Nanodegraders Based on Proteolysis-Targeting Chimeric Molecules, Preparation Methods Thereof, and Applications

Abstract

The present invention relates to the technical field of nanomaterials, and particularly relates to a nano-degrader based on proteolysis-targeting chimeras, and a preparation method and application thereof. The nano-degrader provided by the present invention targets the BRD4 protein, utilizes the natural cationic side groups of polylysine, connects two ligands through a condensation reaction, optimizes the ligand ratio, and then modifies dimethyl maleic anhydride to endow it with acid-responsive charge inversion ability, thereby enhancing material uptake, degrading the BRD4 protein, and inducing the death of tumor cells. Through the technical scheme of the present invention, the dynamic response of the tumor microenvironment is utilized to overcome the problems of poor permeability and insufficient selectivity of traditional proteolysis-targeting chimeras, and provide a more efficient and safe nano-degrader system for the targeted treatment of tumors and other diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and particularly to a nano-degrader based on proteolysis-targeting chimeras and its preparation method and application. Background Art

[0002] The proteolysis-targeting chimera technology aims to utilize small molecule compounds or biological macromolecules to induce proteins to be recognized and degraded by the intracellular degradation system, thereby achieving specific regulation of proteins. Generally, a proteolysis-targeting chimera is a small molecule connected by a linker to a ligand targeting a target protein and a ligand targeting an E3 ubiquitin ligase. By bringing the two closer, the proteolysis-targeting chimera can label the target protein with ubiquitin tags, enabling it to be further degraded through the ubiquitin-proteasome system. Different from the "occupation-driven" mode of traditional small molecule inhibitors, the proteolysis-targeting chimera can target "undruggable" proteins in the traditional sense through the "event-driven" catalytic degradation effect, and is expected to overcome the drug resistance of small molecule inhibitors, which has received extensive attention in the field of drug research and development. Currently, this technology has multiple advantages in treating diseases such as tumors and neurodegenerative diseases: (1) it can overcome the drug resistance of the body 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 fast and reversible chemical knockout method.

[0003] Although a variety of small molecule proteolysis-targeting chimeras have been approved in the United States and shown effective treatment for various cancers, they also expose challenges in aspects such as linker screening, pharmacokinetics, cell permeability, and tissue cell selectivity. On the one hand, the design of proteolysis-targeting chimeras does not conform to the "rule of five" principle, and the relatively large molecular weight results in disadvantages such as poor water solubility and poor membrane permeability. On the other hand, the ability of proteolysis-targeting chimeras to efficiently catalyze the degradation of target proteins may cause unpredictable harm to normal organs during systemic drug administration. Therefore, how to improve the membrane permeability, targeting, and stability of proteolysis-targeting chimeras, especially to achieve efficient 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 bromodomains and belongs to the BET (Bromodomain and Extra-Terminal) family. BRD4 specifically binds to the acetylated histone tails through its bromodomains, thereby affecting the open state of chromatin and transcriptional activity, and regulating key processes such as the 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), which can promote the proliferation and metastasis of tumor cells, BRD4 has become an important target in anti-tumor therapy.

[0005] Traditional small molecule proteolysis-targeting chimeras cannot target tumor tissues, so they may cause off-target effects. And they face disadvantages such as poor water solubility, poor permeability, and the "hook effect" of being difficult to form ternary complexes, which hinder their clinical research. Nanotechnology, due to its advantages such as improving drug concentration at the lesion site, spatiotemporally controllable drug release, high specific surface area, and the possibility of multivalent binding, provides a solution for the clinical transformation of proteolysis-targeting chimeras. Although some progress has been made in the research of macromolecular proteolysis-targeting chimeras, however, the development of these nano-degraders is hindered by their unfavorable physicochemical properties, including high molecular weight, poor membrane permeability, and instability. In addition, the undifferentiated distribution and continuous biological activity of small molecule and macromolecule degraders may lead to uncontrollable degradation of proteins at non-target sites, thus limiting the in vivo efficacy. Therefore, developing a strategy for high-efficiency and specific macromolecular proteolysis-targeting chimeras for tumor-related proteins has 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 proteolysis-targeting chimeras and its preparation method and application. The present invention provides a polymer designed to be a dual-headed targeting polymer itself, using its high specific surface area and the possibility of multivalent binding to make it a nano-reactor, thereby enhancing the in vivo stability, water solubility of proteolysis-targeting chimeras and optimizing the degradation ability, and improving the effectiveness of tumor treatment.

[0007] The present invention provides a nano-degrader 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 group, 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 nano - degrader provided by the present invention connects two ligands of the proteolysis - targeting chimera through a linker, respectively recruiting the target protein and the E3 ubiquitin ligase, thereby tagging the target protein with ubiquitin labels and making it degraded by the proteasome. Different from the "site - occupancy" mode of traditional small - molecule inhibitors, it is like a catalyst that can continuously form ternary complexes and efficiently degrade the target protein through the intracellular ubiquitin - proteasome system. Although traditional small - molecule proteolysis - targeting chimeras have the advantages of targeting and degrading "undruggable" proteins and high - efficiency degradation ability, they often face insufficient ability to penetrate cell membranes in practical applications, thus affecting the delivery efficiency in tissues such as tumors. In addition, the non - specific distribution and continuous activity of small - molecule proteolysis - targeting chimeras easily lead to the degradation of non - target proteins, increasing the potential toxicity to normal tissues. Nanotechnology, due to its advantages such as improving the drug concentration at the lesion site, spatiotemporally controllable drug release, high specific surface area, and the possibility of multivalent binding, provides a solution for the clinical transformation of proteolysis - targeting chimeras.

[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, the 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 preparation method of the nano - degrader, comprising the following steps:

[0020] Step 1: Take N(ε) - carbobenzoxy - L - lysine, anhydrous tetrahydrofuran and triphosgene and react them to obtain N(ε) - carbobenzoxy - L - lysine cyclic anhydride monomer;

[0021] Step 2: React the N(ε) - carbobenzoxy - L - lysine cyclic anhydride monomer with amino - terminated methoxypolyethylene glycol in the presence of a solvent to obtain methoxypolyethylene glycol - b - poly(N - carbobenzoxy - lysine);

[0022] Step 3: Mix poly(ethylene glycol) monomethyl ether-b-poly(N-carbobenzoxy-lysine), trifluoroacetic acid, and hydrobromic acid in acetic acid solution for reaction to obtain poly(ethylene glycol) monomethyl ether-b-polylysine;

[0023] Step 4: Couple the groups of R1 and R2 in the nanodegrader to poly(ethylene glycol) monomethyl ether-b-polylysine to obtain a cationic polylysine proteolytic targeting chimera molecule;

[0024] Step 5: Mix and react the cationic polylysine proteolytic targeting chimera molecule with the group of R3 in the nanodegrader to obtain the nanodegrader.

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

[0026] Preferably, it further includes subjecting the mixture after the mixing reaction to sedimentation with excess n-hexane, separation, washing, recrystallization, and vacuum drying to obtain the N(ε)-carbobenzoxy-L-lysine cyclic anhydride monomer.

[0027] In some embodiments, in Step 2,

[0028] The preparation method of the amino-functionalized poly(ethylene glycol) monomethyl ether includes:

[0029] S1: Take mPEG-OH, potassium hydroxide, and p-toluenesulfonyl chloride, dissolve them in dichloromethane, react, wash, and separate to obtain an organic phase. The obtained organic phase is filtered, concentrated, sedimented with cold ethyl ether, and filtered to obtain the intermediate mPEG-OTs;

[0030] S2: Take mPEG-OTs, ammonium chloride, and ammonia monohydrate, perform a mixing reaction, extract the organic phase with dichloromethane. The obtained organic phase is washed with water, dried, filtered, rotary evaporated and concentrated, sedimented with cold ethyl ether, and dried by suction to obtain the amino-functionalized poly(ethylene glycol) monomethyl ether;

[0031] After azeotropically removing water with toluene in a vacuum environment, the amino-functionalized poly(ethylene glycol) monomethyl ether is distilled under reduced pressure to remove toluene, and then mixed and reacted with the N(ε)-carbobenzoxy-L-lysine cyclic anhydride monomer. The solvent is N,N-dimethylformamide. The temperature of the reaction is 15-35 °C, and the time of the mixing reaction is 2-4 days;

[0032] Preferably, it further includes subjecting the reaction solution after the reaction to sedimentation with cold ethyl ether and vacuum drying to obtain poly(ethylene glycol) monomethyl ether-b-poly(N-carbobenzoxy-lysine).

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

[0034] Preferably, it further includes subjecting the reaction solution after the reaction to sedimentation with cold ethyl ether and centrifugation, and dissolving the obtained precipitate in N,N-dimethylformamide, dialyzing, and freeze-drying to obtain methoxypolyethylene glycol-b-poly(N-carbobenzoxy-lysine).

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

[0036] Preferably, it further includes subjecting the reaction solution after the coupling to sedimentation with cold ethyl ether and centrifugation, dissolving the obtained precipitate by vacuum drying in N,N-dimethylformamide, and dialyzing and freeze-drying to obtain a cationic polylysine proteolytic targeting chimera molecule.

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

[0038] Preferably, it further includes dialyzing and freeze-drying the reaction solution after the coupling to obtain the nano-degrading agent.

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

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

[0041] Compared with the prior art, the nano-degrading agent provided by the present invention does not adopt the form of loading small molecule proteolysis-targeting chimeras on nanoparticles, or is designed as a bonded drug of a polymer carrier and a small molecule proteolysis-targeting chimera. Instead, targeting the BRD4 protein, using the natural cationic side groups of polylysine, two ligands are attached through a condensation reaction, the ligand ratio is optimized, and then dimethyl maleic anhydride is modified to endow it with acid-responsive charge reversal ability, thereby enhancing material uptake, degrading the BRD4 protein, and inducing the death of tumor cells. By precisely regulating the release and activity of the proteolysis-targeting chimera locally in the tumor, the present invention achieves tumor-specific protein degradation, improves the selectivity for the targeted tissue, and reduces the non-specific toxic side effects on normal tissues. 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, providing a more efficient and safe nano-degrading agent system for the targeted treatment of tumors and other diseases. Description of the Drawings

[0042] Figure 1 Showing the NMR spectrum, infrared and mass spectrometry results of N(ε)-carbobenzoxy-L-lysine cyclic anhydride monomer, where Figure A is 1 the 1H spectrum, Figure B is 13 the 13C spectrum, Figure C is the infrared spectrum, and Figure D is the mass spectrum;

[0043] Figure 2 Showing the NMR 1 1H spectrum (left) of the end-aminated methoxypolyethylene glycol and 13 the 13C spectrum (right);

[0044] Figure 3 Showing the NMR spectrum of methoxypolyethylene glycol-b-polylysine;

[0045] Figure 4 Showing the preparation reaction formula of the cationic polylysine proteolysis-targeting chimera targeting BRD4 and the NMR spectra of polylysine proteolysis-targeting chimeras with different grafting ratios. Among them, Figure A is the preparation reaction formula, and Figure B is the NMR spectrum;

[0046] Figure 5 Showing the NMR spectra of SA non-responsive proteolysis-targeting chimeras with different grafting ratios;

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

[0048] Figure 7 Showing the particle sizes and zeta potentials of three nano-proteolysis-targeting chimeras of JT2-1, JT2-1-SA and JT2-1-DMMA;

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

[0050] Figure 9 Show the CCK-8 cytotoxicity assay of nanoprotein hydrolysis targeting chimeras with different grafting ratios against MCF-7 cells;

[0051] Figure 10 Show the CCK-8 cytotoxicity assay of JQ1 and nanoprotein hydrolysis targeting chimeras against MCF-7 cells under different pH conditions;

[0052] Figure 11 Show the flow cytometry analysis of the endocytosis of acid-responsive polylysine protein hydrolysis targeting chimeras;

[0053] Figure 12 Show the CLSM analysis of the endocytosis of JT2-1-SA (left) and JT2-1-DMMA (right);

[0054] Figure 13 Show the WB of the expression of BRD4 and c-MYC in cells induced by different concentrations of JT2-1-SA;

[0055] Figure 14 Show the CLSM (left) and flow cytometry results (right) of the expression of BRD4 in cells induced by different nanoprotein hydrolysis targeting chimeras;

[0056] Figure 15 Show the CLSM and flow cytometry analysis diagrams of the degradation mechanism of BRD4 in cells induced by different nanoprotein hydrolysis targeting chimeras;

[0057] Figure 16 Show the cell cycle results diagrams of polylysine protein hydrolysis targeting chimeras modified with different anhydrides;

[0058] Figure 17 Show the in vivo imaging distribution map after the tail vein injection of Cy5.5-JT2-1-DMMA;

[0059] Figure 18 Show the results of the mouse treatment experiment after the tail vein injection of different nanoprotein hydrolysis targeting chimeras. Among them, Figure A is the experimental flow chart, Figure B is the mouse tumor tissue map after the tail vein injection, Figure C is the statistical result of the mouse tumor weight after the tail vein injection, Figure D is the statistical result of the mouse tumor volume after the tail vein injection, and Figure E is the statistical result of the mouse body weight after the tail vein injection. Detailed implementation method

[0060] The present invention provides a nano-degrader based on proteolysis targeting chimera, and its preparation method and application. Those skilled in the art can draw on 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 regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate alterations and combinations to the methods and applications in 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] Term Explanation:

[0062] Proteolysis Targeting Chimera, Proteolysis Targeting Chimera

[0063] A proteolysis targeting chimera is a bifunctional small molecule that degrades target proteins by utilizing the ubiquitin-proteasome system within cells through linking a ligand targeting the protein and a ligand of the E3 ubiquitin ligase, thereby achieving the regulation of specific proteins. This technology is widely used in the targeted degradation of "undruggable" proteins and has advantages of high selectivity and potential drug resistance.

[0064] BRD4 (Bromodomain-containing Protein 4)

[0065] BRD4 (Bromodomain-containing protein 4) is a transcriptional regulatory protein containing bromodomains and belongs to the BET (Bromodomain and Extra-Terminal) family. It is involved in transcriptional regulation and plays a key role in processes such as tumor growth and cell proliferation. BRD4 specifically binds to the acetylated histone tail through its bromodomain, thereby affecting the open state of chromatin and transcriptional activity, and regulating key processes such as the 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), which can promote the proliferation and metastasis of tumor cells, BRD4 has become an important target in anti-tumor therapy.

[0066] E3 Ubiquitin Ligase

[0067] An 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 targeting chimera technology, the target protein is linked to the E3 ligase through a ligand, and 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 polymeric nanomaterials in the present invention will undergo a structural transformation 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 the polymerization reaction through the action of an initiator to form a polymer with biocompatibility and water solubility. The 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 an acidic environment, making it suitable as the backbone of 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, marking the target proteins and transferring them to the proteasome for degradation. Proteolysis-targeting chimeras selectively clear specific proteins by using the ubiquitin-proteasome system to mark target proteins for degradation.

[0076] Nano-degrader

[0077] A nano-degrader is a nanoscale material that can achieve the degradation of specific proteins through the connection of target proteins and degrading enzymes. The nano-degrader of the present invention combines the characteristics of proteolysis-targeting chimeras and acid-responsive nanomaterials, enabling it to act more efficiently on the target protein BRD4 in the tumor microenvironment.

[0078] Ligand

[0079] A ligand refers to a chemical molecule that can specifically bind to a target molecule (such as a protein). In proteolysis-targeting chimeras, one ligand targets the target protein (such as BRD4), and the other ligand targets the E3 ubiquitin ligase. The ligand guides 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 structures formed through physical or chemical interactions between molecules. The polymeric nano-degradants of the present invention self-assemble through mPEG and polylysine to form stable nanoparticles, improving the stability, targeting, and bioavailability of drugs.

[0082] The present invention aims to address multiple key challenges in the clinical application of existing proteolysis-targeting chimera (PROTAC) technologies, particularly the off-target effects caused by their inability to target tumor tissues, as well as problems such as poor membrane permeability and poor water solubility. PROTACs connect two ligands through a linker to recruit a target protein and an E3 ubiquitin ligase respectively, thereby tagging the target protein with ubiquitin labels and enabling its degradation by the proteasome. This technology is different from the "site occupancy" mode of traditional small molecule inhibitors. It acts like a catalyst that can continuously form ternary complexes and efficiently degrade target proteins through the cell's endogenous ubiquitin-proteasome system. Although traditional small molecule PROTACs have the advantages of targeting and degrading "undruggable" proteins and high degradation efficiency, they often face insufficient ability to penetrate cell membranes in practical applications, thus affecting the delivery efficiency in tissues such as tumors. In addition, the non-specific distribution and sustained activity of small molecule PROTACs easily lead to the degradation of off-target proteins, increasing the potential toxicity to normal tissues. Nanotechnology, due to its advantages such as improving drug concentration at the lesion site, spatiotemporally controllable drug release, high specific surface area, and the possibility of multivalent binding, provides a solution for the clinical translation of PROTACs. Currently, nanoscale PROTAC technologies mainly improve the in vivo distribution of PROTACs and reduce side effects through methods such as designing multi-responsive prodrugs, nanodelivery systems, split-and-mix nanotechnology, and drug co-assembly. Despite many emerging methods, there is still a long way to go for this nanoscale PROTAC technology in clinical applications, and the key is to enhance tumor accumulation and exert the degradation effect of PROTACs. Therefore, how to increase the effective concentration of PROTACs in the targeted tissue, reduce off-target effects, and simultaneously maintain high degradation efficiency of target proteins is a major challenge in this technical field. To this end, we propose a scheme of multi-headed PROTAC mode + acid-responsive nanoparticles. On the one hand, as the saying goes, many hands make light work. The mode of multi-target head modification using polymer side chains is theoretically superior to the efficiency of a single small molecule. On the other hand, tumor accumulation is achieved through the enhanced permeability and retention (EPR) effect of nanoparticles, and further charge reversal is realized in the acidic microenvironment of tumors to enhance tumor penetration and uptake.

[0083] To address these issues, the present invention constructs a polymeric proteolysis-targeting chimera nanoparticle that responds to the tumor acidic microenvironment and targets BRD4 protein degradation, aiming to enhance the in vivo stability, targeting ability, and membrane permeability of proteolysis-targeting chimeras through polymer nanotechnology and improve the effectiveness of tumor treatment. Our goal is to target the BRD4 protein, develop an acid-responsive polylysine nanoproteolysis-targeting chimera, 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, attach two ligands through a condensation reaction, optimize the ligand ratio, and then modify it with dimethyl maleic anhydride to endow it with acid-responsive charge inversion ability, thereby enhancing material uptake, degrading the BRD4 protein, and inducing the death of tumor cells.

[0084] In the present invention, polylysine initiated by PEG5000 is used as a template, and two small molecule ligands of the proteolysis-targeting chimera are grafted onto the polylysine side chain through the condensation reaction of amino and carboxyl groups, and the excess side chain amino groups are blocked with 2,3-dimethyl maleic anhydride to form a stable nanoparticle structure. In the acidic microenvironment of the tumor, the maleic anhydride groups on the nanoparticle surface are responsively removed, triggering the secondary structure transformation and charge inversion of the polymer, enhancing the uptake efficiency of the nanoparticles in tumor cells, and further exposing the side chain targeting module. Inside the tumor cells, the nanoparticles recruit BRD4 protein and E3 ubiquitin ligase, and achieve the degradation of the target protein through the ubiquitin-proteasome pathway. By precisely regulating the release and activity of the proteolysis-targeting chimera at the tumor site, the present invention realizes tumor-specific protein degradation, improves the selectivity for the targeted tissue, and reduces the non-specific toxicity to 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 nano-degrading agent system for the targeted treatment of tumors and other diseases.

[0085] The present invention proposes a polymeric proteolysis-targeting chimera nano-delivery system based on acid microenvironment response for the specific degradation of BRD4 protein to achieve efficient and precise tumor treatment. This system overcomes the deficiencies of traditional proteolysis-targeting chimera technology in terms of permeability, targeting ability, and stability by responsively releasing proteolysis-targeting chimera molecules in the tumor microenvironment. The core of the present invention is to construct a PEG5000-initiated hydrophilic polylysine template through ring-opening polymerization reaction, graft two small molecule ligands of the proteolysis-targeting chimera (a ligand targeting BRD4 protein and a ligand targeting E3 ligase) onto the polylysine side chain in a certain proportion through the condensation reaction of amino and carboxyl groups, and finally block the excess side chain amino groups with acid-sensitive 2,3-dimethyl maleic anhydride, thereby forming a polymeric proteolysis-targeting chimera nanoparticle with acid response and targeted degradation functions.

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

[0087] Formula (I);

[0088] Wherein,

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

[0090] The materials used in the present invention are all ordinary commercially available products and can be purchased on the market. The present invention will be further described below in conjunction with examples.

[0091] Example 1 Preparation and Characterization of Acid-Responsive Polylysine Proteolysis-Targeting Chimera

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

[0093] In an atmosphere of nitrogen at 25 °C, 10.0 g of N(ε)-carbobenzoxy-L-lysine was mixed with 100 mL of anhydrous tetrahydrofuran, and 7.0 g of triphosgene was added. The mixture was heated to 55 °C and reacted for 2 h. After the reaction, the reaction mixture was precipitated in excess n-hexane, separated, washed, recrystallized, and dried in vacuo to obtain a white solid of N(ε)-carbobenzoxy-L-lysine cyclic anhydride monomer. Figure 1 1H NMR, IR and MS spectra of N(ε)-carbobenzoxy-L-lysine cyclic anhydride monomer.

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

[0095] 2. Amination of the hydroxyl terminus of methoxypolyethylene glycol

[0096] Weigh 25.0 g of mPEG-OH and dissolve it in 250 mL of dichloromethane. Then add 5.6 g of potassium hydroxide and 9.5 g of p-toluenesulfonyl chloride, and stir the reaction at room temperature. After 9 days, the reaction was stopped, and the mixture was poured into a separatory funnel, washed with saturated brine solution, and the organic phase was separated, dried over anhydrous magnesium sulfate overnight. The filtered and concentrated solution was precipitated with cold ether, filtered through a Buchner funnel, and dried to obtain a white product, the intermediate mPEG-OTs. Weigh the white product mPEG-OTs, add an equal mass of ammonium chloride solid, mix them in a round-bottom flask, and then add 250 mL of aqueous ammonia. Stir the reaction at room temperature. After 7 days, the reaction was stopped, the organic phase was extracted with dichloromethane, washed with water, dried, filtered, and concentrated by rotary evaporation. It was precipitated with cold ether and dried to obtain a white product mPEG-NH2. In the present invention, the number-average molecular weight of the methoxypolyethylene glycol is preferably 1000 - 20000. Figure 2 1H NMR spectra of the aminated methoxypolyethylene glycol (left) and 1 13C NMR spectra (right). 13 13C NMR spectra (right).

[0097] Summary: To prove the synthesis of the initiator PEG5000.

[0098] 3. Preparation of methoxypolyethylene glycol-b-poly(N-carbobenzoxy-lysine)

[0099] Weigh the aminated methoxypolyethylene glycol (M w(= 5000) 4.88 g was added to a vacuum reaction flask with a side port and azeotroped with 400 mL of toluene to remove water. Then, it was azeotroped in an oil bath at 130 °C for 2 hours to remove excess water, and then toluene was removed by vacuum distillation. The 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 cyclic anhydride monomer was added thereto under nitrogen protection, and stirred at 25 °C for 72 hours. The reaction solution was precipitated in 10-fold ice ether, and the ether on the surface of the material was pumped dry by a vacuum drying pump to obtain a white solid of polyethylene glycol monomethyl ether-b-poly(N-benzyloxycarbonyl-lysine).

[0100] 4. Preparation of 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), 30.0 mL of 33 wt.% HBr / acetic acid solution was added to the solution, and the reaction was carried out at room temperature for 2 h. After the reaction, it was precipitated in 10-fold volume of ice ether. The precipitate obtained by centrifugation was dissolved in DMF, loaded into a dialysis bag with Mw3500, dialyzed with deionized water for 3 days, and finally freeze-dried to obtain a white solid of polyethylene glycol monomethyl ether-b-polylysine (Plys 60 ). Figure 3 is the NMR spectrum of polyethylene glycol monomethyl ether-b-polylysine.

[0102] 5. Preparation of Cationic Polylysine Proteolysis Targeting Chimera Targeting BRD4

[0103] Weighed 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 and dissolved them in 15 mL of anhydrous DMF. 59.0 mg of DMAP was dissolved in 3 mL of anhydrous DMF and then added to the reaction solution. Finally, 52.3 mg of DIC was diluted with 5 mL of anhydrous DMF, and slowly dropped into the reaction solution under ice-water bath conditions and in a nitrogen atmosphere. After returning to room temperature, the reaction was carried out for 2 days. After the reaction, the reaction solution was precipitated in 10-fold volume of ice ether, and the white precipitate obtained by centrifugation was vacuum dried, redissolved in DMF, loaded into a dialysis bag with Mw3500, dialyzed with deionized water for 3 days, and finally freeze-dried to obtain a white solid of cationic polylysine proteolysis targeting chimera (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 to Thalidomide is i:j. The adjustment of the grafting ratio of the two is controlled by the calculation and design of the feeding ratio. The grafting rate can be obtained by calculating the integral of the corresponding hydrogen from the 1H NMR spectrum. Figure 4 1H NMR spectra of different grafting ratio polylysine proteolytic targeting chimeras targeting BRD4, 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 at 7.7 and 5.6 in the 1H NMR spectrum can represent the grafting amounts of thalidomide (Tha) and JQ1 respectively, proving that the control of the grafting ratio can be achieved.

[0105] 6. Preparation of anhydride-modified polylysine proteolytic targeting chimeras

[0106] Weigh 311.2 mg of P(lys 0.8 -JQ1 i -Tha j ) 60 dissolve it in 5 mL of pure water, and use 1.0 M NaOH aqueous solution to adjust the pH value to 8.6. Subsequently, add twice the amount of succinic anhydride (acid non-responsive, SA) or 2,3-dimethylmaleic anhydride (acid-responsive, DMMA) to the reaction solution, and use 1.0 M NaOH to maintain the pH value in the range of 8-9. When the pH value is constant, the reaction continues at room temperature for 8 h. Then, dialyze with a dialysis bag with MWCO = 3500 Da for 6 h. The dialysis water is NaOH aqueous solution (pH 8-9), and the water is changed every 1 h. Finally, freeze-dry to obtain P(SA 0.8 -JQ1 i -Tha j ) 60 white solid. Figure 5 1H NMR spectra of JT2-1-SA, JT1-1-SA, and JT1-2-SA non-responsive proteolytic targeting chimeras. Figure 6 1H NMR spectrum of JT2-1-DMMA acid-responsive proteolytic targeting chimera.

[0107] 7. Preparation and characterization of nano proteolytic targeting chimeras

[0108] Weigh 1 mol of the freeze-dried material, dissolve it in 1 mL of DMF, and use a constant-flow pump to drip it into 4 mL of PBS within 1 h. Subsequently, dialyze it in PBS for 6 h, changing the PBS once per hour. Finally, ultrafiltrate and concentrate it, and make the volume up to 200 μM with PBS. The prepared nanoparticles are tested for their particle size and zeta potential by DLS, the size is photographed by TEM, the secondary structure is tested by circular dichroism spectroscopy, and the response ability of the acid-responsive nanoparticles is verified. Figure 7 The particle size and zeta potential of three nano-protein hydrolysis targeting chimeras. Figure 8 The circular dichroism spectra of three nano-protein hydrolysis targeting chimeras under the condition of pH 6.8.

[0109] Summary: DLS and TEM characterized the particle sizes of the three nano-protein hydrolysis targeting chimeras. By DLS measurement, JT2-1 has a positive zeta potential, and after shielding the amino group, both JT2-1-SA and JT2-1-DMMA have negative zeta potentials. Circular dichroism spectroscopy characterized the secondary structures of the three nano-protein hydrolysis targeting chimeras. Lys 60 and JT2-1 are random coils, and after grafting SA and DMMA, both JT2-1-SA and JT2-1-DMMA have α-helix structures. As time prolongs, the structure of JT2-1-DMMA changes from α-helix to random coil, proving the acid-responsive ability of the material.

[0110] Effect Example 1 In vitro cytotoxicity of nano-protein hydrolysis targeting chimeras

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

[0112] Culture Mcf-7 cells until logarithmic growth, digest the cells with trypsin, seed 5000 cells per well in a 96-well plate, add 0.2 mL of medium per well, incubate overnight at 37 °C with 5% CO2 to allow the cells to adhere. After discarding the medium, wash the cells twice with PBS, add the nano-protein hydrolysis targeting chimeras with different ligand grafting ratios and anhydride-modified polylysine, and gradually dilute the drug concentration by the dichotomy method. Add 100 μL per well to the 96-well plate. Discard the medium, wash the plate three times with PBS, add 100 μL of diluted CCK-8 detection solution per well, and incubate at 37 °C for 0.5 h. Detect with an enzyme-labeled instrument at a wavelength of 450 nm. Calculate according to the following formula.

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

[0114] Figure 9 CCK-8 cytotoxicity detection of nano-protein hydrolysis targeting chimeras with different grafting ratios on MCF-7 cells. Figure 10CCK-8 cytotoxicity assay of JQ1 and nanoprotein hydrolysis targeting chimeras on MCF-7 cells under different pH conditions.

[0115] Summary: Using MCF7 cells rich in BRD4 protein as the research object, through the CCK-8 experiment, it was found that the material with a JQ1:Tha ratio of 2:1 screened from cationic protein hydrolysis targeting chimeras and SA-screened protein hydrolysis targeting chimeras had the best cytotoxicity. By exploring cytotoxicity under different pH conditions, it was found that the acid-responsive protein hydrolysis targeting chimera JT2-1-DMMA had an IC50 value 13.7 times lower at pH 6.8 than at pH 7.4, 14.8 times lower than that of the small molecule JQ1, and 24 times lower than that of the non-responsive protein hydrolysis targeting chimera JT2-1-SA at pH 6.8. This indicates that the acid-responsive protein hydrolysis targeting chimera has better tumor cell cytotoxicity than the small molecule BRD4 inhibitor JQ1 and the non-responsive nanoprotein hydrolysis targeting chimera.

[0116] Effect Example 2 Cellular Uptake Characterization of Acid-Responsive Polylysine Protein Hydrolysis Targeting Chimeras

[0117] The breast cancer cell line Mcf-7 was used to evaluate the in vitro cellular uptake efficiency of three polylysine protein hydrolysis targeting chimeras.

[0118] The steps for flow cytometry detection are as follows:

[0119] Cultivate Mcf-7 cells until they reach logarithmic growth. Use trypsin to make the cells into a single-cell suspension, with 100,000 cells per well in a 6-well plate. The next day, after discarding the culture medium, wash the cells twice with PBS, and add 2 mL of culture medium containing 1 μM Cy5.5-JT2-1-DMMA, 1 μM Cy5.5-JT2-1-SA, and 1 μM Cy5.5-JT2-1. After co-incubating for 0.5, 2, 4, 8, 12, and 24 h, discard the culture medium, wash the cells three times with PBS to remove un-uptaken nanoparticles. Digest the cells with trypsin and transfer them to a flow cytometry tube. After centrifugation, discard the supernatant, and resuspend the cells in 0.5 mL of PBS to make a single-cell suspension. Use a flow cytometer to detect the endocytosis of the three materials by the cells. Figure 11 Flow analysis of material endocytosis.

[0120] The steps for CLSM detection are as follows:

[0121] Cultivate Mcf-7 cells until logarithmic growth phase, and use trypsin to make the cells into a single-cell suspension. Seed 100,000 cells in each confocal dish. The next day, after discarding the culture medium, wash the cells twice with PBS, and add 2 mL of culture medium containing 1 μM Cy5.5-JT2-1-DMMA, 1 μM Cy5.5-JT2-1-SA, and 1 μM Cy5.5-JT2-1. After co-incubating for 2, 4, 8, 12, and 24 h, discard the culture medium, and wash the cells three times with PBS to remove the nanoparticles not taken up by the cells. Fix the cells with paraformaldehyde (4%) for 10 min, and wash the cells three times with PBS. Add 500 μL of the prepared DIO staining working solution to each well and stain for 5 min, then wash the cells three times with PBS again. Add 6 drops of anti-fluorescence quenching mounting medium containing DAPI, and incubate at room temperature for 5 minutes. Imaging can be performed within one week. Figure 12 CLSM analysis of endocytosis of JT2-1-SA (left) and JT2-1-DMMA (right).

[0122] Summary: A key reason for the action of acid-responsive proteolytic targeting chimeras is that charge reversal enhances endocytosis. Therefore, we compared the endocytosis of JT2-1-SA and JT2-1-DMMA. The results from flow cytometry and laser confocal microscopy showed that acid-responsive proteolytic targeting chimeras were endocytosed by cells faster than non-responsive ones.

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

[0124] 1. Use breast cancer cell MCF-7 to evaluate the degradation effect of nanoproteolytic targeting chimera on BRD4 protein in vitro cells.

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

[0126] Cultivate Mcf-7 cells until logarithmic growth phase, use trypsin to make the cells into a single-cell suspension, inoculate 200,000 cells per well in a 6-well plate, and add 2 mL of complete DMEM medium to each well. After culturing the cells for 1 day, discard the medium in the 6-well plate, wash the cells 2 times with PBS buffer, add complete DMEM medium containing different concentrations of JT2-1-SA to each well, and induce for 1 day, with 3 replicate wells in each group. After the induction treatment, carefully aspirate the medium, quickly wash the cells 2 times with pre-cooled PBS buffer, and aspirate as much PBS as possible. Scrape the cell lysate with a cell scraper and collect it into a pre-cooled centrifuge tube. Add an appropriate amount of pre-cooled RIPA lysis buffer (containing protease inhibitor and phosphatase inhibitor, added according to the ratio in the reagent instruction manual) to each tube, and lyse on ice for 30 min. After lysis, centrifuge at 12,000 - 14,000 rpm at 4°C for 15 min, carefully aspirate the supernatant and transfer it to a new pre-cooled centrifuge tube. Use a BCA protein quantification kit to measure the protein concentration, dilute to the same concentration, add 5×SDS-PAGE loading buffer, mix well, and boil in a boiling water bath for 10 min to denature the protein. The WB experimental samples are subjected to electrophoresis, membrane transfer, BSA blocking, incubation with primary antibodies (anti-BRD4 antibody, anti-c-MYC antibody, anti-GAPDH antibody), and incubation with secondary antibody (HRP-labeled goat anti-rabbit). After washing the membrane, incubate it in a chemiluminescent substrate solution for 1 - 2 minutes, and use a chemiluminescent imaging system for exposure imaging to obtain the target protein band image. Figure 13 WB of the expression of BRD4 and c-MYC in cells induced by different concentrations of JT2-1-SA.

[0127] Summary: To exclude cation interference, we used materials grafted with succinic anhydride for verification and found that its degradation of BRD4 was concentration-dependent, and the down-regulation of BRD4 expression also successfully down-regulated the expression of its downstream c-MYC.

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

[0129] Cultivate Mcf-7 cells until logarithmic growth phase, and use trypsin to make the cells into a single-cell suspension, with 100,000 cells seeded in each confocal dish. After culturing the cells for 1 day, discard the original culture medium, wash the cells 2 times with PBS buffer, and add 5 μM of JT2-1, JT2-1-SA, and JT2-1-DMMA to each well. After 1 day of induction treatment, discard the culture medium, wash 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 h, then incubate with the primary antibody (anti-BRD4 antibody) at 4 °C overnight. Wash 3 times with PBS, add the secondary antibody (FITC-labeled goat anti-rabbit), and incubate at room temperature for 2 h. Wash 3 times with PBS, add 6 drops of anti-fluorescence quenching mounting medium containing DAPI, incubate at room temperature for 5 minutes, and then perform imaging on the machine.

[0130] The flow cytometry procedure is as follows:

[0131] Cultivate Mcf-7 cells until logarithmic growth phase, and use trypsin to make the cells into a single-cell suspension, with 100,000 cells seeded in each well of a 12-well plate. After culturing the cells for 1 day, discard the original culture medium, wash the cells 2 times with PBS buffer, and add 5 μM of JT2-1, JT2-1-SA, and JT2-1-DMMA to each well, with 3 replicates. After 1 day of induction treatment, discard the culture medium. Digest the cells with trypsin, transfer them to a flow cytometry tube, centrifuge to discard the culture medium, and wash 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 h, then incubate with the primary antibody (anti-BRD4 antibody) at 4 °C overnight. Wash 3 times with PBS, add the secondary antibody (FITC-labeled goat anti-rabbit), and incubate at room temperature for 2 h. Wash 3 times with PBS, and finally add 400 μL of PBS and perform testing on the machine. Figure 14 The CLSM and flow cytometry results of BRD4 expression in cells after induction with different nanoprotein hydrolysis targeting chimeras.

[0132] Summary: By comparing several nanoparticles, it was found that the acid-responsive protein hydrolysis targeting chimera has the best BRD4 degradation effect. This may be because of effective cell uptake and lysosomal escape, enabling it to exert its function.

[0133] 3. To evaluate the degradation mechanism of the nanoprotein hydrolysis targeting chimera, the CLSM procedure is as follows:

[0134] Cultivate Mcf-7 cells until logarithmic growth phase, and use trypsin to make the cells into a single-cell suspension, inoculating 100,000 cells per well in a confocal dish. After culturing the cells for 1 day, discard the original culture medium and wash the cells 2 times with PBS buffer. Pretreat the two groups of cells with 50 nM MG132 (proteasome inhibitor) and 50 nM MLN4924 (ubiquitination inhibitor) for 30 min respectively, then discard the culture medium, wash the cells 3 times with PBS buffer, and add 25 μM JT2-1-SA to each well. After 1 day of induction treatment, discard the culture medium, wash 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 h, then incubate with the primary antibody (anti-BRD4 antibody) at 4 °C overnight. Wash 3 times with PBS, add the secondary antibody (FITC-labeled goat anti-rabbit), and incubate at room temperature for 2 h. Wash 3 times with PBS, add 6 drops of anti-fluorescence quenching mounting medium containing DAPI, incubate at room temperature for 5 minutes, and then perform imaging on the machine.

[0135] The flow cytometry procedure is as follows:

[0136] Cultivate Mcf-7 cells until logarithmic growth phase, and use trypsin to make the cells into a single-cell suspension, inoculating 100,000 cells per well in a 12-well plate. After culturing the cells for 1 day, discard the original culture medium and wash the cells 2 times with PBS buffer. Pretreat the two groups of cells with 50 nM MG132 (proteasome inhibitor) and 50 nM MLN4924 (ubiquitination inhibitor) for 30 min respectively, then discard the culture medium, wash the cells 3 times with PBS buffer, and add 25 μM JT2-1-SA to each well. After 1 day of induction treatment, discard the culture medium. Digest the cells with trypsin, transfer them to a flow cytometry tube, centrifuge to discard the culture medium, and wash 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 h, then incubate with the primary antibody (anti-BRD4 antibody) at 4 °C overnight. Wash 3 times with PBS, add the secondary antibody (FITC-labeled goat anti-rabbit), and incubate at room temperature for 2 h. Wash 3 times with PBS, and finally add 400 μL PBS and perform testing on the machine. Figure 15 It is the CLSM and flow cytometry analysis diagrams of the BRD4 degradation mechanism in cells induced by different nanoprotein hydrolysis targeting chimeras.

[0137] Summary: In cells pretreated with proteasome inhibitor and ubiquitination inhibitor, the expression of BRD4 returns to normal, indicating that the degradation process depends on the ubiquitin-proteasome pathway.

[0138] Effect Example 4: Effect of acid-responsive polylysine 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, JQ1 group, as well as reagents such as the cell apoptosis and cell cycle kit from Beyotime, 70% ethanol, and trypsin. Culture Mcf-7 cells in a 12-well plate, with 100,000 cells seeded in each well. After 24 hours, add drugs according to the grouping. Dilute the concentration to 5 μM with DMEM complete medium in each group, and add 1 mL of drug-containing medium to each well, and induce for 24 hours. Then collect the cells. Digest the cells with trypsin to make them detach from the surface of the culture dish, collect them into a centrifuge tube, and centrifuge to discard the supernatant. After that, fix the cells. Add pre-cooled 70% ethanol and fix at 4°C overnight. The next day, centrifuge to discard the ethanol and wash the cells twice with PBS. Then perform PI staining and RNase A treatment. Prepare the propidium iodide staining working solution according to the ratio in the instruction manual. Add 0.5 mL of propidium iodide staining solution to each tube of cell sample, slowly and fully resuspend the cell pellet, and incubate at 37°C in the dark for 30 minutes. Complete the flow cytometry detection within 24 hours after staining. Use FlowJo software to analyze the distribution of each stage of the cell cycle. Figure 16 It is a cell cycle result graph of polylysine proteolysis-targeted chimeras modified with different anhydrides.

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

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

[0142] Demonstrate the passive targeting of the nanomaterials through in vivo imaging and ex vivo imaging. Perform quantitative analysis through a standard curve. Inject nanoparticles Cy5.5-JT2-1-DMMA with the same fluorescence intensity into the tail vein of each mouse, and take pictures at 12 h, 24 h, and 36 h. Figure 17 It is an in vivo imaging distribution graph after tail vein injection of Cy5.5-JT2-1-DMMA.

[0143] Summary: The acid-responsive nanoproteolysis-targeted chimeras are enriched at the tumor site through the EPR effect. In the in vivo experiment, it was first observed that the material reached the maximum enrichment at the tumor site at about 24 hours, and then the signal attenuated.

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

[0145] The inhibitory effects of the nanoprotein hydrolysis targeting chimera JT2-1-SA and JT2-1-DMMA were evaluated using the MCF-7 tumor model in nude mice. The mice were divided into 4 groups and were given normal saline, JQ1, JT2-1-SA, and JT2-1-DMMA respectively. The doses of JT2-1-SA and JT2-1-DMMA were 20 mg / kg body weight, and the equivalent dose of JQ1 was used. Administration was via tail vein injection. When the tumors grew to approximately 100 mm 3 , the first treatment was carried out, and then injections were given once every 3 days for a total of 6 injections over 16 days. During the experiment, the body weight and tumor volume were recorded every 2 days. Figure 18 This is the result of the treatment experiment on mice after tail vein injection.

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

[0147] The results showed that the tumor inhibition rate of the JQ1 group was only 14.4%, without showing significant differences, indicating that the therapeutic effect of the single JQ1 small molecule was weak and may not have fully targeted tumor cells. The JT2-1-SA group also showed a relatively significant tumor inhibition effect, with a tumor inhibition rate of 41.4%. Compared with the JQ1 group, JT2-1-SA may achieve enrichment in the tumor part through the EPR effect and continuously play the degradation role of the protein hydrolysis targeting chimera intracellularly, thereby more effectively inhibiting tumor growth. The tumor inhibition rate of the JT2-1-DMMA group reached 76.7%, significantly superior to other groups, which may be the result of effective tumor enrichment and enhanced tumor penetration and uptake after the removal of DMMA under the acidic conditions of the tumor microenvironment. In addition, there were no significant differences in the body weight changes of the mice in each group during the treatment process, indicating that the treatment doses used did not cause obvious toxic side effects.

[0148] The above is only the preferred implementation mode of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A nano-degrading agent having a structure shown in formula (I): Formula (I); Among them, The structure of R1 is shown in formula (II), the structure of R2 is shown in formula (III), and the structure of R3 is shown in formula (IV); Formula (II) Formula (III) Formula (IV) m is the degree of polymerization, m = 10 - 500, n = 10 - 200, x = 1 - 100; y = 1 - 100; z = 8 - 500; x:y = (1 - 5):(1 - 5).

2. The preparation method of the nano-degrading agent according to claim 1, characterized in that, It includes the following steps: Step 1: Take N(ε)-benzyloxycarbonyl-L-lysine, anhydrous tetrahydrofuran and triphosgene and react them to obtain N(ε)-benzyloxycarbonyl-L-lysine cyclic anhydride monomer; Step 2: React the N(ε)-benzyloxycarbonyl-L-lysine cyclic anhydride monomer with amino-terminated methoxypolyethylene glycol in the presence of a solvent to obtain methoxypolyethylene glycol-b-poly(N-benzyloxycarbonyl-lysine); Step 3: Mix and react methoxypolyethylene glycol-b-poly(N-benzyloxycarbonyl-lysine), trifluoroacetic acid and hydrobromic acid acetic acid solution to obtain methoxypolyethylene glycol-b-polylysine; Step 4: Couple the groups of R1 and R2 in the nano-degrading agent according to claim 1 to methoxypolyethylene glycol-b-polylysine to obtain a cationic polylysine proteolytic targeting chimera molecule; Step 5: Mix and react the cationic polylysine proteolytic targeting chimera molecule with 2,3-dimethylmaleic anhydride to obtain the nano-degrading agent.

3. The preparation method according to claim 2, wherein, In step 1, the temperature of the mixing reaction is 50 - 60 °C, and the time of the mixing reaction is 1.5 - 3 h; It also includes subjecting the mixture after the mixing reaction to sedimentation with excess n-hexane, separation, washing, recrystallization, and vacuum drying to obtain the N(ε)-benzyloxycarbonyl-L-lysine cyclic anhydride monomer.

4. The preparation method according to claim 2 or 3, characterized in that, In step 2, The preparation method of the amino-terminated methoxypolyethylene glycol includes: S1: Take mPEG-OH, potassium hydroxide and p-toluenesulfonyl chloride and dissolve them in dichloromethane, react, wash, and separate to obtain an organic phase. The obtained organic phase is filtered and concentrated, sedimented with cold diethyl ether, and filtered to obtain the intermediate mPEG-OTs; S2: Take mPEG-OTs, ammonium chloride and ammonia monohydrate, carry out a mixing reaction, extract the organic phase with dichloromethane. The obtained organic phase is washed with water, dried, filtered, rotary evaporated and concentrated, sedimented with cold diethyl ether, and dried by suction to obtain amino-terminated methoxypolyethylene glycol; After the amino-terminated methoxypolyethylene glycol is azeotropically dehydrated with toluene under a vacuum environment, toluene is removed by vacuum distillation, and then it is mixed and reacted with the N(ε)-benzyloxycarbonyl-L-lysine cyclic anhydride monomer. The solvent is N,N-dimethylformamide. The temperature of the reaction is 15 - 35 °C, and the time of the mixing reaction is 2 - 4 days; It also includes subjecting the reaction solution after the reaction to sedimentation with cold diethyl ether and vacuum drying to obtain methoxypolyethylene glycol-b-poly(N-benzyloxycarbonyl-lysine).

5. The preparation method according to claim 2, characterized in that, In step 3, the temperature of the mixing is 15 - 35 °C, and the time of the mixing reaction is 1.5 - 3 h; It also includes subjecting the reacted reaction solution to sedimentation with cold ethyl ether and centrifugation, and dissolving, dialyzing, and freeze-drying the obtained precipitate with N,N-dimethylformamide to obtain methoxypolyethylene glycol-b-poly(N-carbobenzoxy-lysine).

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

7. The preparation method according to claim 2, wherein In the 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; It also includes subjecting the reaction solution after the mixed reaction to dialysis and freeze-drying to obtain the nano-degrading agent.

8. Use of the nano-degrading agent according to claim 1 or the nano-degrading agent prepared by the preparation method according to any one of claims 2-7 in the preparation of a drug for treating breast cancer.

9. A drug for treating breast cancer, characterized in that, It includes the nano-degrading agent according to claim 1 or the nano-degrading agent prepared by the preparation method according to any one of claims 2-7.

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