosome targeting polypeptide is shown in SEQ ID NO. 4; (3) The target protein includes one or more of programmed death receptor-ligand 1, beta-amyloid protein, advanced glycation end product ligand, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, prostate-specific membrane antigen, and mitochondrial p32 molecular chaperone protein; wherein, When the target protein includes programmed death receptor-ligand 1, the target protein-specific ligand includes a programmed death receptor-ligand 1-specific polypeptide ligand, and the amino acid sequence of the programmed death receptor-ligand 1-specific polypeptide ligand is shown in SEQ ID NO.1; And / or, when the target protein includes β-amyloid protein, the target protein-specific ligand includes a β-amyloid protein-specific polypeptide ligand, and the amino acid sequence of the β-amyloid protein-specific polypeptide ligand is shown in SEQ ID NO.2; And / or, when the target protein includes an advanced glycation ligand end product, the target protein-specific ligand includes an advanced glycation ligand end product-specific polypeptide ligand, and the amino acid sequence of the advanced glycation ligand end product-specific polypeptide ligand is shown in SEQ ID NO.

3.

6. The method for preparing the protein degradation agent according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: The monomers are dispersed in an aqueous solution at a set concentration, and the monomers self-assemble to form the assembly.

7. The preparation method according to claim 6, characterized in that Contains at least one of the following characteristics: (1) The set concentration is not lower than the critical concentration for micelle formation; (2) Specific steps include: S1. preparing a phagosome targeting polypeptide and a modified endosomal membrane inserting polypeptide to obtain an endosomal anchored phagosome targeting peptide; preparing a target protein-specific ligand containing the modification; S2, mixing the endosomal-anchored phagosome targeting peptide and the target protein-specific ligand in the presence of a solvent to react, so that the modified portion on the endosomal membrane-inserting polypeptide and the target protein-specific ligand is connected to obtain the monomer; (3) After the self-assembly is completed, a purification step is also included.

8. The preparation method according to claim 7, characterized in that Contains at least one of the following characteristics: (1) The molar ratio of the endosomal anchored phagosome targeting peptide to the target protein-specific ligand is 1:(1-20); (2) The solvent is an organic solvent that can dissolve the polypeptide; the organic solvent includes N , N -dimethylformamide or dimethyl sulfoxide; (3) The purification step includes dialysis.

9. Use of the protein degrader according to any one of claims 1 to 5 or the protein degrader obtained by the preparation method according to any one of claims 6 to 8 in the preparation of preventive or therapeutic drugs, characterized in that: The medical uses of the preventive or therapeutic drug include at least one of the following: (1) Prevention or treatment of solid tumors; (2) Prevention or treatment of neurological diseases; (3) Prevention or treatment of inflammatory diseases.

10. A pharmaceutical composition, characterized in that The invention comprises the protein degrading agent according to any one of claims 1 to 5 or the protein degrading agent obtained by the preparation method according to any one of claims 6 to 8.

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