CN113456828B

CN113456828B - Redox-responsive sequential drug delivery system and preparation method and application thereof

Info

Publication number: CN113456828B
Application number: CN202110739502.6A
Authority: CN
Inventors: 袁友永; 刘晔; 姜茂麟; 罗诗维; 杨蕊梦; 江新青
Original assignee: Guangzhou First Peoples Hospital
Current assignee: Guangzhou First Peoples Hospital
Priority date: 2021-06-30
Filing date: 2021-06-30
Publication date: 2022-08-16
Anticipated expiration: 2041-06-30
Also published as: CN113456828A

Abstract

The invention discloses a redox-responsive sequential drug delivery system and a preparation method and application thereof. The redox-responsive sequential drug release system is a dual-response sequential drug release system synthesized based on active oxygen-sensitive thiol ketal bonds and glutathione-sensitive disulfide bonds and used for sequentially delivering CyNH ₂ And chemotherapeutic drugs, to enhance the therapeutic effect of cancer. The redox-responsive sequential drug release system integrates the characteristics of response to a tumor microenvironment, sequential drug release, cooperative treatment, exogenous ROS production and the like, and well overcomes the limitations of the prior art.

Description

Redox-responsive sequential drug delivery system and preparation method and application thereof

Technical Field

The invention relates to the technical field of high molecular biomaterials, in particular to a redox-responsive sequential drug delivery system and a preparation method and application thereof.

Background

At present, malignancies remain a major health burden worldwide and effective therapeutic strategies are urgently needed. The tumor microenvironment is a complex system distinct from the normal tissue environment and plays a crucial role in tumorigenesis. Hypoxia, acidosis, high interstitial fluid pressure, elevated glutathione levels and active oxygen, specific expression of certain proteases, and immune/inflammatory responses constitute the biological characteristics of tumors. The multiple stromal cells and the secreted factors thereof are used as soil and fertilizer of tumor cells, and the tumor interacts with the tumor microenvironment, so that favorable conditions are provided for the proliferation, invasion and metastasis of the tumor. However, the specificity of the tumor microenvironment also brings opportunities for the treatment of tumors, and the tumors can be used as targets for drug delivery design to design various responsive nanoreactors so as to enhance the drug uptake of tumor cells and the deposition, penetration and release of drugs at the tumor sites.

The prodrug of high molecular polymer has been widely studied due to its good biocompatibility, biodegradability and strong designability. Researchers have designed various responsive polymeric prodrug systems, such as acid-sensitive responsive polymers. The polymer can reverse the charge in the circulation process, separate the PEG shell of the polymer, activate the ligand, and change the size once the polymer reaches the tumor site, thereby realizing programmed tumor targeting and drug delivery. In addition to acid-responsive drug carriers, some responsive polymeric prodrugs that are capable of responding to high redox environments in tumor tissues (e.g., compounds such as glutathione and active oxygen) have also been widely developed.

However, due to the high complexity and heterogeneity of tumor tissues, a single responsive drug delivery system may have inefficient drug release due to insufficient exogenous stimulation, and complete tumor clearance is difficult due to the chemotherapeutic effect of a single drug. One solution is to select a load of two drugs, with the hope that the synergistic effect between the two drugs will increase the inhibition of cancer. Dosing regimens such as by chronologically applying single drug loaded nanoparticles have been shown to be synergistic in anticancer effects by sensitizing or remodeling tumor cells to the tumor vasculature and microenvironment to enhance tumor accumulation of a second drug. However, achieving timed-programmed drug delivery of synergistic chemotherapy from a single carrier remains a significant challenge. Co-administered combination chemotherapy may elicit antagonism due to mismatch of dose combinations. In addition, the simultaneous administration of two or more drugs may cause overlapping side effects.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a redox-responsive sequential drug delivery system.

Another object of the present invention is to provide a method for preparing the redox-responsive, sequential drug delivery system.

It is a further object of the present invention to provide the use of the redox-responsive, sequential drug delivery system described above.

The purpose of the invention is realized by the following technical scheme: a preparation method of a redox-responsive sequential drug delivery system comprises the following steps:

(1) synthetic polymer mPEG-CPDB

Dissolving polyethylene glycol monomethyl ether (mPEG), 4-cyano-4- (thiobenzylthio) pentanoic acid (CPDB), 4- (dimethylamino) pyridine (DMAP) and Dicyclohexylcarbodiimide (DCC) in a solvent, and reacting to obtain mPEG-CPD;

(2) synthesis of monomer M _TK

Mixing mercaptoacetic acid and acetone, reacting to obtain TK-COOH and sodium borohydride, dissolving in Tetrahydrofuran (THF), adding iodine, reacting for the second time, quenching, purifying, dissolving TK-OH and triethylamine in Dichloromethane (DCM), adding methacryloyl chloride, reacting again, purifying, and drying to obtain M _TK ；

(3) Synthesis of monomer M _SS

Mixing methacryloyl chloride with 2,2' -dithiodiethanol and triethylamine which are dissolved in dichloromethane, reacting, purifying and drying to obtain M _SS ；

(4) Synthesis of Polymer mPEG-PTK with active oxygen responsive bond

The mPEG-CPDB in the step (1) and the M in the step (2) are added _TK And Azobisisobutyronitrile (AIBN) are dissolved in dioxane for reaction and separation to obtain mPEG-PTK;

(5) synthesis of polymer mPEG-PSS with glutathione response bond

The mPEG-CPDB in the step (1) and the M in the step (3) are added _SS Dissolving AIBN in 1, 4-dioxane, reacting, and separating to obtain mPEG-PTK;

(6) synthesis of drug-loaded polymer systems with ROS-responsive linkages

Dissolving mPEG-PTK and P-nitrophenylchloroformic acid (NPC) in DCM in the step (4), reacting, separating to obtain a crude product TEA, dissolving the crude product TEA and a chemotherapeutic drug in N, N-Dimethylformamide (DMF), reacting again, and purifying to obtain mPEG-P _Durg ；

(7) Synthesis of drug-loaded polymer mPEG-PCy with glutathione response bond

Bis (trichloromethyl) carbonate and CyNH ₂ Mixing N, N-Diisopropylethylamine (DIPEA) with acetonitrile, reacting, removing the solvent, adding the mPEG-PSS dissolved in DCM in the step (5), reacting again, and purifying to obtain mPEG-PCy;

(8) PPDCCy (p-phenylene diamine tetraacetic acid) synthesized redox-responsive sequential drug delivery system

Subjecting the mPEG-P in the step (6) _Durg And (5) dissolving the mPEG-PCy in dimethyl sulfoxide (DMSO) in the step (7), mixing with water, reacting, and dialyzing to obtain PPDCY.

Preferably, the mPEG, CPDB, DMAP and DCC are used in step (1) in a molar ratio of 1: 1-5: 0.1-1: 1-5 proportion calculation; more preferably, the molar ratio of 1: 4: 0.4: 4, calculating the mixture ratio.

Preferably, the solvent in step (1) is at least one of dichloromethane, chloroform and tetrahydrofuran.

Preferably, the amount of the solvent used in step (1) is calculated according to the proportion of 10-35mL of the solvent in 1g of mPEG.

Preferably, the DCC is added in step (1) by dissolving it in a solvent and adding it dropwise to the other reactants.

Preferably, the reaction in step (1) is a stirring reaction for 20-30 h.

Preferably, the mPEG-CPD in step (1) is filtered, precipitated with cold ether and dried in vacuo before proceeding to the next reaction.

Preferably, the molar ratio of the thioglycolic acid to the acetone in the step (2) is 1: 1-2;

preferably, the reaction time in step (2) is 4-8 h.

Preferably, the reaction in step (2) is carried out in N ₂ Stirring was carried out under an atmosphere.

Preferably, the TK-COOH and sodium borohydride in the step (2) are cooled, filtered and washed before being dissolved in tetrahydrofuran.

Preferably, the washing is with hexane and water, respectively.

Preferably, the TK-COOH and sodium borohydride in the step (2) are mixed according to a mass ratio of 1: 1-5 proportion; more preferably, the mass ratio of 1: 1 proportion.

Preferably, the tetrahydrofuran and TK-COOH in the step (2) are calculated according to the ratio of 1g TK-COOH to 5-30mL tetrahydrofuran.

Preferably, in the step (2), the iodine and the sodium borohydride are mixed according to a mass ratio of 1-4: 1, proportioning; more preferably, the mass ratio of 4: 1 proportion.

Preferably, the second reaction in the step (2) is a reflux reaction for 24-48 h.

Preferably, the quenching reaction in step (2) is a quenching reaction by adding methanol under ice bath.

Preferably, the step of purifying in step (2): the re-reacted product was freed of solvent in vacuo, the mixture was dissolved in sodium hydroxide solution, extracted with ethyl acetate, dried, concentrated in vacuo, purified on a silica gel column and dried in vacuo overnight.

Preferably, the silica gel column purification is performed in a volume ratio of 1: 1-3 DCM/EtOAc as eluent.

Preferably, the molar ratio of TK-OH and triethylamine in the step (2) is 1-3: 1; more preferably 2: 1.

preferably, the molar ratio of the methacryloyl chloride to the TK-OH in the step (2) is 1: 1-2; more preferably, the molar ratio is 1: 2.

preferably, the dichloromethane and TK-OH in step (2) are calculated according to the ratio of 1g TK-OH to 20-50mL dichloromethane.

Preferably, the re-reaction in step (2) is a stirring reaction for 7-15 h.

Preferably, the purification in step (2) is a silica gel column purification.

Preferably, the silica gel column purification is performed in a volume ratio of 1: 1 DCM/ethyl acetate as eluent.

Preferably, the molar ratio of the 2,2' -dithiodiethanol and triethylamine in the step (3) is 1-5: 1; more preferably 2: 1.

preferably, the molar ratio of methacryloyl chloride to 2,2' -dithiodiethanol in step (3) is 1: 1-2; more preferably, the molar ratio is 1: 2.

preferably, the dichloromethane and triethylamine in step (3) are calculated according to the ratio of 1g triethylamine to 40-80mL dichloromethane.

Preferably, the reaction in step (3) is a stirred reaction for 7-15 h.

Preferably, the purification in step (3) is performed using a silica gel column.

Preferably, the mPEG-CPDB and M in the step (4) _TK The mass ratio of (1): 1-3.

Preferably, the mass ratio of mPEG-CPDB to AIBN in the step (4) is 1: 0.01-0.02.

Preferably, the ratio of dioxane to mPEG-CPDB in step (4) is calculated according to the proportion of 1g of mPEG-CPDB being 10-50 mL.

Preferably, oxygen is removed before the reaction in step (4) is carried out, and repeated freezing and thawing are performed.

Preferably, the reaction in step (4) is a stirring reaction at 65-85 ℃ for 12-24 h.

Preferably, the separation in step (4) is performed by precipitation with hexane, collecting the precipitate by filtration, and vacuum drying.

Preferably, the mPEG-CPDB and M in the step (5) _SS The mass ratio of (1): 1-3.

Preferably, the mass ratio of mPEG-CPDB to AIBN in the step (5) is 1: 0.01-0.02.

Preferably, the ratio of dioxane to mPEG-CPDB in step (5) is calculated according to the proportion of 1g of mPEG-CPDB being 10-50 mL.

Preferably, the reaction in step (5) is a stirring reaction at 65-85 ℃ for 12-24 h.

Preferably, the separation in step (5) is performed by precipitation with cold hexane, collecting the precipitate by filtration and drying in vacuum.

Preferably, the chemotherapeutic agent in step (6) is Doxorubicin (DOX).

Preferably, the mass ratio of mPEG-PTK to NPC in the step (6) is 1: 0.5-2.

Preferably, the molar ratio of TEA to chemotherapeutic drug in step (6) is 1-3: 1.

preferably, the DCM in step (6) is mixed with p-nitrophenyl chloroformate in a ratio of 1g p-nitrophenyl chloroformate to 20-50mL DCM.

Preferably, the DMF and the mPEG-PTK in the step (6) are calculated according to the ratio of 1g of mPEG-PTK to 20-50mL of DMF.

Preferably, the reaction in step (6) is a stirring reaction for 12-48 h.

Preferably, the re-reaction in step (6) is stirred for 24-48 h.

Preferably, the separation in step (6) is by precipitation with cold ether, collection of the precipitate by filtration and drying in vacuo.

Preferably, the purification in step (6) is dialysis with DMF followed by pure water, followed by lyophilization.

Preferably, the dialysis bag cut-off (MWCO) for dialysis is 3500 Da.

Preferably, said CyNH is step (7) ₂ In an amount of 1g CyNH ₂ The mixture ratio is 0.25-1.5mL DIPEA.

Preferably, said bis (trichloromethyl) carbonate and CyNH are used in step (7) ₂ The mass ratio of (A) to (B) is 1-2: 1.

preferably, the acetonitrile in step (7) is reacted with CyNH ₂ According to 1g of CyNH ₂ The mixture ratio is calculated by 20-100mL acetonitrile.

Preferably, the reaction in step (7) is a reflux reaction for 4 to 8 hours.

Preferably, the re-reaction in step (7) is a stirring reaction for 24-48 h.

Preferably, the purification in step (7) is dialysis with DMF followed by pure water, followed by lyophilization.

Preferably, the dialysis bag cut-off (MWCO) of the dialysis is 3500 Da.

Preferably, the mPEG-P in step (8) _Durg And the mass ratio of mPEG-PCy is 1: 0.8-1.2.

The mPEG-P in the step (8) _Durg And dimethyl sulfoxide in an amount of 1g mPEG-P _Durg The mixture ratio is calculated by 100mL of dimethyl sulfoxide.

Preferably, the mPEG-P in step (8) _Durg And water as 1g mPEG-P _Durg The ratio is 1900-2100mL water.

Preferably, the mixing with water in step (8) is dropwise adding into water under stirring.

Preferably, the reaction in step (8) is a stirred reaction for 1 to 48 hours.

Preferably, the dialysis in step (8) is light-shielding dialysis, and the molecular weight cut-off of the dialysis bag is 14000.

A redox-responsive sequential drug delivery system, which is prepared by the preparation method.

The redox-responsive sequential drug delivery system is applied to the preparation of drugs for treating tumors.

CyNH ₂ Is prepared in the literature "Liu, s.y.; xiong, h.; li, r.r.; yang, w.c.; yang, G.F.Activity-Based near-isolated fluorescent Probe for enhancing viral and in Vivo Profiling of Neutrophil elastomer, term.2019, 91(6), 3877-.

Compared with the prior art, the invention has the following beneficial effects:

the invention relates to a redox-responsive sequential drug delivery system based on active oxygen-sensitive thiol ketal bond and glutathione-sensitive disulfideA dual-response sequential release system synthesized by sulfur bonds, which utilizes glutathione-sensitive CyNH ₂ Amplifying ROS signaling for sequential delivery of thiol ketal bond responsive chemotherapeutic drugs (e.g., DOX), synergistically enhanced to enhance cancer treatment efficacy. Under the higher redox environment in tumor cells, the disulfide bond of the redox-responsive sequential drug release system can be rapidly disintegrated firstly, so that fluorescent molecules in the inner core of the granule can be rapidly released, cell mitochondria can be targeted and initially damaged, intracellular oxidative stress is caused, and a large amount of active oxygen can be generated. At this point, the thiol ketal bond, which is sensitive to active oxygen, will break, releasing the chemotherapeutic agent. Because mitochondria are damaged in advance, the toxicity of the chemotherapy drugs to cells is further increased, and finally, the comprehensive inhibition to tumors is realized under the synergistic effect of the two drugs. The technology of the invention integrates the characteristics of response to a Tumor Microenvironment (TME), sequential drug release, cooperative treatment, exogenous ROS production and the like, and the dual redox-responsive sequential drug release system well overcomes the limitations of the prior art.

Drawings

FIG. 1 shows mPEG ₁₁₃ -synthetic route to CPDB.

FIG. 2 is M _TK The synthetic route of (1).

FIG. 3 is M _SS The synthetic route of (1).

FIG. 4 shows mPEG ₁₁₃ Synthetic routes to PTK.

FIG. 5 shows mPEG ₁₁₃ -synthetic route of PSS.

FIG. 6 shows mPEG ₁₁₃ -synthetic route to PDOX.

FIG. 7 shows mPEG ₁₁₃ -synthetic route to PCy.

FIG. 8 shows mPEG ₁₁₃ -CPDB、TK-OH、M _SS And M _TK Nuclear magnetic resonance spectrum of (a).

FIG. 9 shows mPEG ₁₁₃ -PTK、mPEG ₁₁₃ -PSS、mPEG ₁₁₃ -PDOX and mPEG ₁₁₃ -nuclear magnetic resonance spectrum of PCy.

FIG. 10 is a graph showing the particle size distribution of PPD, PPCy and PPDCCy nanoparticles in an aqueous solution.

FIG. 11 shows mPEG ₁₁₃ -PDOX and mPEG ₁₁₃ of-PCyUltraviolet and visible absorption spectrum.

FIG. 12 shows the concentration of H in PPD and PPDCY nanoparticles ₂ O ₂ A statistical plot of the release rate of DOX in solution; wherein A is PPD and B is PPDCy.

FIG. 13 shows CyNH after PPCy and PPDCCy nanoparticles were treated with different concentrations of glutathione ₂ A fluorescence spectrum of (a); wherein A is PPCy and B is PPDCCy.

FIG. 14 is a graph showing fluorescence spectra of DOX, PPDCY nanoparticles, and DOX in the PPDCY + glutathione-treated group.

FIG. 15 is CyNH of PPCy and PPDCCy nanoparticles in glutathione solutions of different concentrations ₂ The drug release rate statistical chart; wherein A is PPCy and B is PPDCCy.

FIG. 16 shows DOX and CyNH ₂ The synergistic effect of the two medicines is shown.

FIG. 17 shows DOX and CyNH ₂ Toxicity profile of the dual drugs on 4T1 cells with different administration modes.

FIG. 18 is a graph showing the results of the PPDCY nanoparticles in the intracellular drug release assay.

FIG. 19 shows CyNH ₂ PPCy, PPCy + BSO and PPDCCy nanoparticles release CyNH in response to intracellular glutathione ₂ Figure (a).

FIG. 20 shows CyNH ₂ The colocalization maps of PPCy and PPDCy nanoparticles with cellular mitochondria.

FIG. 21 shows CyNH ₂ And a mitochondrial membrane potential map of the cells after the PPCy nanoparticles and the PPCy + BSO treatment.

FIG. 22 shows CyNH ₂ Expression profile of cytochrome C after PPCy, PPCy + BSO and PPDCy nanoparticle treatment.

FIG. 23 shows CyNH ₂ Graph of intracellular reactive oxygen species levels after PPCy, PPCy + BSO, PPD and PPDCy nanoparticle treatment.

FIG. 24 is a graph showing the results of experiments on accumulation of DOX in cells of DOX, PPD and PPDCCy-treated groups.

FIG. 25 is a graph of the toxic effect of PPD, PPCy and PPDCCy nanoparticles on cells.

FIG. 26 is a graph showing the tumor changes in mice of each experimental group in an in vivo treatment experiment.

FIG. 27 is a graph showing the change in body weight of mice in each experimental group in an in vivo treatment experiment.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 Synthesis and characterization of Redox-responsive materials

First step, Synthesis of Polymer mPEG ₁₁₃ -CPDB

1) Synthesis of mPEG via the synthetic route of FIG. 1 ₁₁₃ -a CPDB. Mixing mPEG ₁₁₃ (1.0g, 0.2mmol), CPDB (0.223g, 0.8mmol) and DMAP (0.010g, 0.08mmol) were added to 10mL dry dichloromethane and transferred to a 50mL round bottom flask. DCC (0.165g, 0.8mmol) dissolved in 5mL of anhydrous dichloromethane was added dropwise to the above solution. The esterification reaction was carried out for 24h at room temperature with stirring, and the opaque solution was then filtered. The product was collected by precipitation in cold ether. After drying under vacuum overnight, a pink powder was obtained in 89% yield.

Second step, synthesis of monomer M _TK 。

2.1) Synthesis of TK-COOH via the synthetic route of FIG. 2. At N ₂ The reaction was stirred at room temperature for 6h under an atmosphere and anhydrous thioglycolic acid (0.92g, 10mmol) and anhydrous acetone (1.16g, 20mmol) were purged with dry HCl gas. The mixture was then cooled in an ice bath, filtered, and washed with ice-cold hexane and water, respectively, to give a white solid in 62% yield.

2.2) TK-OH was synthesized via the synthetic route of FIG. 2. Sodium borohydride (5.0g, 0.132mol) and TK-COOH (5.0g, 0.022mol) obtained in step 2.1) were dissolved in 50mL anhydrous THF. Subsequently, a solution of iodine (20.0g, 0.057mol) in 60mL THF was slowly added dropwise via a funnel on an ice bath, and the mixture was refluxed for 24 h. Next, under ice bath, 50mL of methanol was carefully added to quench the reaction. The solvent was removed thoroughly under vacuum and the mixture was further dissolved in aqueous NaOH (25%, 200 mL). The resulting solution was stirred for 5h and extracted with ethyl acetate (3X 100 mL). The organic layer obtained was dried and concentrated in vacuo and purified by silica gel column chromatography using DCM/ethyl acetate (1: 1, v/v) as eluent. After drying overnight in vacuo, a colorless oil was finally obtained (2.88g, 66%).

2.3) Synthesis of M by the synthetic route of FIG. 2 _TK . TK-OH (1.96g, 0.02mol) obtained in step 2.2) and triethylamine (0.51g, 0.01mol) were dissolved in 50mL dry DCM. Methacryloyl chloride (0.52g, 0.01mol) mixed in 20mL of anhydrous DCM was added under ice bath. The mixture was then stirred at room temperature overnight. The product was purified by silica gel column chromatography using DCM/EtOAc (1: 1, v/v). After drying overnight in vacuo, a colorless oil (0.79g, 60%) was finally obtained.

Thirdly, synthesizing monomer M _SS 。

3) Synthesis of monomer M by the synthetic route of FIG. 3 _SS .2, 2' -Dithiodiethanol (3.08g, 0.02mmol) and triethylamine (1.01g, 0.01mmol) were dissolved in 50mL of dry DCM. Methacryloyl chloride (1.04g, 0.01mmol) in 20mL of anhydrous DCM was added to the solution under ice bath. The mixture was then stirred at room temperature overnight. The product was purified by silica gel column chromatography using DCM/EtOAc (1: 1, v/v). After drying overnight in vacuo, a colorless oil was finally obtained (1.47g, 66%).

The above products are passed through FIG. 8 ¹ H NMR confirmed the successful synthesis of polymer and monomer.

Fourthly, synthesizing polymer mPEG with active oxygen response bond ₁₁₃ -PTK。

4) Synthesis of mPEG via the synthetic route of FIG. 4 ₁₁₃ -PTK. Synthesis of mPEG by reversible addition-fragmentation chain transfer polymerization (RAFT) polymerization ₁₁₃ -PTK. Mixing mPEG ₁₁₃ -CPDB(0.20g，1.0eqv)，M _TK (396mg, 30eqv) and AIBN (3.1mg, 0.33eqv) were dissolved in 80mL anhydrous dioxane. Freezing and thawing several times to remove oxygen, and stirring the mixture at 75 deg.C12h and then precipitated into hexane. The precipitate was collected by filtration. After drying overnight in vacuo, the product was obtained as a pink solid in 82% yield.

Step five, synthesizing polymer mPEG with glutathione response bonds ₁₁₃ -PSS。

5) Synthesis of mPEG via the synthetic route of FIG. 5 ₁₁₃ -PSS. mPEG Synthesis by RAFT polymerization ₁₁₃ -PSS. Mixing mPEG ₁₁₃ -CPDB(0.20g，1.0eqv)，M _SS (333mg, 30eqv) and AIBN (3.1mg, 0.33eqv) were dissolved in 80mL of anhydrous 1, 4-dioxane. After freezing and thawing several times to remove oxygen, the mixture was stirred at 75 ℃ for 12h and then precipitated into cold hexane. The precipitate was collected by filtration. After drying overnight in vacuo, the product was obtained as a pink solid in 81% yield.

Sixthly, synthesizing a drug-loaded polymer mPEG with glutathione response bonds ₁₁₃ -PDOX。

6) Synthesis of mPEG by the synthetic route of FIG. 6 ₁₁₃ -PDOX. Mixing mPEG ₁₁₃ PTK (110mg, 1.0eqv), NPC (180mg, 90eqv) were dissolved in 8mL of anhydrous DCM, and the mixture was stirred at room temperature for 12 h. The solution was then precipitated into cold ether and the precipitate was collected by filtration. After drying in vacuo overnight, the crude product TEA (220. mu.L, 1.6mmol) and DOX (240mg, 0.8mmol) were dissolved in 4mL anhydrous DMF and stirred at room temperature for 24 h. After the reaction, the mixture was dialyzed against DMF followed by pure water (MWCO: 3500 Da). Finally, the solution was lyophilized to obtain the product in 62% yield.

Seventhly, synthesizing a drug-loaded polymer mPEG with glutathione response bonds ₁₁₃ -PCy。

7) Synthesis of mPEG via the synthetic route of FIG. 7 ₁₁₃ -PCy. CyNH was added dropwise to a mixture of bis (trichloromethyl) carbonate (320mg) and acetonitrile (10mL) in an ice bath ₂ A mixture of (200mg) and DIPEA (166 μ L) in acetonitrile (20 mL). The resulting solution was refluxed for 4h and the solvent was removed thoroughly under vacuum. Next, mPEG113-PSS (0.10g) dissolved in anhydrous DCM was added and stirred at room temperature for 24 h. After the reaction, the mixture was dialyzed against DMF and water (MWCO: 3500 Da). Finally, the solution was lyophilized to 6The product was obtained in 0% yield.

The above products are obtained by nuclear magnetic resonance ¹ H NMR confirmed the successful synthesis of the polymer, and the spectra are shown in fig. 8 and 9.

EXAMPLE 2 preparation and application of Redox-responsive progressive drug Release System

(Redox-responsive sequential drug delivery system hereinafter referred to as Biredox-responsive nanoparticle)

Preparation of mono-redox and di-redox response nanoparticles

The double redox-responsive nanoparticles are prepared by a Nano precipitation method (Nano precipitation method), the adopted chemotherapeutic drug is adriamycin, and the specific method is as follows:

first, mPEG prepared in example 1 was weighed ₁₁₃ -PCy (10.0mg) and mPEG ₁₁₃ PDOX (10.0mg) was dissolved in DMSO (1.0mL), respectively, and 50. mu.L of mPEG was pipetted ₁₁₃ -PCy and 50. mu.L of mPEG ₁₁₃ PDOX solutions were mixed together, and the mixture was added dropwise to 20mL of pure water and stirred for 1 hour. Subsequently, the particle solution was transferred to a dialysis bag (MWCO 14000) and dialyzed in ultrapure water away from light for 24h to remove DMSO, yielding a double redox-responsive nanoparticle PPDCy. After the dialysis is finished, the DOX and CyNH in PPDCy are measured by a microplate reader ₂ Absorption value according to DOX and CyNH ₂ The encapsulation efficiency of the two drugs was calculated from the standard curve of (a).

Wherein, control material PPCy nanoparticles were prepared from 10 μ L of mPEG prepared in example 1 ₁₁₃ PCy (10mg/mL) and 100. mu.L of polyethylene glycol-polylactic acid (PEG-PLA, 10mg/mL, Shanghai Biotech Co., Ltd., D-L-2k5k) were prepared and synthesized by the same procedure as described above. Control Material PPD nanoparticles 50 μ L of mPEG prepared in example 1 ₁₁₃ -PDOX(10mg.mL ^-1 ) And 50. mu.L of PEG-PLA (10 mg.mL) ^-1 ) The preparation and synthesis steps are the same as the above.

Characterization of double redox response nanoparticles

The particle sizes of three drug-loaded nanoparticles PPD, PPCy and PPDCy were measured by Dynamic Light Scattering (DLS). As a result, as shown in FIG. 10, the particle diameters of PPCy and PPD were about 105.8nm and 158.3nm, respectively,the particle size of the PPDCY nano-particles is about 130.9 nm. Measurement of mPEG ₁₁₃ -PDOX and mPEG ₁₁₃ PCy UV-visible absorption Spectroscopy, results are shown in FIG. 11, mPEG ₁₁₃ -PDOX and mPEG ₁₁₃ -PCy contains DOX and CyNH ₂ Absorption peaks for SSOH, demonstrating the successful synthesis of both materials.

Third, verification of in vitro redox responsiveness

1. Active oxygen responsiveness of nanoparticles

The active oxygen responsiveness of the nanoparticles was verified using drug release experiments. By different concentrations of H ₂ O ₂ (0, 0.01, 0.1, 1 and 10mM) solutions were tested for DOX release in PPD and PPDCY nanoparticles. 1mL of PPD and PPDCY nanoparticles ([ DOX ] respectively]＝25μg mL ^-1 ) Put into a dialysis bag (

Float-a-Lyzer, MWCO 3500), and the dialysis bag was placed in five groups of 15mL H of different concentrations ₂ O ₂ In solution, drug release was performed on a 37 ℃ water bath shaker (80 rpm). 3mL of the released external solution were withdrawn each time at the indicated time point and supplemented with an equal amount of fresh H ₂ O ₂ And (3) solution. The fluorescence intensity of the collected released external liquid was measured by a microplate reader (Ex 488nm, Em 587 nm). The results are shown in FIG. 12, with PPDCY nanoparticles at concentrations of 0mM and 0.01mM H ₂ O ₂ The DOX release in the solution was only 3.8% and 11.4%, respectively, which is H in normal tissue ₂ O ₂ The concentration of (a), which proves that it can well avoid drug release in normal tissues; when H is present ₂ O ₂ The dosage of DOX is increased when the concentration is gradually increased, and when H is increased ₂ O ₂ At a concentration of 10mM (usually H at the tumor site) ₂ O ₂ Concentration level), the release amount of DOX can reach 47.6%. The same trend is true for PPD nanoparticles. The above results indicate that both PPDCCy and PPD have hydrogen peroxide-responsive release, and we speculate that these two nanoparticles have a large number of copper dithiol bonds at high H ₂ O ₂ The environment can break down, resulting in the release of DOX.

2. Glutathione responsiveness of nanoparticles

After PPDCY or PPCy and glutathione (0, 0.01, 1, 5 and 10mM) at different concentrations were reacted for 4 hours in a 37 ℃ water bath shaker, respectively, CyNH in PPDCY or PPCy was measured using a fluorescence spectrophotometer ₂ Fluorescence spectrum of (2). As a result, as shown in FIG. 13, the fluorescence spectrum of PPDCY nanoparticles tends to be weak under the glutathione concentration of 0mM or 0.01 mM. This is probably because of CyNH ₂ Modified by disulfide bonds to mask its own fluorescence. CyNH in PPDCy nanoparticles as glutathione concentration was gradually increased ₂ The fluorescence spectrum of (2) gradually recovers and becomes stronger. This result indicates that PPDCy does not cause premature ejaculation in the drug in normal tissues (2-10. mu.M glutathione); when PPDCY reaches a high-concentration glutathione site, the disulfide bond can rapidly respond to glutathione and release CyNH ₂ . PPCy nanoparticles have a similar trend to PPDCy.

Fluorescence spectra of DOX (Solambio/Solebao, D8740-25mg) and PPDCY nanoparticles were measured separately using a spectrofluorometer, and the results are shown in FIG. 14. At the same concentration of DOX, little fluorescence of DOX was detected in PPDCy nanoparticles, probably because of the fluorescent molecule CyNH in PPDCy ₂ And DOX produces a fluorescence resonance energy transfer that attenuates the fluorescence intensity of DOX. After PPDCY and 10mM glutathione react for 24h in a 37 ℃ water bath shaking table, the fluorescence spectrum of DOX is measured to find that the fluorescence spectrum of DOX in PPDCY is restored to a level similar to PPD, which proves that glutathione sufficiently breaks the disulfide bond in PPDCY to cause CyNH ₂ The distance from DOX increases, so that the fluorescence resonance energy transfer phenomenon disappears, and DOX recovers the fluorescence thereof.

Detection of CyNH of PPD and PPDCy nanoparticles in glutathione (0, 0.01, 1, 5 and 10mM) solutions at different concentrations ₂ And (4) releasing the situation. 1mL of PPCy and PPDCCy nanoparticles ([ CyNH ], respectively ₂ ]＝32.7μg mL ^-1 ) Put into a dialysis bag (

Float-a-Lyzer, MWCO 3500), and the dialysis bags were placed in five groups of 15mL glutathione with different concentrationsIn the peptide solution, drug release was performed at 37 ℃ in a water bath shaker (80 rpm). 3mL of the released external solution was removed at each time point and supplemented with an equal amount of fresh glutathione solution. The absorbance of the collected released external liquid was measured at 670nm by a microplate reader. The results are shown in FIG. 15, in which CyNH was observed at glutathione concentrations of 0mM and 0.01mM for PPDCY nanoparticles ₂ The release amounts of the compounds are respectively only 3.2 percent and 7.3 percent, which are the concentrations of glutathione in normal tissues, and prove that the compounds can well avoid the release of drugs in the normal tissues; when the concentration of glutathione is gradually increased, CyNH ₂ The dosage of CyNH is increased when the dosage of CyNH is increased ₂ CyNH when the concentration reached 10mM (usually the glutathione concentration level at the tumor site) ₂ The release amount of (A) can reach 51.5%. The same trend is also true for PPCy nanoparticles. The results show that both PPDCCy and PPCy have glutathione-responsive release, and the two nanoparticles have a large number of disulfide bonds and can be broken under the high glutathione environment to cause CyNH ₂ Is released.

Four, in vitro cell experiment

1. Toxicity of double-drug synergy and administration mode on 4T1 cells

Mouse breast cancer cells (4T1) are selected to explore the toxicity of the double-drug administration ratio and the administration mode to the cells. For the experiment of determining the administration ratio of the two drugs, CyNH is set respectively ₂ And total DOX concentration of 0-100 μ M, CyNH ₂ Ten equal proportions of 0-100% molar ratio. The double drugs with different proportions and different concentrations are cultured together with the 4T1 cell line for 48 hours respectively, finally, the cytotoxicity of each group is detected by using an MTT method, and the synergy index of the double drugs in each proportion is calculated by using CompuSyn software. The results are shown in FIG. 16, where the synergy index of both drugs is less than 1 at ten ratios, demonstrating DOX and CyNH ₂ Has a good synergistic effect, and when DOX and CyNH are used ₂ The molar ratio is 1: 1, the synergy index is minimal, demonstrating that at this ratio they have the best ability to kill cells. Subsequently, the molar ratio of the two drugs was set to 1: 1, administered separately at the same time and sequentially at 4h intervals (first CyNH) ₂ post-DOX) was incubated with the cells for 48h, and the results are shown in fig. 17. Administered sequentially at 4h intervalsThe mode is more toxic to cells than the mode of simultaneous administration, probably because of CyNH ₂ The mitochondria in the cells are damaged firstly, and the sensitivity of the cells to DOX is increased.

2. Intra-cellular programmed drug release of PPDCy nanoparticles

Observing DOX and CyNH in PPDCY nanoparticles by using laser confocal scanning microscope ₂ Release sequence in 4T1 cells. PPDCY nanoparticles (5. mu.M DOX, 5. mu.M CyNH) ₂ ) After 0.5, 1, 2, 3, 4h co-incubation with 4T1 cells, respectively, followed by three washes with 1 XPBS, nuclei were stained with Hoechst 33342 (10. mu.M) for 15min at room temperature. As shown in FIG. 18, CyNH appeared first in cytoplasm when PPDCy nanoparticles were incubated with cells for 0.5h ₂ Fluorescence of (2) to prove CyNH ₂ Release into the cell precedes DOX, probably because disulfide bonds respond to glutathione faster. After 2h incubation with cells, fluorescence of DOX appeared in the cytoplasm, probably due to CyNH ₂ The release of (a) eliminates the fluorescence resonance energy transfer phenomenon, so that the fluorescence is restored again by the unreleased DOX in the PPDCy nanoparticles. After incubation of the PPDCY nanoparticles with the cells for 4h, co-localization of DOX to the nucleus was observed, confirming that DOX was released from the PPDCY nanoparticles and entered the nucleus. The above results indicate that when PPDCy nanoparticles enter cells, the disulfide bonds rapidly respond to intracellular high glutathione levels to convert CyNH ₂ The nano particles are released into cytoplasm and release DOX in response to intracellular active oxygen after a period of time, thereby realizing CyNH ₂ And DOX in intracellular order, maximizing damage to the cells.

3. Effect of nanoparticles on mitochondria

To understand the effect of nanoparticles on mitochondria, the inventors further explored the responsive release of CyNH by nanoparticles in 4T1 cells against glutathione ₂ And (6) behaviors. Firstly, CyNH is respectively added ₂ PPCy, PPCy + BSO (glutathione inhibitor, 100. mu.M) and PPDCy (20. mu.M CyNH) ₂ ) After incubation of the nanoparticles with the cells for 4h, nuclei were stained with Hoechst 33342(10 μ M) for 15min at room temperature. The results are shown in FIG. 19, CyNH ₂ The PPCy and PPDCCy experimental groups can observe strong fluorescence in cytoplasm, and prove that the PPCy and PPDCCy nanoparticles can well respond to intracellular glutathione and release CyNH ₂ . When cells were pre-treated with the glutathione inhibitor BSO, no significant fluorescence was observed in cells co-incubated with PPCy nanoparticles, demonstrating that at lower glutathione levels, disulfide bonds cannot be cleaved to release CyNH ₂ 。

Next, the inventors explored the nanoparticle release of CyNH ₂ Then, CyNH ₂ The intracellular site of action of (a). Respectively reacting CyNH ₂ PPCy and PPDCy (5. mu.M CyNH) ₂ ) After incubation of the nanoparticles with 4T1 cells for 4h, the nuclei and mitochondria were labeled with Hoechst 33342 and mitochondrial green probes, respectively. The results are shown in FIG. 20, CyNH ₂ CyNH in PPCy and PPDCy nanoparticles ₂ The signals are well localized to mitochondria.

The effect of the nanoparticles on mitochondrial membrane potential was subsequently explored. Since the fluorescence spectra of the mitochondrial membrane potential red fluorescent probe and DOX overlap, the inventors set only CyNH ₂ Three experimental groups PPCy and PPCy + BSO. Respectively mixing the above materials with CyNH ₂ After incubating with cells at a concentration of 5 μ M for 4h, the mitochondrial membrane potential of each group of cells was monitored using a mitochondrial membrane potential assay kit. The results are shown in FIG. 21, using CyNH ₂ And PPCy-treated cells were able to significantly reduce mitochondrial membrane potential and thus damage mitochondria. When cells were treated with BSO, the membrane potential of mitochondria did not decrease significantly compared to the control.

Finally, the expression of cytochrome C in cells after treatment in each experimental group was investigated. Respectively reacting CyNH ₂ PPCy, PPCy + BSO and PPDCy (5. mu.M CyNH) ₂ ) After incubation of the nanoparticles with the cells for 12h, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min. Subsequently, 0.5% Triton-100 was added and incubated with the cells at room temperature for 10min to permeabilize the cells, and then the cells were blocked with 5% Bovine Serum Albumin (BSA) for 1 h. After removal of the blocking solution, the cells were incubated with cytochrome C primary antibody (Yojanbio/Utiliaceae, 3025-100) (1:80) at 3After 1h incubation at 7 ℃ the cells were washed 3 times with 1 XPBS, and goat anti-mouse FITC secondary antibody (Invitrogen, F2761-0.5ML) (1:80) was incubated with the cells for 1h at 37 ℃. Nuclei were stained with Hoechst 33342 (10. mu.M). Finally, the cells were observed by CLSM imaging system. The results are shown in FIG. 22, using CyNH ₂ The PPCy and PPDCCy nanoparticle treated cells can induce the expression level of intracellular cytochrome C to be increased, thereby mediating the apoptosis of the cells. Similarly, after pre-treatment of cells with BSO, PPCy failed to achieve intracellular drug release and also failed to produce significant cellular toxicity.

4. Toxicity of nanoparticles to cells

First, the intracellular ROS production was detected using the DCF-DA kit (Beyotime/Biyunyan, S0033S). Reacting CyNH ₂ PPCy, BSO (100. mu.M) + PPCy, PPD and PPDCy (5. mu.M CyNH) ₂ 5 μ M DOX) nanoparticles were incubated with cells for 4 h. Subsequently, the cells were washed 3 times with 1 XPBS and stained with 10. mu.M DCF-DA at 37 ℃ for 30 min. Finally, qualitative studies were performed by observing the cells with a confocal microscope. The results are shown in FIG. 23, where the cells are in contact with CyNH ₂ PPCy and PPDCCy were able to produce large amounts of ROS after co-incubation without the presence of CyNH ₂ PPD group of (1) and the failure to realize CyNH ₂ No significant ROS production was observed for the drug released PPCy + BSO group, probably due to the CyNH in the above three groups ₂ The components cause oxidative stress in the cell and thereby cause a surge of intracellular ROS.

Next, 4T1 cells were incubated for 8h with DOX, PPD and PPDCy and then observed in confocal laser experiments. As a result, as shown in FIG. 24, more DOX was released and accumulated in cells in the PPDCy-treated group than in the PPD-treated group, both because of CyNH in the PPDCy-treated group ₂ After being rapidly released into cells, the DOX release agent can cause oxidative stress of the cells, thereby increasing the intracellular reactive oxygen species level and leading the DOX release depending on the release of the reactive oxygen species to be more complete.

Finally, the toxic effect of each nanoparticle on cells was tested using the MTT method. After incubating the cells with PPD, PPCy and PPDCCy nanoparticles at different concentration gradients for 48h, 10. mu.L of MTT solution (5 mg.mL) was added to each well ^-1 ) Incubation was continued for 4 h. 4After h, the supernatant from each well was aspirated, 150. mu.L of DMSO solution was added to each well, and the absorbance of each well was measured at 490nm using a microplate reader. As shown in FIG. 25, the toxic effect of PPDCY on cells was the greatest, and particularly, when the drug concentration reached 10. mu.M, the cell survival rate of the PPDCY-treated group was only 2.86%, whereas the cell survival rates of the PPCy-and PPD-treated groups were 62.53% and 28.2%, respectively. This indicates that there is no CyNH ₂ When present, the efficiency of DOX release in the PPD group was poor resulting in poor treatment. Although the PPCy group also had a better killing effect, the treatment efficiency of the two drugs was clearly much higher than that of the single drug when the drug concentrations were the same as in the PPCy-treated group.

Fifth, animal level experiment

1. In vivo antitumor therapy experiment

20 BALB/C mice, implanted with a 4T1 subcutaneous tumor model, were randomly divided into 4 groups of 5 mice each. Tail vein injections of 1 XPBS (100. mu.L), PPD ([ DOX)]＝2.5mg/kg)、PPCy([CyNH ₂ ]2.5mg/kg) and PPDCy ([ DOX)]＝1.25mg/kg，[CyNH ₂ ]1.25mg/kg), the drug was given every two days for 4 consecutive times, followed by one week of observation. The tumor volume was measured with a vernier caliper every two days throughout the treatment and the weight change of the mice in each experimental group was examined. The formula for tumor volume is as follows: volume (mm) ³ ) 0.5 x length x width ² 。

As a result, as shown in fig. 26, the tumors of the mice in the PBS group and the PPD group grew rapidly, the PPCy-treated group had a certain tumor growth inhibition effect, and the PPDCy group had the best tumor inhibition effect. This is due to the fact that the ROS in the tumor tissue is not sufficient to completely release DOX from the PPD group, and thus the tumor-inhibiting effect is limited; although the PPCy group also showed some tumor suppression, the therapeutic efficiency of the dual drug administration was much higher than that of the single drug administration at the same drug concentration compared to the PPCy group. The body weights of the mice are shown in fig. 27, and the body weights of the mice of all groups do not change obviously in the whole treatment process, so that the fact that all experimental components do not cause serious systemic toxicity to the mice is proved, and the fact that the material has good biocompatibility is reflected.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

Claims

1. A method for preparing a redox-responsive, sequential drug delivery system, comprising the steps of:

(1) synthetic polymer mPEG-CPDB

Dissolving polyethylene glycol monomethyl ether mPEG, 4-cyano-4- (thiobenzylthio) pentanoic acid CPDB, 4- (dimethylamino) pyridine DMAP and dicyclohexylcarbodiimide DCC in a solvent for reaction to obtain mPEG-CPD;

(2) synthesis of monomer M _TK

Mixing mercaptoacetic acid and acetone, reacting to obtain TK-COOH and sodium borohydride, dissolving in tetrahydrofuran, adding iodine, reacting for the second time, quenching, purifying to obtain TK-OH and triethylamine, dissolving in dichloromethane, adding methacryloyl chloride, reacting again, purifying, and drying to obtain M _TK ；

(3) Synthesis of monomer M _SS

(4) Synthesis of Polymer mPEG-PTK with active oxygen responsive bond

The mPEG-CPDB in the step (1) and the M in the step (2) are added _TK And azobisisobutyronitrile AIBN are dissolved in dioxane to react and be separated to obtain mPEG-PTK;

(5) synthesis of polymer mPEG-PSS with glutathione response bond

The mPEG-CPDB in the step (1) and the M in the step (3) are added _SS And AIBN are dissolved in 1, 4-dioxane to react and separate to obtain mPEG-PSS;

(6) synthesis of drug-loaded polymer systems with ROS-responsive linkages

The mPEG-PTK and the paranitro in the step (4) are reactedDissolving NPC in DCM, reacting, separating to obtain crude product TEA, dissolving it and chemotherapeutic drug in N, N-dimethylformamide DMF, reacting again, and purifying to obtain mPEG-P _Durg ；

(7) Synthesis of drug-loaded polymer mPEG-PCy with glutathione response bond

Bis (trichloromethyl) carbonate and CyNH ₂ Mixing N, N-diisopropylethylamine DIPEA with acetonitrile, reacting, removing the solvent, adding the mPEG-PSS dissolved in DCM in the step (5), reacting again, and purifying to obtain mPEG-PCy;

Subjecting the mPEG-P in the step (6) _Durg And (7) dissolving the mPEG-PCy in dimethyl sulfoxide, mixing with water, reacting, and dialyzing to obtain PPDCCy.

2. The method of claim 1, wherein the redox-responsive, sequential drug delivery system,

the dosages of mPEG, CPDB, DMAP and DCC in the step (1) are as follows according to a molar ratio of 1: 1-5: 1-5: 1-5 proportion calculation;

the solvent in the step (1) is at least one of dichloromethane, chloroform and tetrahydrofuran;

the dosage of the solvent in the step (1) is calculated according to the proportion of 1g mPEG to 10-35mL of solvent;

in the step (2), the molar ratio of the thioglycolic acid to the acetone is 1: 1-2;

the TK-COOH and sodium borohydride in the step (2) are mixed according to a mass ratio of 1: 1-5 proportion;

calculating the tetrahydrofuran and TK-COOH in the step (2) according to the ratio of 1g TK-COOH to 5-30mL tetrahydrofuran;

in the step (2), iodine and sodium borohydride are mixed according to a mass ratio of 1-4: 1, proportioning;

in the step (2), the molar ratio of TK-OH to triethylamine is 1-3: 1;

in the step (2), the molar ratio of the methacryloyl chloride to the TK-OH is 1: 1-2;

and (3) calculating the dichloromethane and TK-OH in the step (2) according to the ratio of 1g TK-OH to 20-50mL dichloromethane.

3. The method of preparing a redox-responsive, sequential drug delivery system according to claim 1 or 2,

the reaction in the step (1) is a stirring reaction for 20-30 h;

the reaction time in the step (2) is 4-8 h;

the second reaction in the step (2) is a reflux reaction for 24-48 h;

the secondary reaction in the step (2) is a stirring reaction for 7-15 h.

4. The method of claim 1, wherein the redox-responsive, sequential drug delivery system,

filtering the mPEG-CPD in the step (1) before the next reaction, precipitating by adopting cold ethyl ether, and drying in vacuum;

the reaction in step (2) is carried out in N ₂ Stirring under atmosphere;

cooling, filtering and washing the TK-COOH and sodium borohydride before dissolving the TK-COOH and the sodium borohydride in tetrahydrofuran;

the washing is respectively washing by adopting hexane and water;

the purification step in step (2): removing the solvent from the product of the secondary reaction in vacuum, dissolving the mixture in a sodium hydroxide solution, extracting with ethyl acetate, drying, concentrating in vacuum, purifying with a silica gel column, and drying in vacuum overnight;

the silica gel column purification is carried out according to a volume ratio of 1: 1-3 DCM/ethyl acetate as eluent;

the purification in the step (2) is performed by adopting a silica gel column;

the silica gel column purification is carried out according to a volume ratio of 1: 1 in DCM/EtOAc as eluent.

5. The method of claim 1, wherein the redox-responsive, sequential drug delivery system,

the molar ratio of the 2,2' -dithiodiethanol to the triethylamine in the step (3) is 1-5: 1;

in the step (3), the molar ratio of the methacryloyl chloride to the 2,2' -dithiodiethanol is 1: 1-2;

in the step (3), the dichloromethane and triethylamine are calculated according to the proportion of 1g triethylamine to 40-80mL dichloromethane;

the mPEG-CPDB and the M in the step (4) _TK The mass ratio of (1): 1-3;

the mass ratio of mPEG-CPDB to AIBN in the step (4) is 1: 0.01-0.02;

calculating the ratio of the dioxane to the mPEG-CPDB in the step (4) according to 1g of the mPEG-CPDB of 10-50 mL;

the mPEG-CPDB and the M in the step (5) _SS The mass ratio of (1): 1-3;

the mass ratio of mPEG-CPDB to AIBN in the step (5) is 1: 0.01-0.02;

calculating the ratio of the dioxane to the mPEG-CPDB in the step (5) according to 1g of the mPEG-CPDB of 10-50 mL;

the mass ratio of mPEG-PTK to NPC in the step (6) is 1: 0.5 to 2;

in the step (6), the molar ratio of the TEA to the chemotherapeutic drug is 1-3: 1;

calculating the DCM and the NPC in the step (6) according to the proportion of 1g of p-nitrophenyl chloroformate to 20-50mL of DCM;

calculating the ratio of DMF to mPEG-PTK in the step (6) according to the ratio of 1g mPEG-PTK to 20-50mL of DMF;

CyNH in the step (7) ₂ In an amount of 1g CyNH ₂ The ratio of DIPEA is 0.25-1.5 mL;

bis (trichloromethyl) carbonate and CyNH in step (7) ₂ The mass ratio of (A) to (B) is 1-2: 1;

the acetonitrile and CyNH in the step (7) ₂ According to 1g of CyNH ₂ Calculating the mixture ratio of 20-100mL acetonitrile;

the mPEG-P in the step (8) _Durg And the mass ratio of mPEG-PCy is 1: 0.8-1.2;

the mPEG-P in the step (8) _Durg And dimethyl sulfoxide in an amount of 1g mPEG-P _Durg Calculating the mixture ratio of 100mL of dimethyl sulfoxide;

the mPEG-P in the step (8) _Durg And water in an amount of 1g mPEG-P _Durg The ratio is 1900-2100mL water.

6. The method of claim 1 or 5, wherein the redox-responsive, sequential drug delivery system,

the reaction in the step (3) is a stirring reaction for 7-15 h;

the reaction in the step (4) is stirred and reacted for 12 to 24 hours at the temperature of 65 to 85 ℃;

the reaction in the step (5) is stirred and reacted for 12 to 24 hours at the temperature of 65 to 85 ℃;

the reaction in the step (6) is a stirring reaction for 12-48 h;

the secondary reaction is carried out in the step (6) and stirred for 24-48 h;

the reaction in the step (7) is a reflux reaction for 4-8 h;

the secondary reaction in the step (7) is a stirring reaction for 24-48 h;

in the step (8), the reaction is stirred for 1-48 h.

7. The method of claim 1, wherein the redox-responsive, sequential drug delivery system,

the purification in the step (3) is performed by adopting a silica gel column;

the silica gel column purification is carried out according to a volume ratio of 1: 1 DCM/ethyl acetate as eluent;

removing oxygen before the reaction in the step (4) is carried out, and adopting a repeated freezing and thawing mode;

in the step (4), the separation is performed by adopting hexane for precipitation, filtering and collecting precipitates, and performing vacuum drying;

the separation in the step (5) is to precipitate by adopting cold hexane, filter and collect precipitates, and carry out vacuum drying;

the separation in the step (6) is to precipitate by adopting cold ether, filter and collect precipitate and carry out vacuum drying;

the purification in the steps (6) and (7) is to dialyze the mixture by DMF and pure water in sequence and then freeze-dry the mixture;

the cut-off molecular weight of the dialysis bag for dialysis is 3500 Da;

the dialysis in the step (8) is dark dialysis, and the molecular weight cut-off of the dialysis bag is 14000.

8. The method for preparing a redox-responsive, sequential drug delivery system according to claim 1, wherein in step (6) the chemotherapeutic agent is doxorubicin.

9. A redox-responsive, sequential drug delivery system, characterized in that it is prepared by the process according to any one of claims 1 to 8.

10. Use of the redox-responsive, sequential drug delivery system of claim 9 in the manufacture of a medicament for the treatment of a tumor.