PH / active oxygen dual-response iron-based-PROTAC nano-drug as well as preparation method and application thereof

Abstract

The invention discloses an iron-based-PROTAC nano-drug with a pH / active oxygen dual-response characteristic. The iron-based-PROTAC nano-drug is a nano-drug prepared from a carrier, zero-valent iron nanoparticles and an active oxygen-responsive PROTAC prodrug through a two-phase stirring method, wherein the carrier is an amphiphilic polyphenol polymer modified by a pH (Potential of Hydrogen) group, and ultra-small-size zero-valent iron nanoparticles and an active oxygen responsive PROTAC prodrug are wrapped in the carrier. The nano-drug developed by the invention shows good hydrophilicity and stability characteristics, and can effectively utilize high permeability and retention effect (EPR effect), thereby realizing long-time accumulation at a tumor site, and accurately releasing an active oxygen responsive PROTAC prodrug in a specific subacid environment of a tumor. In addition, the iron-based nano material catalyzes hydrogen peroxide to generate hydroxyl free radicals through Fenton catalytic activity of the iron-based nano material, the prodrug can be further activated, and then tumor cells are induced to generate apoptosis. Meanwhile, the novel iron-based nano material also has the capability of inducing ferroptosis.

Description

Technical Field

[0001] The present invention relates to biomedical nanomedicines, and in particular to a pH / active oxygen dual-responsive iron-based PROTAC nanomedicine, as well as a preparation method and application of the nanomedicine. Background Art

[0002] As an emerging tumor treatment strategy, proteolysis-targeted chimeras (PROTACs) effectively reduce the risk of drug resistance by uniquely utilizing the ubiquitin-proteasome system (UPS) to degrade target proteins (POIs), providing new treatment methods for traditional "difficult-to-drug" targets and significantly broadening the horizons of drug development. However, the clinical application of PROTACs still faces challenges such as significant systemic toxicity and poor water solubility. To solve these problems, stimulus-responsive PROTACs came into being. They can achieve activity regulation under endogenous (such as enzymes, hydrogen peroxide, etc.) or exogenous (such as light, X-rays, etc.) stimulation to ensure that their therapeutic effects are activated only at the tumor site.

[0003] On the other hand, ferroptosis, as an iron-dependent programmed cell death, is characterized by an imbalance in the level of reactive oxygen species (ROS) in tumor cells, leading to the accumulation of lipid peroxides (LPO) and the inhibition of glutathione peroxidase 4 (GPX4) activity. A large number of studies have shown that iron-based nanomaterials have excellent catalytic properties, can effectively promote the Fenton reaction, accelerate the decomposition of hydrogen peroxide (H2O2), and thus generate toxic hydroxyl radicals (·OH) in the tumor microenvironment, significantly enhancing their therapeutic potential.

[0004] In view of the above, there is an urgent need for an innovative nanomedicine that can overcome the shortcomings of PROTACs, achieve site-specific activation of PROTACs, and also exert ferroptosis, thereby more effectively treating tumors. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a new method for preparing pH / reactive oxygen dual-responsive iron-based-PROTAC nanodrugs. The core of this method is to use pH group-modified amphiphilic polyphenol polymers as carriers to encapsulate ultra-small zero-valent iron nanoparticles and reactive oxygen responsive PROTAC prodrugs. By adopting a two-phase stirring method, an iron-based-PROTAC nanodrug with pH / reactive oxygen dual-responsive properties is effectively assembled. The drug exhibits superior biosafety and can effectively target and accumulate in tumor sites, thereby synergistically enhancing the therapeutic effect of tumors.

[0006] Technical solution: The iron-based PROTAC nanodrug with pH / active oxygen dual response characteristics described in the present invention is a nanodrug composed of a carrier, zero-valent iron nanoparticles, and active oxygen responsive PROTAC prodrug through a two-phase stirring method; wherein the carrier is an amphiphilic polyphenol polymer modified with a pH group, and the carrier encapsulates ultra-small zero-valent iron nanoparticles and active oxygen responsive PROTAC prodrug.

[0007] The iron-based PROTAC nanomedicine with dual response characteristics of pH / active oxygen species, the nanomedicine has a size of 100-200 nm; the particle size of the ultra-small zero-valent iron nanoparticles is 3-5 nm;

[0008] The pH group-modified amphiphilic polyphenol polymer is PEG-polyphenol; the reactive oxygen species-responsive PROTAC prodrug is DHBC-MZ1, and the structural formula is shown below:

[0009]

[0010] Preferably, the PEG-polyphenol is PEG 50000 -Polyphenols.

[0011] The iron-based PROTAC nanomedicine with pH / active oxygen dual response characteristics, the structural formula of the PEG-polyphenol is:

[0012]

[0013] Wherein, m is selected from 100-130, and n is selected from 20-50. Preferably, m is selected from 113, and n is selected from 20-50.

[0014] A method for preparing an iron-based PROTAC nanodrug with pH / active oxygen dual response characteristics comprises the following steps:

[0015] (1) adding ammonium bromide and sodium oleate to a mixture of oleylamine and hexadecyltrimethyl bromide and heating the mixture for reaction, then adding carbonyl iron to the mixture after the temperature is further increased, cooling the mixture to room temperature, and purifying the nanoparticles to obtain ultra-small zero-valent iron nanoparticles (USINPs);

[0016] (2) 3,5-dihydroxybenzoic acid methyl ester and DIPEA are mixed in an organic solvent, bromomethyl methyl ether is added dropwise, and the generated HCl gas is blown off to obtain compound 1; then, compound 1 is reacted with lithium aluminum hydride in tetrahydrofuran, and water and sodium hydroxide are added for precipitation to obtain a colorless oily liquid compound 2; then, compound 2 is reacted with DIPEA and 4-nitrophenyl chloroformate in an organic solvent to obtain DHBC; then, MZ1 is mixed with DIPEA, DMAP and DHBC in an organic solvent for reaction, and compound 4 is obtained after purification; finally, compound 4 is deprotected and purified to obtain the active oxygen-responsive PROTAC prodrug DHBC-MZ1;

[0017]

[0018] (3) Synthesis of amphiphilic polyphenol polymers (PEG-polyphenols): A hydrophobic NCA group was introduced onto the hydrophilic PEG-NH2 segment by chemical grafting technology to generate compound 6; subsequently, an amination reaction was performed using hydrazine hydrate to introduce an amino functional group at the end of each hydrophobic group; then, these amino groups reacted with 3,4-dihydroxybenzaldehyde to form PEG-polyphenols containing pH-responsive acylhydrazone bonds;

[0019]

[0020] (4) Ultra-small zero-valent iron nanoparticles dissolved in organic solvent A are mixed with DHBC-MZ1 and PEG-polyphenol dissolved in organic solvent B, water and an emulsifier are added, and a two-phase stirring method is used to promote the self-assembly of the components to form the target nanomedicine.

[0021] In the preparation method, in step (1), the mass ratio of ammonium bromide, sodium oleate, oleylamine and octadecene is 1:5-10; 150-250; 400-500; and the mass ratio of carbonyl iron to octadecene is 1:15-20.

[0022] In the preparation method, the purification in step (1) is to purify the nanoparticles using n-hexane and ethanol.

[0023] In the preparation method, the organic solvent in step (2) is selected from dichloromethane.

[0024] In the preparation method, the organic solvent A in step (3) is selected from chloroform, and the organic solvent B is selected from dimethyl sulfoxide.

[0025] In the preparation method, in step (4), the mass ratio of the amphiphilic polyphenol polymer PEG-polyphenol to the ultra-small iron nanoparticles is 1:3-1:7.

[0026] The preparation method, step (4) is specifically as follows: the ultra-small size zero-valent iron nanoparticles USINPs in the oil phase are washed by centrifugal separation, and then redissolved in chloroform, and then the solution is mixed with a DMSO solution containing DHBC-MZ1 and PEG-polyphenol and an SDS aqueous solution, and then water is added; the mixed solution is ultrasonically emulsified and then stirred; after the chloroform evaporates, the precipitate is collected by high-speed centrifugation and dispersed in water; DOPA-PEG is then added to improve the stability of the obtained product; finally, the product is purified and concentrated to obtain a pH / active oxygen dual-responsive iron-based-PROTAC nanodrug (USINAs@DHBC-MZ1).

[0027] The use of the iron-based PROTAC nanomedicine with pH / active oxygen dual response characteristics in the preparation of a drug for treating tumors. Preferably, the tumor includes breast cancer.

[0028] The present invention discloses a pH / active oxygen dual-responsive iron-based PROTAC nanomedicine and a preparation method thereof, and applies the same to tumor treatment. The nanomedicine system is constructed on an amphiphilic polyphenol polymer matrix modified with pH-sensitive groups, encapsulating ultra-small zero-valent iron nanoparticles and an active oxygen-sensitive PROTAC prodrug, which together constitute a complex nanoassembly structure.

[0029] The size of the successfully assembled pH / reactive oxygen dual-responsive iron-based-PROTAC nanodrug is 100-200 nm, and the particle size of the ultrasmall iron nanoparticles is 3-5 nm.

[0030] Further, the specific synthesis steps are as follows:

[0031] (1) Ammonium bromide and sodium oleate are added to a mixture of oleylamine and hexadecyltrimethyl bromide and heated to react, and then carbonyl iron is added to react after the temperature is further increased. After the mixture is cooled to room temperature, the nanoparticles are purified using n-hexane and ethanol to obtain ultra-small zero-valent iron nanoparticles (USINPs).

[0032] (2) First, methyl 3,5-dihydroxybenzoate and DIPEA were mixed in dichloromethane, bromomethyl methyl ether was added dropwise, and the generated HCl gas was blown off to obtain compound 1. Next, compound 1 was reacted with lithium aluminum hydride in tetrahydrofuran, and water and sodium hydroxide were added to precipitate to obtain a colorless oily liquid compound 2. Then, compound 2 was reacted with DIPEA and 4-nitrophenyl chloroformate in dichloromethane to obtain DHBC. Subsequently, MZ1 (a PROTAC) was mixed with DIPEA, DMAP and DHBC in dichloromethane for reaction, and compound 4 was obtained after purification. Finally, compound 4 was treated with 4M HCl aqueous solution to remove the protecting group. After methanol was spin-dried, it was purified to obtain the active oxygen-responsive PROTAC prodrug DHBC-MZ1. Throughout the process, purification was performed by extraction, drying and concentration.

[0033] (3) Synthesis of amphiphilic polyphenol polymers (PEG 5000 -polyphenols). Through chemical grafting technology, hydrophilic PEG 5000 The hydrophobic NCA group was introduced into the -NH2 segment to generate compound 6. Subsequently, an amination reaction was carried out using hydrazine hydrate to introduce an amino functional group at the end of each hydrophobic group. These amino groups then reacted with 3,4-dihydroxybenzaldehyde to form a PEG containing a pH-responsive acylhydrazone bond. 5000 - Polyphenols, polymers with a specific pH sensitivity.

[0034] (4) USINPs dissolved in chloroform and DHBC-MZ1 and PEG dissolved in DMSO were mixed. 5000 -polyphenols, add a certain amount of deionized water and emulsifier, and use a two-phase stirring method to promote the self-assembly of each component to form the target nanomedicine.

[0035] Wherein, in step (1), the mass ratio of ammonium bromide, sodium oleate, oleylamine and octadecene is 1:5-10; 150-250; 400-500; and the mass ratio of carbonyl iron to octadecene is 1:15-20.

[0036] Wherein, step (1) is specifically as follows: first, ammonium bromide and sodium oleate are added to a mixture of oleylamine and hexadecyltrimethylammonium bromide, heated to 120°C and degassed for 1 hour. Then, the mixture is heated to 260°C and carbon-based iron is injected under an inert atmosphere. After reacting for 30 minutes, the solution turns black, indicating that nanoparticles are formed. After cooling to room temperature, ethanol is added for precipitation, and then purified with n-hexane and ethanol at least three times to remove impurities.

[0037] Wherein, in step (2), the molar mass ratio of methyl 3,5-dihydroxybenzoate, bromomethyl methyl ether and DIPEA is 1:2:3; the molar mass ratio of compound 1 and lithium aluminum hydride is 1:1.5; and the molar mass ratio of compound 2, 4-nitrophenyl chloroformate and DIPEA is 1:3:3.

[0038] Wherein, step (2) is specifically as follows: first, 3,5-dihydroxybenzoic acid methyl ester is dissolved in dichloromethane, DIPEA is added and precooled to 0°C, then bromomethyl methyl ether is added dropwise, and the generated HCl gas is blown off with an ear bulb. The solution is restored to room temperature and stirred for 4 hours to obtain compound 1. Next, compound 1 is dissolved in anhydrous tetrahydrofuran and precooled to 0°C, then a suspension made of lithium aluminum hydride is added dropwise, and stirred for 2 hours after returning to room temperature. A small amount of 15% NaOH and water are added to precipitate the remaining lithium aluminum hydride, filtered and spin-dried to obtain a colorless oily liquid compound 2. Compound 2 is dissolved in dichloromethane, DIPEA is added, 4-nitrophenyl chloroformate is added dropwise at 0°C, returned to room temperature and stirred for 2 hours, and purified to obtain DHBC. Subsequently, MZ1 is dissolved in dichloromethane, DIPEA and DMAP are added and precooled to 0°C, then DHBC is added dropwise, returned to room temperature and stirred for 18 hours, and purified to obtain compound 4. Finally, compound 4 was dissolved in methanol, 4M HCl aqueous solution was added at 0°C, and the mixture was returned to room temperature and stirred for 2 hours. After the methanol was spin-dried, the white solid product DHBC-MZ1 was obtained by purification.

[0039] Wherein, in step (3), the 5000 The molar mass ratio of -NH2 and (S)-benzyl 2-(2,5-dioxooxazolidin-4-yl) acetate is 1:50; the molar mass ratio of compound 6, hydrazine hydrate and DIPEA is 1:100:100; the molar mass ratio of compound 7 and 3,4-dihydroxybenzaldehyde is 1:500.

[0040] Wherein, step (3) is specifically: at 35°C, PEG 5000 -NH2 and (S)-benzyl 2-(2,5-dioxooxazolidin-4-yl) acetate were added to a 10:1 mixed solution of anhydrous dichloromethane and N,N-dimethylformamide, and heated under reflux for 3 days to prepare compound 6, which was then purified by ether; then, compound 6 dissolved in DMSO was reacted with hydrazine hydrate at 35°C for 12 hours under the catalysis of DIPEA to achieve a large amount of amino group grafting, and compound 7 was obtained by purification by dialysis and freeze-drying; finally, the aqueous solution of compound 7 was reacted with 3,4-dihydroxybenzaldehyde dissolved in methanol at a pH of about 5 for 12 hours to obtain the final pH-responsive PEG 5000 -Polyphenols.

[0041] Wherein, in step (4), the mass ratio of the amphiphilic polyphenol polymer to the ultra-small iron nanoparticles is 1:5.

[0042] Step (4) is as follows: First, the oil phase USINPs are washed by centrifugation and then redissolved in 200 μL chloroform. The solution is then mixed with a mixture containing DHBC-MZ1 and PEG 5000 -50 μL DMSO solution of polyphenols and 100 μL SDS aqueous solution were mixed, and then 1.85 mL of deionized water was added. The mixed solution was ultrasonically emulsified for 20 minutes and then magnetically stirred for 2 hours. After chloroform was evaporated, the precipitate was collected by high-speed centrifugation and dispersed in deionized water. DOPA-PEG was then added 2000 , in order to improve the stability of the obtained product. Finally, the product was purified and concentrated by ultrafiltration (molecular weight cutoff value was 30 kDa) to obtain pH / active oxygen dual-responsive iron-based-PROTAC nanodrug (USINAs@DHBC-MZ1).

[0043] The key technical points of the present invention are: (1) The pH / reactive oxygen dual-responsive iron-based PROTAC nanodrug in the present invention has a nanoscale size and can accurately target and effectively accumulate in the tumor area. When responding under the specific pH conditions of the tumor microenvironment, the prodrug it carries will be released. At the same time, the iron-based nanoparticles can catalyze hydrogen peroxide to generate hydroxyl radicals, which not only induces the ferroptosis process, but also activates the prodrug to promote tumor cell apoptosis, thereby achieving a synergistic killing effect on tumor cells. (2) The present invention uses mild reaction conditions to prepare pH / reactive oxygen dual-responsive iron-based PROTAC nanodrugs with good stability. (3) By using amphiphilic polyphenol polymers modified with pH-sensitive groups as carriers, the present invention successfully encapsulates prodrugs and ultra-small zero-valent iron nanoparticles to form a stable nanoassembly. This design not only optimizes the pharmacokinetic properties of the prodrug and improves its accumulation efficiency in the tumor site, but also significantly reduces the systemic toxicity that PROTAC may cause, showing good biocompatibility, and providing possibilities for future clinical transformation applications.

[0044] The pH / reactive oxygen dual-responsive iron-based PROTAC nanodrug in the present invention can respond to the specific pH environment of tumor tissue and release the loaded reactive oxygen-responsive PROTAC prodrug. At the same time, the iron-based nanomaterial, with its intrinsic Fenton catalytic activity, effectively catalyzes hydrogen peroxide in the tumor microenvironment to generate highly toxic hydroxyl radicals. This process not only promotes the activation of PROTAC prodrugs, but also gives the ability to induce ferroptosis of tumor cells, thereby achieving a synergistic anti-tumor effect of multiple mechanisms.

[0045] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the iron-based PROTAC nanodrug with dual response to pH / reactive oxygen species, which is assembled based on hydrophilic and hydrophobic forces, developed by the present invention, exhibits good hydrophilicity and stability characteristics. The primary advantage is that the nanoassembly has a nanoscale size and can effectively utilize high permeability and retention effect (EPR effect) to achieve long-term accumulation at the tumor site. Once it reaches the tumor area, the nanodrug will show responsiveness to pH, ensuring the precise release of the reactive oxygen-responsive PROTAC prodrug in the slightly acidic environment unique to the tumor. In addition, the iron-based nanomaterial catalyzes the hydroxyl radicals generated by hydrogen peroxide through its Fenton catalytic activity, which can further activate the prodrug and induce apoptosis of tumor cells. At the same time, the new iron-based nanomaterial also has the ability to induce ferroptosis. Through the synergistic effect of these two mechanisms, effective treatment of tumors is achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 TME diagram of ultra-small zero-valent iron nanoparticles USINPs;

[0047] Figure 2 The nuclear magnetic resonance hydrogen spectrum (A) and mass spectrum (B) of the reactive oxygen species-responsive PROTAC prodrug DHBC-MZ1;

[0048] Figure 3 is the H NMR spectrum of the amphiphilic polyphenol polymer (PEG5000-polyphenol);

[0049] Figure 4 Schematic diagram of the synthesis of pH / reactive oxygen dual-responsive iron-based PROTAC nanodrug USINAs@DHBC-MZ1;

[0050] Figure 5 The particle size distribution diagram (A) and TME diagram (B) of the pH / reactive oxygen dual-responsive iron-based PROTAC nanodrug USINAs@DHBC-MZ1;

[0051] Figure 6 The size and PDI of the pH / ROS dual-responsive iron-based PROTAC nanodrug USINAs@DHBC-MZ1 were incubated in DMEM (37°C) supplemented with 10% FBS within 72 hours (A) and the particle size distribution after incubation in PBS (pH 7.4, 37°C) for 72 hours (B);

[0052] Figure 7 The TMB method was used to detect the production of reactive oxygen species by the pH / reactive oxygen dual-responsive iron-based PROTAC nanodrug USINAs@DHBC-MZ1 in the presence of different concentrations of hydrogen peroxide at pH 5.5.

[0053] Figure 8 The particle size distribution diagram (A) and TME diagram (B) of the pH / reactive oxygen dual-responsive iron-based PROTAC nanodrug USINAs@DHBC-MZ1 that responded to disintegration at pH 5.5;

[0054] Fig. 9 The release curves of the pH / ROS dual-responsive iron-based PROTAC nanodrug USINAs@DHBC-MZ1 at pH 5.5 and 7.4 for the ROS-responsive PROTAC prodrug (DHBC-MZ1);

[0055] Fig.10 Schematic diagram of the activation of the active oxygen-responsive PROTAC prodrug of the pH / active oxygen dual-responsive iron-based PROTAC nanodrug USINAs@DHBC-MZ1 under conditions of pH 5.5 and different concentrations of hydrogen peroxide, and the release curve of the active PROTAC (MZ1);

[0056] Fig.11 Figure 2 shows the cytotoxicity results of pH / ROS dual-responsive iron-based PROTAC nanodrug USINAs@DHBC-MZ1 and other experimental and control groups (A) MDA-MB-231 cells, (B) L02 cells;

[0057] Fig.12 The pH / ROS dual-responsive iron-based PROTAC nanodrug USINAs@DHBC-MZ1 and other experimental and control groups produced ROS in MDA-MB-231 cells;

[0058] Fig.13 The effect of pH / reactive oxygen dual-responsive iron-based PROTAC nanodrug on MDA-MB-231 cells; (A is the degradation of BRD4 protein in MDA-MB-231 cells by pH / reactive oxygen dual-responsive iron-based PROTAC nanodrug USINAs@DHBC-MZ1; B is the GPX4 protein level in MDA-MB-231 cells after treatment with USINAs@DHBC-MZ1 and other experimental and control groups);

[0059] Fig.14 The distribution of the fluorescently labeled pH / reactive oxygen dual-responsive iron-based PROTAC nanodrug USINAs@DHBC-MZ1 after intravenous injection into MDA-MB-231 tumor-bearing nude mice;

[0060] Fig.15 This is a curve of the change of tumor volume over time in nude mice within 14 days after administration of pH / reactive oxygen dual-responsive iron-based PROTAC nanodrug USINAs@DHBC-MZ1 and other control groups of tumor-bearing nude mice;

[0061] Fig.16 This is a graph showing the changes in nude mouse body weight over time within 14 days after administration of the pH / reactive oxygen dual-responsive iron-based PROTAC nanodrug USINAs@DHBC-MZ1 and other control group tumor-bearing nude mice. DETAILED DESCRIPTION

[0062] Example 1

[0063] Preparation of pH / reactive oxygen dual-responsive iron-based PROTAC nanomedicine:

[0064] (1) Synthesis of ultra-small zero-valent iron nanoparticles

[0065] 0.04 g of ammonium bromide and 0.3 g of sodium oleate were mixed in a solution of 12 mL of octadecene and 5 mL of oleylamine. Under an inert gas atmosphere, the mixture was heated to 120 ° C to remove moisture and oxygen, and the temperature was maintained for 60 minutes. Subsequently, under the protection of an inert gas, the temperature was raised to 220 ° C and kept warm for 30 minutes. After the temperature dropped to 220 ° C, 500 mL of carbonyl iron was added, and then heated to 240 ° C at a rate of 5 ° C per minute and maintained for 30 minutes. After the reaction was completed, the system was allowed to cool naturally to room temperature. The nanoparticle precipitate was collected by precipitating with 40 mL of anhydrous ethanol and centrifuging at 8000 rpm for 10 minutes, removing the supernatant. The precipitate was dissolved in 3 mL of n-hexane, precipitated with 40 mL of anhydrous ethanol again, and centrifuged at 8000 rpm for 10 minutes to successfully prepare ultra-small zero-valent iron nanoparticles (USINPs).

[0066] 0.5 g of the prepared USINPs was dispersed in 1 mL of n-hexane, 20 μL was dropped onto a copper grid and air-dried, and then observed by transmission electron microscopy (TEM). Figure 1 The TEM images shown show that the obtained USINPs have uniform particle size ranging from 3 to 5 nm.

[0067] (2) Synthesis of ROS-responsive PROTAC prodrugs

[0068] Synthesis steps diagram

[0069]

[0070] First, 1.0 g of methyl 3,5-dihydroxybenzoate was dissolved in 20 ml of dichloromethane, 2.33 g of DIPEA was added, and the mixture was precooled to 0° C. Then, 1.0 mL of bromomethyl methyl ether dissolved in 5 mL of dichloromethane was added dropwise, and the generated HCl gas was blown away with an ear bulb, and the mixture was stirred at room temperature for 4 hours. 0.8 g of compound 1 was obtained by extraction, washing, drying and concentration purification. Next, 0.8 g of compound 1 was dissolved in anhydrous tetrahydrofuran, precooled to 0° C., 0.175 g of lithium aluminum hydride was added dropwise, and the mixture was stirred at room temperature for 2 hours. After treatment with a small amount of 15% NaOH solution and water, the mixture was filtered under reduced pressure to obtain compound 2. Then, the unpurified compound 2 (3.1 mmol) was dissolved in anhydrous dichloromethane, 1.2 g DIPEA (9.33.1 mmol) was added, 1.87 g (9.33.1 mmol) was added dropwise, and the mixture was reacted at 0°C and stirred at room temperature for 2 hours. After washing, drying and concentration, 0.96 g DHBC was purified by silica gel column chromatography. Finally, 50 mg MZ1 was dissolved in anhydrous dichloromethane, 20 mg DIPEA and 6.1 mg DMAP were added, precooled to 0°C, 40 mg DHBC was added dropwise, and stirred at room temperature for 18 hours. After washing, drying and concentration, 35.8 mg product 4 was purified. 30 mg compound 4 was dissolved in methanol, an equal volume of 4M HCl aqueous solution was added, and the mixture was stirred at 0°C for 2 hours. The methanol was decompressed and dried, and after three extractions and washings, the white solid product DHBC-MZ1 was purified by silica gel column chromatography.

[0071] Take 5 mg of the final product DHBC-MZ1 and dissolve it in 600 mL of deuterated DMSO. Perform H NMR analysis. The result is 1H NMR (400MHz, DMSO-d6) δ9.32 (s, 2H), 8.98 (s, 1H), 8.65 (t, J = 6.0Hz, 1H), 8.28 (t, J = 5.6Hz, 1H), 7.48 (d, J = 1.7Hz, 1H), 7.47-7.35 (m, 8H ), 6.20 (d, J=2.2Hz, 2H), 6.16 (d, J=2.2Hz, 1H), 5.24 (s, 1H), 5.00-4.90 (m, 2H), 4.52-4.35 (m, 4H), 4.27 (dd, J=15.7, 5.6Hz, 1H), 4.03 ( d, J=12.1Hz, 1H), 3.97 (s, 2H), 3.86 (dd, J=12.1, 3.9Hz, 1H), 3.63-3.51 (m, 8H), 3.43 (t, J=5.8Hz, 2H), 3.24 (dt, J=10.6, 6.4Hz, 4H), 2. 58 (s, 3H), 2.43 (s, 3H), 2.40 (s, 3H), 2.36-2.29 (m, 1H), 2.14 (ddd, J=13.7, 9.3, 4.7Hz, 1H), 1.61 (s, 3H), 0.95 (s, 9H). HRMS (ESI+): m / z calculated for C57H67ClN9O12S2([M+H]+): 1168.34, Found: 1168.40. Such as Figure 2 The H NMR spectrum and mass spectrum of DHBC-MZ1 are shown.

[0072] (3) Synthesis of amphiphilic polyphenol polymers

[0073]

[0074] At 35°C, 250 mg PEG 5000-NH2 and 625 mg (S)-benzyl 2-(2,5-dioxoxazolidin-4-yl) acetate were added to a 10:1 mixed solution of anhydrous dichloromethane and N,N-dimethylformamide (volume 8-10 mL), and heated under reflux for 3 days to prepare compound 6. After the reaction, it was purified three times using 15 mL of ether (ether precipitation, centrifugation at 8000 rpm to remove the supernatant, and then the precipitate was dissolved in tetrahydrofuran); then, 200 mg of compound 6 was dissolved in 2 mL of DMSO, and reacted with 298 μL of hydrazine hydrate at 35°C for 12 hours under the catalysis of 86 mg of DIPEA to achieve a large amount of grafting of amino groups. Compound 7 was collected by dialysis purification and freeze drying; finally, 50 mg of compound 7 was dissolved in 1 mL of water and reacted with a solution of 197 mg of 3,4-dihydroxybenzaldehyde dissolved in 4 mL of methanol at a pH of about 5 (adjusted by adding 20 μL of glacial acetic acid) for 12 hours to obtain the final pH-responsive PEG. 5000 -Polyphenol polymers.

[0075] Take 5 mg of the final product PEG 5000 -The polyphenol was dissolved in 600 mL of deuterated DMSO and analyzed by nuclear magnetic resonance hydrogen spectrum. The results were as follows. 1H NMR (400 MHz, DMSO-d6) δ 11.14 (s, 40H), 9.28 (s, 80H), 8.05 (d, J = 206.4 Hz, 80H), 7.60-6.07 (m, 120H), 4.68 (s, 40H), 3.50 (s, 452H), 3.23 (s, 3H), 2.93-2.53 (m, 40H), 1.23 (s, 2H).

[0076] like Figure 3 PEG displayed 5000 -H NMR spectra of polyphenols.

[0077] (4) Preparation and characterization of pH / ROS dual-responsive iron-based PROTAC nanomedicines

[0078] 5 mg of hydrophobic USINPs were first precipitated by centrifugation and then redissolved in 200 μL chloroform. The chloroform solution was then mixed with 0.25 mg DHBC-MZ1 and 1 mg PEG 5000 -50mL of DMSO solution of polyphenol and an aqueous solution containing 1 mg SDS were mixed, and then the ratio was adjusted to chloroform: H2O = 1:10 with deionized water. The mixture was ultrasonically emulsified for 20 minutes and then magnetically stirred for 2 hours. After the chloroform evaporated, the precipitate was collected by high-speed centrifugation and dispersed in deionized water. 5-10 mg DOPA-PEG was added 2000Finally, the product was purified and concentrated by ultrafiltration (30 kDa cutoff) to obtain USINAs@DHBC-MZ1. The same process can be used to obtain USINAs without prodrug.

[0079] like Figure 4 Shown is a schematic diagram of the process for preparing pH / reactive oxygen dual-responsive iron-based PROTAC nanomedicines.

[0080] Characterization: USINAs@DHBC-MZ1 was prepared with deionized water into a solution containing 30 μg / mL of Fe, and detected by a nanoparticle size analyzer; 20 mL of the reaction solution was dropped on a copper mesh, dried, and then scanned by a transmission electron microscope.

[0081] The characterization results are as follows Figure 5 As shown, the particle size of USINAs@DHBC-MZ1 is approximately 110 nm, indicating that a responsive nanoassembly with uniform particle size has been formed.

[0082] Example 2

[0083] The nanodrug was prepared into a solution containing 30 μg / mL of iron in phosphate buffered saline (PBS) and incubated at 37°C for 72 hours, followed by detection using a nanoparticle size analyzer. Similarly, USINAs@DHBC-MZ1 was prepared into a solution containing 30 μg / mL of iron in DMEM medium containing 10% fetal bovine serum, and the particle size was analyzed at different time points during incubation at 37°C for 72 hours.

[0084] like Figure 6 As shown, neither the particle size nor the PDI changed significantly, which indicates that USINAs@DHBC-MZ1 has good stability under physiological conditions.

[0085] Example 3

[0086] In vitro verification of ROS production by pH / ROS dual-responsive nanomedicine

[0087] Different concentrations of H2O2 were added to a pH 5.5 PBS solution (to prepare solutions with H2O2 concentrations of 1 mM, 5 mM, and 10 mM), and appropriate amounts of TMB stock solution and USINAs@DHBC-MZ1 were added (to prepare a solution with a final iron concentration of 40 μg / mL). Subsequently, the mixtures were incubated at 37°C for 30 minutes, and the UV-visible absorbance spectra of oxidized TMB were measured. Figure 7 As shown, the absorbance at 562 nm increased significantly, indicating that USINAs@DHBC-MZ1 was able to generate ROS.

[0088] Example 3

[0089] In vitro verification of the release and activation of pH / ROS dual-responsive nanomedicines.

[0090] A USINAs@DHBC-MZ1 PBS (pH 5.5) solution containing an iron concentration of 30 μg / mL was prepared and incubated at 37°C for 3 to 4 hours before detection using a nanoparticle size analyzer. 20 μL of the reaction solution was then dropped onto a copper grid and, after drying, scanned using a transmission electron microscope (TEM).

[0091] like Figure 8 As shown, the particle size of USINAs@DHBC-MZ1 decreased, and TEM images showed that USINAs@DHBC-MZ1 was dispersed, indicating that USINAs@DHBC-MZ1 was able to disintegrate in response to pH changes.

[0092] In the release study, 500 μL of concentrated USINAs@DHBC-MZ1 was loaded into a dialysis bag with a molecular weight cutoff of 2000 Da and then immersed in 15 mL of PBS (pH 5.5 and 7.4) containing 10% acetonitrile. The temperature was maintained at 37 ° C and stirred at 200 rpm. At the preset time points (0, 2, 4, 8, 12, 24, 48 and 72 hours), 1 mL of samples were drawn from the PBS outside the dialysis bag and the content of DHBC-MZ1 was analyzed by HPLC. At the same time, an activation experiment was performed, and different concentrations of H2O2 were added to PBS (pH 5.5) as a source of active oxygen, and the same procedure was used to quantify the released MZ1 by HPLC.

[0093] like Fig. 9 As shown, under pH 7.4, only 6-7% of DHBC-MZ1 was released within 72 hours, while under pH 5.5, about 80% of DHBC-MZ1 was released, indicating that USINAs@DHBC-MZ1 can disintegrate in response to pH changes and release ROS-responsive PROTAC prodrug.

[0094] like Fig.10 As shown in B, compared with only under acidic conditions, about 70-80% of MZ1 was released when USINAs@DHBC-MZ1 was additionally added with hydrogen peroxide. This indicates that after USINAs@DHBC-MZ1 disintegrated in response to pH changes, it was able to release ROS-responsive PROTAC prodrugs and was successfully activated into active PROTACs by ROS generated by USINAs@DHBC-MZ1. The activation mechanism is detailed in Fig.10 A.

[0095] Example 4

[0096] Cytotoxicity assay

[0097] Inoculate an appropriate amount of MDA-MB-231 and L02 cells in a 96-well plate. When the cells grow to 70-80% confluence, replace the culture medium and incubate with different concentrations of USINAs@DHBC-MZ1 and other experimental and control groups, with three replicate wells for each group. After 24 hours, remove the culture medium and wash with PBS to remove residual drugs, then add MTT solution. After 1 to 3 hours, aspirate the supernatant, add DMSO, and use a microplate reader to measure the absorbance value (OD value) of each well at 492nm. The formula for calculating cell survival rate is as follows:

[0098] Cell viability = OD sample / OD control ×100%

[0099] like Fig.11 As shown, USINAs@DHBC-MZ1 had no significant toxicity to L02 cells, but showed obvious killing effect on MDA-MB-231 cells.

[0100] Example 5

[0101] Intracellular reactive oxygen species production level

[0102] 2′,7′-dichlorodihydrofluorescein (DCFH-DA) was used to stain the cells treated with nanoparticles. MDA-MB-231 cells were selected, seeded in a 48-well plate, and cultured overnight. Subsequently, USINAs@DHBC-MZ1 and other experimental and control groups were added to the cell culture medium and incubated at 37°C for 12 hours. After the incubation, the culture medium was removed and the cells were stained with DCFH-DA. After staining, the fluorescence changes of the cells were observed using a fluorescence microscope.

[0103] like Fig.12 As shown, compared with other experimental groups and the control group, cells treated with USINAs@DHBC-MZ1 showed significantly enhanced green fluorescence. This result indicates that USINAs@DHBC-MZ1 can promote the production of ROS in tumor cells and thereby synergistically enhance the death effect of tumor cells.

[0104] Example 6

[0105] Verify the effect of pH / ROS dual-responsive nanomedicine on the expression levels of different proteins in cells

[0106] MDA-MB-231 cells were seeded into 6-well culture plates and cultured overnight. Subsequently, the cells were treated with different conditions at 37°C and incubated for 12 hours. After the treatment, the cells were lysed to extract proteins, which were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were then incubated with primary antibodies such as anti-BRD4, anti-GPX4, and anti-GAPDH and their corresponding secondary antibodies at 4°C overnight. The protein expression levels of the target protein bands were detected using chemiluminescent signals (Tanon-4600 SF).

[0107] As a key transcription factor, BRD4 plays an important role in many tumor cells, and its degradation can induce apoptosis. Glutathione peroxidase 4 (GPX4) is a widely recognized biomarker of ferroptosis, and the downregulation of GPX4 is one of the key pathways that trigger ferroptosis.

[0108] like Fig.13 As shown in A, USINAs@DHBC-MZ1 can degrade BRD4 protein in a concentration-dependent manner. Fig.13 B shows that the level of GPX4 in the USINAs@DHBC-MZ1-treated group was significantly decreased compared with other experimental groups and the control group. The above results indicate that USINAs@DHBC-MZ1 can induce cell apoptosis and ferroptosis simultaneously, providing a new strategy for anti-tumor treatment.

[0109] Example 7

[0110] In vivo distribution of fluorescently labeled nanomedicines

[0111] The prepared USINAs@DHBC-MZ1 was mixed with DPA-PEG-NH2 and Cy5-NHS at the same time, and the labeling was achieved by shaking overnight, and then the unbound fluorescent dye was removed by ultrafiltration. When the volume of the MDA-MB-231 tumor implanted in nude mice grew to about 200mm 3 At the same time, fluorescently labeled nanodrugs (dose of Cy5: 0.5 mg / kg) were injected through the tail vein, and the distribution of nanodrugs in nude mice was observed using a small animal in vivo imaging system (Carestream Kodak Multimodal Imaging System IS2000MM).

[0112] like Fig.14The results showed that nude mice injected with USINAs@DHBC-MZ1 via the tail vein showed obvious fluorescence signals in the tumor area after 6 hours, reaching the strongest level at about 12 hours, and then gradually weakened due to the EPR effect. This shows that the nanoassembly has significantly improved tumor targeting and retention capabilities, providing important conditions for subsequent anti-tumor treatment.

[0113] Example 8

[0114] Evaluation of the inhibitory effect of USINAs@DHBC-MZ1 on tumor-bearing nude mice

[0115] Female BALB / c nude mice (5 weeks old) weighing 18 grams were selected and a suspension of 8×10^6 MDA-MB-231 cells was subcutaneously injected into the right mammary fat pad to establish a nude mouse tumor model. 16 to 20 days after inoculation, when the tumor volume grew to about 100 mm 3 At 14:00, mice were randomly divided into groups and administered (5 mice per group). The experimental group was injected with pH / reactive oxygen dual-responsive iron-based PROTAC nanodrugs, while the control group was injected with an equal volume of phosphate buffered saline (PBS), MZ1 or USINAs. Over the next 14 days, the tumor volume and body weight changes of the mice were recorded and monitored.

[0116] like Fig.15 As shown, the changing trend of mouse tumor volume during treatment is demonstrated; Fig.16 The figure shows the changes in the weight of mice. Compared with other experimental groups and the control group, the pH / reactive oxygen dual-responsive iron-based PROTAC nanomedicine showed a significant tumor inhibition effect, and there was no significant change in the weight of mice during the treatment.

Claims

1. An iron-based PROTAC nanodrug with dual pH / active oxygen response characteristics, characterized in that: It is a nanodrug composed of a carrier, zero-valent iron nanoparticles, and reactive oxygen species-responsive PROTAC prodrug through a two-phase stirring method; wherein the carrier is an amphiphilic polyphenol polymer modified with a pH group, and the carrier encapsulates ultra-small zero-valent iron nanoparticles and reactive oxygen species-responsive PROTAC prodrug.

2. The iron-based PROTAC nanodrug with pH / active oxygen dual response characteristics according to claim 1, characterized in that The size of the nano drug is 100-200 nm; the particle size of the ultra-small zero-valent iron nanoparticles is 3-5 nm; the pH group-modified amphiphilic polyphenol polymer is PEG-polyphenol; the active oxygen-responsive PROTAC prodrug is DHBC-MZ1, and the structural formula is as follows:

3. The iron-based PROTAC nanodrug with pH / active oxygen dual response characteristics according to claim 2, characterized in that The structural formula of the PEG-polyphenol is: Wherein, m is selected from 100-130, and n is selected from 20-50.

4. A method for preparing the iron-based PROTAC nanodrug with pH / active oxygen dual response characteristics according to claim 1, characterized in that: The following steps are involved: (1) adding ammonium bromide and sodium oleate to a mixture of oleylamine and hexadecyltrimethyl bromide and heating the mixture for reaction, then adding carbonyl iron to react after the temperature is further increased, cooling the mixture to room temperature, and purifying the nanoparticles to obtain ultra-small zero-valent iron nanoparticles; (2) 3,5-dihydroxybenzoic acid methyl ester and DIPEA are mixed in an organic solvent, bromomethyl methyl ether is added dropwise, and the generated HCl gas is blown off to obtain compound 1; then, compound 1 is reacted with lithium aluminum hydride in tetrahydrofuran, and water and sodium hydroxide are added for precipitation to obtain a colorless oily liquid compound 2; then, compound 2 is reacted with DIPEA and 4-nitrophenyl chloroformate in an organic solvent to obtain DHBC; then, MZ1 is mixed with DIPEA, DMAP and DHBC in an organic solvent for reaction, and compound 4 is obtained after purification; finally, compound 4 is deprotected and purified to obtain the active oxygen-responsive PROTAC prodrug DHBC-MZ1; (3) Synthesis of amphiphilic polyphenol polymers: A hydrophobic NCA group was introduced onto the hydrophilic PEG-NH2 chain segment by chemical grafting technology to generate compound 6. Subsequently, an amino functional group was introduced at the end of each hydrophobic group by an amination reaction using hydrazine hydrate. Subsequently, these amino groups reacted with 3,4-dihydroxybenzaldehyde to form PEG-polyphenol containing pH-responsive acylhydrazone bonds. (4) Ultra-small zero-valent iron nanoparticles dissolved in organic solvent A are mixed with DHBC-MZ1 and PEG-polyphenol dissolved in organic solvent B, water and an emulsifier are added, and a two-phase stirring method is used to promote the self-assembly of the components to form the target nanomedicine.

5. The preparation method according to claim 4, characterized in that: In step (1), the mass ratio of ammonium bromide, sodium oleate, oleylamine and octadecene is 1:5-10; 150-250; 400-500; the mass ratio of carbonyl iron and octadecene is 1:15-20.

6. The preparation method according to claim 4, characterized in that: The purification in step (1) is to purify the nanoparticles using n-hexane and ethanol.

7. The preparation method according to claim 4, characterized in that: The organic solvent in step (2) is selected from dichloromethane.

8. The preparation method according to claim 4, characterized in that: In step (4), the organic solvent A is selected from chloroform, and the organic solvent B is selected from dimethyl sulfoxide; the mass ratio of the amphiphilic polyphenol polymer PEG-polyphenol to the ultra-small iron nanoparticles is 1:3-1:

7.

9. The preparation method according to claim 4, characterized in that: Step (4) is specifically as follows: the ultra-small size zero-valent iron nanoparticles USINPs in the oil phase are washed by centrifugation, and then redissolved in chloroform, and then the solution is mixed with a DMSO solution containing DHBC-MZ1 and PEG-polyphenol and an SDS aqueous solution, and then water is added; the mixed solution is ultrasonically emulsified and then stirred; after the chloroform evaporates, the precipitate is collected by high-speed centrifugation and dispersed in water; DOPA-PEG is then added to improve the stability of the resulting product; finally, the product is purified and concentrated to obtain a pH / active oxygen dual-responsive iron-based-PROTAC nanodrug.

10. Use of the iron-based PROTAC nanodrug with pH / active oxygen dual response characteristics according to claim 1 in the preparation of drugs for treating tumors.

Images