A lactate reactor capable of inhibiting intervertebral disc degeneration and a preparation method thereof

Abstract

This invention discloses a lactate reactor capable of inhibiting intervertebral disc degeneration and its preparation method. The lactate reactor is an injectable gel microsphere loaded with lactate-catalyzing nanoparticles and matrix cell-derived factor SDF-1α. The lactate-catalyzing nanoparticles are mesoporous silica nanoparticles, whose mesopores are coated with lactate dehydrogenase, nicotinamide adenine dinucleotide, alanine aminotransferase, and glutamate, and whose surface is coated with an oxidized sodium alginate layer via a Schiff base reaction. This invention can efficiently convert pathological lactate in the nucleus pulposus microenvironment into alanine, which inhibits degeneration, while simultaneously recruiting stem cells to synergistically inhibit the process of intervertebral disc degeneration.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and tissue engineering technology, specifically relating to an injectable lactate reactor for the treatment of intervertebral disc degeneration (IVDD) and its preparation method. Background Technology

[0002] Intervertebral disc degeneration (IVDD) is one of the leading causes of low back pain. Its pathological features include a reduction in nucleus pulposus cells (NPCs), degradation of the extracellular matrix, and the formation of an inflammatory microenvironment. Studies have shown that in the nucleus pulposus tissue of degenerated intervertebral discs, the anaerobic glycolysis of NPCs is abnormally activated, producing large amounts of pathological lactic acid, with concentrations reaching up to 10 times the normal level. High concentrations of lactic acid not only activate the NLRP3 inflammasome, exacerbating the local inflammatory response, but also upregulate reactive oxygen species (ROS) levels, directly inducing NPC degeneration and aging, forming a vicious cycle and significantly accelerating the progression of IVDD.

[0003] Currently, clinical treatments for IVDD, such as analgesia, physical therapy, and surgery, primarily focus on symptom relief and are unlikely to reverse the degenerative process or intervene in the core pathological processes related to lactate. At the research level, some studies have attempted to regulate the lactate microenvironment. For example, some researchers have used biomaterials to load lactate oxidase, catalyzing lactate into pyruvate. However, pyruvate, as an intermediate product of anaerobic glycolysis, can still be converted back into lactate, failing to fundamentally eliminate lactate. Other studies have introduced calcium carbonate to neutralize the hydrogen ions (H⁺) generated by lactate ionization, but failed to eliminate lactate ions. Lactate formed by the combination of lactate ions with sodium ions in the body still has the ability to activate inflammatory pathways.

[0004] Therefore, developing a strategy that can convert pathological lactate in situ, efficiently, and thoroughly into protective or neutral substances, while simultaneously intervening in intervertebral disc degeneration, is of great significance for the reversal treatment of IVDD. Based on a multi-enzyme cascade catalytic system, lactate is converted in two steps into harmless or even beneficial metabolites (such as alanine) for NPCs, and combined with stem cell recruitment technology, providing a novel approach to the treatment of IVDD. Summary of the Invention

[0005] To address the shortcomings of existing technologies that lack the ability to efficiently and thoroughly transform pathological lactic acid in the microenvironment of intervertebral disc degeneration while simultaneously achieving tissue repair, this invention provides a lactic acid reactor that can inhibit intervertebral disc degeneration and its preparation method.

[0006] The technical solution adopted in this invention is: a lactate reactor that can inhibit intervertebral disc degeneration, which is an injectable gel microsphere. The gel microsphere is loaded with lactate-catalyzing nanoparticles and stromal cell-derived factor SDF-1α. The lactate-catalyzing nanoparticles are mesoporous silica nanoparticles, which are loaded with lactate dehydrogenase (LDH), nicotinamide adenine dinucleotide (NAD⁺), alanine aminotransferase (ALT), and glutamate (Glu) in their mesopores. The surface of the nanoparticles is coated with sodium alginate through a Schiff base reaction.

[0007] As a further improvement of the present invention, the mesoporous silica nanoparticles have a particle size of 50–200 nm and a pore size of 2–10 nm. This size range is beneficial for achieving efficient loading of enzymes and reaction substrates and efficient diffusion of products. The mass of the sodium alginate coating is 1%–10% of the mass of the nanoparticles. This ratio achieves an optimal balance between effectively preventing enzyme leakage and maintaining sufficient mass transfer efficiency.

[0008] As a further improvement of the present invention, the matrix material of the gel microspheres is chondroitin sulfate with good biocompatibility, which possesses injectability and photocrosslinking properties. The particle size of the gel microspheres is 100–500 μm, a size suitable for delivery by syringe and residence in intervertebral disc tissue. The loading amount of SDF-1α in the gel microspheres is 5–20 μg / mL to ensure effective stem cell chemotaxis.

[0009] The present invention also provides a method for preparing the above-mentioned lactic acid reactor, characterized by comprising the following steps: S1. Preparation of amino-modified mesoporous silica nanoparticles: Mesoporous silica nanoparticles were reacted with silane coupling agent KH550 in anhydrous ethanol to obtain nanoparticles with amino-modified surfaces. S2. Preparation of enzyme- and substrate-loaded nanoparticles: Using negative pressure adsorption, a mixed solution containing LDH, NAD⁺, ALT and Glu was loaded into the mesopores of aminated mesoporous silica nanoparticles. S3. Constructing a sodium alginate oxide coating layer: The loaded nanoparticles are mixed with a sodium alginate oxide solution, and a dense coating layer is formed by the Schiff base reaction between the amino groups on the surface of the nanoparticles and the aldehyde groups of sodium alginate oxide, thus obtaining nanoparticles that can catalyze lactic acid. S4. Preparation of aqueous and oil phases: Add lactic acid catalytic nanoparticles, SDF-1α, and photoinitiator to an aqueous solution of chondroitin sulfate with methacrylamide to form an aqueous phase; dissolve emulsifier Span 80 in liquid paraffin to form an oil phase; S5. Preparation of gel microspheres: Microfluidics is used to form microdroplets in the aqueous phase under shearing of the oil phase, and cross-linking is initiated by ultraviolet light irradiation, which then solidifies to form gel microspheres. S6. Post-processing: Clean the gel microspheres to remove the oil phase and emulsifier, and freeze-dry to obtain the lactic acid reactor.

[0010] As a further improvement of the present invention, in step S1, the mass ratio of the mesoporous silica nanoparticles to the silane coupling agent KH550 is 1:0.2-0.6. This ratio ensures sufficient amination modification and provides enough reaction sites for subsequent coating.

[0011] As a further improvement of the present invention, in step S2, the concentration of LDH in the mixed solution is 0.5–2 KU / mL, the concentration of NAD⁺ is 5–20 mg / mL, the concentration of ALT is 0.5–2 KU / mL, and the concentration of Glu is 5–20 mg / mL. This concentration range has been optimized and determined to achieve high loading capacity of enzyme and substrate within the mesoporous structure while maintaining activity, thus ensuring catalytic efficiency.

[0012] As a further improvement of the present invention, in step S3, the mass concentration of the oxidized sodium alginate solution is 0.05% to 0.5%. This concentration helps to form a dense and stable coating layer.

[0013] As a further improvement of the present invention, in step S4, the mass concentration of the chondroitin sulfate aqueous solution is 5%–10%, which ensures that the precursor solution has a viscosity suitable for microfluidic processing and the mechanical strength of the final gel. The concentration of the lactic acid-catalyzing nanoparticles in the aqueous phase is 5–20 mg / mL, which ensures that the reactor has sufficient lactic acid conversion capacity. The concentration of SDF-1α in the aqueous phase is 5–20 μg / mL, which is used to provide an effective stem cell recruitment concentration. The photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), and its mass concentration in the aqueous phase is 0.1%–0.5%, which ensures a highly efficient photocrosslinking reaction.

[0014] As a further improvement of the present invention, in step S4, the volume fraction of Span 80 in the oil phase is 1% to 10% to ensure the formation of a stable water-in-oil emulsion.

[0015] As a further improvement of the present invention, in step S5, the flow rate of the aqueous phase in the microfluidic device is 10–50 μL / min, and the flow rate of the oil phase is 200–600 μL / min. This flow rate ratio is crucial for obtaining uniformly sized microdroplets. The wavelength of the ultraviolet light irradiation is 365 nm, the light intensity is >1500 mw / cm², and the irradiation time is 5–30 minutes. These conditions ensure sufficient cross-linking of the gel.

[0016] As a further improvement of the present invention, in step S6, the organic solvent is isopropanol and ethanol, and the washing is performed 2 to 5 times to thoroughly remove oil phase residue. The freeze-drying conditions are: pre-freezing temperature below -40°C, freeze-drying time 24 to 72 hours, to obtain structurally intact gel microspheres.

[0017] The present invention also discloses a lactic acid reactor that can inhibit intervertebral disc degeneration, which is prepared by the preparation method of the present invention.

[0018] The present invention also discloses the use of the lactic acid reactor that can inhibit intervertebral disc degeneration in the preparation of drugs or medical devices for treating or preventing intervertebral disc degeneration.

[0019] The beneficial effects of this invention are: 1) Catalytic Pathway Innovation and Synergistic Therapy: For the first time, the "lactic acid → pyruvate" step catalyzed by LDH / NAD⁺ and the "pyruvate → alanine" step catalyzed by ALT / Glu are cascaded and integrated into a single nanocarrier, achieving the direct and complete conversion of harmful lactic acid into alanine, which can inhibit intervertebral disc degeneration. Simultaneously, the stem cell recruitment function of SDF-1α is integrated, forming a novel synergistic therapeutic strategy of "clearing harmful metabolites - alleviating inflammation and replenishing repair cells - inhibiting intervertebral disc degeneration".

[0020] 2) High system stability: Mesoporous silica nanoparticles are used to encapsulate enzymes and reaction substrates, and sodium alginate is used to form a coating layer through chemical cross-linking, which significantly improves the stability of bioactive components in complex physiological environments, reduces enzyme leakage, and ensures long-lasting catalytic ability.

[0021] 3) Superior delivery system: It uses injectable and photocurable chondroitin sulfate microspheres as the final carrier. The microsphere size is controllable, with good tissue compatibility and in-situ implantation ability, which facilitates clinical minimally invasive injection application.

[0022] 4) Controllable and scalable process: The preparation method combines mature nanomaterial surface modification technology, negative pressure adsorption technology and microfluidic-photocrosslinking molding technology. The parameters of each step are clear and the repeatability is good, which lays the foundation for large-scale production. Attached Figure Description

[0023] Figure 1 These are data on the conversion rate and reaction efficiency of lactic acid to alanine in the lactic acid reactor (OTC MSs@SDF-1α-1) of the specific implementation method.

[0024] Figure 2This is the imaging data of the lactate reactor (OTC MSs@SDF-1α-1) in specific embodiments inhibiting intervertebral disc degeneration in vivo. (AC) X-ray and magnetic resonance imaging images of the healthy (control) group, the degenerated (Puncture+Lactate) group, the blank gel microsphere (MSs) group, the gel microspheres loaded with SDF-1α only (MSs@SDF-1α) group, and the lactate reactor (OTC MSs@SDF-1α-1) group at 4 and 8 weeks after implantation. (D, E) Relative intervertebral disc height and grayscale values ​​of the above 5 groups at 4 and 8 weeks after implantation. (n=5, *p<0.05, ***p<0.001, ****p<0.0001, NS means no significant difference).

[0025] Figure 3 This section presents histological and extracellular matrix component staining data on the inhibition of intervertebral disc degeneration in vivo by the lactate reactor (OTC MSs@SDF-1α-1) of a specific embodiment. (AC) control, Puncture+Lactate, MSs, MSs@SDF-1α, and OTC MSs@SDF-1α-1 groups at 4 and 8 weeks after implantation, H&E, AGG, and COL II staining images, scale bars at 400 μm and 40 μm, respectively. (DG) Relative fluorescence intensities of AGG and COL II at 4 and 8 weeks after implantation. (n=5, ****p<0.0001).

[0026] Figure 4 This section presents histological and inflammatory factor staining data on the in vivo inhibition of intervertebral disc degeneration by the lactate reactor (OTC MSs@SDF-1α-1) of the specific implementation method. (A, B) Safranin O, Fast Green, TNF-α, and IL-1β staining images of the control, Puncture+Lactate, MSs, MSs@SDF-1α, and OTCMSs@SDF-1α-1 groups at 4 and 8 weeks after implantation, with scale bars at 400 μm and 40 μm, respectively. (CF) Relative fluorescence intensities of TNF-α and IL-1β at 4 and 8 weeks after implantation. (n=5, ****p<0.0001, NS means no significant difference). Detailed Implementation

[0027] The present invention will be further described below with reference to embodiments.

[0028] To enable those skilled in the art to better understand and implement the present invention, specific embodiments are described below, but the present invention is not limited thereto. Unless otherwise specified, all raw materials used in the embodiments are commercially available products.

[0029] The specifications of the main raw materials used are as follows: Mesoporous silica nanoparticles: particle size 100-200 nm, pore size 2-6 nm, purchased from Xianfeng Nanomaterials Technology Co., Ltd.

[0030] Silane coupling agent KH550: purity ≥99%, purchased from Shanghai Aladdin Reagent Co., Ltd.

[0031] Lactate dehydrogenase (LDH): ≥300 U / mg protein, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0032] Nicotinamide adenine dinucleotide (NAD⁺): 98% purity, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0033] Alanine aminotransferase (ALT): ≥75 units / mg protein, purchased from Shanghai Maclean Biotechnology Co., Ltd.

[0034] Glutamic acid (Glu): purity ≥99%, purchased from Shanghai Aladdin Reagent Co., Ltd.

[0035] Sodium alginate oxide: aldehyde content 35%, purchased from Sigma-Aldrich.

[0036] Methacrylamide chondroitin sulfate: labeling rate 30-50%, purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0037] Stromal cell-derived factor SDF-1α: purity ≥95%, purchased from PeproTech.

[0038] Lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP): 98% purity, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0039] Span 80: viscosity 1000-2000 mPa·s, purchased from Shanghai Aladdin Reagent Co., Ltd.

[0040] Ultraviolet crosslinking equipment: UPP1010, UVATA, China.

[0041] Example 1: This embodiment provides a lactic acid reactor that can inhibit intervertebral disc degeneration and its preparation method. The specific steps are as follows: S1. Preparation of aminated mesoporous silica nanoparticles: 0.1 g of mesoporous silica nanoparticles and 0.04 g of silane coupling agent KH550 were weighed and dispersed together in 10 mL of anhydrous ethanol. The mixture was placed in an oil bath at 75 °C and magnetically stirred for 8 hours. After the reaction was completed, the nanoparticles were collected by centrifugation (12000 rpm, 10 min), washed three times with anhydrous ethanol, and dried overnight in a vacuum drying oven at 40 °C to obtain aminated mesoporous silica nanoparticles.

[0042] S2. Preparation of enzyme- and substrate-loaded nanoparticles: Prepare mixed solutions of LDH (1 KU / mL) and NAD⁺ (10 mg / mL) and mixed solutions of ALT (1 KU / mL) and Glu (10 mg / mL).

[0043] Weigh 0.1 g of the aminated mesoporous silica nanoparticles obtained in step S1 and place them in a 50 mL flask. Connect the flask to a vacuum pump, and under negative pressure, slowly inject 200 μL of a mixed solution of LDH and NAD⁺ and 200 μL of a mixed solution of LALT and Glu into the flask using a syringe, ensuring complete liquid saturation of the nanoparticles. Release the vacuum and allow the flask to stand at room temperature (25 °C) for 1 hour for adsorption. After adsorption is complete, collect the adsorption by centrifugation (12000 rpm, 15 min), and gently wash once with phosphate-buffered saline (PBS, pH 7.4) to obtain the primary loaded nanoparticles.

[0044] S3. Constructing an oxidized sodium alginate coating layer: Prepare a 0.1 wt% sodium alginate aqueous solution. Disperse 0.1 g of the primary loaded nanoparticles obtained in step S2 in 10 mL of the above sodium alginate aqueous solution. Place the mixture in a shaker and react for 3 days at 25 °C and 100 rpm. After the reaction is complete, collect the nanoparticles by centrifugation (12000 rpm, 15 min), wash three times with deionized water, and freeze-dry to obtain lactic acid catalytic nanoparticles (denoted as OTC NPs-1).

[0045] S4. Preparation of aqueous and oil phases: Aqueous phase preparation: Weigh 1.0 g of chondroitin sulfate (methacrylamide) and dissolve it in 10 mL of deionized water to prepare a 10 wt% solution. Weigh 100 mg of OTC NPs-1 nanoparticles obtained in step S3, 100 μg of SDF-1α, and photoinitiator LAP (0.5 wt%), add them to the above solution, and vortex to mix thoroughly and disperse evenly to obtain the aqueous phase.

[0046] Oil phase preparation: Measure 95 mL of liquid paraffin, add 5 mL of Span 80, stir well to obtain an oil phase containing 5 vol% Span 80.

[0047] S5. Preparation of gel microspheres: The aqueous and oil phases were injected separately into the syringes of a coaxial microfluidic device (inner tube diameter 0.25 mm, outer tube diameter 0.8 mm). The flow rate of the aqueous phase was set to 30 μL / min, and the flow rate of the oil phase was set to 400 μL / min. Under the shearing action of the oil phase, the aqueous phase formed uniform microdroplets at the outlet. The generated microdroplets were collected in a cryostat pre-cooled to -40°C for rapid freezing and solidification.

[0048] The container containing the frozen microdroplets was placed under a 365 nm ultraviolet lamp (intensity >1500 mw / cm²) at a distance of 10 cm for 15 minutes to induce photocrosslinking of chondroitin sulfate with methacrylamide, forming gel microspheres.

[0049] S6. Post-processing: The UV-crosslinked gel microspheres, along with the oil phase, were poured into a centrifuge tube and centrifuged (1000 rpm, 3 min) to collect the microspheres. The supernatant was discarded, and the microspheres were washed three times each with isopropanol, anhydrous ethanol, and deionized water to remove liquid paraffin and Span 80 from the oil phase. After the final wash, the microspheres were allowed to precipitate, the supernatant was removed, and the microspheres were frozen in liquid nitrogen for 10 minutes and then freeze-dried for 48 hours to obtain the lactic acid reactor (denoted as OTC MSs@SDF-1α-1).

[0050] Example 2: The difference between this embodiment and Embodiment 1 is that the concentration of the enzyme / substrate loaded on the nanoparticles, the concentration of the gel precursor, and the microfluidic control parameters were adjusted. The specific steps are as follows: S1, Same as Example 1.

[0051] S2. Preparation of enzyme- and substrate-loaded nanoparticles: Prepare mixed solutions: Prepare mixed solutions of LDH (0.8 KU / mL) and NAD⁺ (8 mg / mL) and ALT (0.8 KU / mL) and Glu (8 mg / mL).

[0052] Using the same negative pressure adsorption method as in Example 1, 0.1 g of aminated mesoporous silica nanoparticles were subjected to an adsorption reaction with 200 μL of a mixed solution of LDH and NAD⁺ and 200 μL of a mixed solution of ALT and Glu, under the same conditions as in Example 1, to obtain primary loaded nanoparticles.

[0053] S3. Same as in Example 1, obtain lactic acid catalytic nanoparticles (denoted as OTC NPs-2).

[0054] S4. Preparation of aqueous and oil phases: Aqueous phase preparation: Weigh 0.75 g of chondroitin sulfate (methacrylamide) and dissolve it in deionized water to prepare a 7.5 wt% solution. Weigh 75 mg of OTC NPs-2 nanoparticles obtained in step S3, 50 μg of SDF-1α, and LAP (0.5 wt%), add them to the above solution, and mix well to obtain the aqueous phase.

[0055] Oil phase preparation: Same as in Example 1.

[0056] S5. Preparation of gel microspheres: The microfluidic parameters were adjusted to: aqueous phase flow rate 20 μL / min, oil phase flow rate 300 μL / min. Microdroplets were collected in a -40°C freezer. The UV crosslinking conditions were the same as in Example 1.

[0057] S6. Post-processing: Same as in Example 1, to obtain a lactic acid reactor (denoted as OTC MSs@SDF-1α-2).

[0058] Example 3: The difference between this embodiment and Embodiment 1 is that the concentration of the sodium alginate coating and the loading of SDF-1α were adjusted. The specific steps are as follows: S1, Same as Example 1.

[0059] S2. Same as in Example 1, primary loaded nanoparticles are obtained.

[0060] S3. Constructing an oxidized sodium alginate coating layer: Prepare an aqueous solution of sodium alginate with a mass concentration of 0.05 wt%. Disperse 0.1 g of primary loaded nanoparticles in 10 mL of this solution and react with shaking at 25 °C and 100 rpm for 2 days. Subsequent processing is the same as in Example 1 to obtain lactic acid catalytic nanoparticles (denoted as OTC NPs-3).

[0061] S4. Preparation of aqueous and oil phases: Aqueous phase preparation: The concentration of the chondroitin sulfate solution with methacrylamide was the same as in Example 1 (10 wt%). Weigh 150 mg of OTC NPs-3 nanoparticles obtained in step S3, 200 μg of SDF-1α and LAP (0.5 wt%), and mix well to obtain the aqueous phase.

[0062] Oil phase preparation: Same as in Example 1.

[0063] S5. Preparation of gel microspheres: The microfluidic parameters were adjusted to: aqueous phase flow rate 40 μL / min, oil phase flow rate 500 μL / min. Microdroplets were collected in a -40℃ freezer. The UV crosslinking time was extended to 20 minutes.

[0064] S6. Post-processing: Same as in Example 1, to obtain lactic acid reactor microspheres (denoted as OTC MSs@SDF-1α-3).

[0065] Example 4: This example is a control experiment of Example 1, the difference being that: the gel microspheres are only loaded with SDF-1α and not with lactic acid catalytic nanoparticles (OTC NPs).

[0066] Specific steps: S1 to S3 are omitted. In S4, no nanoparticles are added during the preparation of the aqueous phase; only 100 μg of SDF-1α and LAP are added to a 10 wt% chondroitin sulfate solution (10 mL). S5 to S6 are the same as in Example 1, to obtain gel microspheres loaded only with SDF-1α (denoted as MSs@SDF-1α). Performance verification experiment of microspheres for lactic acid reactor: 1. Performance characterization of the lactic acid reactor: To investigate the conversion rate and reaction efficiency of lactic acid to alanine catalyzed by the lactic acid reactor prepared in Example 1, a 10 mM lactic acid solution was prepared using PBS buffer. The lactic acid reactor was then added to the lactic acid solution, and after reacting for 36, 60, 84, 108, and 132 hours, the mixture was centrifuged and the supernatant was collected. The concentrations of lactic acid and alanine at the above time points were detected using a high-performance liquid chromatograph (HPLC). The HPLC instrument was a Shimadzu LC-20AT; the chromatographic column was a C18-WP; the detector was ultraviolet (222 nm); and the injection volume was 10 μL.

[0067] 2. Characterization of lactic acid reactor inhibiting intervertebral disc degeneration: A model of intervertebral disc degeneration was constructed in the tail of SD rats using a puncture method. Different groups were established, including a healthy control group, a degenerated (Puncture + Lactate) group, a blank gel microsphere (MSs) group, a gel microsphere loaded only with SDF-1α (MSs@SDF-1α) group, and an OTC MSs@SDF-1α-1 group. Intervertebral disc samples were collected for analysis 4 and 8 weeks after in vivo transplantation.

[0068] The height and water content of intervertebral disc tissue were measured using an ultra-high resolution X-ray imaging system (UltraFoucs) and a small animal magnetic resonance imaging system (BioSpec 94 / 30 USR). Intervertebral disc tissue samples were further fixed, decalcified, embedded, and sectioned. H&E, AGG, COL II, Safranin O, Fast Green, TNF-α, and IL-1β staining were performed to detect intervertebral disc tissue degeneration and regeneration behavior, as well as inflammation levels. Experimental results: Figure 1This is the result of testing the conversion rate and reaction efficiency of lactic acid to alanine in a lactic acid reactor. Figure 1 It can be seen that the conversion rate of lactic acid to alanine by the lactic acid reactor can reach 73%, and the reaction efficiency is maintained at around 71%, indicating that the lactic acid reactor can efficiently catalyze the conversion of lactic acid to alanine.

[0069] Figure 2 The imaging data show that the lactate reactor inhibits intervertebral disc degeneration in vivo. X-ray and magnetic resonance imaging (MRI) data show that only the intervertebral disc height and water content of the SDF-1α-loaded gel microsphere group are lower than those of the lactate reactor group. After 8 weeks of implantation, the intervertebral disc height and water content of the lactate reactor group are significantly increased and close to those of the healthy group, indicating that the lactate reactor can significantly inhibit intervertebral disc degeneration. Figure 3 and Figure 4 These are histological, extracellular matrix component, and inflammatory factor staining data on the role of lactate reactors in inhibiting intervertebral disc degeneration in vivo. Figure 3 and Figure 4 Further evidence demonstrates that the lactate reactor group of this invention significantly promotes the expression of proteoglycans (AGG) and type II collagen (COL II) in the nucleus pulposus matrix compared to the degeneration group and the group containing only SDF-1α-loaded gel microspheres. Eight weeks after in vivo transplantation, the expression levels of AGG and COL II in the lactate reactor group reached 68% and 75% of those in the healthy group, respectively. Simultaneously, the lactate reactor group inhibited the expression of pro-inflammatory factors (TNF-α and IL-1β) in vivo, and the inflammation level in the lactate reactor group was close to that in the healthy group, with no statistically significant difference.

[0070] In summary, the lactic acid reactor developed in this invention has achieved excellent results in inhibiting intervertebral disc degeneration, and is expected to provide a new material, a new strategy, and a new approach for the clinical treatment of intervertebral disc degeneration.

Claims

1. A lactic acid reactor capable of inhibiting intervertebral disc degeneration, characterized in that, The lactic acid reactor is an injectable gel microsphere loaded with lactic acid catalytic nanoparticles and matrix cell-derived factor SDF-1α. The lactic acid catalytic nanoparticles are mesoporous silica nanoparticles loaded with lactate dehydrogenase, nicotinamide adenine dinucleotide, alanine aminotransferase and glutamate within their mesopores, and the nanoparticles are coated with an oxidized sodium alginate layer on their surface via a Schiff base reaction.

2. The lactic acid reactor for inhibiting intervertebral disc degeneration according to claim 1, characterized in that, The mesoporous silica nanoparticles have a particle size of 50–200 nm and a pore size of 2–10 nm; the mass of the sodium alginate oxidized coating is 1%–10% of the mass of the nanoparticles.

3. The lactic acid reactor for inhibiting intervertebral disc degeneration according to claim 1, characterized in that, The matrix material of the gel microspheres is chondroitin sulfate with methacrylamide, and the particle size of the gel microspheres is 100-500 μm; the loading amount of SDF-1α in the gel microspheres is 5-20 μg / mL.

4. A method for preparing a lactic acid reactor capable of inhibiting intervertebral disc degeneration as described in any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Preparation of aminated mesoporous silica nanoparticles: Mesoporous silica nanoparticles and silane coupling agent KH550 are dispersed in anhydrous ethanol and reacted at 60-85℃ for 4-10 hours. After centrifugation, washing and drying, aminated mesoporous silica nanoparticles are obtained. S2. Preparation of enzyme- and substrate-loaded nanoparticles: The aminated mesoporous silica nanoparticles obtained in step S1 are placed in a negative pressure environment to adsorb a mixed solution containing LDH, NAD⁺, ALT and Glu. The adsorption temperature is 15-30℃ and the adsorption time is 0.5-2 hours. The primary loaded nanoparticles are collected by centrifugation. S3. Constructing the sodium oxidized alginate coating layer: The primary loaded nanoparticles obtained in step S2 are dispersed in a sodium oxidized alginate solution with a mass concentration of 0.05% to 0.5% and reacted at 15 to 30°C for 2 to 4 days. After centrifugation, washing, and drying, nanoparticles that can catalyze lactic acid are obtained. S4. Preparation of aqueous and oil phases: Add the lactic acid catalytic nanoparticles, SDF-1α, and photoinitiator obtained in step S3 to an aqueous solution of chondroitin sulfate methacrylamide, mix well to obtain the aqueous phase; dissolve Span 80 in liquid paraffin to obtain the oil phase; S5. Preparation of gel microspheres: The aqueous phase and oil phase obtained in step S4 are injected into a microfluidic device, and the flow rate is controlled so that the aqueous phase forms microdroplets under the shear of the oil phase. The microdroplets are collected in a low-temperature environment and then placed under ultraviolet light for cross-linking to form gel microspheres. S6. Post-processing: The gel microspheres obtained in step S5 are washed sequentially with organic solvent and deionized water to remove the oil phase and emulsifier, and then freeze-dried to obtain the lactic acid reactor.

5. The preparation method according to claim 4, characterized in that, In step S1, the mass ratio of the mesoporous silica nanoparticles to the silane coupling agent KH550 is 1:0.2-0.6; in step S2, the concentration of LDH in the mixed solution is 0.5-2 KU / mL, the concentration of NAD⁺ is 5-20 mg / mL, the concentration of ALT is 0.5-2 KU / mL, and the concentration of Glu is 5-20 mg / mL.

6. The preparation method according to claim 4, characterized in that, In step S4, the mass concentration of the methylacrylamide chondroitin sulfate aqueous solution is 5%–10%; the concentration of the lactic acid catalyzing nanoparticles in the aqueous phase is 5–20 mg / mL; the concentration of SDF-1α in the aqueous phase is 5–20 μg / mL; and the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), with a mass concentration of 0.1%–0.5% in the aqueous phase.

7. The preparation method according to claim 4, characterized in that, In step S4, the volume fraction of Span 80 in the oil phase is 1% to 10%; in step S5, the flow rate of the aqueous phase in the microfluidic device is 10 to 50 μL / min, and the flow rate of the oil phase is 200 to 600 μL / min; the wavelength of the ultraviolet light irradiation is 365 nm, the light intensity is >1500 mw / cm², and the irradiation time is 5 to 30 minutes.

8. The preparation method according to claim 4, characterized in that, In step S6, the organic solvent is isopropanol and ethanol, and the number of washing cycles is 2 to 5; the freeze-drying conditions are: pre-freezing temperature below -40°C, and freeze-drying time of 24 to 72 hours.

9. A lactic acid reactor capable of inhibiting intervertebral disc degeneration, prepared by the method according to any one of claims 4 to 8.

10. The use of the lactate reactor for inhibiting intervertebral disc degeneration as described in claims 1 to 3 or claim 9 in the preparation of a medicament or medical device for treating or preventing intervertebral disc degeneration.

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