pH / active oxygen dual-responsive iron-based-protac nanodrugs, and preparation method and application thereof

Abstract

The application discloses a kind of pH / active oxygen dual-response characteristics iron-based-PROTAC nano-drug, is by carrier, zero-valent iron nanoparticles, active oxygen response PROTAC prodrug is formed by two-phase stirring method Nanomedicine;Among them, the carrier is the amphiphilic polyphenol polymer of pH group modification, and the active oxygen response PROTAC prodrug of super-small size zero-valent iron nanoparticles is wrapped in carrier.This nano-drug developed in the application shows good hydrophilicity and stability characteristics, can effectively use high permeability and retention effect (EPR effect), so as to realize long-time accumulation in tumor site, and in the micro-acidic environment of tumor, active oxygen response PROTAC prodrug is accurately released.In addition, iron-based nanomaterials can further activate prodrug by catalyzing hydroxyl radicals generated by hydrogen peroxide through its Fenton catalytic activity, and then induce tumor cells to undergo apoptosis.Meanwhile, the new iron-based nanomaterial also has the ability to induce iron death.

Description

Technical Field

[0001] The present invention relates to biopharmaceutical nanomedicines, and more particularly 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 strategy for tumor treatment, protein-targeting chimeras (PROTACs) uniquely utilize the ubiquitin-proteasome system (UPS) to degrade target proteins (POIs), effectively reducing the risk of drug resistance, providing new treatment options 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 address these problems, stimulus-responsive PROTACs have emerged. They can achieve activity regulation under endogenous (such as enzymes, hydrogen peroxide, etc.) or exogenous (such as light, X-rays, etc.) stimuli, ensuring that their therapeutic effects are activated only at the tumor site.

[0003] Ferroptosis, an iron-dependent programmed cell death, is characterized by an imbalance in reactive oxygen species (ROS) levels within tumor cells, leading to the accumulation of lipid peroxides (LPO) and the inhibition of glutathione peroxidase 4 (GPX4) activity. Numerous studies have demonstrated the excellent catalytic properties of iron-based nanomaterials, effectively promoting the Fenton reaction and accelerating the decomposition of hydrogen peroxide (H2O2), thereby generating 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 present invention aims to provide a novel method for preparing pH / ROS dual-responsive iron-based PROTAC nanodrugs. The core of this method is to utilize amphiphilic polyphenol polymers modified with pH groups as carriers to encapsulate ultrasmall zero-valent iron nanoparticles and ROS-responsive PROTAC prodrugs. Using a two-phase mixing method, the iron-based PROTAC nanodrug with pH / ROS dual-responsive properties is effectively assembled. This drug exhibits excellent biosafety and can effectively target and accumulate in tumors, thereby synergistically enhancing tumor therapeutic efficacy.

[0006] Technical Solution: The pH / ROS dual-responsive iron-based PROTAC nanodrug described in the present invention is a nanodrug composed of a carrier, zero-valent iron nanoparticles, and an ROS-responsive PROTAC prodrug through a two-phase mixing 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 the ROS-responsive PROTAC prodrug.

[0007] The iron-based PROTAC nanodrug with pH / active oxygen dual-responsiveness has a size of 100-200 nm; the ultra-small zero-valent iron nanoparticles have a particle size of 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 its structural formula is shown below:

[0009]

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

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

[0012]

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

[0014] A method for preparing an iron-based PROTAC nanodrug with pH / active oxygen dual-responsiveness 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 after further heating, cooling the mixture to room temperature, and purifying the nanoparticles to obtain ultrasmall zero-valent iron nanoparticles (USINPs);

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

[0017]

[0018] (3) Synthesis of amphiphilic polyphenol polymers (PEG-polyphenols): Hydrophobic NCA groups were introduced onto the hydrophilic PEG-NH2 segments via chemical grafting to generate compound 6. Subsequently, amino functional groups were introduced at the end of each hydrophobic group via an amination reaction using hydrazine hydrate. These amino groups then reacted with 3,4-dihydroxybenzaldehyde to form PEG-polyphenols containing pH-responsive acylhydrazone bonds.

[0019]

[0020] (4) Ultrasmall zero-valent iron nanoparticles dissolved in organic solvent A are mixed with DHBC-MZ1 and PEG-polyphenol dissolved in organic solvent B, water and emulsifier are added, and a two-phase stirring method is used to promote the self-assembly of each component 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 ultrasmall iron nanoparticles is 1:3-1:7.

[0026] The preparation method, step (4) is specifically as follows: the ultrasmall 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 is evaporated, 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 / reactive oxygen dual-responsiveness in the preparation of a drug for treating tumors. Preferably, the tumor includes breast cancer.

[0028] This invention discloses a pH / ROS dual-responsive iron-based PROTAC nanodrug and its preparation method, with applications in tumor treatment. The nanodrug system is constructed on an amphiphilic polyphenol polymer matrix modified with pH-sensitive groups. It encapsulates ultrasmall zero-valent iron nanoparticles and an ROS-sensitive PROTAC prodrug, forming 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] Furthermore, 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. Carbonyl iron is then added after further heating. After the mixture is cooled to room temperature, the nanoparticles are purified using n-hexane and ethanol to obtain ultrasmall 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 for precipitation 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 carried out through steps such as 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 the components 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 and octadecene is 1:15-20.

[0036] Step (1) specifically comprises the following steps: 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. The mixture is then heated to 260°C and carbon-based iron is injected under an inert atmosphere. After 30 minutes of reaction, the solution turns black, indicating the formation of nanoparticles. After cooling to room temperature, ethanol is added for precipitation, and then purified at least three times with n-hexane and ethanol 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, methyl 3,5-dihydroxybenzoate 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 returned 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 the mixture is returned to room temperature and stirred for 2 hours. A small amount of 15% NaOH and water is 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, and 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, 4 M aqueous HCl was added at 0°C, and the mixture was returned to room temperature and stirred for 2 hours. The methanol was spin-dried and purified to obtain the white solid product DHBC-MZ1.

[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; and the molar mass ratio of compound 7 and 3,4-dihydroxybenzaldehyde is 1:500.

[0040] Step (3) is as follows: 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 ultrasmall 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 of chloroform. The solution is then mixed with a mixture containing DHBC-MZ1 and PEG. 5000 50 μL of DMSO solution of polyphenols and 100 μL of 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 Finally, the product was purified and concentrated by ultrafiltration (molecular weight cutoff value of 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 / active 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 it responds under the specific pH conditions of the tumor microenvironment, it will release the prodrug it carries. 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 / active 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 translation applications.

[0044] The pH / ROS dual-responsive iron-based PROTAC nanodrug of this invention responds to the specific pH environment of tumor tissue, releasing its loaded ROS-responsive PROTAC prodrug. Simultaneously, the iron-based nanomaterial, through its intrinsic Fenton catalytic activity, effectively catalyzes hydrogen peroxide in the tumor microenvironment to generate highly toxic hydroxyl radicals. This process not only activates the PROTAC prodrug but also imparts the ability to induce ferroptosis in tumor cells, thereby achieving a synergistic anti-tumor effect through multiple mechanisms.

[0045] Beneficial effects: Compared with the existing technology, the present invention has the following significant advantages: The iron-based-PROTAC nanodrug with a pH / reactive oxygen dual response developed by the present invention, which is assembled based on hydrophilic and hydrophobic forces, exhibits good hydrophilicity and stability properties. The primary advantage is that the nanoassembly has a nanoscale size and can effectively utilize high permeability and retention effects (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 ultrasmall zero-valent iron nanoparticles USINPs;

[0047] Figure 2 H NMR spectrum (A) and mass spectrum (B) of the reactive oxygen species-responsive PROTAC prodrug DHBC-MZ1;

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

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

[0050] Figure 5 Particle size distribution (A) and TME (B) of pH / ROS 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 supplemented with 10% FBS (37°C) for 72 h (A) and the particle size distribution after incubation in PBS (pH 7.4, 37°C) for 72 h (B).

[0052] Figure 7 The TMB method was used to detect the production of reactive oxygen species by the pH / ROS 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 (A) and TME (B) of the pH / ROS dual-responsive iron-based PROTAC nanodrug USINAs@DHBC-MZ1 that disintegrated in response to pH 5.5.

[0054] Figure 9 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] Figure 10 Schematic diagram of the activation of the active oxygen-responsive PROTAC prodrug and the release curve of the active PROTAC (MZ1) of the pH / ROS dual-responsive iron-based nanoparticle USINAs@DHBC-MZ1 at pH 5.5 and different concentrations of hydrogen peroxide;

[0056] Figure 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] Figure 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] Figure 13 The effects of pH / reactive oxygen dual-responsive iron-based-PROTAC nanodrugs 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] Figure 14 The distribution of the fluorescently labeled pH / ROS dual-responsive iron-based PROTAC nanodrug USINAs@DHBC-MZ1 after intravenous injection into MDA-MB-231 tumor-bearing nude mice.

[0060] Figure 15 This is a graph showing the changes in 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 to tumor-bearing nude mice;

[0061] Figure 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 / ROS dual-responsive iron-based PROTAC nanomedicines:

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

[0065] 0.04g of ammonium bromide and 0.3g of sodium oleate were mixed in a solution of 12mL of octadecene and 5mL 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 inert gas, the temperature was raised to 220°C and kept warm for 30 minutes. After the temperature dropped to 220°C, 500mL 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 40mL of anhydrous ethanol and centrifuging at 8000 rpm for 10 minutes, removing the supernatant. The precipitate was dissolved in 3mL of n-hexane, precipitated again with 40mL of anhydrous ethanol, and centrifuged at 8000 rpm for 10 minutes to successfully prepare ultra-small zero-valent iron nanoparticles (USINPs).

[0066] 0.5 g of 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 TEM images 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. The generated HCl gas was blown away with an ear bulb. The mixture was stirred at room temperature for 4 hours. 0.8 g of compound 1 was purified by extraction, washing, drying, and concentration. 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. Unpurified compound 2 (3.1 mmol) was then dissolved in anhydrous dichloromethane, 1.2 g of DIPEA (9.33.1 mmol) was added, and 1.87 g (9.33.1 mmol) was added dropwise. The mixture was reacted at 0°C and stirred at room temperature for 2 hours. After washing, drying, and concentration, 0.96 g of DHBC was purified by silica gel column chromatography. Finally, 50 mg of MZ1 was dissolved in anhydrous dichloromethane, 20 mg of DIPEA and 6.1 mg of DMAP were added. The mixture was precooled to 0°C, 40 mg of DHBC was added dropwise, and the mixture was stirred at room temperature for 18 hours. After washing, drying, and concentration, 35.8 mg of product 4 was purified. 30 mg of compound 4 was dissolved in methanol, an equal volume of 4 M aqueous HCl was added, and the mixture was stirred at 0°C for 2 hours. The methanol was then evaporated to dryness under reduced pressure. After three extractions and washings, the mixture was purified by silica gel column chromatography to obtain DHBC-MZ1 as a white solid.

[0071] 5 mg of the final product DHBC-MZ1 was dissolved in 600 mL of deuterated DMSO and analyzed by H NMR. The results were 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 of PEG 5000-NH2 and 625 mg of (S)-benzyl 2-(2,5-dioxooxazolidin-4-yl) acetate were added to a 10:1 mixture of anhydrous dichloromethane and N,N-dimethylformamide (volume 8-10 mL) and heated under reflux for 3 days to produce compound 6. After the reaction, the product was purified three times using 15 mL of diethyl ether (diethyl ether precipitation, centrifugation at 8000 rpm to remove the supernatant, and then the precipitate was dissolved in tetrahydrofuran). Next, 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 amino group grafting. Compound 7 was collected by dialysis purification and lyophilization; finally, 50 mg of compound 7 was dissolved in 1 mL of water and reacted with 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 5mg of the final product PEG 5000 The polyphenol was dissolved in 600 mL of deuterated DMSO and analyzed by proton nuclear magnetic resonance spectroscopy. H 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 of chloroform. The chloroform solution was then mixed with 0.25 mg of DHBC-MZ1 and 1 mg of PEG 5000 Mix 50 mL of a dimethyl sulfoxide solution of DOPA-polyphenols with an aqueous solution containing 1 mg of SDS, then adjust the ratio of chloroform to H₂O to 1:10 with deionized water. Ultrasonic emulsification of the mixture was performed for 20 minutes, followed by magnetic stirring for 2 hours. After evaporation of the chloroform, the precipitate was collected by high-speed centrifugation and dispersed in deionized water. 5–10 mg of DOPA-PEG was added. 2000To stabilize the product. Finally, 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 prodrugs.

[0079] As Figure 4 The process diagram for preparing the pH / ROS dual-responsive iron-based-PROTAC nanodrugs is shown.

[0080] Characterization: USINAs@DHBC-MZ1 was configured into a solution with a Fe concentration of 30 μg / mL using deionized water, and was detected using a nanoparticle size analyzer; 20 mL of the reaction solution was dropped on a copper mesh, and after air-drying, transmission electron microscopy scanning was performed.

[0081] The characterization results are shown in Figure 5 The particle size of USINAs@DHBC-MZ1 was about 110 nm, indicating that a responsive nanassembler with uniform particle size was formed.

[0082] Example 2

[0083] The nanodrug was configured into a solution with an iron concentration of 30 μg / mL using phosphate buffer solution (PBS), and was incubated at 37°C for 72 hours, after which it was detected using a nanoparticle size analyzer. Similarly, USINAs@DHBC-MZ1 was configured into a solution with an iron concentration of 30 μg / mL using DMEM medium containing 10% fetal bovine serum, and was incubated at 37°C for 72 hours, and the particle size was analyzed at different time points.

[0084] As Figure 6 shown, the particle size and PDI did not change significantly, indicating that USINAs@DHBC-MZ1 has good stability under physiological conditions.

[0085] Example 3

[0086] In vitro verification of ROS production of the pH / ROS dual-responsive nanodrug

[0087] Different concentrations of H2O2 were added to a PBS solution at pH 5.5 (solutions with H2O2 concentrations of 1 mM, 5 mM, and 10 mM were prepared), and an appropriate amount of TMB stock solution and USINAs@DHBC-MZ1 (a solution with a final iron concentration of 40 μg / mL was prepared) were added, respectively. Subsequently, these mixtures were incubated at 37°C for 30 minutes, and the ultraviolet-visible absorbance spectrum of the oxidized TMB was determined. As Figure 7 shown, the absorbance at 562 nm increased significantly, indicating that USINAs@DHBC-MZ1 can produce ROS.

[0088] Example 3

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

[0090] A USINAs@DHBC-MZ1 PBS (pH 5.5) solution with an iron concentration of 30 pg / mL was prepared and detected using a nanoparticle size analyzer after incubation at 37 °C for 3-4 h; then 20 pL of the reaction solution was dropped on a copper mesh, dried, and then scanned by transmission electron microscopy (TEM).

[0091] As shown in FIG. 4A, the particle size of USINAs@DHBC-MZ1 decreased, and the TEM image showed that USINAs@DHBC-MZ1 dispersed, indicating that USINAs@DHBC-MZ1 could disintegrate in response to pH changes. Figure 8

[0092] In the release study, 500 pL 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 h), 1 mL of sample was drawn from the PBS outside the dialysis bag, and the content of DHBC-MZ1 was analyzed by HPLC. At the same time, the activation experiment was carried out, different concentrations of H2O2 were added to PBS (pH 5.5) as active oxygen source, and the same procedure was used to quantitatively analyze the released MZ1 by HPLC.

[0093] As shown in FIG. 4B, under the condition of pH 7.4, only 6-7% of DHBC-MZ1 was released within 72 h, while under the condition of pH 5.5, about 80% of DHBC-MZ1 was released, indicating that USINAs@DHBC-MZ1 could disintegrate in response to pH changes and release ROS-responsive PROTAC prodrugs. Figure 9 As shown in FIG. 4C, compared with only under acidic conditions, about 70-80% of MZ1 was released when hydrogen peroxide was additionally added to USINAs@DHBC-MZ1. This indicates that after USINAs@DHBC-MZ1 disintegrates in response to pH changes, it can release ROS-responsive PROTAC prodrugs, which are successfully activated to active PROTAC by the ROS produced by USINAs@DHBC-MZ1. The activation mechanism is described in detail in

[0094] A. Figure 10 Figure 10 Example 4

[0095] Cytotoxicity experiment

[0096] Cytotoxicity experiment ​​

[0097] MDA-MB-231 and L02 cells were seeded in appropriate amounts in a 96-well plate. When the cells grew to 70-80% confluence, the culture medium was replaced and incubated with different concentrations of USINAs@DHBC-MZ1 and other experimental and control groups, with three replicate wells set up for each group. After 24 hours, the culture medium was removed and washed with PBS to remove residual drugs, followed by the addition of MTT solution. After 1 to 3 hours, the supernatant was aspirated, DMSO was added, and the absorbance (OD value) of each well at 492 nm was measured using a microplate reader. The cell viability was calculated as follows:

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

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

[0100] Example 5

[0101] Intracellular reactive oxygen species production levels

[0102] Nanoparticle-treated cells were stained with 2′,7′-dichlorodihydrofluorescein (DCFH-DA). MDA-MB-231 cells were seeded in 48-well plates 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 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 Figure 12 As shown in the figure, 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 plates and cultured overnight. Subsequently, the cells were treated with various conditions and incubated for 12 hours at 37°C. After treatment, the cells were lysed and proteins were extracted. These proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was then incubated with primary antibodies (anti-BRD4, anti-GPX4, and anti-GAPDH) and their corresponding secondary antibodies overnight at 4°C. Protein expression levels of the target protein bands were detected using chemiluminescence (Tanon-4600 SF).

[0107] BRD4, as a key transcription factor, 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 downregulation of GPX4 is one of the key pathways that trigger ferroptosis.

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

[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, and labeled by shaking overnight. Unbound fluorescent dyes were then removed by ultrafiltration. 3 At the same time, fluorescently labeled nanomedicine (dose of Cy5: 0.5 mg / kg) was injected through the tail vein, and the distribution of nanomedicine in nude mice was observed using a small animal in vivo imaging system (Carestream Kodak Multimodal Imaging System IS2000MM).

[0112] like Figure 14As shown in the results, the nude mice injected with USINAs@DHBC-MZ1 via tail vein showed obvious fluorescence signals in the tumor area after 6 hours, reached the strongest at about 12 hours, and then gradually weakened due to the EPR effect. This indicates that the nanoassemblies have significantly improved tumor targeting and retention capacity, 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] Select 18 grams of female BALB / c nude mice (5 weeks old), construct a nude mouse tumor model by subcutaneously injecting 100 μL of a suspension containing 8 x 10^6 MDA-MB-231 cells into the right mammary fat pad of the mice. After 16 to 20 days of inoculation, when the tumor volume grows to about 100 mm 3 When the tumor volume grows to about 100 mm

[0116] As shown in the results, the nude mice injected with USINAs@DHBC-MZ1 via tail vein showed obvious fluorescence signals in the tumor area after 6 hours, reached the strongest at about 12 hours, and then gradually weakened due to the EPR effect. This indicates that the nanoassemblies have significantly improved tumor targeting and retention capacity, providing important conditions for subsequent anti-tumor treatment. Figure 15 As shown in the results, the nude mice injected with USINAs@DHBC-MZ1 via tail vein showed obvious fluorescence signals in the tumor area after 6 hours, reached the strongest at about 12 hours, and then gradually weakened due to the EPR effect. This indicates that the nanoassemblies have significantly improved tumor targeting and retention capacity, providing important conditions for subsequent anti-tumor treatment. Figure 16 As shown in the results, the nude mice injected with USINAs@DHBC-MZ1 via tail vein showed obvious fluorescence signals in the tumor area after 6 hours, reached the strongest at about 12 hours, and then gradually weakened due to the EPR effect. This indicates that the nanoassemblies have significantly improved tumor targeting and retention capacity, providing important conditions for subsequent anti-tumor treatment. Compared with other experimental groups and control groups, the pH / active oxygen dual-responsive iron-based-PROTAC nanomedicine showed obvious tumor inhibition effect, and the body weight of the mice did not change significantly during the treatment.

Claims

1. A pH / reactive oxygen dual-responsive iron-based PROTAC nanodrug, characterized in that: The nanomedicine is composed of a carrier, zero-valent iron nanoparticles, and a reactive oxygen species-responsive PROTAC prodrug through a two-phase mixing method; wherein the carrier is a pH-group-modified amphiphilic polyphenol polymer, which encapsulates ultra-small zero-valent iron nanoparticles and the reactive oxygen species-responsive PROTAC prodrug; the pH-group-modified amphiphilic polyphenol polymer is PEG-polyphenol; the reactive oxygen species-responsive PROTAC prodrug is DHBC-MZ1, and its structural formula is as follows: ； The structural formula of the PEG-polyphenol is: ； wherein m is selected from 100-130, and n is selected from 20-50.

2. The iron-based PROTAC nanomedicine 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.

3. 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 octadecene and heating the mixture for reaction, then adding carbonyl iron to the mixture after further heating, cooling the mixture to room temperature, and purifying the nanoparticles to obtain ultra-small zero-valent iron nanoparticles; (2) Methyl 3,5-dihydroxybenzoate and DIPEA were mixed in an organic solvent, bromomethyl methyl ether was added dropwise, and the generated HBr gas was blown off to obtain compound 1; then, compound 1 was reacted with lithium aluminum hydride in tetrahydrofuran, and water and sodium hydroxide were added for precipitation to obtain a colorless oily liquid compound 2; then, compound 2 was reacted with DIPEA and 4-nitrophenyl chloroformate in an organic solvent to obtain DHBC; subsequently, MZ1 was mixed with DIPEA, DMAP and DHBC in an organic solvent for reaction, and compound 4 was obtained after purification; finally, compound 4 was deprotected and purified to obtain the active oxygen-responsive PROTAC prodrug DHBC-MZ1; ； (3) Synthesis of amphiphilic polyphenol polymers: Hydrophobic NCA groups were introduced onto the hydrophilic PEG-NH2 chain segment by chemical grafting technology to generate compound 6. Subsequently, amino functional groups were introduced at the end of each hydrophobic group by amination reaction using hydrazine hydrate. These amino groups then reacted with 3,4-dihydroxybenzaldehyde to form PEG-polyphenol containing pH-responsive acylhydrazone bonds. ； (4) Ultrasmall zero-valent iron nanoparticles dissolved in organic solvent A are mixed with DHBC-MZ1 and PEG-polyphenol dissolved in organic solvent B, water and emulsifier are added, and a two-phase stirring method is used to promote the self-assembly of each component to form the target nanomedicine.

4. The preparation method according to claim 3, characterized in that The purification described in step (1) is to purify the nanoparticles using n-hexane and ethanol.

5. The preparation method according to claim 3, characterized in that The organic solvent in step (2) is dichloromethane.

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

7.

7. The preparation method according to claim 3, characterized in that Step (4) is specifically as follows: the ultrasmall zero-valent iron nanoparticles USINPs in the oil phase are washed by centrifugation and then redissolved in chloroform. The solution is then mixed with a DMSO solution containing DHBC-MZ1 and PEG-polyphenol and an SDS aqueous solution, and 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.

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

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