Nanometer degradation agent based on proteolysis targeting chimeric molecules as well as preparation method and application of nanometer degradation agent

Abstract

The invention relates to the technical field of nano materials, in particular to a nano degradation agent based on a proteolysis targeting chimera as well as a preparation method and application of the nano degradation agent. According to the nanometer degradation agent provided by the invention, BRD4 protein is taken as a target spot, natural cationic side groups of polylysine are utilized, two ligands are connected through condensation reaction, the proportion of the ligands is optimized, and then dimethyl maleic anhydride is modified to enable the nanometer degradation agent to have acid response charge reversal capability, so that material uptake is enhanced, the BRD4 protein is degraded, and death of tumor cells is induced. According to the technical scheme, through dynamic response of a tumor microenvironment, the problems that a traditional proteolysis targeting chimera is poor in permeability, insufficient in selectivity and the like are solved, and a more efficient and safer nano degradation agent system is provided for targeted therapy of diseases such as tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and in particular to a nano-degrading agent based on protein hydrolysis targeting chimera, and a preparation method and application thereof. Background Art

[0002] Proteolysis-targeted chimera technology aims to use small molecule compounds or biomacromolecules to induce proteins to be recognized and degraded by the intracellular degradation system, thereby achieving specific regulation of proteins. Generally, proteolysis-targeted chimeras are small molecules that connect a ligand targeting a target protein and a ligand targeting an E3 ubiquitin ligase by a linker. By bringing the two together, proteolysis-targeted chimeras can tag the target protein with a ubiquitin tag, allowing it to be further degraded through the ubiquitin-proteasome system. Unlike the traditional "occupancy-driven" model of small molecule inhibitors, proteolysis-targeted chimeras can target proteins that are traditionally "undruggable" through an "event-driven" catalytic degradation effect, which is expected to overcome the drug resistance of small molecule inhibitors and has attracted widespread attention in the field of drug research and development. At present, this technology has many advantages in the treatment of diseases such as tumors and neurodegenerative diseases: (1) It can overcome the body's resistance to drugs; (2) It has high target selectivity and safety; (3) It has a wider range of action, higher activity, and can target "undruggable" targets; (4) It provides a new type of rapid and reversible chemical knockout method.

[0003] Although a variety of small molecule proteolysis-targeted chimeras have been approved in the United States and have shown effective treatment for a variety of cancers, they have also exposed challenges in linker screening, pharmacokinetics, cell permeability, and tissue cell selectivity. Although a variety of small molecule proteolysis-targeted chimeras have been approved in the United States and have shown effective treatment for a variety of cancers, they have also exposed challenges in linker screening, pharmacokinetics, cell permeability, and tissue cell selectivity. On the one hand, the design of proteolysis-targeted chimeras does not conform to the "five-drug-like" principle, and their large molecular weight leads to their poor water solubility and poor membrane permeability. On the other hand, the ability of proteolysis-targeted chimeras to efficiently catalyze the degradation of target proteins may cause unpredictable damage to normal organs when used systemically. Therefore, how to improve the membrane permeability, targeting, and stability of proteolysis-targeted chimeras, especially to achieve efficient and specific protein degradation in the tumor microenvironment, is a key problem in this technical field.

[0004] BRD4 (Bromodomain-containing protein 4) is a transcriptional regulatory protein containing a bromodomain and belongs to the BET (Bromodomain and Extra-Terminal) family. BRD4 specifically binds to acetylated histone tails through its bromodomain, thereby affecting the open state of chromatin and transcriptional activity, regulating key processes such as cell cycle, proliferation and DNA repair. Due to the abnormal expression and activation of BRD4 in various cancers (such as breast cancer, prostate cancer and lung cancer), it can promote tumor cell proliferation and metastasis, so BRD4 has become an important target in anti-tumor therapy.

[0005] Traditional small-molecule proteolysis-targeted chimeras cannot target tumor tissues and may therefore lead to off-target effects. They also face disadvantages such as poor water solubility, poor permeability, and difficulty in forming a ternary complex "hook effect", which hinders their clinical research. Nanotechnology, with its advantages such as improving drug concentration at the lesion, spatiotemporally controlled drug release, high specific surface area, and multivalent binding possibility, provides a solution for the clinical transformation of proteolysis-targeted chimeras. Although some progress has been made in the study of macromolecular proteolysis-targeted chimeras, the development of these nanodegraders is hampered by their unfavorable physicochemical properties, including high molecular weight, poor membrane permeability, and instability. In addition, the indiscriminate distribution and sustained biological activity of small and macromolecular degraders may lead to uncontrollable degradation of proteins at non-target sites, thereby limiting the in vivo efficacy. Therefore, the development of a high-molecular proteolysis-targeted chimera strategy with high specificity for tumor-related proteins is of great scientific significance and practical application value. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a nano-degrader based on a protein hydrolysis targeting chimera, and a preparation method and application thereof. The present invention provides a method of designing the high molecular weight polymer itself into a double-headed targeted polymer, which is made into a nano-reactor by utilizing the high specific surface area and the possibility of multivalent binding, thereby improving the in vivo stability and water solubility of the protein hydrolysis targeting chimera and optimizing the degradation ability, thereby improving the effectiveness of tumor treatment.

[0007] The present invention provides a nano-degradation agent having a structure shown in formula (I):

[0008] Formula (I);

[0009] Wherein, the structure of R1 is shown in formula (II), and the structure of R2 is shown in formula (III);

[0010] Formula (II); Formula (III);

[0011] R3 is selected from any one of amino, succinic anhydride and maleic anhydride;

[0012] m is the degree of polymerization, m=10~500, n=10~200, x=1~100; y=1~100; z=8~500.

[0013] The nanodegrader provided by the present invention connects two ligands of the proteolysis targeting chimera through a linker, recruits the target protein and E3 ubiquitin ligase respectively, thereby affixing ubiquitin tags to the target protein so that it can be degraded by the proteasome. Different from the "site occupancy" mode of traditional small molecule inhibitors, it is like a catalyst, which can continuously form a ternary complex and efficiently degrade the target protein through the ubiquitin-proteasome system in the cell. Although traditional proteolysis targeting chimera small molecules have the advantages of targeted degradation of "undruggable" proteins and efficient degradation capabilities, they often face insufficient ability to penetrate the cell membrane in practical applications, thereby affecting the delivery efficiency in tissues such as tumors. In addition, the nonspecific distribution and continuous activity of small molecule proteolysis targeting chimeras easily lead to the degradation of non-targeted proteins, increasing the potential toxicity to normal tissues. Nanotechnology provides a solution for the clinical transformation of proteolysis targeting chimeras because of its advantages such as being able to improve drug concentration at the lesion, spatiotemporally controllable drug release, high specific surface area and multivalent binding possibilities.

[0014] In some embodiments, x:y=(1-5):(1-5).

[0015] In some specific embodiments, the x:y=2:1, 1:1 or 1:2.

[0016] Preferably, x:y=2:1.

[0017] In some specific embodiments, the R3 is 2,3-dimethylmaleic anhydride.

[0018] In some specific embodiments, m=60, n=113, x=8; y=4; z=48.

[0019] The present invention provides a method for preparing the nano-degradation agent, comprising the following steps:

[0020] Step 1, taking N(ε)-benzyloxycarbonyl-L-lysine, anhydrous tetrahydrofuran and triphosgene and reacting them to obtain N(ε)-benzyloxycarbonyl-L-lysine intracyclic anhydride monomer;

[0021] Step 2, reacting N(ε)-benzyloxycarbonyl-L-lysine intracyclic anhydride monomer with aminated polyethylene glycol monomethyl ether in the presence of a solvent to obtain polyethylene glycol monomethyl ether-b-poly(N-benzyloxycarbonyl-lysine);

[0022] Step 3, mixing polyethylene glycol monomethyl ether-b-poly (N-benzyloxycarbonyl-lysine), trifluoroacetic acid and hydrogen bromide acetic acid solution to obtain polyethylene glycol monomethyl ether-b-polylysine;

[0023] Step 4, coupling the R1 and R2 groups in the nanodegrader to polyethylene glycol monomethyl ether-b-polylysine to obtain a cationic polylysine proteolysis targeting chimera molecule;

[0024] Step 5: Mix the cationic polylysine protein hydrolysis targeting chimera molecule with the R3 group in the nanodegrader to obtain the nanodegrader.

[0025] In some embodiments, in step 1, the temperature of the mixed reaction is 50-60° C., and the time of the mixed reaction is 1.5-3 h;

[0026] Preferably, the method further comprises subjecting the mixture after the mixed reaction to precipitation with n-hexane, separation, washing, recrystallization and vacuum drying to obtain the N(ε)-benzyloxycarbonyl-L-lysine intracyclic anhydride monomer.

[0027] In some embodiments, in step 2,

[0028] The preparation method of the amination polyethylene glycol monomethyl ether comprises:

[0029] S1, dissolving mPEG-OH, potassium hydroxide and p-toluenesulfonyl chloride in dichloromethane, reacting, washing and separating to obtain an organic phase, filtering and concentrating the obtained organic phase, precipitating with cold ether and filtering to obtain the intermediate product mPEG-OTs;

[0030] S2, taking mPEG-OTs, ammonium chloride and ammonia monohydrate, mixing and reacting, extracting the organic phase with dichloromethane, washing the obtained organic phase with water, drying, filtering, rotary evaporation concentration, cold ether precipitation, and draining to obtain amino polyethylene glycol monomethyl ether;

[0031] The aminated polyethylene glycol monomethyl ether and toluene are subjected to azeotropic dehydration under vacuum conditions, toluene is removed by reduced pressure distillation, and then mixed and reacted with N(ε)-benzyloxycarbonyl-L-lysine intracyclic anhydride monomer, wherein the solvent is N,N-dimethylformamide, the reaction temperature is 15-35° C., and the mixing reaction time is 2-4 days;

[0032] Preferably, the method further comprises subjecting the reaction solution after the reaction to cold ether precipitation and vacuum drying to obtain polyethylene glycol monomethyl ether-b-poly(N-benzyloxycarbonyl-lysine).

[0033] In some embodiments, in step 3, the mixing temperature is 15-35° C., and the mixing reaction time is 1.5-3 h;

[0034] Preferably, the method further comprises subjecting the reaction solution after the reaction to cold ether sedimentation and centrifugation, dissolving the obtained precipitate with N,N-dimethylformamide, dialyzing, and freeze-drying to obtain polyethylene glycol monomethyl ether-b-poly(N-benzyloxycarbonyl-lysine).

[0035] In some embodiments, in step 4, the coupling conditions include: using N,N-dimethylformamide as solvent, DMAP and DIC as catalysts, and reacting at a temperature of 15-35° C. for 2-3 days;

[0036] Preferably, the reaction solution after the coupling is precipitated with cold ether and centrifuged, the obtained precipitate is vacuum dried and then dissolved in N,N-dimethylformamide, and the cationic polylysine protein hydrolysis targeting chimera molecule is obtained after dialyzation and freeze drying.

[0037] In some embodiments, in step 5, the pH of the mixed reaction is 8-9, the temperature of the mixed reaction is 15-35° C., and the time of the mixed reaction is 7-10 h;

[0038] Preferably, the nano-degradation agent is obtained by dialyzing and freeze-drying the reaction solution after the coupling.

[0039] The present invention provides the use of the nano-degrading agent or the nano-degrading agent prepared by the preparation method in preparing drugs for preventing and / or treating tumors.

[0040] The present invention provides a drug for preventing and / or treating tumors, comprising the nano-degrading agent or the nano-degrading agent prepared by the preparation method.

[0041] Compared with the prior art, the nanodegrader provided by the present invention does not adopt the form of nanoparticle-supported small molecule protein hydrolysis targeting chimera, or is designed as a bonded drug of a polymer carrier and a small molecule protein hydrolysis targeting chimera, but takes BRD4 protein as the target, utilizes the natural cationic side group of polylysine, connects two ligands through condensation reaction, optimizes the ligand ratio, and then modifies dimethyl maleic anhydride to make it have acid-responsive charge reversal ability, thereby enhancing material uptake, degrading BRD4 protein, and inducing tumor cell death. By precisely regulating the release and activity of protein hydrolysis targeting chimera in the local tumor, the present invention realizes tumor-specific protein degradation, improves the selectivity of targeted tissues, and reduces nonspecific toxic side effects to normal tissues. The technical solution of the present invention overcomes the problems of poor permeability and insufficient selectivity of traditional protein hydrolysis targeting chimeras through the dynamic response of the tumor microenvironment, and provides a more efficient and safe nanodegrader system for the targeted treatment of diseases such as tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The NMR, IR and mass spectra of N(ε)-benzyloxycarbonyl-L-lysine anhydride monomer are shown, where Figure A is 1 H spectrum, B spectrum 13 C spectrum, C is the infrared spectrum, D is the mass spectrum;

[0043] Figure 2 NMR showing terminal amino group of polyethylene glycol methyl ether 1 H spectrum (left) and 13 C spectrum (right);

[0044] Figure 3 shows the NMR spectrum of polyethylene glycol monomethyl ether-b-polylysine;

[0045] Figure 4 The figure shows the preparation reaction formula of the cationic polylysine protein hydrolysis targeting chimera targeting BRD4 and the nuclear magnetic resonance spectra of the polylysine protein hydrolysis targeting chimera with different grafting ratios, wherein Figure A is the preparation reaction formula and Figure B is the nuclear magnetic resonance spectrum;

[0046] Figure 5 The NMR spectra of SA-unresponsive proteolysis-targeted chimeras with different grafting ratios are shown;

[0047] Figure 6 shows the NMR spectrum of the JT2-1-DMMA acid-responsive proteolysis targeting chimera;

[0048] Figure 7 The particle size and potential of three nanoproteolysis targeting chimeras, JT2-1, JT2-1-SA and JT2-1-DMMA, are shown;

[0049] Figure 8 Circular dichroism spectra of three nanoprotein hydrolysis targeting chimeras, JT2-1, JT2-1-SA and JT2-1-DMMA, at pH 6.8 are shown;

[0050] Fig. 9 The CCK-8 cytotoxicity test of nano-protein hydrolysis targeting chimeras with different grafting ratios on MCF-7 cells is shown;

[0051] Fig.10 The CCK-8 cytotoxicity test of JQ1 and nanoprotein hydrolysis targeting chimera on MCF-7 cells under different pH conditions is shown;

[0052] Fig.11 Flow cytometric analysis of cellular endocytosis of the acid-responsive poly-lysine proteolysis-targeting chimera;

[0053] Fig.12 CLSM analysis of JT2-1-SA (left) and JT2-1-DMMA (right) endocytosis is shown;

[0054] Fig.13 WB showing the expression of BRD4 and c-MYC in cells after induction with different concentrations of JT2-1-SA;

[0055] Fig.14 The CLSM (left) and flow cytometry results (right) of cell BRD4 expression after induction of different nanoprotein proteolysis targeting chimeras are shown;

[0056] Fig.15 CLSM and flow cytometry analysis of the cell BRD4 degradation mechanism after induction of different nanoprotein proteolysis targeting chimeras;

[0057] Fig.16 Figure 2 shows the cell cycle results of poly-lysine proteolysis targeting chimeras modified with different anhydrides;

[0058] Fig.17 Shows the in vivo imaging distribution diagram after tail vein injection of Cy5.5-JT2-1-DMMA;

[0059] Fig.18 Figure 2 shows the experimental results of mouse treatment after tail vein injection of different nanoprotein hydrolysis targeted chimeras, where Figure A is the experimental flow chart, Figure B is the tumor tissue diagram of mice after tail vein injection, Figure C is the statistical results of mouse tumor weight after tail vein injection, Figure D is the statistical results of mouse tumor volume after tail vein injection, and Figure E is the statistical results of mouse body weight after tail vein injection. DETAILED DESCRIPTION

[0060] The present invention provides a nanodegrader based on a proteolytic targeting chimera and a preparation method and application thereof. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0061] Terminology explanation:

[0062] Proteolysis Targeting Chimera

[0063] Proteolysis targeting chimeras are bifunctional small molecules that degrade target proteins by connecting ligands of targeted proteins and ligands of E3 ubiquitin ligases, thereby achieving regulation of specific proteins through the ubiquitin-proteasome system in cells. This technology is widely used in the targeted degradation of "undruggable" proteins, with high selectivity and potential drug resistance advantages.

[0064] BRD4 (Bromodomain-containing Protein 4)

[0065] BRD4 (Bromodomain-containing protein 4) is a transcriptional regulatory protein containing a bromodomain. It belongs to the BET (Bromodomain and Extra-Terminal) family. It participates in transcriptional regulation and plays a key role in tumor growth, cell proliferation and other processes. BRD4 specifically binds to acetylated histone tails through its bromodomain, thereby affecting the open state of chromatin and transcriptional activity, regulating key processes such as cell cycle, proliferation and DNA repair. Due to the abnormal expression and activation of BRD4 in various cancers (such as breast cancer, prostate cancer and lung cancer), it can promote tumor cell proliferation and metastasis, so BRD4 has become an important target in anti-tumor therapy.

[0066] E3 Ubiquitin Ligase

[0067] E3 ubiquitin ligase is an enzyme responsible for specifically attaching ubiquitin tags to target proteins, thereby marking them for degradation in the proteasome system. In the proteolysis-targeted chimera technology, the target protein is connected to the E3 ligase through a ligand, so that the target protein is ubiquitinated and then degraded.

[0068] Acid-responsive

[0069] Acid response refers to the structural or functional changes of materials in an acidic environment. The acid-responsive polymer nanomaterials of the present invention undergo structural changes under the acidic conditions of the tumor microenvironment, thereby increasing the exposure of the targeting ligand and improving the uptake and targeted degradation efficiency of tumor cells.

[0070] mPEG (Methoxy Polyethylene Glycol)

[0071] mPEG is a hydrophilic polymer that can guide polymerization through the action of an initiator to form a biocompatible and water-soluble polymer. Polylysine initiated by mPEG is used in the present invention to prepare acid-responsive nanoparticles to enhance the water solubility and in vivo stability of the material.

[0072] Poly-L-lysine (Plys)

[0073] Polylysine is a polymer formed by the polymerization of lysine monomers and is commonly used in the biomedical field. It contains multiple active amino sites, is easy to graft functional molecules, and exhibits structural changes in acidic environments, making it suitable as a skeleton for acid-responsive materials.

[0074] Ubiquitin-Proteasome System

[0075] The ubiquitin-proteasome system is a protein degradation pathway in which ubiquitin molecules are attached to target proteins by E3 ligases, causing the target proteins to be tagged and transferred to the proteasome for degradation. Proteolysis targeting chimeras selectively eliminate specific proteins by utilizing the ubiquitin-proteasome system to tag target proteins for degradation.

[0076] Nano-degrader

[0077] Nanodegraders are nanoscale materials that can degrade specific proteins by connecting target proteins to degradative enzymes. The nanodegraders of the present invention combine the properties of proteolysis-targeted chimeras and acid-responsive nanomaterials, enabling them to act more efficiently on the target protein BRD4 in the tumor microenvironment.

[0078] Ligand

[0079] A ligand is a chemical molecule that can specifically bind to a target molecule (such as a protein). In a proteolysis targeting chimera, one ligand targets the target protein (such as BRD4) and the other ligand targets the E3 ubiquitin ligase, and the ligand directs the degradation of the target by connecting the target and the enzyme.

[0080] Self-assembled Nanoparticles

[0081] Self-assembled nanoparticles are a type of stable structure formed by physical or chemical forces between molecules. The polymer nanodegradation agent of the present invention forms stable nanoparticles through the self-assembly of mPEG and polylysine, thereby improving the stability, targeting and bioavailability of the drug.

[0082] The present invention aims to solve several key challenges of existing proteolysis-targeted chimera technology in clinical applications, especially the off-target effects caused by its inability to target tumor tissues, as well as the problems of poor membrane permeability and poor water solubility. The proteolysis-targeted chimera connects two ligands through a linker, recruiting the target protein and E3 ubiquitin ligase respectively, thereby labeling the target protein with ubiquitin so that it can be degraded by the proteasome. This technology is different from the "site occupancy" mode of traditional small molecule inhibitors. It is like a catalyst that can continuously form a ternary complex and efficiently degrade the target protein through the ubiquitin-proteasome system in the cell. Although traditional proteolysis-targeted chimera small molecules have the advantages of targeted degradation of "undruggable" proteins and efficient degradation capabilities, they often face insufficient ability to penetrate the cell membrane in practical applications, thereby affecting the delivery efficiency in tumor and other tissues. In addition, the nonspecific distribution and sustained activity of small molecule proteolysis-targeted chimeras easily lead to the degradation of non-targeted proteins, increasing the potential toxicity to normal tissues. Nanotechnology provides a solution for the clinical transformation of proteolysis-targeted chimeras because of its advantages such as improving drug concentration at the lesion, spatiotemporally controlled drug release, high specific surface area and multivalent binding possibility. At present, nano-proteolysis-targeted chimera technology mainly improves the body distribution of proteolysis-targeted chimeras and reduces side effects by designing multi-level responsive prodrugs, nano-delivery systems, split hybrid nanotechnology and drug co-assembly. Although there are many emerging methods, this nano-proteolysis-targeted chimera technology still has a long way to go in clinical application, and the key is to enhance tumor enrichment and exert the degradation effect of proteolysis-targeted chimeras. Therefore, how to increase the effective concentration of proteolysis-targeted chimeras in targeted tissues, reduce non-targeted effects, and maintain efficient degradation of target proteins is a major problem in this technical field. To this end, we propose a scheme of multi-head proteolysis-targeted chimera mode + acid-responsive nanoparticles. On the one hand, the more people contribute, the higher the flame. The mode of using polymer side chains for multi-target head modification is theoretically better than the efficiency of a small molecule. On the other hand, tumor enrichment is achieved through the EPR effect of nanoparticles, and charge reversal is further achieved in the acidic microenvironment of the tumor, thereby enhancing tumor penetration and uptake.

[0083] In response to these problems, the present invention constructs a high-molecular-weight protein hydrolysis-targeted chimera nanoparticle that responds to the acidic microenvironment of tumors and targets the degradation of BRD4 protein, aiming to improve the in vivo stability, targeting and membrane permeability of the protein hydrolysis-targeted chimera through polymer nanotechnology, thereby improving the effectiveness of tumor treatment. Our goal is to target BRD4 protein, develop acid-responsive polylysine nanoprotein hydrolysis-targeted chimeras, optimize the surface ligand ratio, and enhance tumor uptake and degradation functions. The basic idea is to utilize the natural cationic side groups of polylysine, connect two ligands through a condensation reaction, optimize the ligand ratio, and then modify dimethylmaleic anhydride to give it an acid-responsive charge reversal ability, thereby enhancing material uptake, degrading BRD4 protein, and inducing tumor cell death.

[0084] The present invention uses polylysine initiated by PEG5000 as a template, grafts two small molecule ligands of the proteolysis targeting chimera onto the polylysine side chain through the condensation reaction of amino and carboxyl groups, and uses 2,3-dimethylmaleic anhydride to block the excess side chain amino groups to form a stable nanoparticle structure. In the acidic microenvironment of the tumor, the maleic anhydride group on the surface of the nanoparticle is responsively removed, triggering the secondary structure transformation and charge reversal of the polymer, enhancing the uptake efficiency of the nanoparticles in tumor cells, and further exposing the side chain targeting module. The nanoparticles enter the tumor cells to recruit BRD4 protein and E3 ubiquitin ligase, and achieve degradation of the target protein through the ubiquitin-proteasome pathway. By precisely regulating the release and activity of the proteolysis targeting chimera in the local tumor, the present invention achieves tumor-specific protein degradation, improves the selectivity for the targeted tissue, and reduces the nonspecific toxic side effects on normal tissues. Therefore, the technical solution of the present invention overcomes the problems of poor permeability and insufficient selectivity of traditional proteolysis targeting chimeras through the dynamic response of the tumor microenvironment, and provides a more efficient and safe nanodegrader system for the targeted treatment of diseases such as tumors.

[0085] The present invention proposes a high molecular protein hydrolysis targeting chimera nano delivery system based on acidic microenvironment response, which is used to target the specific degradation of BRD4 protein and achieve efficient and precise tumor treatment. The system overcomes the shortcomings of traditional protein hydrolysis targeting chimera technology in terms of permeability, targeting and stability by responsively releasing protein hydrolysis targeting chimera molecules in the tumor microenvironment. The core of the present invention is to construct a PEG5000-triggered hydrophilic polylysine template through a ring-opening polymerization reaction, and graft two small molecule ligands of the protein hydrolysis targeting chimera (a ligand targeting BRD4 protein and a ligand targeting E3 ligase) to the side chain of polylysine in a certain proportion through the condensation reaction of amino and carboxyl groups, and finally block the redundant side chain amino groups through acid-sensitive 2,3-dimethylmaleic anhydride, thereby forming a high molecular protein hydrolysis targeting chimera nanoparticle with acid response and targeted degradation function.

[0086] The present invention provides a high molecular protein hydrolysis targeting chimera based on a polyethylene glycol monomethyl ether-b-polylysine hydrophilic template, having a structure shown in formula (I). The initiator of the polylysine acid initiated by mPEG is mPEG with an amino group at the end, which can have different molecular weights. The monomer unit of the polyamino acid is lysine, and the total polymerization degree m is 10-500. The polyamino acid synthesis method is the ring-opening polymerization of N-carboxylic acid ring anhydride (NCA) monomer well known to those skilled in the art. The total polymerization degree can be controlled by changing the feed ratio of the initiator to the monomer. Different proportions of R1 and R2 molecules are grafted onto the lysine side group, and R1 and R2 are respectively the ligand of the target degradation protein BRD4 and the E3 ubiquitin ligase ligand, and the R1 and R2 ligand molecules are as follows. By changing the feed ratio of R1 and R2, polylysine protein hydrolysis targeting chimera molecules with different grafting ratios can be obtained, and the degradation effect of the target protein can be regulated. R3 can be an amino group, succinic anhydride, maleic anhydride or other acid response groups. By changing the feed ratio of initiator and monomer, monodisperse polyamino acid nanoparticles with uniform and stable particle size can be obtained. Polymer proteolysis targeting chimeras can obtain water-soluble single-molecule nanoparticles with positive or negative surface charge in aqueous solution or phosphate buffer solution, and the particle size is precisely controlled between 4-120 nm. Polylysine proteolysis targeting chimera nanoparticles can effectively degrade BRD4 protein and promote tumor cell apoptosis.

[0087] Formula (I);

[0088] in,

[0089] R1= ; R2 = ; R3= or .

[0090] The test materials used in the present invention are all common commercial products and can be purchased in the market. The present invention is further described below in conjunction with the embodiments.

[0091] Example 1 Preparation and characterization of acid-responsive polylysine proteolysis-targeted chimeras

[0092] 1. Preparation of N(ε)-benzyloxycarbonyl-L-lysine intracyclic anhydride monomer

[0093] At 25 °C and in a nitrogen atmosphere, 10.0 g of N(ε)-benzyloxycarbonyl-L-lysine was mixed with 100 mL of anhydrous tetrahydrofuran, and 7.0 g of triphosgene was added, and the mixture was heated to 55 °C for 2 h. After the reaction, the reaction mixture was precipitated in excess n-hexane, and a white N(ε)-benzyloxycarbonyl-L-lysine cyclic anhydride monomer solid was obtained after separation, washing, recrystallization, and vacuum drying. Figure 1 The NMR, IR and mass spectra of N(ε)-benzyloxycarbonyl-L-lysine intracyclic anhydride monomer.

[0094] Summary: Demonstrated the synthesis of lysine NCA monomer.

[0095] 2. Amination of the hydroxyl terminal of polyethylene glycol methyl ether

[0096] Weigh 25.0 g of mPEG-OH, dissolve it in 250 mL of dichloromethane, add 5.6 g of potassium hydroxide and 9.5 g of p-toluenesulfonyl chloride, and stir the reaction at room temperature. Stop the reaction after 9 days, pour it into a separatory funnel, wash it with saturated saline solution, separate the organic phase, add anhydrous magnesium sulfate and dry overnight. The filtered and concentrated solution is precipitated with cold ether, filtered with a Buchner funnel, and dried to obtain a white intermediate product mPEG-OTs. Weigh the white product mPEG-OTs, add an equal amount of ammonium chloride solid, mix in a round-bottom flask, add 250 mL of monohydrated ammonia, and stir the reaction at room temperature. Stop the reaction after 7 days, extract the organic phase with dichloromethane, wash with water, dry, filter, concentrate by rotary evaporation, precipitate with cold ether, and drain, and finally successfully prepare the white product mPEG-NH 2 In the present invention, the number average molecular weight of the polyethylene glycol monomethyl ether is preferably 1,000 to 20,000. Figure 2 The NMR of polyethylene glycol methyl ether terminally amino 1 H spectrum (left) and 13 C spectrum (right).

[0097] Summary: Demonstrated the synthesis of initiator PEG5000.

[0098] 3. Preparation of polyethylene glycol monomethyl ether-b-poly (N-benzyloxycarbonyl-lysine)

[0099] Weigh the amount of amino-modified polyethylene glycol monomethyl ether (M w= 5000) 4.88 g was added to a vacuum reaction bottle with a branch and azeotroped with 400 mL toluene to remove water, then azeotroped in an oil bath at 130 °C for 2 hours to remove excess water, and then toluene was removed by reduced pressure distillation. Anhydrous amino-terminated polyethylene glycol monomethyl ether was dissolved in 100 mL of anhydrous N,N-dimethylformamide, and then 17.9 g of N(ε)-benzyloxycarbonyl-L-lysine ring anhydride monomer was added thereto under nitrogen protection, and stirred at 25 °C for 72 hours. The reaction solution was precipitated in 10 times ice ether, and the ether on the surface of the material was drained by a vacuum drying pump to obtain polyethylene glycol monomethyl ether-b-poly (N-benzyloxycarbonyl-lysine) as a white solid.

[0100] 4. Polyethylene glycol monomethyl ether-b-polylysine (Plys 60 )

[0101] 10 g of polyethylene glycol monomethyl ether-b-poly (N-benzyloxycarbonyl-lysine) was dissolved in 100.0 mL of trifluoroacetic acid (TFA), and 30.0 mL of HBr / acetic acid 33 wt.% solution was added to the solution. The mixture was reacted at room temperature for 2 h. After the reaction, the mixture was precipitated in 10 times the volume of ice ether. The precipitate obtained by centrifugation was dissolved in DMF, loaded into a dialysis bag with Mw3500, and dialyzed with deionized water for 3 days. Finally, white polyethylene glycol monomethyl ether-b-polylysine (Plys 60 )solid. Figure 3 This is the NMR spectrum of polyethylene glycol monomethyl ether-b-polylysine.

[0102] 5. Preparation of cationic polylysine proteolysis targeting chimeras targeting BRD4

[0103] 311.0 mg of polyethylene glycol monomethyl ether-b-polylysine, 79.7 mg of commercially available (+)-JQ1 carboxylic acid molecule (CasNo: 202592-23-2) and 33.7 mg of commercially available Thalidomide-O-COOH (CasNo: 1061605-21-7) molecule were weighed and dissolved in 15 mL of anhydrous DMF. 59.0 mg of DMAP was dissolved in 3 mL of anhydrous DMF and added to the reaction solution. Finally, 52.3 mg of DIC was diluted with 5 mL of anhydrous DMF and slowly dripped into the reaction solution in an ice-water bath and nitrogen atmosphere. After returning to room temperature, the reaction was allowed to react for 2 days. After the reaction, the reaction solution was precipitated with 10 volumes of ice ether, and the white precipitate obtained by centrifugation was vacuum dried, re-dissolved with DMF, loaded into a dialysis bag with Mw3500, dialyzed with deionized water for 3 days, and finally freeze-dried to obtain a white cationic polylysine protein hydrolysis targeted chimera solid (P(lys 0.8-JQ1 i -Tha j ) 60 ), where the total grafting rate of JQ1 and Thalidomide is 20%, and the grafting ratio of JQ1 and Thalidomide is i:j. The adjustment of the grafting ratio between the two is controlled by the calculation and design of the feed ratio. The grafting rate can be obtained by calculating the integral of the corresponding hydrogen from the nuclear magnetic resonance hydrogen spectrum. Figure 4 To target BRD4, NMR spectra of poly-lysine proteolysis targeting chimeras with different grafting ratios, where JT2-1 represents a grafting ratio of i:j of 2:1, JT1-1 represents a grafting ratio of i:j of 1:1, and JT1-2 represents a grafting ratio of i:j of 1:2.

[0104] Summary: The integrals of the H-NMR spectrum at 7.7 and 5.6 can represent the grafting amounts of thalidomide (Tha) and JQ1, respectively, proving that the grafting ratio can be controlled.

[0105] 6. Preparation of anhydride-modified polylysine protein hydrolysis-targeted chimeras

[0106] Weigh 311.2 mg P(lys 0.8 -JQ1 i -Tha j ) 60 Dissolve in 5 mL pure water, and adjust the pH to 8.6 with 1.0 M NaOH aqueous solution. Then, add twice the amount of succinic anhydride (no acid response, SA) or 2,3-dimethylmaleic anhydride (acid response, DMMA) to the reaction solution, and use 1.0 M NaOH to keep the pH value in the range of 8-9. When the pH value is constant, the reaction is continued at room temperature for 8 h, and then dialyzed for 6 h using a MWCO = 3500Da dialysis bag, and the dialysis water is replaced with NaOH aqueous solution (pH 8-9) every 1 h, and finally freeze-dried to obtain P(SA 0.8 -JQ1 i -Tha j ) 60 White solid. Figure 5 The NMR spectra of JT2-1-SA, JT1-1-SA, and JT1-2-SA unresponsive protein hydrolysis targeted chimeras. Figure 6 This is the NMR spectrum of the JT2-1-DMMA acid-responsive proteolysis targeting chimera.

[0107] 7. Preparation and characterization of nanoprotein hydrolysis-targeted chimeras

[0108] Weigh 1 mol of freeze-dried material, dissolve it with 1 mL of DMF, use a constant flow pump, drop it into 4 mL of PBS within 1 hour, then dialyze in PBS for 6 hours, change PBS once every hour, and finally ultrafilter and concentrate and dilute to 200 μM with PBS. The prepared nanoparticles were tested for their particle size potential by DLS, and their size was photographed by TEM, their secondary structure was tested by circular dichroism, and the responsiveness of the acid-responsive nanoparticles was verified. Figure 7 Particle size and electric potential of three nanoproteolytic targeting chimeras. Figure 8 Circular dichroism spectra of three nanoprotein proteolysis targeting chimeras at pH 6.8.

[0109] Summary: DLS and TEM characterized the particle size of the three nano-protein hydrolysis targeting chimeras. The DLS test showed that JT2-1 had a positive potential, while JT2-1-SA and JT2-1-DMMA had a negative potential after shielding the amino group. Circular dichroism spectroscopy characterized the secondary structure of the three nano-protein hydrolysis targeting chimeras. Lys 60 and JT2-1 are random. After grafting SA and DMMA, JT2-1-SA and JT2-1-DMMA are both α-helical structures. As time goes by, the structure of JT2-1-DMMA changes from α-helical to random, which proves the acid response ability of the material.

[0110] Effect Example 1 In vitro cytotoxicity of nanoprotein hydrolysis-targeted chimeras

[0111] The toxicity of the nano-protein hydrolysis targeting chimeras with different grafting ratios and SA modification on MCF-7 cells was detected by CCK-8 method. The detection steps are as follows:

[0112] Mcf-7 cells were cultured until logarithmic growth and then digested with trypsin. 5000 cells were plated per well of a 96-well plate with 0.2 mL of culture medium per well in a 5% CO 2 , 37℃, overnight incubation to allow cells to adhere. After discarding the culture medium, wash twice with PBS, add polylysine proteolysis targeting chimeras with different ligand grafting ratios and anhydride modification, and dilute the drug concentration stepwise by dichotomy, and add 100 μL per well to a 96-well plate. Discard the culture medium, wash three times with PBS, add 100 μL of diluted CCK-8 detection solution to each well, and incubate at 37℃ for 0.5 h. Detect by microplate reader at a wavelength of 450 nm. Calculate by the following formula.

[0113] Cell survival rate = [(absorbance of experimental well - absorbance of blank well) / (absorbance of control well - absorbance of blank well)] × 100%.

[0114] Fig. 9 CCK-8 cytotoxicity test of nanoprotein hydrolysis targeting chimeras with different grafting ratios on MCF-7 cells. Fig.10 CCK-8 cytotoxicity test of JQ1 and nanoprotein hydrolysis targeting chimera on MCF-7 cells under different pH conditions.

[0115] Summary: We used MCF7 cells rich in BRD4 protein as the research object, and screened out JQ1:Tha=2:1 materials from cationic proteolysis targeting chimeras and SA-shielded proteolysis targeting chimeras through CCK-8 experiments, which had the best cytotoxicity. Cytotoxicity was investigated under different pH conditions, and it was found that the IC50 of the acid-responsive proteolysis targeting chimera JT2-1-DMMA at pH 6.8 was 13.7 times lower than that at pH 7.4, 14.8 times lower than that of the small molecule JQ1, and 24 times lower than that of the unresponsive proteolysis targeting chimera JT2-1-SA at 6.8. This indicates that the acid-responsive proteolysis targeting chimera has better tumor cell toxicity than the small molecule BRD4 inhibitor JQ1 and the unresponsive nanoproteolysis targeting chimera.

[0116] Example 2 Characterization of cellular uptake of acid-responsive poly-lysine proteolysis-targeted chimeras

[0117] Breast cancer cells Mcf-7 were used to evaluate the in vitro cellular uptake efficiency of three poly-lysine proteolysis-targeting chimeras.

[0118] The flow detection steps are as follows:

[0119] Mcf-7 cells were cultured until logarithmic growth, and the cells were made into a single cell suspension with trypsin, with 100,000 cells per well of a 6-well plate. The next day, the culture medium was discarded, and the cells were washed twice with PBS, and 2 mL of culture medium containing Cy5.5-JT2-1-DMMA 1 μM, Cy5.5-JT2-1-SA 1 μM, and Cy5.5-JT2-1 1 μM was added. After incubation for 0.5, 2, 4, 8, 12, and 24 h, the culture medium was discarded and the cells were washed three times with PBS to wash away the nanoparticles that were not taken up by the cells. The cells were digested with trypsin and transferred to a flow tube. After centrifugation, the supernatant was discarded and 0.5 mL PBS was added to resuspend the cells to make a single cell suspension. The internalization of the three materials by the cells was detected by flow cytometry. Fig.11 Flow cytometric analysis of material internalization.

[0120] The CLSM detection steps are as follows:

[0121] Mcf-7 cells were cultured until logarithmic growth, and the cells were made into a single cell suspension with trypsin, and 100,000 cells were plated on each confocal dish. The next day, after discarding the culture medium, wash twice with PBS, and add 2 mL of culture medium containing Cy5.5-JT2-1-DMMA 1 μM, Cy5.5-JT2-1-SA 1 μM, and Cy5.5-JT2-1 1 μM. After incubation for 2, 4, 8, 12, and 24 h, the culture medium was discarded and washed three times with PBS to wash away the nanoparticles that were not taken up by the cells. Fix with paraformaldehyde (4%) for 10 min and wash three times with PBS. Add 500 μL of the prepared DIO staining working solution to each well for 5 min, and wash again with PBS three times. Add 6 drops of anti-fluorescence quenching sealing solution containing DAPI, incubate at room temperature for 5 minutes, and can be photographed within one week. Fig.12 CLSM analysis of endocytosis of JT2-1-SA (left) and JT2-1-DMMA (right).

[0122] Summary: A key reason why the acid-responsive proteolysis targeting chimera works is that charge reversal enhances cellular endocytosis, so we compared the cellular endocytosis of JT2-1-SA and JT2-1-DMMA. Flow cytometry and laser confocal microscopy results showed that the acid-responsive proteolysis targeting chimera was internalized faster than the non-responsive one.

[0123] Effect Example 3 Degradation effect and degradation mechanism of nanoprotein hydrolysis targeting chimera on BRD4 protein

[0124] 1. Breast cancer cells MCF-7 were used to evaluate the effect of nanoprotein hydrolysis targeting chimera on cell BRD4 protein degradation in vitro.

[0125] Western Blot (WB) experimental steps are as follows:

[0126] Mcf-7 cells were cultured until logarithmic growth, and the cells were made into single-cell suspension with trypsin. 200,000 cells were inoculated in each well of a 6-well plate, and 2 mL of DMEM complete medium was added to each well. After 1 day of cell culture, the medium in the 6-well plate was discarded, the cells were washed twice with PBS buffer, and DMEM complete medium with different concentrations of JT2-1-SA was added to each well. The induction treatment was carried out for 1 day, and 3 replicates were added to each group. After the induction treatment, the medium was carefully aspirated, and the cells were quickly washed twice with pre-cooled PBS buffer, and the PBS was aspirated as much as possible. The cell lysate was scraped off with a cell scraper and collected in a pre-cooled centrifuge tube. An appropriate amount of pre-cooled RIPA lysis buffer (containing protease inhibitors and phosphatase inhibitors, added according to the proportions in the reagent instructions) was added to each tube, and lysed on ice for 30 min. After lysis, centrifuge at 4°C, 12000-14000 rpm for 15 min, carefully transfer the supernatant to a new pre-cooled centrifuge tube, use the BCA protein quantification kit to determine the protein concentration, dilute to the same concentration, add 5×SDS-PAGE loading buffer, mix thoroughly, and boil in a boiling water bath for 10 min to denature the protein. WB experimental samples were subjected to electrophoresis, transfer, BSA blocking, primary antibody (anti-BRD4 antibody, anti-c-MYC antibody, anti-GAPDH antibody) incubation and secondary antibody (HRP-labeled goat anti-rabbit) incubation, and the washed membrane was placed in a chemiluminescent substrate solution for 1-2 minutes, and exposed and imaged using a chemiluminescent imaging system to obtain the target protein band image. Fig.13 Western blot analysis of BRD4 and c-MYC expression in cells after induction of different concentrations of JT2-1-SA.

[0127] Summary: To eliminate cationic interference, we used materials grafted with succinic anhydride for verification and found that it was concentration-dependent for BRD4 degradation, and the downregulation of BRD4 expression also successfully downregulated the expression of its downstream c-MYC.

[0128] 2. CLSM experiments were used to evaluate the degradation effects of different nanoprotein hydrolysis targeting chimeras on BRD4 protein in vitro at the same concentration. The steps are as follows:

[0129] Mcf-7 cells were cultured until logarithmic growth, and the cells were made into single-cell suspension with trypsin, and 100,000 cells were inoculated per confocal dish. After 1 day of cell culture, the original culture medium was discarded, the cells were washed twice with PBS buffer, and 5 μM JT2-1, JT2-1-SA and JT2-1-DMMA were added to each well. After 1 day of induction treatment, the culture medium was discarded, washed 3 times with PBS, fixed with paraformaldehyde (4%) for 10 min, and washed 3 times with PBS. Permeabilized with Triton X-100 for 10 min and washed 3 times with PBS. Blocked with protein blocking solution for 1 hour, followed by incubation with primary antibody (anti-BRD4 antibody) at 4°C overnight. Washed three times with PBS, added secondary antibody (FITC-labeled goat anti-rabbit), and incubated at room temperature for 2 hours. Washed three times with PBS, added 6 drops of anti-fluorescence quenching sealing solution containing DAPI, incubated at room temperature for 5 minutes, and photographed on the machine.

[0130] The streaming steps are as follows:

[0131] Mcf-7 cells were cultured until logarithmic growth, and the cells were made into single-cell suspension with trypsin, and 100,000 cells were seeded in each well of a 12-well plate. After 1 day of cell culture, the original culture medium was discarded, and the cells were washed twice with PBS buffer. 5 μM JT2-1, JT2-1-SA and JT2-1-DMMA were added to each well, and 3 replicates were added. After 1 day of induction treatment, the culture medium was discarded. The cells were digested with trypsin, transferred to a flow tube, centrifuged and the culture medium was discarded, and washed 3 times with PBS. Fix with paraformaldehyde (4%) for 10 min and wash 3 times with PBS. Permeabilize with Triton X-100 for 10 min and wash 3 times with PBS. Block with protein blocking solution for 1 hour, and then incubate with primary antibody (anti-BRD4 antibody) at 4°C overnight. Wash three times with PBS, add secondary antibody (FITC-labeled goat anti-rabbit), and incubate at room temperature for 2 hours. Wash 3 times with PBS, and finally add 400 μL PBS and test on the machine. Fig.14 CLSM and flow cytometry results of BRD4 expression in cells after induction of different nanoparticle proteolysis targeting chimeras.

[0132] Summary: By comparing several nanoparticles, it was found that the acid-responsive proteolysis-targeted chimera had the best BRD4 degradation effect, which may be due to its effective cellular uptake and lysosomal escape.

[0133] 3. To evaluate the degradation mechanism of the nanoprotein hydrolysis-targeted chimera, the CLSM steps are as follows:

[0134] Mcf-7 cells were cultured to logarithmic growth, and the cells were made into single-cell suspension with trypsin. 100,000 cells were seeded per confocal dish. After 1 day of cell culture, the original culture medium was discarded and the cells were washed twice with PBS buffer. The two groups of cells were pretreated with 50nM MG132 (proteasome inhibitor) and 50nM MLN4924 (ubiquitination inhibitor) for 30min, respectively, and then the culture medium was discarded, the cells were washed 3 times with PBS buffer, and 25μM JT2-1-SA was added to each well. After 1 day of induction treatment, the culture medium was discarded, washed 3 times with PBS, fixed with paraformaldehyde (4%) for 10 min, and washed 3 times with PBS. Permeabilized with Triton X-100 for 10 min and washed 3 times with PBS. Blocked with protein blocking solution for 1h, followed by incubation with primary antibody (anti-BRD4 antibody) at 4℃ overnight. Washed three times with PBS, added secondary antibody (FITC-labeled goat anti-rabbit), and incubated at room temperature for 2h. Wash three times with PBS, add 6 drops of anti-fluorescence quenching sealing solution containing DAPI, incubate at room temperature for 5 minutes, and take pictures on the machine.

[0135] The streaming steps are as follows:

[0136] Mcf-7 cells were cultured until logarithmic growth, and the cells were made into single-cell suspension with trypsin. 100,000 cells were seeded in each well of a 12-well plate. After 1 day of cell culture, the original culture medium was discarded and the cells were washed twice with PBS buffer. The two groups of cells were pretreated with 50nM MG132 (proteasome inhibitor) and 50nM MLN4924 (ubiquitination inhibitor) for 30min, respectively, and then the culture medium was discarded, the cells were washed three times with PBS buffer, and 25μM JT2-1-SA was added to each well. After 1 day of induction treatment, the culture medium was discarded. The cells were digested with trypsin, transferred to a flow tube, centrifuged and the culture medium was discarded, and washed three times with PBS. Fix with paraformaldehyde (4%) for 10 min and wash three times with PBS. Permeabilize with Triton X-100 for 10 min and wash three times with PBS. Block with protein blocking solution for 1h, and then incubate with primary antibody (anti-BRD4 antibody) at 4℃ overnight. Wash three times with PBS, add secondary antibody (FITC-labeled goat anti-rabbit), incubate at room temperature for 2 hours, wash three times with PBS, and finally add 400 μL PBS and test on the instrument. Fig.15 CLSM and flow cytometry analysis of the cellular BRD4 degradation mechanism after induction by different nanoproteolytic targeting chimeras.

[0137] Summary: After cells were pretreated with proteasome inhibitors and ubiquitination inhibitors, BRD4 expression returned to normal, indicating that the degradation process depends on the ubiquitin-proteasome pathway.

[0138] Effect Example 4 Effect of acid-responsive poly-lysine proteolysis-targeting chimera on cell cycle

[0139] Prepare the materials for the Control group (PBS), JT2-1 group, JT2-1-SA group, JT2-1-DMMA group, and JQ1 group, as well as the cell apoptosis and cell cycle kit, 70% ethanol, trypsin and other reagents of Biyuntian. Mcf-7 cells were cultured in 12-well plates, with 100,000 cells in each well. After 24 hours, the drug was added according to the grouping. Each group was diluted to 5μM with DMEM complete medium, and 1mL of drug-containing medium was added to each well for induction for 24 hours. Then the cells were collected, the cells were digested with trypsin to detach them from the surface of the culture dish, collected in a centrifuge tube, and centrifuged and discarded the supernatant. After that, the cells were fixed, pre-cooled 70% ethanol was added to fix them at 4℃ overnight, and the ethanol was centrifuged and discarded the next day, and the cells were washed twice with PBS. Then PI staining and RNase A treatment were performed, and the propidium iodide staining working solution was prepared according to the proportion in the instruction manual. 0.5mL of propidium iodide staining solution was added to each tube of cell sample, and the cell pellet was slowly and fully resuspended, and the cells were warmed at 37℃ in the dark for 30 minutes. Flow cytometry was performed within 24 hours after staining, and FlowJo software was used to analyze the distribution of each phase of the cell cycle. Fig.16 Cell cycle results of poly-lysine proteolysis targeting chimeras modified with different anhydrides.

[0140] Summary: Inhibition of BRD4 protein will inhibit the transition of cell cycle G1 to S phase. The study found that low doses of acid-responsive proteolysis targeting chimeras can significantly inhibit cell cycle arrest in G1 phase, thereby inhibiting cell proliferation.

[0141] Effect Example 5 In vivo imaging distribution of acid-responsive poly-lysine proteolysis-targeted chimeras

[0142] The passive targeting of nanomaterials was demonstrated by in vivo and in vitro imaging. Quantitative analysis was performed using a standard curve. Nanoparticles Cy5.5-JT2-1-DMMA with the same fluorescence intensity were injected into the tail vein of each mouse and images were taken at 12h, 24h, and 36h. Fig.17 This is the in vivo imaging distribution diagram after tail vein injection of Cy5.5-JT2-1-DMMA.

[0143] Summary: The acid-responsive nanoprotein hydrolysis targeting chimera was enriched in the tumor through the EPR effect. In vivo experiments first observed that the material reached maximum enrichment in the tumor in about 24 hours, and then the signal attenuated.

[0144] Effect Example 6 In vivo therapeutic effect of acid-responsive polylysine proteolysis-targeted chimera

[0145] The inhibitory effect of the nanoprotein hydrolysis targeting chimeras JT2-1-SA and JT2-1-DMMA was evaluated in the nude mouse MCF-7 tumor model. The mice were divided into 4 groups and given saline, JQ1, JT2-1-SA, and JT2-1-DMMA, respectively. The dose of JT2-1-SA and JT2-1-DMMA was 20 mg / kg body weight, and JQ1 was given at an equivalent dose through tail vein injection. When the tumor grew to about 100 mm 3 The first treatment was carried out at 1:00 pm, and then injections were given every 3 days for a total of 6 injections over 16 days. During the experiment, body weight and tumor volume were recorded every 2 days. Fig.18 These are the results of the mouse treatment experiment after tail vein injection.

[0146] The tumor inhibition rate was calculated based on the tumor weight of the treatment group and the tumor weight of the control group. The calculation formula was: tumor inhibition rate (%) = (1-tumor weight of the treatment group / tumor weight of the control group) × 100%.

[0147] The results showed that the tumor inhibition rate of the JQ1 group was only 14.4%, showing no significant difference, indicating that the single JQ1 small molecule treatment effect was weak and may not fully target tumor cells. The JT2-1-SA group also showed a significant tumor inhibition effect, with a tumor inhibition rate of 41.4%. Compared with the JQ1 group, JT2-1-SA may achieve tumor partial enrichment through the EPR effect and continue to play a proteolytic targeting chimera degradation role in cells, thereby more effectively inhibiting tumor growth. The tumor inhibition rate of the JT2-1-DMMA group reached 76.7%, which was significantly better than other groups. This may be the result of effective tumor enrichment and enhanced tumor penetration and uptake after DMMA was removed under acidic conditions in the tumor microenvironment. In addition, there was no significant difference in the weight changes of mice in each group during the treatment, indicating that the therapeutic dose used did not cause obvious toxic side effects.

[0148] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A nano-degradation agent having a structure shown in formula (I): Formula (I); in, The structure of R1 is shown in formula (II), and the structure of R2 is shown in formula (III); Formula (II) Formula (III) R3 is selected from any one of amino, succinic anhydride and maleic anhydride; m is the degree of polymerization, m=10~500, n=10~200, x=1~100; y=1~100; z=8~500.

2. The nano-degradation agent according to claim 1, characterized in that: x:y=(1~5):(1~5).

3. The method for preparing the nano-degradation agent according to claim 1 or 2, characterized in that: The steps include: Step 1, taking N(ε)-benzyloxycarbonyl-L-lysine, anhydrous tetrahydrofuran and triphosgene and reacting them to obtain N(ε)-benzyloxycarbonyl-L-lysine intracyclic anhydride monomer; Step 2, reacting N(ε)-benzyloxycarbonyl-L-lysine intracyclic anhydride monomer with aminated polyethylene glycol monomethyl ether in the presence of a solvent to obtain polyethylene glycol monomethyl ether-b-poly(N-benzyloxycarbonyl-lysine); Step 3, mixing polyethylene glycol monomethyl ether-b-poly (N-benzyloxycarbonyl-lysine), trifluoroacetic acid and hydrogen bromide acetic acid solution to obtain polyethylene glycol monomethyl ether-b-polylysine; Step 4, coupling the R1 and R2 groups in the nanodegradation agent according to claim 1 or 2 to polyethylene glycol monomethyl ether-b-polylysine to obtain a cationic polylysine proteolysis targeting chimera molecule; Step 5: Mix the cationic polylysine protein hydrolysis targeting chimera molecule with the group of R3 in the nanodegrader according to claim 1 or 2 to obtain the nanodegrader.

4. The preparation method according to claim 3, characterized in that: In step 1, the temperature of the mixed reaction is 50-60° C., and the time of the mixed reaction is 1.5-3 h; The method also includes subjecting the mixture after the mixed reaction to n-hexane precipitation, separation, washing, recrystallization and vacuum drying to obtain the N(ε)-benzyloxycarbonyl-L-lysine intracyclic anhydride monomer.

5. The preparation method according to claim 3 or 4, characterized in that: In the step 2, The preparation method of the amination polyethylene glycol monomethyl ether comprises: S1, dissolving mPEG-OH, potassium hydroxide and p-toluenesulfonyl chloride in dichloromethane, reacting, washing and separating to obtain an organic phase, filtering and concentrating the obtained organic phase, precipitating with cold ether and filtering to obtain the intermediate product mPEG-OTs; S2, taking mPEG-OTs, ammonium chloride and ammonia monohydrate, mixing and reacting, extracting the organic phase with dichloromethane, washing the obtained organic phase with water, drying, filtering, rotary evaporation concentration, cold ether precipitation, and draining to obtain amino polyethylene glycol monomethyl ether; The aminated polyethylene glycol monomethyl ether and toluene are subjected to azeotropic dehydration under vacuum conditions, toluene is removed by reduced pressure distillation, and then mixed and reacted with N(ε)-benzyloxycarbonyl-L-lysine intracyclic anhydride monomer, wherein the solvent is N,N-dimethylformamide, the reaction temperature is 15-35° C., and the mixing reaction time is 2-4 days; The method also includes subjecting the reaction solution after the reaction to cold ether precipitation and vacuum drying to obtain polyethylene glycol monomethyl ether-b-poly (N-benzyloxycarbonyl-lysine).

6. The preparation method according to claim 3, characterized in that: In step 3, the mixing temperature is 15-35° C., and the mixing reaction time is 1.5-3 h; The method also includes subjecting the reaction solution after the reaction to cold ether sedimentation and centrifugation, dissolving the obtained precipitate with N,N-dimethylformamide, dialysis, and freeze-drying to obtain polyethylene glycol monomethyl ether-b-poly (N-benzyloxycarbonyl-lysine).

7. The preparation method according to claim 3, characterized in that: In step 4, the coupling conditions include: using N,N-dimethylformamide as solvent, DMAP and DIC as catalysts, and reacting at a temperature of 15-35° C. for 2-3 days; The method also includes subjecting the reaction solution after the coupling to cold ether sedimentation and centrifugation, vacuum drying the obtained precipitate, dissolving it with N,N-dimethylformamide, and dialysis and freeze drying to obtain the cationic polylysine protein hydrolysis targeting chimera molecule.

8. The preparation method according to claim 3, characterized in that: In step 5, the pH of the mixed reaction is 8-9, the temperature of the mixed reaction is 15-35° C., and the time of the mixed reaction is 7-10 hours; The method also includes dialyzing and freeze-drying the reaction solution after the coupling to obtain the nano degradation agent.

9. Use of the nanodegrader according to claim 1 or 2 or the nanodegrader prepared by the preparation method according to any one of claims 3 to 8 in the preparation of drugs for preventing and / or treating tumors.

10. A drug for preventing and / or treating tumors, characterized in that: The invention comprises the nano-degradation agent according to claim 1 or 2 or the nano-degradation agent prepared by the preparation method according to any one of claims 3 to 8.

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