An amphiphilic enzyme-sensitive polymer, and a preparation method and application thereof

Abstract

This invention provides an amphiphilic enzyme-sensitive polymer, its preparation method, and its applications. The amphiphilic enzyme-sensitive polymer is a neutrophil elastase-binding peptide-polyethylene glycol-matrix metalloproteinase 2-responsive peptide-polylactic acid block copolymer. This invention prepares Mal-PEG-GPLGIAGQ through a substitution reaction, then prepares Mal-PEG-GPLGIAGQ-PLA through an amidation reaction, and finally prepares NE-PEG-GPLGIAGQ-PLA through a Michael addition reaction. When this polymer is applied to prepare a nanomicelle drug system, it can self-assemble into structurally stable nanomicelles in aqueous solution and possesses targeting functionality. The prepared nanomicelle drug system exhibits excellent enzyme responsiveness, and after drug loading, it can achieve efficient and safe therapeutic effects.

Description

Technical Field

[0001] This invention belongs to the field of polymer technology, and particularly relates to an amphiphilic enzyme-sensitive polymer, its preparation method, and its application. Background Technology

[0002] Traditional cancer treatments primarily rely on chemotherapy and radiotherapy. Conventional chemotherapy drugs often lack selectivity, causing severe damage to normal tissues while killing tumor cells, leading to systemic toxic side effects. Furthermore, due to the rapid metabolism of these drugs in the body, it is difficult to achieve effective therapeutic concentrations at the tumor site. To address these issues, nanotechnology-based intelligent drug delivery systems (DDS) have emerged. These nanomedicine carriers utilize the enhanced penetration and retention (EPR) effect in tumor tissue to passively target and accumulate at the tumor site, thereby improving efficacy and reducing side effects.

[0003] The complexity and heterogeneity of the tumor microenvironment are core bottlenecks restricting the efficacy of traditional anti-tumor therapies. Its hypoxic, acidic, and protease-overexpressing characteristics provide important targets for the design of intelligent nanomedicine delivery systems. Matrix metalloproteinase-2 (MMP-2) is highly expressed in tumor tissues and tumor-associated stromal cells, and its specific enzymatic properties have been widely used for the targeted release regulation of nanomedicines. Modifying nanoparticles with MMP-2-sensitive peptides can achieve precise responsive drug release at the tumor site, avoiding damage to normal tissues. Studies have confirmed that enzyme-responsive nanosystems can significantly increase drug concentration in tumor regions and reduce systemic toxicity, providing a new technological pathway for anti-tumor therapy. However, traditional nanoparticles suffer from insufficient targeting, toxic side effects due to drug burst release, and limited tumor penetration, severely hindering their clinical translation.

[0004] Neutrophils, as a key component of the innate immune system, possess a strong tumor chemotaxis ability and can actively home to tumor sites in response to inflammatory factors, making them highly promising in vivo drug delivery systems. Research on neutrophils has largely focused on constructing nanoparticle-neutrophil hybrid systems in vitro. However, as terminally differentiated mature cells, neutrophils cannot proliferate in vitro and have an extremely short natural lifespan. This inherent characteristic not only significantly compresses the operational time window for in vitro experiments but also significantly weakens their chemotactic response to inflammatory sites, severely limiting the functional stability of in vitro neutrophil delivery systems. More critically, neutrophils undergo bone marrow homing and rapid apoptosis in the later stages of their lifespan, further drastically shortening the effective duration of such delivery systems in vivo, severely limiting their practical applications and translational potential. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an amphiphilic enzyme-sensitive polymer, its preparation method, and its applications. The amphiphilic enzyme-sensitive polymer constructed in this invention is an MMP-2-responsive neutrophil-targeting polymer. Its surface is modified with a neutrophil elastase-binding peptide (NE peptide) to target the neutrophil elastase receptor, enabling it to bind to neutrophils in vivo and enhance tumor penetration by leveraging the natural chemotaxis of neutrophils. After reaching the tumor site, the polymer can achieve precise drug release through enzymatic hydrolysis of the MMP-2-sensitive peptide, avoiding damage to normal tissues.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: On the one hand, the present invention provides an amphiphilic enzyme-sensitive polymer, the chemical structure of which is shown in Formula 1; Formula 1; In Equation 1, n = 22~136; m = 55~111.

[0007] Preferably, n=45; m=55.

[0008] In the technical solution of the present invention, the amphiphilic enzyme-sensitive polymer is a neutrophil elastase-binding peptide-polyethylene glycol-matrix metalloproteinase 2 responsive peptide-polylactic acid block copolymer, wherein the molecular weight of polyethylene glycol is 1000~6000, preferably 2000; the molecular weight of polylactic acid is 4000~8000, preferably 4000; the amino acid sequence of matrix metalloproteinase 2 responsive peptide is GPLGIAGQ; the molecular formula of the amphiphilic enzyme-sensitive polymer is NE-PEG-GPLGIAGQ-PLA; wherein NE is a neutrophil elastase-binding peptide.

[0009] In another aspect, the present invention provides a method for preparing the above-mentioned amphiphilic enzyme-sensitive polymer, comprising the following steps: (1) Maleimide-polyethylene glycol-succinimide propionate (Mal-PEG-SPA) was subjected to a nucleophilic substitution reaction with matrix metalloproteinase 2 responsive peptide (GPLGIAGQ) to obtain Mal-PEG2000-GPLGIAGQ; wherein, the chemical structure of Mal-PEG-SPA is shown in Formula 2; the chemical structure of matrix metalloproteinase 2 responsive peptide is shown in Formula 3; and the chemical structure of Mal-PEG-GPLGIAGQ is shown in Formula 4. Formula 2; Formula 3; Equation 4; (2) Mal-PEG-GPLGIAGQ and NH2-PLA are amidated to obtain Mal-PEG-GPLGIAGQ-PLA; wherein the chemical structure of NH2-PLA is shown in Formula 5; and the chemical structure of Mal-PEG-GPLGIAGQ-PLA is shown in Formula 6. Formula 5; Formula 6; (3) Mal-PEG-GPLGIAGQ-PLA is subjected to Michael addition reaction with neutrophil elastase-binding peptide (NE peptide) to obtain NE-PEG-GPLGIAGQ-PLA, which is the amphiphilic enzyme-sensitive polymer.

[0010] Further, step (1) specifically includes: nucleophilic substitution reaction of matrix metalloproteinase 2 responsive peptide (GPLGIAGQ) and Mal-PEG-SPA in an organic solvent containing triethylamine.

[0011] Preferably, the organic solvent is selected from at least one of N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO).

[0012] Preferably, the organic solvent containing triethylamine is an anhydrous and oxygen-free solvent.

[0013] Preferably, the nucleophilic substitution reaction takes 24-48 h, more preferably 24-36 h.

[0014] Preferably, the nucleophilic substitution reaction is carried out at room temperature in the dark.

[0015] Preferably, the molar ratio of matrix metalloproteinase 2 responsive peptide (GPLGIAGQ) to triethylamine is 1:2-5, more preferably 1:2-3.

[0016] Preferably, the molar ratio of triethylamine to Mal-PEG2000-SPA is 3-6:1.

[0017] In some specific embodiments, the nucleophilic substitution reaction is followed by dialysis and freeze-drying post-treatment; the molecular weight cutoff for dialysis is 1000 Da; the dialysis solution used is deionized water; and the dialysis time is 48-96 h.

[0018] Further, step (2) specifically includes: performing an amidation reaction of Mal-PEG-GPLGIAGQ, PLA-NH2 and a polypeptide condensation reagent in an organic solvent containing triethylamine.

[0019] Preferably, the organic solvent is selected from at least one of N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO).

[0020] Preferably, the amidation reaction is carried out under light-protected conditions.

[0021] Preferably, the polypeptide condensation reagent is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU).

[0022] Preferably, the molar ratio of Mal-PEG-GPLGIAGQ to the polypeptide condensation reagent is 1:1-4, and more preferably 1:1.5-2.

[0023] Preferably, the molar ratio of the polypeptide condensing reagent to triethylamine is 1-2:3.

[0024] Preferably, the molar ratio of triethylamine to PLA4000-NH2 is 1-5:1, more preferably 2:1.

[0025] Preferably, the amidation reaction takes 8-24 h, more preferably 8-12 h.

[0026] Further, step (3) specifically includes: performing a Michael addition reaction between Mal-PEG-GPLGIAGQ-PLA and neutrophil elastase-binding peptide (NE peptide) in a mixed solution of tris(2-carboxyethyl)phosphine (TCEP), dimethyl sulfoxide (DMSO), and PBS buffer.

[0027] Preferably, the Michael addition reaction is carried out in an inert atmosphere.

[0028] Preferably, the molar ratio of Mal-PEG-GPLGIAGQ-PLA to neutrophil elastase-binding peptide (NE peptide) is 1:1-2.

[0029] Preferably, the molar ratio of the neutrophil elastase-binding peptide (NE peptide) to tris(2-carboxyethyl)phosphine (TCEP) is 1-6:1, more preferably 3:1.

[0030] Preferably, the Michael addition reaction takes 24-120 h, more preferably 48 h.

[0031] In another aspect, the present invention provides the application of the above-mentioned amphiphilic enzyme-sensitive polymer in the preparation of drug delivery systems.

[0032] Preferably, its application in the preparation of enzyme-responsive antitumor drug delivery systems.

[0033] The beneficial effects of the present invention are at least as follows: The amphiphilic enzyme-sensitive polymer provided by this invention can self-assemble into structurally stable nanomicelles in aqueous solution, while also possessing targeting capabilities. These nanomicelles exhibit excellent enzyme responsiveness, enabling efficient and safe therapeutic effects when loaded with drugs. This invention overcomes the limitations of traditional drug delivery systems in terms of tumor targeting efficiency and biosafety, achieving precise delivery. Firstly, traditional nanocarriers suffer from the problem of ineffective delivery to lesion sites. The amphiphilic enzyme-sensitive polymer provided by this invention actively targets neutrophils, reaching the tumor site along with them. Subsequently, leveraging the high expression of matrix metalloproteinases (MMPs) at the tumor site, it responds to enzyme concentration to promote nanoparticle release, achieving precise delivery to deep lesions while avoiding non-specific drug release in normal tissues and reducing systemic toxicity. This system ingeniously solves the problem of traditional carriers' inability to penetrate tumor cells, achieving on-demand drug delivery to lesion sites and significantly improving anti-tumor efficacy. Attached Figure Description

[0034] Figure 1 The infrared spectrum of intermediate product 1 Mal-PEG2000-GPLGIAGQ in Example 1 of this invention is shown.

[0035] Figure 2 This is the intermediate product 1 Mal-PEG2000-GPLGIAGQ of Example 1 of the present invention. 1 H-NMR spectrum, solvent is d -CDCl3.

[0036] Figure 3 The infrared spectrum of intermediate product 2 Mal-PEG2000-GPLGIAGQ-PLA4000 from Example 1 of this invention is shown.

[0037] Figure 4 This is the intermediate product 2 Mal-PEG2000-GPLGIAGQ-PLA4000 of Example 1 of the present invention. 1 H-NMR spectrum, solvent is d -CDCl3.

[0038] Figure 5 The NE-PEG2000-GPLGIAGQ-PLA4000 in Embodiment 1 of this invention 1 H-NMR spectrum, solvent is d -CDCl3.

[0039] Figure 6 This is the GPC diagram of NE-PEG2000-GPLGIAGQ-PLA4000 in Embodiment 1 of the present invention.

[0040] Figure 7This is a CMC test chart of NE-PEG2000-GPLGIAGQ-PLA4000 in Embodiment 2 of the present invention.

[0041] Figure 8 This is a particle size distribution diagram of the drug-loaded nanoparticles prepared based on polymer NE-PEG2000-GPLGIAGQ-PLA4000 in Example 3 of the present invention.

[0042] Figure 9 The graph shows the results of the enrichment of NE-NPs prepared in Example 4 of this invention at the tumor site compared with the non-targeted NPs.

[0043] Figure 10 The graph shows the test results of the high-efficiency anti-tumor efficacy of the nanoparticles NE-NPs prepared in Example 5 of this invention. Detailed Implementation

[0044] The following embodiments are merely some, not all, of the embodiments of the present invention. Therefore, the detailed descriptions of the embodiments provided below are not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0045] In this invention, unless otherwise specified, all equipment and raw materials are commercially available or commonly used in the industry. The methods described in the following embodiments are conventional methods in the art, unless otherwise specified.

[0046] Example 1: This embodiment provides an amphiphilic enzyme-sensitive polymer NE-PEG2000-GPLGIAGQ-PLA4000, the preparation method of which includes the following steps: (1) Place matrix metalloproteinase 2 responsive peptide (GPLGIAGQ) in a reaction flask, and after nitrogen protection, inject anhydrous and oxygen-free DMF solution containing triethylamine, followed by anhydrous and oxygen-free DMF solution containing Mal-PEG2000-SPA; mix well and react at room temperature in the dark for 48 h; transfer the resulting reaction solution to a dialysis bag with a molecular weight cutoff of 1000 Da, dialyze with deionized water for 48 h, and freeze-dry to obtain the solid, which is Mal-PEG2000-GPLGIAGQ (infrared spectrum as shown in Figure 1). Figure 1 As shown, 1 H-NMR such as Figure 2 (As shown). In this step, the molar ratio of triethylamine to Mal-PEG2000-SPA is 3:1; the molar ratio of matrix metalloproteinase 2 responsive peptide (GPLGIAGQ) to triethylamine is 1:3.

[0047] The chemical reaction process in this step is shown in Equation 7: Formula 7 (2) Dissolve Mal-PEG2000-GPLGIAGQ and HATU in DMSO, add a DMSO solution containing triethylamine, and stir for 5 min until the solution turns noticeably yellow; then add a DMSO solution containing PLA4000-NH2, mix well, and react overnight under light-protected conditions to obtain Mal-PEG2000-GPLGIAGQ-PLA4000 (infrared spectrum as shown in Figure 1). Figure 3 As shown, 1 H-NMR such as Figure 4 (As shown). In this step, the molar ratio of Mal-PEG2000-GPLGIAGQ to HATU is 1:2; the molar ratio of HATU to triethylamine is 2:3; and the molar ratio of triethylamine to PLA4000-NH2 is 2:1. The chemical reaction process in this step is shown in Equation 8: Formula 8 (3) Mal-PEG2000-GPLGIAGQ-PLA4000 and NE peptide were placed in a reaction flask, protected with nitrogen, and then DMSO solution of tris(2-carboxyethyl)phosphine (TCEP) and PBS buffer were added. After mixing evenly, the mixture was reacted under nitrogen protection for 48 h to obtain MMP-2 responsive neutrophil-targeting nanoparticles NE-NPs (NE-PEG2000-GPLGIAGQ-PLA4000). 1 H-NMR such as Figure 5 (As shown); In this step, the molar ratio of Mal-PEG2000-GPLGIAGQ-PLA4000 to NE peptide is 1:2; the molar ratio of NE peptide to tris(2-carboxyethyl)phosphine (TCEP) is 3:1; The chemical reaction process in this step is shown in Equation 9:

[0048] Formula 9 Example 2 In this embodiment, the critical micelle concentration (CMC) of the amphiphilic enzyme-sensitive polymer NE-PEG2000-GPLGIAGQ-PLA4000 prepared in Example 1 was tested using a fluorescent probe method.

[0049] (1) Preparation of pyrene solution: Dissolve pyrene in acetone to prepare a solution with a concentration of 12×10⁻⁶. -5 M's pyrene solution; (2) Preparation of sample solution: Weigh 5 mg of NE-PEG2000-GPLGIAGQ-PLA4000 and dissolve it in 10 mL of acetone. Quickly add the solution to 50 mL of deionized water and stir for 24 hours to evaporate the acetone, obtaining a polymer mother liquor with a concentration of 0.1 mg / mL. Dilute it to a series of concentrations (concentration range: 0.0001-0.1 mg / mL). Take 20 10 mL volumetric flasks, add 0.1 mL of the pyrene solution prepared in step (1) to each flask, and then add the copolymer solutions of different concentrations to make up to the volume. Shake well to obtain the sample solution. The concentration of pyrene in the sample solution is 12 × 10⁻⁶. -7 M; (3) Fluorescence spectroscopy test: The fluorescence excitation spectrum of the sample solution was scanned at 300-350 nm with an emission wavelength of 373 nm. The intensity ratio (Ig) at wavelengths of 330 nm and 331 nm was taken. 330 / I 331 Plotting the logarithm of the polymer concentration, the abscissa corresponding to the point of abrupt change in the curve is lg(CMC).

[0050] like Figure 7 As shown, the critical micelle concentration of NE-PEG2000-GPLGIAGQ-PLA4000 was measured to be 10.0 μg / L in this embodiment.

[0051] Example 3: This embodiment provides a NE-PEG2000-GPLGIAGQ-PLA4000 drug-loaded nanoformulation, and the preparation process is as follows: (1) Dissolve 5 mg of the anticancer drug doxorubicin in dimethyl sulfoxide and mix it with a dimethyl sulfoxide solution containing 30 mg of NE-PEG2000-GPLGIAGQ-PLA4000. Then add the mixed solution to a dialysis bag and dialyze with deionized water for 48-72 h to prepare nano micelles by dialysis. The obtained suspension is freeze-dried to obtain the target micelles. (2) The particle size, distribution, and zeta potential of the prodrug micelles were determined by dynamic light scattering (DLS). The particle size of the nano-drug-loaded formulation was found to be 117.53 nm, and the PDI was 0.21 ( ). Figure 8 The zeta potential is +7.54 mV.

[0052] Example 4: This embodiment explores the targeting effect of a neutrophil-mediated drug delivery system on tumor tissues, as follows: Five mg of desalted doxorubicin (DOX) and 20 mg of NE-PEG2000-GPLGIAGQ-PLA4000 and Mal-PEG2000-GPLGIAGQ-PLA4000 prepared in Example 1 were placed in clean centrifuge tubes. A mixed solvent of 3 mL acetone and 3 mL methanol was added, and the mixture was sonicated to completely dissolve the polymer and drug, forming a homogeneous and clear oil phase solution. Subsequently, under constant temperature magnetic stirring at 40°C, the oil phase solution was slowly added dropwise to 20 mL of phosphate buffer, and stirring was continued for 2 h to achieve thorough mixing and initial dispersion of the system. Next, the mixture was sonicated using a cell disruptor with the following parameters: power 200 W, operating mode 5 s on / 5 s off, and total sonication time 10 min, to further refine the droplets and accelerate the evaporation of the organic solvent. After sonication, the resulting dispersion was concentrated by ultracentrifugation, and unencapsulated free doxorubicin was removed by filtration, finally yielding drug-loaded amphiphilic polymer nanoparticle dispersions NE-NPs and untargeted NPs.

[0053] In a mouse E0771 orthotopic breast cancer xenograft model, intratumoral injection of lipopolysaccharide (LPS, 0.9 mg / kg) solution successfully established a local acute inflammation model. In this model, LPS injection promoted neutrophil migration by enhancing local inflammatory signals. Twelve hours after local inflammation, unmodified NE peptide nanoparticles (NPs) and NE-NPs prepared in Example 1 (both at a dose of 37.6 mg / kg) were intravenously injected, with mice without local inflammation receiving intravenous injection of NE-NPs as a control group. In vivo imaging was used to investigate the enrichment of the nanodelivery system at the tumor site at different time points. The results showed that the enrichment of the NE-NPs group in the tumor significantly increased within 6 hours after local inflammation. Furthermore, analysis of the fluorescence intensity at the tumor site quantitatively demonstrated that LPS pretreatment synergistically with NE-NPs effectively promoted the enrichment of the formulation at the tumor site. Figure 9 ).

[0054] Example 5 A mouse model of E0771 orthotopic breast cancer xenograft was established. The experimental animals were then randomly assigned to six different groups and received corresponding interventions, as detailed below: PBS group (G1): Injected with PBS.

[0055] LPS+PBS group (G2): PBS and 0.9 mg / kg LPS were injected.

[0056] LPS+DOX group (G3): 0.9 mg / kg LPS and 4 mg / kg DOX were injected.

[0057] LPS+NPs group (G4): 0.9 mg / kg LPS and 37.6 mg / kg NPs were injected.

[0058] The NE-NPs group without LPS stimulation (G5): 37.6 mg / kg NE-NPs were injected.

[0059] LPS+NE-NPs group (G6): 0.9 mg / kg LPS and 37.6 mg / kg NE-NPs were injected.

[0060] Tumor growth in each group of tumor-bearing mice was continuously monitored and growth curves were plotted. Figure 10 Compared with the PBS group (G1), LPS+PBS group (G2), LPS+DOX group (G3), LPS+NPs group (G4) and NE-NPs group without LPS stimulation (G5), the tumor growth inhibition effect of the G6 group was significantly superior.

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An amphiphilic enzyme-sensitive polymer, characterized in that, The chemical structure of the amphiphilic enzyme-sensitive polymer is shown in Formula 1. Formula 1 In Equation 1, n = 22~136; m = 55~111; Preferably, n=45; m=55.

2. The method for preparing the amphiphilic enzyme-sensitive polymer according to claim 1, characterized in that, Includes the following steps: (1) Maleimide-polyethylene glycol-succinimide propionate (Mal-PEG-SPA) was subjected to a nucleophilic substitution reaction with matrix metalloproteinase 2 responsive peptide (GPLGIAGQ) to obtain Mal-PEG2000-GPLGIAGQ; wherein, the chemical structure of Mal-PEG-SPA is shown in Formula 2; the chemical structure of matrix metalloproteinase 2 responsive peptide is shown in Formula 3; and the chemical structure of Mal-PEG-GPLGIAGQ is shown in Formula 4. Formula 2; Formula 3; Equation 4; (2) Mal-PEG-GPLGIAGQ and NH2-PLA are amidated to obtain Mal-PEG-GPLGIAGQ-PLA; wherein the chemical structure of NH2-PLA is shown in Formula 5; and the chemical structure of Mal-PEG-GPLGIAGQ-PLA is shown in Formula 6. Formula 5; Formula 6; (3) Mal-PEG-GPLGIAGQ-PLA is subjected to Michael addition reaction with neutrophil elastase-binding peptide (NE peptide) to obtain NE-PEG-GPLGIAGQ-PLA, which is the amphiphilic enzyme-sensitive polymer.

3. The preparation method according to claim 2, characterized in that, Step (1) specifically includes: nucleophilic substitution reaction of matrix metalloproteinase 2 responsive peptide and Mal-PEG-SPA in an organic solvent containing triethylamine.

4. The preparation method according to claim 3, characterized in that, The organic solvent is selected from at least one of N,N-dimethylformamide and dimethyl sulfoxide; Preferably, the organic solvent containing triethylamine is an anhydrous and oxygen-free solvent; Preferably, the nucleophilic substitution reaction takes 24-48 h, more preferably 24-36 h; Preferably, the nucleophilic substitution reaction is carried out at room temperature in the dark; Preferably, the molar ratio of the matrix metalloproteinase 2 responsive peptide to triethylamine is 1:2-5, more preferably 1:2-3; Preferably, the molar ratio of triethylamine to Mal-PEG2000-SPA is 3-6:

1.

5. The preparation method according to claim 2, characterized in that, Step (2) specifically includes: performing an amidation reaction of Mal-PEG-GPLGIAGQ, PLA-NH2 and peptide condensation reagent in an organic solvent containing triethylamine.

6. The preparation method according to claim 5, characterized in that, The organic solvent is selected from at least one of N,N-dimethylformamide and dimethyl sulfoxide; Preferably, the amidation reaction is carried out under light-protected conditions; Preferably, the polypeptide condensing agent is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; Preferably, the molar ratio of Mal-PEG-GPLGIAGQ to the polypeptide condensation reagent is 1:1-4, more preferably 1:1.5-2; Preferably, the molar ratio of the polypeptide condensing agent to triethylamine is 1-2:3; Preferably, the molar ratio of triethylamine to PLA4000-NH2 is 1-5:1, more preferably 2:1; Preferably, the amidation reaction takes 8-24 h, more preferably 8-12 h.

7. The preparation method according to claim 2, characterized in that, Step (3) specifically includes: performing a Michael addition reaction between Mal-PEG-GPLGIAGQ-PLA and neutrophil elastase-binding peptide in a mixed solution containing tris(2-carboxyethyl)phosphine, dimethyl sulfoxide, and PBS buffer.

8. The preparation method according to claim 7, characterized in that, The Michael addition reaction is carried out in an inert atmosphere; Preferably, the molar ratio of Mal-PEG-GPLGIAGQ-PLA to neutrophil elastase-binding peptide is 1:1-2; Preferably, the molar ratio of the neutrophil elastase-binding peptide to tris(2-carboxyethyl)phosphine is 1-6:1, more preferably 3:1; Preferably, the Michael addition reaction takes 24-120 h, more preferably 48 h.

9. The use of the amphiphilic enzyme-sensitive polymer of claim 1 in the preparation of a drug delivery system.

10. The application according to claim 9, characterized in that, Application in the preparation of enzyme-responsive antitumor drug delivery systems.

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