Enzyme-responsive composite scaffold for muscle injury repair and preparation method thereof

Abstract

The application discloses an enzyme response type composite scaffold for muscle injury repair and a preparation method thereof, and belongs to the technical field of polymer scaffolds. The application takes a decellularized matrix as the scaffold, and combines with MMP-2 responsive nanomicelles loaded with lipoxin LxA4 and SDF-1 alpha, so that precise and long-acting release of drugs at the injury site is realized. Through the synergistic effect of LxA4 and SDF-1 alpha, the inflammatory reaction at the injury site is reduced, and the recruitment and migration of endogenous stem cells are enhanced, so that the repair cycle is shortened. In addition, the composite scaffold of the application also ensures the synchronism of the degradation speed of the scaffold and the tissue regeneration speed, and ensures the effective support of the scaffold in the tissue healing process. The application provides a safer and more effective solution for the treatment of muscle tissue defects, and promotes the application of tissue engineering in clinical practice.

Description

Technical Field

[0001] The present invention belongs to the technical field of polymer scaffolds, and in particular relates to an enzyme-responsive composite scaffold for repairing muscle damage and a preparation method thereof. Background Art

[0002] Trauma and tumor resection can lead to volumetric muscle loss (VML). This type of injury not only results in permanent loss of contractile tissue but is also accompanied by significant strength loss. Currently, there is no standardized regenerative treatment. VML injuries commonly occur in the limbs, abdomen, and facial muscles. Existing treatment strategies focus on implanting bioactive substitutes (such as satellite cells or basal lamina components) to activate the host's regenerative potential, promote integration of new muscle tissue with the remaining muscle groups, and restore functional function.

[0003] Among numerous therapeutic approaches, acellular bioscaffolds have garnered significant attention due to their unique advantages. These biomaterials, derived from processing natural extracellular matrices, avoid the donor-site damage associated with autologous muscle transplantation and have been FDA-approved for safety in soft tissue repair. Preclinical animal studies and small-scale human studies have demonstrated that these scaffolds can provide both physical support and a biosignaling microenvironment for muscle regeneration. However, their clinical translation remains a key bottleneck: the degree of muscle regeneration after implantation is generally below physiological levels. This may be due to the scaffold design's inability to accurately mimic the spatiotemporal regulation of skeletal muscle regeneration. The natural regeneration process involves the precise spatiotemporal regulation of multiple steps, including satellite cell activation, inflammatory cell recruitment, and angiogenesis, and existing scaffolds are unable to fully replicate this complex biological process. Therefore, the development of novel enzyme-responsive composite scaffolds for muscle repair has become a critical and pressing research topic. Summary of the Invention

[0004] To address the problems of poor release precision, asynchronous degradation and tissue regeneration, and inadequate control of postoperative chronic inflammation in existing composite drug-loaded stents for muscle injury repair, the present invention provides an enzyme-responsive composite scaffold for muscle injury repair and its preparation method. This scaffold utilizes a decellularized extracellular matrix (dECM) as a scaffold combined with MMP-2-responsive nanomicelles loaded with lipoxin A4 (LxA4) and SDF-1α to achieve precise and long-lasting drug release at the injury site. The synergistic effect of LxA4 and SDF-1α reduces inflammatory responses at the injury site while enhancing the recruitment and migration of endogenous stem cells, thereby shortening the repair cycle. Furthermore, the composite scaffold synchronizes its degradation rate with tissue regeneration, ensuring effective support for tissue healing. This invention provides a safer and more effective solution for the treatment of VML, promotes the application of tissue engineering in clinical practice, and significantly improves patients' postoperative recovery and quality of life.

[0005] The object of the present invention is to achieve the following goals:

[0006] In a first aspect, the present invention provides a method for preparing MMP-2 enzyme-responsive nanomicelles, comprising the following steps:

[0007] (1) Dissolve 1-vinyl-1H-imidazole and 4-bromomethylphenylboronic acid in acetonitrile and react at 70-90°C for 5-24 hours to obtain compound 3-(4-boronic acid benzyl)-1-vinyl-1H-imidazole (BVII);

[0008] (2) A THF solution containing 1-(tert-butyl) 5-(2,5-dioxopyrrolidin-1-yl)(tert-butoxycarbonyl) glutamate was added dropwise to an alkaline aqueous solution of 2-aminobutyric acid and reacted for 5 to 24 hours to obtain compound 1; ethylenediamine was dissolved in water, the pH was adjusted to 8 to 9, and the solution was placed in an ice bath, and then a chloroform solution containing acryloyl chloride was added dropwise, and the reaction was stirred for 2 to 10 hours to obtain compound 2; compound 1 and N , N' -Carbonyldiimidazole is dissolved in dichloromethane and reacted at room temperature for 1-5 hours, compound 2 and triethylamine are added, and the reaction is carried out at room temperature for 5-24 hours to obtain compound 3; compound 3 is dissolved in dichloromethane, and trifluoroacetic acid is added, and the reaction is carried out at room temperature for 5-24 hours to obtain N-(1-((2-acrylamidoethyl)amino)-1-oxobutane-2-yl)glutamine trifluoroacetate (NAOGT);

[0009] (3) Dissolve the MMP-2 responsive peptide PLGLAG and triethylamine in N , N-dimethylformamide, slowly add acryloyl chloride to react to obtain compound 4; compound 4, 2-(7-azabenzotriazole)- N , N , N' , N' -Tetramethyluronium hexafluorophosphate was dissolved in DMF and then added N , N -diisopropylethylamine, react at room temperature for 20-60 minutes, add DMF containing 3,6,9-trioxaundecane-1,11-diamine, and react at room temperature for 5-24 hours to obtain an MMP-2 responsive compound (POP);

[0010] (4) The lipophilic small molecule LxA4 and the hydrophilic protein SDF-1α were dissolved in sterile PBS buffer to obtain LxA4 solution and SDF-1α solution, respectively. The LxA4 solution and SDF-1α solution were slowly added dropwise at a molar ratio of 1:10 to 10:1 and mixed. The mixture was incubated at room temperature in the dark for 20 to 60 minutes to spontaneously assemble into LxA4 / SDF-1α nanomedicines.

[0011] (5) The LxA4 / SDF-1α nanodrug obtained in step (4) is evenly mixed with an aqueous solution containing 3-(4-boronic acid benzyl)-1-vinyl-1H-imidazole (BVII) monomer, so that the BVII monomer is uniformly adsorbed on the surface of the LxA4 / SDF-1α nanodrug. Then, an MMP-2 responsive compound (POP) and N-(1-((2-acrylamidoethyl)amino)-1-oxobutane-2-yl)glutamine trifluoroacetate (NAOGT) monomer are added. An initiator is added to initiate a free radical polymerization reaction of 3-(4-boronic acid benzyl)-1-vinyl-1H-imidazole (BVII), MMP-2 responsive compound (POP), and N-(1-((2-acrylamidoethyl)amino)-1-oxobutane-2-yl)glutamine trifluoroacetate (NAOGT) monomer on the surface of the nanodrug to form nanomicelles, which are then freeze-dried to obtain the nanomicelles.

[0012] Based on the above technical solution, further, the molar ratio of 1-vinyl-1H-imidazole to 4-bromomethylphenylboronic acid in step (1) is 1~10:1.

[0013] Based on the above technical solution, further, the molar ratio of 2-aminobutyric acid and 1-(tert-butyl) 5-(2,5-dioxopyrrolidin-1-yl)(tert-butoxycarbonyl) glutamate described in step (2) is 1~5:1; the alkaline substance in the alkaline aqueous solution of 2-aminobutyric acid is sodium bicarbonate, and the molar ratio of 2-aminobutyric acid and sodium bicarbonate is 1:1~5.

[0014] Based on the above technical solution, further, the volume ratio of ethylenediamine and water in step (2) is 1:10-20, and the molar ratio of ethylenediamine and acryloyl chloride is 1:1-5;

[0015] Based on the above technical solution, further, the compound 1 and N , N' The molar ratio of the compound 1 to the carbonyldiimidazole is 1:1-2, and the molar ratio of the compound 1, the compound 2 and the triethylamine is 1:1-2:1-5.

[0016] Based on the above technical solution, further, the molar ratio of the MMP-2 responsive peptide PLGLAG, acryloyl chloride and triethylamine in step (3) is 1:1~3:2~10; compound 4, 2-(7-azabenzotriazole)- N , N , N' , N' -Tetramethyluronium hexafluorophosphate, N , N - the molar ratio of diisopropylethylamine to 3,6,9-trioxaundecane-1,11-diamine is 1:1~3:3~10:0.5~1;

[0017] Based on the above technical solution, further, the concentrations of the lipophilic small molecule LxA4 and the hydrophilic protein SDF-1α in sterile PBS buffer (pH 7.4) described in step (4) are both 0.5~10 mM.

[0018] Based on the above technical solution, further, an ultrasonic water bath is used to assist in the mixing process described in step (4), the ultrasonic frequency is 25~50 kHz, and the ultrasonic duration is 5~30 minutes.

[0019] Based on the above technical solution, further, the molar ratio of the LxA4 / SDF-1α nanodrug, 3-(4-boronic acid benzyl)-1-vinyl-1H-imidazole (BVII), MMP-2 responsive compound (POP), and N-(1-((2-acrylamidoethyl)amino)-1-oxobutane-2-yl)glutamine trifluoroacetate (NAOGT) monomer described in step (5) is 1:10~3000:0.1~30:10~3000.

[0020] Based on the above technical solution, further, the initiator described in step (5) is 1-5% by mass of ammonium persulfate and 3-10% by mass of tetramethylethylenediamine.

[0021] In a second aspect, the present invention provides MMP-2 enzyme-responsive nanomicelles prepared by the above preparation method.

[0022] In a third aspect, the application provides a preparation method of the MMP-2 enzyme-responsive composite scaffold, mainly comprising the following steps:

[0023] 1) Submerge the porcine dermis in a 0.3-0.7 M NaOH aqueous solution, oscillate at 50-200 rpm at room temperature for 5-12 h, wash, vacuum freeze-dry, and sterilize to obtain a decellularized matrix scaffold (dECM);

[0024] 2) Resuspend the MMP-2 enzyme-responsive nanomicelles in the above step in a PBS buffer (pH 7.4); immerse the decellularized matrix scaffold (dECM) obtained in step 1) in an ethanol solution of amino silane coupling agent with a volume percentage of 0.5-5% for 20-60 min, crosslink and solidify at 40-80℃, mix the crosslinked and solidified decellularized matrix scaffold with the MMP-2 enzyme-responsive nanomicelle solution, and rotate and co-incubate for 1-3 h, remove the free nanomicelles, and then vacuum freeze-dry to obtain the MMP-2 enzyme-responsive composite scaffold.

[0025] Based on the above technical solution, further, the washing process in step 1) is as follows: first wash with 0.1-0.3 M PBS buffer for 10-40 min, then ultrasonically treat with ultrapure water (25-50 kHz, 20-30℃) for 5-15 min, and finally rinse with deionized water for 5-15 min.

[0026] Based on the above technical solution, further, the mass ratio of the crosslinked and solidified decellularized matrix scaffold to the MMP-2 enzyme-responsive nanomicelles in step 2) is 100000:1-1000:1.

[0027] Based on the above technical solution, further, the incubation temperature in step 2) is 35-38℃, and the rotation speed is 30-100 rpm.

[0028] In a fourth aspect, the application provides the MMP-2 enzyme-responsive composite scaffold prepared by the above preparation method.

[0029] In a fifth aspect, the application provides the use of the above MMP-2 enzyme-responsive composite scaffold as a muscle injury repair material.

[0030] The application has the following beneficial effects compared with the prior art:

[0031] (1) The muscle scaffold based on the decellularized matrix (dECM) in the application realizes the synergistic release of LxA4 and SDF-1α in combination with the MMP-2-responsive nanomicelles, and a patch material with excellent biocompatibility, controllable degradation rate, and long-acting anti-inflammatory and tissue repair function is designed; this material has innovation in morphology, composition, and responsiveness, can precisely control the long-acting release of drugs, and optimizes the healing environment of muscle defects.

[0032] (2) The present application utilizes the synergistic effect of LxA4 and SDF-1α to reduce the inflammatory response at the injury site, while enhancing the recruitment and migration of endogenous stem cells, thereby shortening the repair period, and having very good application potential in regenerative medicine. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present application, the drawings involved in the embodiments will be briefly introduced below.

[0034] Figure 1 The synthesis process of the monomers required for the LSBPN nanomicelles in Example 1, wherein A is the synthesis process of 3-(4-benzyloxy)-1-ethenyl-1H-imidazole, B is the synthesis process of N-(1-((2-acrylamidoethyl)amino)-1-oxobutan-2-yl) glutamine trifluoroacetate, and C is the synthesis process of the MMP-2 responsive compound monomer.

[0035] Figure 2 TEM images (A-B) and corresponding DLS particle size distribution graphs (C-D) of the LSBPN nanomicelles obtained by different feeding ratios of the monomers in Example 1.

[0036] Figure 3 H&E staining tissue morphology of porcine dermis before decellularization (A) and after decellularization (B) in Example 2, and SEM images of longitudinal section (C) and cross section (D) after decellularization.

[0037] Figure 4 SEM images of the composite scaffold before nanodrug loading (A) and after loading (B) in Example 3.

[0038] Figure 5 Flow cytometry detection of the in vitro anti-inflammatory results of LSBPN NMs / dECM composite scaffolds in Example 4.

[0039] Figure 6 qPCR detection of the in vitro anti-inflammatory results of LSBPN NMs / dECM composite scaffolds in Example 4.

[0040] Figure 7 In vitro degradation results of LSBPN NMs / dECM composite scaffolds in Example 5.

[0041] Figure 8 Pictures of composite scaffolds implanted in muscle defects in Example 6.

[0042] Figure 9 MRI examination pictures of the composite scaffold implantation group and the defect group 6 weeks after the operation in Example 6. DETAILED DESCRIPTION

[0043] The application will be described in detail below with reference to the embodiments, but the embodiments of the application are not limited thereto. It is obvious that the embodiments described below are only some of the embodiments of the application, and other similar embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0044] Embodiment 1

[0045] The embodiment provides a preparation method of MMP-2 enzyme-responsive nanomicelles with different particle sizes, and the method comprises the following steps:

[0046] (1) Synthesis of 3-(4-benzyloxy) benzyl)-1-vinyl-1H-imidazole (BVII): 1-vinyl-1H-imidazole (1.88 g, 20 mmol) and 4-bromomethylphenylboronic acid (1.07 g, 5 mmol) were dissolved in 20 mL acetonitrile (ACN) and reacted at 80 ℃ overnight, ACN was removed by rotary evaporation, 25 mL deionized water was added to dissolve the product, the water layer was washed with DCM (25 mL x 3), and the product was concentrated and dried to obtain compound BVII (1.07 g, yield 69%) (A in Figure 1 ).

[0047] (2) Synthesis of N-(l-((2-acrylamidoethyl)amino)-l-oxobutan-2-yl) glutamine trifluoroacetate (NAOGT): 2-aminobutyric acid (0.62 g, 6 mmol) and sodium bicarbonate (1.01 g, 12 mmol) were dissolved in 20 mL water, l-(tert-butyl) 5-(2,5-dioxopyrrolidin-l-yl)(tert- butoxycarbonyl) glutamate (1.20 g, 3 mmol) was dissolved in 5 mL tetrahydrofuran (THF), then the THF solution of l-(tert-butyl) 5-(2,5-dioxopyrrolidin-l-yl)(tert- butoxycarbonyl) glutamate was added dropwise into the aqueous solution of 2- aminobutyric acid and reacted overnight, THF was removed by rotary evaporation, the pH was adjusted to about 3-5, ethyl acetate (EA) was extracted, the organic layer was washed with 1M hydrochloric acid and saturated sodium chloride solution twice, respectively, the organic layer was dried over anhydrous sodium sulfate, EA was removed by rotary evaporation, and the compound 1 (1.03 g, yield 89%) was obtained by drying. Ethylenediamine (5 mL, 75 mmol) was dissolved in 75 mL deionized water, the pH was adjusted to about 8-9 with concentrated hydrochloric acid, and then the solution was placed in an ice bath. Acryloyl chloride (6.7 mL, 82.5 mmol) was dissolved in 50 mL chloroform, and then it was added dropwise into the above aqueous solution. After several hours of reaction, the stirring was stopped, the layers were separated, the water layer was washed with chloroform (50 mL x 3) to remove excess acryloyl chloride, and the water layer was concentrated to obtain a white solid. 50 mL of methanol (MeOH) was added, and the mixture was filtered, and the filter cake was washed with methanol several times to obtain compound 2 (5.3 g, yield 47%).

[0048] Compound 1 (0.78 g, 2 mmol) and N , N' carbonyldiimidazole (CDI) (0.36 g, 2.22 mmol) were dissolved in 10 mL dichloromethane (DCM) and reacted at room temperature for 2 hours. Compound 2 (0.43 g, 2.82 mmol), TEA (0.7 mL, 5 mmol) and 5 mL DCM were added in sequence, and the mixture was reacted at room temperature overnight. The organic layer was washed with 1M hydrochloric acid (25 mL x 3) and saturated sodium bicarbonate solution (25 mL x 2), respectively, dried over anhydrous sodium sulfate, and purified by column separation (DCM:MeOH=95:5) to obtain compound 3 (0.76 g, yield 79%). Compound 3 (0.57 g, 1.17 mmol) was dissolved in 5 mL DCM, 5 mL trifluoroacetic acid (TFA) was added, and the mixture was reacted at room temperature overnight. The solvent was removed by rotary evaporation, and ethyl ether was added to precipitate a white solid (0.36 g, yield 72%), which was N-(l-((2-acrylamidoethyl)amino)-l-oxobutan-2-yl) glutamine trifluoroacetate (NAOGT) (B in Figure 1 ).

[0049] (3) Synthesis of MMP-2-responsive compound monomer (POP): MMP-2-responsive peptide PLGLAG (150.0 mg, 0.285 mmol) and 200 μL of triethylamine (TEA) were dissolved in 5 mL of N,N-dimethylformamide (DMF), acryloyl chloride (35 μL, 0.4275 mmol) was slowly added, and thin layer chromatography (TLC) analysis showed that the PLGLAG reaction was complete. Compound 4 (120.8 mg, yield 73%) was obtained by column separation and purification. Compound 4 (116.1 mg, 0.2 mmol), 2-(7-azabenzotriazol)-4- carbonitrile (HATU) (152.1 mg, 0.4 mmol), and diisopropylethylamine (DIPEA) (175 μL, 1 mmol) were dissolved in 2.5 mL of DMF and reacted at room temperature. After 30 minutes, 3,6,9-trioxaundecane-1,11-diamine (19.2 mg, 0.1 mmol) dissolved in 2.5 mL of DMF was added to the above reaction solution, and the reaction was carried out at room temperature overnight. The solvent was removed, and MMP-2-responsive compound (POP) (56.7 mg, yield 43%) was obtained by column separation and purification (C in FIG. 1). N , N N , N N' , N' N , N Figure 1

[0050] ​​​​​(4) Preparation and characterization of nanomicelles: Lipophilic small molecule LxA4 and hydrophilic protein SDF-1a were dissolved in sterile PBS buffer (pH 7.4) at a concentration of 1 mM and 1 mM, respectively. Then, they were slowly mixed at a molar ratio of 1:1, with the aid of an ultrasonic water bath (frequency 40 kHz, duration 10 min) to promote the self-assembly of molecules. After incubation at room temperature for 30 min in the dark, the spontaneous assembly of LxA4 / SDF-1a nanodrugs (referred to as LS) was completed, which relied on the hydrophobic interaction and non-covalent bond interaction between molecules to form stable nanostructures. The prepared LS was diluted to a final concentration of 1 nmol / mL (total volume 500 μL), and then slowly mixed with an equal volume of 500 nmol BVII monomer aqueous solution (i.e. each solution volume was 500 μL). During the slow addition process, gentle vortex was maintained to ensure uniform mixing. This process utilized the electrostatic interaction between the positively charged functional groups on the BVII molecule and the negative charge on the surface of LS to uniformly adsorb BVII monomers on the surface of LS, providing a platform for subsequent radical polymerization. After mixing, the mixture was allowed to stand at room temperature for 15 min to ensure complete complexation. Then, 0.05 nmol of POP and 10 nmol of NAOGT monomer were introduced into the system, and 2 μL of 2% ammonium persulfate (APS) and 2 μL of 5% tetramethyl ethylenediamine (TEMED) were added to initiate radical polymerization on the surface of the nanodrug, forming nanomicelles (LSBPN NMs). By adjusting the feed ratio of each component to the nanodrug, a series of nanomicelles with different particle sizes and tertiary amine oxide surface densities were prepared. The morphology of the nanomicelles was observed by TEM, and the hydrodynamic radius and particle size distribution were determined by DLS. Under the condition that the input amounts of LS, POP and NAOGT were kept constant, the input amount of BVII was increased from 500 nmol (A in FIG. 6, Figure 2 C in FIG. 6, Figure 2 D in FIG. 6), the particle size of the nanomicelles increased. After freeze-drying, LSBPN NMs were obtained. Figure 2 Figure 2 Example 2: Preparation of dECM

[0051] Example 2: Preparation of dECM

[0052] ​The present invention uses a 0.5 M NaOH aqueous solution to immerse porcine dermis and shake at 120 rpm at room temperature for 8 hours to achieve gentle decellularization of porcine dermal tissue. A gradient washing procedure is then performed: first, vortex washing is performed twice with 0.2 M PBS buffer (4°C) for 15 minutes to remove alkaline residues, followed by ultrapure water ultrasonic treatment (40 kHz, 25°C) for 10 minutes, and finally a final rinse with deionized water for 10 minutes to complete the sterilization pretreatment. The dehydration and drying stage uses vacuum freeze-drying technology (-40°C, 10 Pa) for ≥48 hours. The final product is sterilized by gamma irradiation (20 kGy dose) and sealed and packaged to obtain dECM. DNA quantitative testing shows that the residual DNA content of the decellularized porcine dermis tissue is 37.24 ± 2.74 ng / mg, and native collagen is retained. H&E staining demonstrates the effectiveness of decellularization, with no residual cell nuclei present and the interconnected ECM network structure preserved ( Figure 3 ).

[0053] Example 3: Loading of LSBPN NMs on dECM

[0054] Freeze-dried LSBPN NMs (800 nmol of BVII was added in Example 1) were reconstituted in 10 mM PBS buffer (pH 7.4) and ultrasonically dispersed (40 kHz, 100 W, 10 min) to obtain a homogeneous suspension (LSBPN concentration 5 mg / mL). The dECM scaffold (see Example 2) was sterilized by gamma irradiation and immersed in an ethanol solution containing 1% (v / v) aminosilane coupling agent for 30 min. The scaffold was then oven-cured for cross-linking at 60°C to enhance the surface positive charge and promote electrostatic adsorption of the nanomicelles. A gradient group of cross-linked and solidified dECM scaffolds and LSBPN mass ratios (5000:1, 7500:1, 10000:1, and 12500:1) was set, with n=3 per group. The LSBPN NMs suspension and dECM scaffold were added to a rotating co-incubation system (37°C, 50 rpm) according to the set ratio for 2 h. Free NMs were removed by centrifugation-displacement washing (3000×g, 10 min / time, repeated 5 times), followed by vacuum freeze-drying (pre-freezing at -80°C for 24 h, sublimation at 0.05 mBar for 48 h). The morphology was observed using SEM. Figure 4 SEM images before and after loading when the mass ratio of dECM to LSBPN NMs is 10000:1.

[0055] Example 4: In vitro anti-inflammatory effect of LSBPN NMs / dECM composite scaffold

[0056] Isolation and Culture of Bone Marrow-Derived Macrophages (BMDMs): 6–8-week-old C57BL / 6 male mice were anesthetized and sacrificed by cervical dislocation. The femurs and tibias were removed under sterile conditions, and the bone marrow cavity was flushed with PBS to obtain bone marrow cells. After cell count, the cells were plated in RPMI 1640 complete medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Recombinant mouse macrophage colony-stimulating factor (M-CSF, 20 ng / mL) was added and incubated at 37°C, 5% CO₂ for 7 days to obtain highly pure adherent M0 BMDMs. Polarized M1 cells were obtained by stimulating M0 cells with 100 ng / mL LPS for 24 hours.

[0057] M1 cells were plated at 500,000 / well in a 6-well plate and four groups (n = 3) were set up: LPS group (100 ng / mL LPS stimulation for 24 h, M1 polarization model), LPS + dECM (100 mg) group (to compare the effect of the decellularized scaffold itself), and LPS + dECM@LSBPN NMs (100 mg) group (to test the anti-inflammatory effect of the composite scaffold in Example 3). M0 cells (no stimulation) were used as a control. After 24 h of co-culture, the cells were collected and blocked with Fc blocker. Surface marker antibodies included PE-CD86 (M1 marker) and APC-CD206 (M2 marker). Flow cytometry (BD Fortessa) was used to detect M1 (CD86) and FlowJo software was used to analyze the expression of M1 (CD86) and M2 (CD206) in the cells. + ) and M2 type (CD206 + ) percentage change.

[0058] qPCR analysis of gene expression: Total RNA was extracted from each group of cells and reverse transcribed into cDNA. SYBR Green method was used for qPCR to detect the expression of the following genes: M1 factor: CD86, M2 factor: CD206, internal reference gene: GAPDH. The ΔΔCt method was used for expression analysis.

[0059] Flow cytometry results showed that CD86 + The proportion of macrophages increased, CD206 + The proportion of macrophages decreased; in the dECM@LSBPN NMs group, CD206 + The number of macrophages increased significantly, indicating that the scaffold promoted the polarization toward M2 type ( Figure 5 ); qPCR results: Compared with the LPS group, the expression of CD86 in the dECM@LSBPN NMs-treated group was significantly decreased, and the expression of CD206 was upregulated ( Figure 6 ), the difference was statistically significant (P<0.01).

[0060] Example 5: In vitro degradation of LSBPN NMs / dECM composite scaffolds

[0061] The in vitro degradation performance of the materials was evaluated by enzymatic hydrolysis, and the specific method was as follows: about 100 mg of sample (dECM of Example 2, LSBPN NMs in Example 3) was weighed into a 10 mL sterile centrifuge tube, 4 mL of 5 U / mL collagenase type I solution was added, and the tube was placed at an inclination at 37°C and oscillated at 70 rpm. At 3h, 6h, 1d, 2d, 4d, 1w, 10d, and 2w, 3 mL of supernatant was taken in a clean bench, and the hydroxyproline detection method was used for quantitative analysis. Fresh collagenase solution was added immediately after each sampling to maintain the stability of the reaction system. All supernatant samples were stored at 4°C, and the hydroxyproline content was determined uniformly. The cumulative degradation rate was calculated by comparing the ratio of the hydroxyproline release amount in the supernatant to the initial total content at each time point (degradation rate%=supernatant hydroxyproline content / initial total content x 100%), and the time-degradation rate curve was drawn to represent the degradation kinetics characteristics of the material. The above experimental samples were sterilized by EO, and the experimental reagents were also sterilized, and the experimental operation was carried out in a sterile environment.

[0062] The results are shown in Figure 7 As shown in the results, the results show that both groups of scaffold materials dECM and LSBPN NMs / dECM exhibit a typical rapid initial release trend during the entire degradation process, followed by a slow release stage, and finally tend to a plateau. From the degradation curve, the hydroxyproline release amount and the corresponding cumulative degradation rate of dECM and LSBPN NMs / dECM at each time point are basically consistent, with no significant difference, and the error range is highly overlapped, indicating that the degradation behavior of the two under enzymatic hydrolysis conditions is highly similar. Especially at key time nodes (such as 1d, 7d and 14d), both groups of samples showed similar degradation rates and cumulative release proportions. The above results show that under the current experimental settings, the in vitro enzymatic degradation performance of dECM and LSBPN NMs / dECM is comparable, with similar degradation kinetics characteristics, and is suitable for similar tissue engineering application scenarios.

[0063] Example 6:

[0064] The animal model was created using rats with bilateral tibialis anterior (TA) muscle defects. The defects were then filled with stents. All experimental rats were anesthetized before surgery, and their physical condition and mental state were assessed. The rats were weighed and recorded. Anesthesia was induced in an isoflurane chamber, and then maintained (2%-2.5%). After that, the rats were transferred to the surgical area. A depilatory cream was applied briefly to the knee joint area of ​​the hind limb to remove hair. A drape was laid, and the surgical area was sprayed with iodine and infused for several minutes for disinfection. Afterwards, the iodine was removed by wiping with medical alcohol to avoid residual irritation. The cut Φ = 6 mm composite stent (Example 3) was removed and rehydrated in sterile saline to fully restore the material's pore structure before removal.

[0065] The specific modeling procedures are as follows: (1) a lateral incision was made on the skin of the lateral lower limb of the rat; (2) the skin and fascia were separated by blunt dissection; (3) the fascia was separated from the muscle by sharp and blunt dissection; (4) the skin and fascia were dissected from the anterior surface of the anterior calf muscle group to expose the tibialis anterior (TA) muscle group; (5) the middle 1 / 3 of the TA was marked; (6) a metal plate was inserted between the TA and the extensor digitorum longus (EDL); (7) a full-thickness muscle defect was made in the middle of the TA using a 5 mm tissue biopsy device, and then the muscle tissue was removed and weighed (reaching 40% to 60% of the theoretical total weight of the TA, which cannot be repaired on its own). Sterile gauze was used to press the injured area to stop bleeding; (8) the prepared composite scaffold was implanted in the defect and fixed. The model was made in the same way on the other side. Three days after surgery, sodium penicillin 4×10 5 UPrevent infection.

[0066] Six weeks after surgery, rats were examined with in vivo MRI to observe the TA defect site. The T2 signal was used to evaluate muscle repair. It was found that the defect group still had some dark signal areas missing, indicating that the new muscle fibers had not healed. However, the dark signal area of ​​the corresponding area in the composite scaffold implantation group was smaller, indicating that some of the defect sites were close to being repaired by regenerated tissue. The composite scaffold helped to rebuild some new muscle tissue. Figure 9 ).

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing MMP-2 enzyme-responsive nanomicelles, characterized in that: The steps include: (1) Dissolve 1-vinyl-1H-imidazole and 4-bromomethylphenylboronic acid in acetonitrile and react at 70-90°C for 5-24 hours to obtain the compound 3-(4-boronic acid benzyl)-1-vinyl-1H-imidazole; (2) A THF solution containing 1-(tert-butyl) 5-(2,5-dioxopyrrolidin-1-yl)(tert-butoxycarbonyl) glutamate was added dropwise to an alkaline aqueous solution of 2-aminobutyric acid and reacted for 5 to 24 hours to obtain compound 1; ethylenediamine was dissolved in water, the pH was adjusted to 8 to 9, and the solution was placed in an ice bath, and then a chloroform solution containing acryloyl chloride was added dropwise, and the reaction was stirred for 2 to 10 hours to obtain compound 2; compound 1 and N , N' -Carbonyldiimidazole was dissolved in dichloromethane and reacted at room temperature for 1-5 hours, compound 2 and triethylamine were added, and the reaction was continued at room temperature for 5-24 hours to obtain compound 3; compound 3 was dissolved in dichloromethane, trifluoroacetic acid was added, and the reaction was continued at room temperature for 5-24 hours to obtain N-(1-((2-acrylamidoethyl)amino)-1-oxobutan-2-yl)glutamine trifluoroacetate; (3) Dissolve the MMP-2 responsive peptide PLGLAG and triethylamine in N , N -dimethylformamide, slowly add acryloyl chloride to react to obtain compound 4; compound 4, 2-(7-azabenzotriazole)- N , N , N' , N' -Tetramethyluronium hexafluorophosphate was dissolved in DMF and added N , N -diisopropylethylamine, react at room temperature for 20-60 minutes, add DMF containing 3,6,9-trioxaundecane-1,11-diamine, and react at room temperature for 5-24 hours to obtain an MMP-2 responsive compound; (4) LxA4 and SDF-1α were dissolved in PBS buffer to obtain LxA4 solution and SDF-1α solution, respectively. The LxA4 solution and SDF-1α solution were slowly added dropwise at a molar ratio of 1:10 to 10:1 and mixed. The mixture was incubated in the dark for 20 to 60 minutes to spontaneously assemble into LxA4 / SDF-1α nanomedicines. (5) The LxA4 / SDF-1α nanomedicine was mixed evenly with an aqueous solution of 3-(4-boronic acid benzyl)-1-vinyl-1H-imidazole monomer, and then the MMP-2 responsive compound and N-(1-((2-acrylamidoethyl)amino)-1-oxobutane-2-yl)glutamine trifluoroacetate monomer were added. An initiator was added to initiate a free radical polymerization reaction to form nanomicelles, which were then freeze-dried to obtain the product.

2. The preparation method according to claim 1, characterized in that The molar ratio of 1-vinyl-1H-imidazole to 4-bromomethylphenylboronic acid in step (1) is 1 to 10:1; The molar ratio of 2-aminobutyric acid and 1-(tert-butyl) 5-(2,5-dioxopyrrolidin-1-yl)(tert-butoxycarbonyl) glutamate in step (2) is 1-5:1; the alkaline substance in the alkaline aqueous solution of 2-aminobutyric acid is sodium bicarbonate, and the molar ratio of 2-aminobutyric acid and sodium bicarbonate is 1:1-5; the volume ratio of ethylenediamine and water is 1:10-20, and the molar ratio of ethylenediamine and acryloyl chloride is 1:1-5; the compound 1 and N , N' The molar ratio of the compound 1 to the carbonyldiimidazole is 1:1-2, and the molar ratio of the compound 1, the compound 2 and the triethylamine is 1:1-2:1-5.

3. The preparation method according to claim 1, characterized in that The molar ratio of the MMP-2 responsive peptide PLGLAG, acryloyl chloride and triethylamine in step (3) is 1:1~3:2~10; compound 4, 2-(7-azabenzotriazole)- N , N , N' , N' -Tetramethyluronium hexafluorophosphate, N , N - the molar ratio of diisopropylethylamine to 3,6,9-trioxaundecane-1,11-diamine is 1:1~3:3~10:0.5~1; The concentrations of LxA4 and SDF-1α in the PBS buffer described in step (4) are both 0.5-10 mM; an ultrasonic water bath is used to assist in the mixing process, the ultrasonic frequency is 25-50 kHz, and the ultrasonication lasts for 5-30 minutes.

4. The preparation method according to claim 1, characterized in that The molar ratio of the LxA4 / SDF-1α nanodrug, 3-(4-boronic acid benzyl)-1-vinyl-1H-imidazole, MMP-2 responsive compound, and N-(1-((2-acrylamidoethyl)amino)-1-oxobutane-2-yl)glutamine trifluoroacetate monomer described in step (5) is 1:10~3000:0.1~30:10~3000; the initiator is 1~5% by mass of ammonium persulfate and 3~10% by mass of tetramethylethylenediamine.

5. MMP-2 enzyme-responsive nanomicelles prepared by the preparation method according to any one of claims 1 to 4.

6. A method for preparing a MMP-2 enzyme-responsive composite scaffold, characterized in that: The steps include: 1) Immerse porcine dermis in 0.3-0.7 M NaOH aqueous solution, shake at room temperature for 5-12 hours, wash, freeze-dry, and sterilize to obtain acellular matrix scaffolds; 2) The MMP-2 enzyme-responsive nanomicelles according to claim 5 are redissolved in PBS buffer; the decellularized matrix scaffold obtained in step 1) is immersed in an ethanol solution containing 0.5% to 5% by volume of an aminosilane coupling agent for 20 to 60 minutes, cross-linked and cured at 40 to 80° C., the cross-linked and cured decellularized matrix scaffold is mixed with the MMP-2 enzyme-responsive nanomicelle solution, and then rotated and incubated for 1 to 3 hours to remove free nanomicelles, and the mixture is freeze-dried in a vacuum to obtain the product.

7. The preparation method according to claim 6, characterized in that The specific cleaning process in step 1) is as follows: first, wash with 0.1-0.3 M PBS buffer for 10-40 minutes, then sonicate with ultrapure water for 5-15 minutes, and finally rinse with deionized water for 5-15 minutes.

8. The preparation method according to claim 6, characterized in that In step 2), the mass ratio of the cross-linked and solidified acellular matrix scaffold to the MMP-2 enzyme-responsive nanomicelles is 100,000:1 to 1,000:1; the incubation temperature is 35 to 38° C., and the rotation speed is 30 to 100 rpm.

9. The MMP-2 enzyme-responsive composite scaffold prepared by the preparation method according to any one of claims 6 to 8.

10. Use of the MMP-2 enzyme-responsive composite scaffold according to claim 9 in preparing muscle damage repair materials.

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