Enzyme response type composite scaffold for muscle injury repair and preparation method of enzyme response type composite scaffold

Abstract

The invention discloses an enzyme response type composite scaffold for muscle injury repair and a preparation method of the enzyme response type composite scaffold, and belongs to the technical field of polymer scaffolds. According to the invention, the acellular matrix is used as a scaffold, and is combined with the MMP-2 responsive nano-micelle loaded with the lipoxin LxA4 and the SDF-1 alpha, so that the precise long-acting release of a drug at an injured part is realized; through the synergistic effect of LxA4 and SDF-1alpha, the inflammatory response of the injured part is reduced, and meanwhile, the collection and migration of endogenous stem cells are enhanced, so that the repair period is shortened; in addition, according to the composite scaffold, the synchronism of the degradation speed of the scaffold and the tissue regeneration speed is guaranteed, and effective support of the scaffold in the tissue healing process is guaranteed. A safer and more effective solution is provided for treatment of muscle tissue defects, and application of tissue engineering in clinical practice is promoted.

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: In a first aspect, the present invention provides a method for preparing MMP-2 enzyme-responsive nanomicelles, comprising the following steps: (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); (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); (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); (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. (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.

[0006] 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.

[0007] 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.

[0008] 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; 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.

[0009] 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; 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.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] In a second aspect, the present invention provides MMP-2 enzyme-responsive nanomicelles prepared by the above preparation method.

[0014] In a third aspect, the present invention provides a method for preparing an MMP-2 enzyme-responsive composite scaffold, which mainly comprises the following steps: 1) Porcine dermis was immersed in a 0.3-0.7 M NaOH aqueous solution and shaken at 50-200 rpm at room temperature for 5-12 hours. The dermis was then washed, freeze-dried, and sterilized to obtain a decellularized ECM scaffold. 2) The MMP-2 enzyme-responsive nanomicelles were redissolved in PBS buffer (pH 7.4). The decellularized matrix scaffold (dECM) obtained in step 1) was immersed in an ethanol solution containing 0.5% to 5% aminosilane coupling agent by volume for 20 to 60 minutes, cross-linked and cured at 40 to 80°C, and the cross-linked and cured decellularized matrix scaffold was mixed with the MMP-2 enzyme-responsive nanomicelle solution and incubated with rotation for 1 to 3 hours to remove free nanomicelles. The dECM was then freeze-dried in a vacuum oven to obtain the product.

[0015] Based on the above technical solution, further, the specific cleaning process in step 1) is: first, use 0.1~0.3 MPBS buffer to wash for 10~40 minutes, then use ultrapure water ultrasonic treatment (25~50 kHz, 20~30℃) for 5~15 minutes, and finally rinse with deionized water for 5~15 minutes.

[0016] Based on the above technical solution, further, 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.

[0017] Based on the above technical solution, further, in step 2), the incubation temperature is 35-38° C., and the rotation speed is 30-100 rpm.

[0018] In a fourth aspect, the present invention provides an MMP-2 enzyme-responsive composite scaffold prepared by the above preparation method.

[0019] In a fifth aspect, the present invention provides the use of the above-mentioned MMP-2 enzyme-responsive composite scaffold as a muscle damage repair material.

[0020] The present invention has the following beneficial effects compared to the prior art: (1) The present invention uses a muscle scaffold based on decellularized matrix (dECM) and combines it with MMP-2 responsive nanomicelles to achieve the synergistic release of LxA4 and SDF-1α, designing a patch material with excellent biocompatibility, controllable degradation rate, and long-term anti-inflammatory and tissue repair functions; this material is innovative in morphology, composition, and responsiveness, and can accurately control the long-term release of drugs and optimize the healing environment of muscle defects.

[0021] (2) The present invention utilizes the synergistic effect of LxA4 and SDF-1α to reduce the inflammatory response at the injury site and enhance the recruitment and migration of endogenous stem cells, thereby shortening the repair cycle. It has great application potential in regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0023] Figure 1 This is the synthesis process of the monomers required for the LSBPN nanomicelles in Example 1, wherein A is the synthesis process of 3-(4-boronic acid benzyl)-1-vinyl-1H-imidazole, B is the synthesis process of N-(1-((2-acrylamidoethyl)amino)-1-oxobutane-2-yl)glutamine trifluoroacetate, and C is the synthesis process of the MMP-2 responsive compound monomer.

[0024] Figure 2 TEM images (AB) and corresponding DLS particle size distribution diagram (CD) of LSBPN nanomicelles obtained with different feed ratios of each monomer in Example 1.

[0025] Figure 3 These are the H&E-stained tissue morphology of the porcine dermis before (A) and after (B) decellularization in Example 2, and the SEM images of the longitudinal section (C) and cross section (D) after decellularization.

[0026] Figure 4 These are SEM images of the composite scaffold before (A) and after (B) nanodrug loading in Example 3.

[0027] Figure 5 The in vitro anti-inflammatory results of the LSBPN NMs / dECM composite scaffold detected by flow cytometry in Example 4 are shown.

[0028] Figure 6 This is the in vitro anti-inflammatory result of qPCR detection of LSBPN NMs / dECM composite scaffold in Example 4.

[0029] Figure 7 This is the in vitro degradation result of the LSBPN NMs / dECM composite scaffold in Example 5.

[0030] Figure 8 This is a picture of the composite scaffold implanted into the muscle defect in Example 6.

[0031] Figure 9 These are MRI examination images of the composite stent implantation group and the defect group in Example 6 6 weeks after surgery. DETAILED DESCRIPTION

[0032] The present invention is described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative work all fall within the scope of protection of the present invention.

[0033] Example 1: This embodiment provides a method for preparing MMP-2 enzyme-responsive nanomicelles of different particle sizes, comprising the following steps: (1) Synthesis of 3-(4-boronic acid 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 of acetonitrile (ACN) and reacted at 80 °C overnight. ACN was removed by rotary evaporation, and 25 mL of deionized water was added to dissolve the product. The aqueous layer was washed with DCM (25 mL × 3) and concentrated to dryness to obtain compound BVII (1.07 g, yield 69%) ( Figure 1 A in ).

[0034] (2) Synthesis of N-(1-((2-acrylamidoethyl)amino)-1-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 of water, 1-(tert-butyl) 5-(2,5-dioxopyrrolidin-1-yl)(tert-butoxycarbonyl) glutamate (1.20 g, 3 mmol) was dissolved in 5 mL of tetrahydrofuran (THF), and then 1-(tert-butyl) A THF solution of 5-(2,5-dioxopyrrolidin-1-yl)(tert-butoxycarbonyl)glutamate was added dropwise to an aqueous solution of 2-aminobutyric acid and allowed to react overnight. THF was removed by rotary evaporation, and the pH was adjusted to about 3-5. The mixture was extracted with ethyl acetate (EA). The organic layer was washed twice with 1 M hydrochloric acid and a saturated sodium chloride solution, respectively. The organic layer was dried over anhydrous sodium sulfate, and EA was removed by rotary evaporation. The mixture was dried to give compound 1 (1.03 g, 89% yield). Ethylenediamine (5 mL, 75 mmol) was dissolved in 75 mL of deionized water, and the pH was adjusted to approximately 8-9 with concentrated hydrochloric acid. The mixture was placed in an ice bath. Acryloyl chloride (6.7 mL, 82.5 mmol) was then dissolved in 50 mL of chloroform and added dropwise to the aqueous solution. After reacting for several hours, stirring was stopped and the mixture was allowed to stand for stratification. The aqueous layer was washed with chloroform (50 mL × 3) to remove excess acryloyl chloride. The aqueous layer was concentrated to obtain a white solid. 50 mL of methanol (MeOH) was added and the solid was filtered. The filter cake was washed with methanol several times to obtain compound 2 (5.3 g, 47% yield).

[0035] Compound 1 (0.78 g, 2 mmol) and N , N' -Carbonyldiimidazole (CDI) (0.36 g, 2.22 mmol) was dissolved in 10 mL of 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 of DCM were added sequentially and reacted at room temperature overnight. The organic layer was then washed with 1 M hydrochloric acid (25 mL × 3) and saturated sodium bicarbonate solution (25 mL × 2), 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 of DCM, and then 5 mL of trifluoroacetic acid (TFA) was added and reacted at room temperature overnight. The solvent was removed by rotary evaporation, and ether was added for precipitation. The mixture was collected by centrifugation to obtain a white solid (0.36 g, yield 72%), which is N-(1-((2-acrylamidoethyl)amino)-1-oxobutan-2-yl)glutamine trifluoroacetate (NAOGT) ( Figure 1 B in ).

[0036] (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), slowly added acryloyl chloride (35 μL, 0.4275 mmol), thin layer chromatography (TLC) analysis showed that the PLGLAG reaction was complete, and column separation and purification gave compound 4 (120.8 mg, yield 73%). Compound 4 (116.1 mg, 0.2 mmol), 2-(7-azabenzotriazole)- N , N , N' , N' -Tetramethyluronium hexafluorophosphate (HATU) (152.1 mg, 0.4 mmol) was dissolved in 2.5 mL DMF and then added N , N -Diisopropylethylamine (DIPEA) (175 μL, 1 mmol) was 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 DMF was added to the above reaction solution and reacted overnight at room temperature. The solvent was removed and the MMP-2 responsive compound (POP) was obtained by column separation and purification (56.7 mg, yield 43%) ( Figure 1C in ).

[0037] (4) Preparation and characterization of nanomicelles: The lipophilic small molecule LxA4 and the hydrophilic protein SDF-1α were dissolved in sterile PBS buffer (pH 7.4) at concentrations of 1 mM and 1 mM, respectively. Subsequently, the mixture was slowly added dropwise at a molar ratio of 1:1, and an ultrasonic water bath (frequency 40 kHz, duration 10 minutes) was used to promote the self-assembly of the molecules. The mixture was incubated at room temperature in the dark for 30 minutes to complete the spontaneous assembly of the LxA4 / SDF-1α nanomedicine (abbreviated as LS). This process relies on the hydrophobic interaction and non-covalent bond interaction between molecules to form a stable nanostructure. The LS prepared above 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., the volume of each solution was 500 μL). During the slow addition, gentle vortexing was maintained to ensure uniform mixing. This process utilizes the electrostatic interaction between the positively charged functional groups on the BVII molecule and the negative charges on the LS surface to uniformly adsorb the BVII monomer onto the LS surface, providing a platform for subsequent free radical polymerization. After mixing, the mixture was allowed to stand at room temperature for 15 minutes to ensure sufficient recombination. 0.05 nmol POP and 10 nmol NAOGT monomers were then introduced into the system, and 2 μL of 2% by mass ammonium persulfate (APS) and 2 μL of 5% by mass tetramethylethylenediamine (TEMED) were added to initiate free radical polymerization on the surface of the nanodrug to form 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 their hydrodynamic radius and particle size distribution were determined by DLS. While keeping the above-mentioned LS, POP, and NAOGT input amounts unchanged, the BVII input amount was increased from 500 nmol ( Figure 2 A in Figure 2 C) was increased to 800 nmol ( Figure 2 B in Figure 2 D in the figure), the particle size of the nanomicelles increased; LSBPN NMs obtained after freeze-drying.

[0038] Example 2: Preparation of dECM 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 ).

[0039] Example 3: Loading of LSBPN NMs on dECM 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.

[0040] Example 4: In vitro anti-inflammatory effect of LSBPN NMs / dECM composite scaffold 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.

[0041] 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.

[0042] 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.

[0043] 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).

[0044] Example 5: In vitro degradation of LSBPN NMs / dECM composite scaffolds The in vitro degradation performance of the materials was evaluated by enzymatic hydrolysis. The specific method was as follows: about 100 mg of sample (dECM in Example 2, LSBPN NMs in Example 3) was weighed and placed in a 10 mL sterile centrifuge tube, 4 mL of 5 U / mL type I collagenase solution was added, and the tube was tilted and incubated at 37°C with shaking at 70 rpm. At each time point of culturing for 3 h, 6 h, 1 d, 2 d, 4 d, 1 w, 10 d, and 2 w, 3 slurries were drawn in a clean bench. mL of supernatant was quantitatively analyzed using the hydroxyproline detection method. An equal amount of fresh collagenase solution was immediately added 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 hydroxyproline release in the supernatant to the initial total content at each time point (degradation rate % = supernatant hydroxyproline content / initial total content × 100%). Time-degradation rate curves were plotted to characterize the degradation kinetics of the material. The above experimental samples were sterilized with EO, and all experimental contact reagents were sterilized. All experimental operations were performed under a sterile environment.

[0045] The results are as follows Figure 7 As shown in the figure, the results showed that both groups of scaffold materials, dECM and LSBPN NMs / dECM, exhibited a typical rapid initial release trend throughout the degradation process, followed by a slow release phase and ultimately a plateau phase. From the degradation curves, the trends of hydroxyproline release and corresponding cumulative degradation rates at each time point for dECM and LSBPN NMs / dECM were basically consistent, with no significant differences and highly overlapping error ranges, indicating that their degradation behaviors under enzymatic hydrolysis conditions were highly similar. In particular, at key time points (such as 1 d, 7 d, and 14 d), both groups of samples exhibited similar degradation rates and cumulative release ratios. These results indicate that, under the current experimental setting, the in vitro enzymatic degradation performance of dECM and LSBPN NMs / dECM is comparable, with similar degradation kinetics, and suitable for similar tissue engineering application scenarios.

[0046] Example 6: 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.

[0047] 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.

[0048] 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 ).

[0049] 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 as a muscle damage repair material.

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