Ateplase based on responsive polypeptide modified PLGA nanoparticles and preparation method thereof

Abstract

The invention discloses ateplase based on responsive polypeptide modified PLGA nano-particles and a preparation method thereof.The ateplase is loaded on the PLGA nano-particles through responsive polypeptide, the responsive polypeptide can be specifically degraded by cerebral apoplexy related enzymes in a blood brain barrier damaged area, and fixed-point release of the ateplase in a focus area is achieved; the preparation method comprises the following steps: S1, preparing a PLGA solution, and adjusting the pH of the PLGA solution to be alkaline; s2, reacting to obtain a PLGA solution grafted with a biological cross-linking agent I; s3, reacting to obtain a responsive polypeptide modified PLGA solution; s4, reacting to obtain an artiplase solution grafted with a biological cross-linking agent II; s5, mixing the solutions in S3 and S4, and reacting to obtain a composite solution; s6, dialyzing, centrifuging, purifying and concentrating to obtain a finished product. According to the present invention, the novel arteplase with characteristics of strong targeting property and high drug stability can be provided, such that the focus specific delivery and the controlled release of the cerebral ischemia region can be achieved, and the safety and the effectiveness of the cerebral apoplexy treatment can be improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to alteplase based on responsive polypeptide-modified PLGA nanoparticles and a preparation method thereof. Background Art

[0002] Stroke, especially ischemic stroke, is one of the leading causes of death and disability worldwide. According to statistics from the World Health Organization, stroke causes millions of deaths and long-term disability each year, placing a heavy burden on patients and their families. The main strategies for the current clinical treatment of ischemic stroke include intravenous thrombolysis and intravascular mechanical thrombectomy. Among them, tissue plasminogen activator (tPA), also known as alteplase, is the only intravenous thrombolytic drug approved by the US FDA for ischemic stroke. Its use within 4.5 hours of onset can significantly improve patient prognosis.

[0003] However, alteplase therapy has several key limitations. First, alteplase has a short half-life and limited circulation time, requiring frequent dosing and increasing the risk of bleeding complications. Second, alteplase lacks targeted distribution, which can lead to systemic depletion of coagulation factors and serious adverse events such as intracranial hemorrhage. Third, the ischemia-reperfusion process following thrombolysis can trigger a surge in reactive oxygen species (ROS), damaging the blood-brain barrier (BBB) and exacerbating neuronal death and brain tissue damage.

[0004] Furthermore, although endovascular mechanical thrombectomy has demonstrated promising efficacy in patients with large vessel occlusion, this method has high technical barriers to entry and relies heavily on hospital facilities and specialized personnel, hindering widespread adoption at the grassroots level. Therefore, intravenous thrombolysis with alteplase remains the preferred option for most stroke patients. However, the current lack of effective delivery strategies that can prolong drug efficacy, improve safety, and provide brain protection severely hinders its clinical adoption and efficacy improvement. Summary of the Invention

[0005] In response to the above problems, the present invention aims to provide alteplase based on responsive polypeptide-modified PLGA nanoparticles and a preparation method thereof.

[0006] The technical solutions of the present invention are as follows:

[0007] On the one hand, an alteplase based on responsive polypeptide-modified PLGA nanoparticles is provided. Alteplase is loaded on the PLGA nanoparticles through the responsive polypeptide. The responsive polypeptide can be specifically degraded by stroke-related enzymes in the damaged area of the blood-brain barrier, thereby achieving the targeted release of alteplase in the lesion area.

[0008] Preferably, the responsive polypeptide is an MMP-9 responsive polypeptide.

[0009] On the other hand, a method for preparing alteplase based on responsive polypeptide-modified PLGA nanoparticles as described above is also provided, comprising the following steps:

[0010] S1: preparing a PLGA solution and adjusting the pH of the PLGA solution to alkaline;

[0011] S2: adding the bio-crosslinking agent 1 to the PLGA solution to react and obtain the PLGA solution grafted with the bio-crosslinking agent 1;

[0012] S3: adding the responsive polypeptide to the PLGA solution grafted with the bio-crosslinking agent 1 to react and obtain a responsive polypeptide-modified PLGA solution;

[0013] S4: preparing a second biocrosslinking agent solution and an alteplase solution respectively, and then mixing the second biocrosslinking agent solution and the alteplase solution to react to obtain an alteplase solution grafted with the second biocrosslinking agent;

[0014] S5: mixing the PLGA solution modified with the responsive polypeptide with the alteplase solution grafted with the second biocrosslinking agent to react to obtain a composite solution;

[0015] S6: dialyzing, centrifuging, purifying, and concentrating the composite solution to obtain the alteplase based on the responsive polypeptide-modified PLGA nanoparticles.

[0016] Preferably, in step S1, an organic pH regulator is used to adjust the pH of the PLGA solution to 8-10.

[0017] Preferably, the organic pH regulator is any one of triethylamine, diisopropylethylamine, pyridine, and potassium tert-butoxide.

[0018] Preferably, in steps S2-S5, the amount of the grafting material is kept in excess compared to the amount of the grafted material.

[0019] Preferably, in step S2, the first biocrosslinker is maleimide-N-hydroxysuccinimide ester or a bifunctional PEG linker.

[0020] Preferably, in step S4, the second biocrosslinking agent is N-hydroxysuccinimidyl-3-(2-pyridyldithio) propionate, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate or dibenzooctyne-azide.

[0021] Preferably, in step S3, the responsive polypeptide is an MMP-9 responsive polypeptide.

[0022] Preferably, in steps S2-S5, each reaction is carried out at room temperature, and step S2 is reacted for 3-6 hours, step S3 is reacted for 6-12 hours, step S4 is reacted for 1-3 hours, and step S5 is reacted for 10-12 hours.

[0023] The beneficial effects of the present invention are:

[0024] The present invention can provide a novel alteplase with strong targeting and high drug stability, achieve lesion-specific delivery and controlled release to the cerebral ischemic area, and improve the safety and effectiveness of stroke treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 Schematic diagram of the preparation principle of alteplase based on responsive polypeptide-modified PLGA nanoparticles of the present invention;

[0027] Figure 2 This is a schematic diagram of the test results of the inhibitory pathway effect in Example 1;

[0028] Figure 3 This is a schematic diagram of the thrombolytic effect test results of Example 1;

[0029] Figure 4 This is a schematic diagram of the targeting effect test results of Example 1;

[0030] Figure 5 Schematic diagram of the stability test results of Example 1. DETAILED DESCRIPTION

[0031] The present invention is further described below with reference to the accompanying drawings and examples. It should be noted that, in the absence of conflict, the embodiments in this application and the technical features in the embodiments can be combined with each other. It should be noted that, unless otherwise specified, all technical and scientific terms used in this application have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs. The use of similar words such as "include" or "comprising" in the present invention means that the elements or objects appearing before the word cover the elements or objects listed after the word and their equivalents, without excluding other elements or objects.

[0032] On the one hand, the present invention provides alteplase based on responsive polypeptide-modified PLGA nanoparticles, in which alteplase is loaded on the PLGA nanoparticles via the responsive polypeptide. The responsive polypeptide can be specifically degraded by stroke-related enzymes in the damaged area of the blood-brain barrier, thereby achieving the targeted release of alteplase in the lesion area.

[0033] In the present invention, alteplase can be precisely released at the ischemic lesion through the responsive polypeptide, thereby enhancing targeting, effectively avoiding systemic nonspecific thrombolysis, and reducing the risk of bleeding; the PLGA nanoparticle encapsulation structure can significantly prolong the circulation time of alteplase in the body and improve the stability of the drug.

[0034] In one specific embodiment, the present invention can also be used with the small molecule anti-inflammatory drug Bindali via oral gavage to target and inhibit the CCL2 / CCR2 inflammatory pathway, reducing neutrophil migration into brain tissue and inhibiting secondary inflammatory damage to the blood-brain barrier by neutrophils, further enhancing neuroprotection and thus improving protection of the blood-brain barrier. This combined strategy achieves a triple effect of thrombolysis, neuroprotection, and inflammation control, significantly extending the therapeutic window and therapeutic benefits for ischemic stroke.

[0035] In a specific embodiment, the responsive polypeptide is an MMP-9 responsive polypeptide. MMP-9 (matrix metalloproteinase 9) itself is an enzyme that can directly damage the endothelial cells of the blood-brain barrier. It is one of the main downstream underlying proteins of many inflammatory pathways, especially the NF-κB pathway, which is known to be upregulated by alteplase. In this embodiment, the use of MMP-9 responsive polypeptide can ensure the stability and efficiency of drug release. Although MMP-2 and the like are also stroke-related enzymes, their distribution and upregulation in neutrophils, the cells with the fastest reaction and recruitment, are not as extensive and rapid as MMP-9. Therefore, this embodiment preferably uses MMP-9 responsive polypeptide as the responsive polypeptide to improve the stability and efficiency of drug release.

[0036] In the above embodiment, after the present invention enters the damaged area of the blood-brain barrier, MMP-9 cleaves the polypeptide linker arm, triggering the precise release of alteplase to perform local thrombolysis.

[0037] On the other hand, the present invention also provides a method for preparing alteplase based on responsive polypeptide-modified PLGA nanoparticles as described in any one of the above, comprising the following steps:

[0038] S1: preparing a PLGA solution and adjusting the pH of the PLGA solution to alkaline;

[0039] S2: adding the bio-crosslinking agent 1 to the PLGA solution to react and obtain the PLGA solution grafted with the bio-crosslinking agent 1;

[0040] S3: adding the responsive polypeptide to the PLGA solution grafted with the bio-crosslinking agent 1 to react and obtain a responsive polypeptide-modified PLGA solution;

[0041] S4: preparing a second biocrosslinking agent solution and an alteplase solution respectively, and then mixing the second biocrosslinking agent solution and the alteplase solution to react to obtain an alteplase solution grafted with the second biocrosslinking agent;

[0042] S5: mixing the PLGA solution modified with the responsive polypeptide with the alteplase solution grafted with the second biocrosslinking agent to react to obtain a composite solution;

[0043] S6: dialyzing, centrifuging, purifying, and concentrating the composite solution to obtain the alteplase based on the responsive polypeptide-modified PLGA nanoparticles.

[0044] In a specific embodiment, in step S1, the pH of the PLGA solution is adjusted to 8-10 using an organic pH regulator. Optionally, the organic pH regulator is any one of triethylamine, diisopropylethylamine, pyridine, and potassium tert-butoxide. In this embodiment, adjusting the pH using an organic pH regulator can improve the success rate and efficiency of grafting between PLGA and the biocrosslinker.

[0045] In one specific embodiment, in steps S2-S5, the amount of grafting agent is maintained in excess relative to the amount of grafted agent. Specifically, in step S2, the amount of biocrosslinker 1 is in excess relative to PLGA; in step S3, the amount of responsive polypeptide is in excess relative to PLGA grafted with biocrosslinker 1; in step S4, the amount of biocrosslinker 2 is in excess relative to alteplase; and in step S5, the amount of alteplase grafted with biocrosslinker 2 is in excess relative to PLGA modified with the responsive polypeptide.

[0046] In a specific embodiment, in step S2, the first biocrosslinker is maleimide-N-hydroxysuccinimide ester (mal-NHS) or a bifunctional PEG linker; in step S4, the second biocrosslinker is N-hydroxysuccinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate or dibenzooctyne-azide; in step S3, the responsive polypeptide is an MMP-9 responsive polypeptide.

[0047] In a specific embodiment, in steps S2-S5, each reaction is carried out at room temperature, and step S2 is reacted for 3-6 hours, step S3 is reacted for 6-12 hours, step S4 is reacted for 1-3 hours, and step S5 is reacted for 10-12 hours.

[0048] Example 1

[0049] An alteplase based on responsive peptide modified PLGA nanoparticles, such as Figure 1 As shown, it is prepared by the following steps:

[0050] (1) Dissolve 5 mg of PLGA in 3 mL of DMSO solution and add 50 μL of triethylamine to adjust the pH to 8;

[0051] (2) Add 0.5 mg of mal-NHS to the DMSO solution of PLGA and stir the mixture at room temperature for 4.5 h;

[0052] (3) Add 0.5 mg of MMP-9 responsive peptide and continue the reaction at room temperature for 6 h;

[0053] (4) SPDP was dissolved in DMSO to prepare a 20 mM solution. Subsequently, 25 μL of the SPDP DMSO solution was added to 1 mg of tPA (alteplase) dissolved in PBS (phosphate-buffered saline) and the mixture was stirred at room temperature for 1 h to obtain an SPDP-tPA solution.

[0054] (5) adding all of the SPDP-tPA solution from step (4) dropwise to the solution after the reaction in step (3), stirring overnight to obtain a tPLGA solution;

[0055] (6) The tPLGA solution of step (5) is dialyzed (Mw=1000Da) for two days, and the suspension is collected for centrifugation, purification and concentration to obtain the alteplase based on the responsive polypeptide-modified PLGA nanoparticles.

[0056] Example 2

[0057] Different from Example 1, the solvent used in step (1) of this embodiment is DMF, the pH adjusting reagent is diisopropylethylamine, and the reaction solution in step (4) is physiological saline.

[0058] Example 3

[0059] Different from Example 1, the amount of PLGA used in step (1) of this example is 10 mg, and the amount of MMP-9 responsive peptide used in step (3) is 1 mg.

[0060] Example 4

[0061] Different from Example 1, the stirring reaction time in step (2) of this example is 6 hours, and the stirring reaction time in step (4) is 3 hours.

[0062] Example 5

[0063] Different from Example 1, the dialysis bag in step (6) of this example has Mw=2000Da and the duration is 1.5 days.

[0064] Test Example 1

[0065] Autologous thrombi were prepared (mouse arterial blood was collected and placed in a PE-50 tube, allowed to stand for 1 hour to form a thrombus, and then refrigerated at 4°C for 23 hours). The thrombi were washed free of red blood cells and suspended in normal saline for later use. C57BL / 6 mice were then anesthetized with 5% isoflurane using a gas anesthesia apparatus, and the neck vessels were exposed. The common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were isolated. The distal end of the ECA was ligated and then ligated with a slipknot. The ICA was clamped with a microvascular clamp, and a slit was created in the distal ECA. A microcatheter containing a thin wire was inserted through the ECA to the bifurcation of the ICA. The catheter was fixed and bent toward the ICA. The wire was then withdrawn, and a 1.5 cm long thrombus was slowly injected. A timer was kept for 1 minute after the thrombus was injected. The catheter was removed, the "Y" opening was quickly ligated, and the slipknot on the CCA was released. The skin was then sutured, and the mice were disinfected and kept warm. Two hours after modeling, four groups of C57BL / 6 mice were injected with 200 μL of normal saline, 200 μL of tPA solution (150 μg / mL) through the tail vein, 200 μL of tPA solution (150 μg / mL) through the tail vein + 200 μL of Bindarit solution (1.25 mg / mL) through oral gavage, 200 μL of tPLGA solution (1.3 mg / mL) through the tail vein + 200 μL of Bindarit solution (1.25 mg / mL) through oral gavage, and a sham operation group were included, namely saline, tPA, tPA+Bindarit, tPLGA+Bindarit and sham groups.

[0066] After anesthetizing and fixing the mouse, apply pressure to the eye socket to promote congestion. Quickly remove the eyeball and collect blood into an anticoagulant or centrifuge tube. Pressure should then be applied to stop bleeding. Serum separation or whole blood processing should be performed as needed. Ensure the depth of anesthesia, control the amount of blood collected, and adhere to ethical standards. Remove the ELISA kit from the refrigerator 30 minutes in advance and equilibrate to room temperature. Wash buffer: Concentrated wash buffer removed from the refrigerator may crystallize. This is normal. Warm and gently shake to completely dissolve the crystals before reconstitution. Dilute the concentrated wash buffer with double-distilled water (1:20). Return any unused portion to 4°C. Standard: Add 0.5 mL of standard / specimen diluent to the lyophilized standard. Once completely dissolved, allow to stand for 15 minutes to mix (concentration of 100,000 pg / mL). Then dilute as needed. The following concentrations are used for the standard curve: 100,000, 50,000, 25,000, 12,500, 6,250, 3,125, 1,562.5, and 0 pg / mL. Diluted standards should not be reused. Unused standards should be divided into aliquots according to the single-use amount and stored at -20 to -70°C for single use to avoid repeated freezing and thawing. Prepare various solutions according to the above preparations. Determine the number of plates required based on the number of samples to be tested and the number of standards, and add one well as a blank control well. Add the specimen and different concentrations of standards (100 μL / well) to the corresponding wells, and add enzyme-labeled antibody working solution (50 μL / well). Seal the reaction wells with sealing tape (only add standard / sample diluent to the zero well) and incubate at room temperature for 180 minutes (except for the blank control well). Thorough mixing is particularly important for the reaction results. Use a micro-oscillator (minimum frequency 700 rpm). Wash the plate 4 times: (1) Automatic plate washer: The required amount of washing solution injected is 350 μL, and the interval between injection and aspiration is 15-30 seconds. (2) Manual plate washing: Shake off all liquid in the wells, add 350 μL of washing solution to each well, let it stand for 30 seconds, shake off all liquid, pat dry on thick absorbent paper, add 100 μL / well of color developer, protect from light, and incubate at room temperature for 20-25 minutes. Add 100 μL / well of stop solution, mix well, and immediately measure the OD450 value (within 5 minutes).

[0067] After obtaining the OD values under gradient concentration, the concentrations of CCL2 and MMP-9 in the samples were determined by plotting the standard curve method, and the parallel samples were also included in the plotting range. The results are as follows: Figure 2 As shown. Figure 2 To evaluate whether the CCL2 pathway and the downstream inflammatory factor MMP-9 that damages the blood-brain barrier caused by cerebral embolism in mice have changed under the action of the present invention. Figure 2 It can be seen that the expression of proteins in the pathway inhibited by Bindarit was significantly inhibited by alteplase based on the responsive polypeptide-modified PLGA nanoparticles of the present invention, and the inhibitory effect was stronger than that of traditional alteplase.

[0068] Test Example 2

[0069] Male C57 / BL6 mice weighing 25-30 g were selected, anesthetized, fixed on an operating table, and the chest skin disinfected with iodine. The skin was incised along the midline of the chest, the muscles and fascia separated, and the common carotid artery was exposed. A rubber gasket was placed beneath the artery to prevent the ferric chloride from damaging other tissues in the body. A 10% by weight FeCl3 solution was prepared, and a small piece of filter paper was soaked in it. The filter paper was then attached to the exposed femoral artery for 5 minutes to induce thrombosis. After the operation, the filter paper and gasket were removed and the incision was sutured. Immediately, 200 μL of normal saline, 200 μL of tPA (150 μg / mL), and 200 μL of tPLGA (1.3 mg / mL) were injected into the tail vein, respectively. The mice were then placed in a warm environment to wake up.

[0070] Thrombosis and thrombus dissolution were evaluated by laser speckle imaging system at different time points (0h, 0.5h, 1h, 1.5h, 2h) after surgery. Figure 3 As shown. Figure 3 As can be seen, tPLGA has a good effect on thrombolysis in vivo, with a basic thrombolytic capacity that is stronger than tPA and significantly superior to tPA. This demonstrates that the thrombolytic capacity of alteplase based on the responsive polypeptide-modified PLGA nanoparticles of the present invention is stronger than that of traditional alteplase.

[0071] Test Example 3

[0072] Two hours after establishing the thrombosis model, four groups of C57BL / 6 mice (n=12) were injected via the tail vein with 200 μL of IR780, 200 μL of tPA solution (150 μg / mL), and 200 μL of tPLGA solution (1.3 mg / mL). Anesthesia was then performed using a gas anesthesia system. After hair removal, the brain was directly observed using a live imaging device at 0, 0.5, 1, 1.5, and 2 hours. The results are shown in Figure 2. Figure 4 shown.

[0073] from Figure 4 It can be seen that tPLGA gradually accumulates in the injured hemisphere within 2 hours and has better, more sustained and targeted fluorescence compared to PLGA, proving that the tPLGA of the present invention has the function of passive targeting.

[0074] Test Example 4

[0075] 5 mg of tPLGA nanoparticles were uniformly dispersed in 5 mL of PBS (pH 7.4), and then placed on a shaker (37°C, 100 rpm). The hydrated particle size and potential at different time points within 7 days were measured by DLS. The results are as follows: Figure 5 shown.

[0076] from Figure 5 It can be seen that the size and potential of the tPLGA nanoparticles did not change significantly over the 7 days, remaining at approximately 132 nm and -70 mV, respectively. This demonstrates that the tPLGA prepared by the present invention has a stable structure and size and is not prone to disintegration or aggregation.

[0077] It should be noted that the above test examples are only partial test results of Example 1 of the present invention. The alteplase based on responsive polypeptide-modified PLGA nanoparticles in other embodiments of the present invention have similar effects to Example 1, all have good stability, can target the release of alteplase, and improve the thrombolytic ability of alteplase.

[0078] In summary, the present invention can release alteplase in a targeted manner and improve the thrombolytic ability of alteplase. Compared with the prior art, the present invention is a significant improvement.

[0079] The above description is merely a representative embodiment of the present invention and does not constitute any form of limitation to the present invention. Any technical personnel familiar with the present invention who, without departing from the scope of the technical solution of the present invention, makes some changes or modifications to the embodiments disclosed above using the technical contents disclosed above are equivalent embodiments of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. An alteplase based on responsive polypeptide-modified PLGA nanoparticles, characterized in that: Alteplase is loaded onto PLGA nanoparticles via a responsive peptide, which can be specifically degraded by stroke-related enzymes in areas where the blood-brain barrier is damaged, thereby achieving the targeted release of alteplase in the lesion area.

2. The alteplase based on responsive polypeptide-modified PLGA nanoparticles according to claim 1, characterized in that: The responsive polypeptide is an MMP-9 responsive polypeptide.

3. The method for preparing alteplase based on responsive polypeptide-modified PLGA nanoparticles according to claim 1 or 2, characterized in that: The following steps are involved: S1: preparing a PLGA solution and adjusting the pH of the PLGA solution to alkaline; S2: adding the bio-crosslinking agent 1 to the PLGA solution to react and obtain the PLGA solution grafted with the bio-crosslinking agent 1; S3: adding the responsive polypeptide to the PLGA solution grafted with the bio-crosslinking agent 1 to react and obtain a responsive polypeptide-modified PLGA solution; S4: preparing a second biocrosslinking agent solution and an alteplase solution respectively, and then mixing the second biocrosslinking agent solution and the alteplase solution to react to obtain an alteplase solution grafted with the second biocrosslinking agent; S5: mixing the PLGA solution modified with the responsive polypeptide with the alteplase solution grafted with the second biocrosslinking agent to react to obtain a composite solution; S6: dialyzing, centrifuging, purifying, and concentrating the composite solution to obtain the alteplase based on the responsive polypeptide-modified PLGA nanoparticles.

4. The method for preparing alteplase based on responsive polypeptide-modified PLGA nanoparticles according to claim 3, characterized in that: In step S1, the pH of the PLGA solution is adjusted to 8-10 using an organic pH regulator.

5. The method for preparing alteplase based on responsive polypeptide-modified PLGA nanoparticles according to claim 4, characterized in that: The organic pH regulator is any one of triethylamine, diisopropylethylamine, pyridine, and potassium tert-butoxide.

6. The method for preparing alteplase based on responsive polypeptide-modified PLGA nanoparticles according to claim 3, characterized in that: In steps S2 to S5, the amount of the grafting material is kept in excess compared to the amount of the grafted material.

7. The method for preparing alteplase based on responsive polypeptide-modified PLGA nanoparticles according to claim 3, characterized in that: In step S2, the first biocrosslinker is maleimide-N-hydroxysuccinimide ester or a bifunctional PEG linker.

8. The method for preparing alteplase based on responsive polypeptide-modified PLGA nanoparticles according to claim 3, characterized in that: In step S4, the second biocrosslinking agent is N-hydroxysuccinimidyl-3-(2-pyridyldithio) propionate, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate or dibenzooctyne-azide.

9. The method for preparing alteplase based on responsive polypeptide-modified PLGA nanoparticles according to claim 3, characterized in that: In step S3, the responsive polypeptide is an MMP-9 responsive polypeptide.

10. The method for preparing alteplase based on responsive polypeptide-modified PLGA nanoparticles according to any one of claims 3 to 9, characterized in that: In steps S2-S5, each reaction is carried out at room temperature, and step S2 is reacted for 3-6 hours, step S3 is reacted for 6-12 hours, step S4 is reacted for 1-3 hours, and step S5 is reacted for 10-12 hours.

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