Nanoparticle composite hydrogel for treating intervertebral disc degeneration and preparation method thereof

Abstract

The invention discloses a nanoparticle composite hydrogel for treating intervertebral disc degeneration and a preparation method thereof, and the nanoparticle composite hydrogel is prepared by selecting an important transcription factor SOX9 expression plasmid for promoting extracellular matrix biosynthesis and a key regulatory factor SIRT1 protein for various adverse factors. And sequentially assembling to a phenylboronic acid functionalized fifth-generation dendritic macromolecule to construct the multifunctional co-delivery nano platform for treating the intervertebral disc. On one hand, the multifunctional nano platform has the capacity of inhibiting various adverse factors and promoting tissue repair in vivo and in vitro. On the other hand, slow release of the multifunctional nano platform is achieved through methacrylated gelatin and aldehyde group modified hyaluronic acid, and the in-vivo action time is further prolonged. The invention provides the nanoparticle composite hydrogel for relieving the progress of the intervertebral disc disease by inhibiting various adverse factors, and a new thinking is brought for developing an intervertebral disc treatment strategy.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical drugs, and in particular relates to a nanoparticle composite hydrogel for treating intervertebral disc degeneration and a preparation method thereof. Background Art

[0002] Intervertebral disc degeneration is a common degenerative spinal disease that is accompanied by structural and functional changes in the intervertebral disc, often leading to chronic back pain, nerve root pain and spinal dysfunction. With the aging of the population and changes in lifestyle, the incidence of this disease has increased year by year, becoming a major global public health issue. Traditional treatment methods include conservative treatment and surgical intervention, but each has its limitations. For example, conservative treatment is difficult to prevent the progression of the disease, while surgical intervention has the problems of large trauma and many postoperative complications. Therefore, there is an urgent need to develop a new treatment method that is minimally invasive and can effectively improve the biomechanical properties of the intervertebral disc and promote its regeneration.

[0003] Bioactive factor delivery is widely used in tissue repair due to its stable therapeutic effects, minimal trauma, simple preparation, low cost and immunogenicity. SIRT1 protein is an NAD+-dependent histone deacetylase that participates in multiple biological processes, including metabolic regulation, anti-oxidative stress, and apoptosis regulation. In the intervertebral disc, SIRT1 is believed to help maintain the metabolic balance and anti-aging state of cells. By increasing the expression of SIRT1 (Sirtuin 1), the aging process of intervertebral disc cells can be delayed, the inflammatory response can be reduced, and the breakdown of the extracellular matrix can be improved, thereby protecting the function of the intervertebral disc. The SOX9 (SexDetermining Region Y Box Protein 9) plasmid encodes a key transcription factor that is widely involved in cell differentiation and function maintenance. In intervertebral disc tissue, especially in nucleus pulposus cells, SOX9 has the function of promoting the production of extracellular matrix (such as proteoglycans and collagen) and maintaining cell phenotype.

[0004] In recent years, advances in nanotechnology and biomaterials have opened up new possibilities for the delivery of bioactive agents. An ideal carrier should possess excellent biocompatibility, mechanical stability, and controlled release capabilities. However, currently used traditional carriers, such as liposomes, poly(lactic-co-glycolic acid) (PLGA), and chitosan, suffer from biocompatibility issues, low delivery efficiency, and poor controlled release. Phenylboronic acid-modified fifth-generation dendrimers have a highly branched structure, providing numerous surface functional groups for further modification and functionalization, thereby improving drug loading capacity and targeting. Phenylboronic acid groups can form reversible complexes with polyhydroxy compounds on the cell membrane surface, enhancing cellular uptake. Furthermore, the abundant groups on the nanoparticle surface can interact with the hydrogel to form a stable nanoparticle-hydrogel composite system, enabling sustained, slow release of SIRT1 protein and SOX9 plasmid within the tissue microenvironment, enhancing therapeutic efficacy. Furthermore, direct drug injection often faces the problem of rapidly elevated local drug concentrations within a short period of time and rapid loss, making long-term therapeutic efficacy difficult. This not only limits the duration of drug efficacy but also can cause damage to local tissues from high drug concentrations. Summary of the Invention

[0005] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a nanoparticle composite hydrogel for treating intervertebral disc degeneration, as well as a preparation method and application thereof.

[0008] To solve the above technical problems, the present invention provides the following technical solution: a nanoparticle composite hydrogel for treating intervertebral disc degeneration, characterized in that: the nanoparticle composite hydrogel comprises a SOX9 expression plasmid, a SIRT1 protein, a fifth-generation dendrimer modified with phenylboronic acid, methacryloylated gelatin, and hyaluronic acid modified with amino and aldehyde groups;

[0009] Among them, SOX9 expression plasmid and SIRT1 protein are used as cargo; phenylboronic acid-modified fifth-generation dendrimers are used as carriers.

[0010] As a preferred embodiment of the nanoparticle composite hydrogel of the present invention, the nitrogen-phosphorus ratio of the SOX9 expression plasmid to the fifth-generation dendrimer modified with phenylboronic acid is 1 to 16:1.

[0011] As a preferred embodiment of the nanoparticle composite hydrogel of the present invention, the mass ratio of the SIRT1 protein to the fifth-generation dendrimer modified with phenylboronic acid is 2 to 8:1.

[0012] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a nanoparticle composite hydrogel for treating intervertebral disc degeneration, which is characterized by comprising:

[0013] The SOX9 expression plasmid and SIRT1 were mixed with the fifth-generation dendrimer modified with phenylboronic acid to obtain multifunctional nanoparticles loaded with dual bioactive factors;

[0014] The multifunctional nanoparticles loaded with dual bioactive factors are cross-linked with methacrylated gelatin and hyaluronic acid modified with amino and aldehyde groups through physical, chemical and photoinitiated cross-linking to obtain the nanoparticle composite hydrogel for treating intervertebral disc degeneration.

[0015] As a preferred embodiment of the preparation method of the present invention, the preparation method of the fifth-generation dendrimer modified with phenylboronic acid comprises:

[0016] The fifth-generation dendrimer is added to anhydrous methanol and mixed evenly to obtain a fifth-generation dendrimer solution;

[0017] Phenylboronic acid was added to the fifth-generation dendrimer solution, heated and stirred, and then cooled to room temperature. The obtained product was dialyzed in anhydrous methanol and deionized water, respectively, and then ultrafiltered and concentrated to obtain the fifth-generation dendrimer modified with phenylboronic acid.

[0018] As a preferred embodiment of the preparation method of the present invention, the molar ratio of the phenylboronic acid to the fifth-generation dendrimer solution is 2 to 10:1.

[0019] As a preferred embodiment of the preparation method of the present invention, the mass ratio of the multifunctional nanoparticles loaded with dual biologically active factors to the methacrylated gelatin is 1:12-48.

[0020] As a preferred embodiment of the preparation method of the present invention, the preparation method of the hyaluronic acid modified with amino groups and aldehyde groups includes dissolving hyaluronic acid in deionized water, stirring thoroughly until dissolved, adding sodium periodate, stirring in the dark, dialyzing the product with deionized water, and freeze-drying.

[0021] As a preferred embodiment of the preparation method of the present invention, the mass ratio of the hyaluronic acid to sodium periodate is 2 to 4:1.

[0022] As a preferred embodiment of the preparation method of the present invention, the ratio of the multifunctional nanoparticles loaded with dual bioactive factors to the aldehyde-modified hyaluronic acid is 1:18-54.

[0023] Beneficial effects of the present invention:

[0024] (1) The composite hydrogel of mixed nanoparticles prepared by the present invention has low cytotoxicity, and this excellent biocompatibility is the basis for it to exert other biological functions. Secondly, SIRT1, as a deacetylase, has an inhibitory effect on various adverse factors faced by intervertebral disc degeneration. The intracellular delivery of SIRT1 protein proves this point. First, its deacetylation effect on FOXO3 activates the antioxidant defense system in the cell, and the expression of various antioxidant proteins is enhanced. In addition, this antioxidant directly promotes the recovery of mitochondrial dysfunction, and the mitochondrial morphology is restored to normal; SOX9 has been proven to be a key transcription factor for extracellular matrix secretion, promoting the secretion of important components such as proteoglycans. After the SOX9 plasmid is effectively delivered into the cell, through a series of processes such as transcription and translation, it promotes the normalization of extracellular matrix metabolism, maintains the function of nucleus pulposus cells, and upregulates the expression of nucleus pulposus cell phenotypic proteins. It was further found that the addition of SIRT1 enhances the function of SOX9 because the deacetylation of SIRT1 also has an effect on SOX 9.

[0025] (2) The present invention simulates the in vivo mode of action by culturing cells in two-dimensional and three-dimensional culture modes. The results showed that the cells grew well and still maintained excellent therapeutic effects; the dynamic cross-linking of nanoparticles and hydrogels facilitated their long-term release and maintained a certain drug concentration locally in the body. Direct injection of nanoparticles in the body is difficult to maintain local concentrations for a long time, while hydrogels are the opposite; due to the sequential assembly of bioactive factors, the SIRT1 protein on its surface often falls off first and can take effect quickly because it has a direct target in the cell. The plasmid falls off later and has to go through the transcription and translation process before it takes effect. The advantage is that SIRT1 can inhibit multiple adverse factors in advance, removing obstacles for the subsequent positive tissue repair function of SOX9; the nanoparticle composite hydrogel has an inhibitory effect on the Nod-like receptor signaling pathway, which is a classic pyroptosis pathway. NLRP3 can promote the aggregation and perforation of GSDMD-NT on the membrane by activating Caspase-1 to cut GSDMD, thereby inducing cell pyroptosis. The results showed that the nanoparticle composite hydrogel had an inhibitory effect on multiple proteins in the apoptosis pathway; the nanoparticle composite hydrogel was injected into an animal model, and imaging and histological results were observed at four and eight weeks. The results showed that it effectively promoted the recovery of intervertebral disc height and water content, and maintained the structure and function of the intervertebral disc. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0027] Figure 1 This is a characteristic diagram of the G5 dendrimer functionalized with phenylboronic acid (PBA) analyzed by 1HNMR spectroscopy.

[0028] Figure 2 The characterization of SOX9@GPNPs, SIRT1@GPNPs and SIRT1 / SOX9@GPNPs nanoparticles of the present invention is shown in FIG. Figure 2 (a) Particle size of SOX9@GPNPs; Figure 2 (b) is the gene transfection efficiency; Figure 2 (c) Quantification of gene expression (Sox9, Col2a1, and Acan) at an n / p ratio of 8:1 by qRT-PCR; Figure 2 (d) is the particle size of SIRT1@GPNPs; Figure 2 (e) Fluorescence image of nucleus pulposus cells treated with BSA-FITC@GPNPs for 6 hours; Figure 2 (f) with PBS, PBS / LPS + 、FREESIRT1 / LPS + and SIRT1@GPNPs / LPS + Reactive oxygen species staining of co-cultured nucleus pulposus cells; Figure 2 (g) Particle size of SIRT1 / SOX9@GP NPs observed by DLS and TEM; Figure 2 (h) Fluorescence imaging of genes and BSA-FITC at different time points after delivery; Figure 2 (i) Particle sizes of GPNPs, SOX9@GPNPs, and SI RT1 / SOX9@GPNPs measured 0, 12, 24, and 36 h after preparation.

[0029] Figure 3 Agarose gel electrophoresis images

[0030] Figure 4 Verify transfection efficiency by fluorescence microscopy

[0031] Figure 5 Schematic diagram and scanning electron microscope image of the synthesis of nanoparticle composite hydrogel of the present invention.

[0032] Figure 6The local concentration of the nanoparticle composite hydrogel of the present invention and direct injection in vivo changes over time.

[0033] Figure 7 This is a biocompatibility test of the nanoparticle composite hydrogels of different components of the present invention.

[0034] Figure 8 The nanoparticle composite hydrogel of the present invention restores the mitochondrial function of nucleus pulposus cells through antioxidant detection.

[0035] Figure 9 The nanoparticle composite hydrogel of the present invention improves the expression of extracellular matrix of nucleus pulposus.

[0036] Figure 10 The invention discloses a two-dimensional and three-dimensional culture method for nucleus pulposus cells using nanoparticle composite hydrogels and the effects on their functions.

[0037] Figure 11 This is the animal experiment of the present invention to promote the repair of rat coccygeal degeneration. DETAILED DESCRIPTION

[0038] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0039] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0040] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0041] Unless otherwise specified, all raw materials used in the examples of the present invention are commercially available. Details are shown in Table 1.

[0042] Table 1

[0043]

[0044]

[0045] Example 1

[0046] This example provides a method for preparing a fifth-generation dendrimer modified with phenylboronic acid:

[0047] (1) Dissolve 100 mg of the fifth-generation dendrimer in anhydrous methanol to ensure a uniform solution to obtain a fifth-generation dendrimer solution.

[0048] (2) Phenylboronic acid was slowly added to the fifth-generation dendrimer solution, wherein the molar ratio of the fifth-generation dendrimer to 4-bromomethylphenylboronic acid was 1:5. After stirring evenly, the reaction system was heated to 70° C. and stirred at this temperature for 24 hours to ensure sufficient reaction.

[0049] (3) After the reaction is complete, the mixture is cooled to room temperature. The product is placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed against anhydrous methanol for 24 hours to remove unreacted phenylboronic acid and solvent impurities. Subsequently, the product is further purified by dialysis against deionized water for 48 hours. Finally, the dialyzed solution is passed through an ultrafiltration membrane for solute concentration to obtain a high-concentration phenylboronic acid-modified fifth-generation dendrimer product.

[0050] (4) The fifth generation dendrimer macromolecules (GPNPs) modified with phenylboronic acid were obtained, and the number of modified phenylboronic acid was detected by 1HNMR. The number of modified phenylboronic acid was calculated to be 55-65 based on the area, as shown in Fig. Figure 1 shown.

[0051] Example 2

[0052] Preparation of SOX9@GPNPs: According to the N / P ratio calculation method, the concentration of SOX9 expression plasmid (0.8 μg) was maintained unchanged, and the N / P ratio (GPNPs: SOX9 expression plasmid) was mixed at an 8:1 ratio. After vortex stirring, the mixture was allowed to stand at room temperature for 30 minutes.

[0053] The prepared SOX9@GPNPs were dispersed in 1 ml of deionized water, and the surface particle size was measured using a Zetasizer Nano-ZS (Malvern, UK). Figure 2 As shown in (a).

[0054] Example 3

[0055] The difference from Example 2 is that the N / P ratios of GPNPs and SOX9 expression plasmid are 0, 1:1, 2:1, 4:1, and 16:1, respectively.

[0056] The binding ability of GPNPs to SOX9 expression plasmid was detected by agarose gel electrophoresis.

[0057] In addition, to evaluate the efficiency of GPNPs in delivering plasmid DNA at different nitrogen-phosphorus ratios, GPNPs were mixed with a plasmid expressing ZFgreen1 green fluorescent protein at N / P ratios of 0, 1, 2, 4, 8, and 16. When the nucleus pulposus cells reached an appropriate density, the mixture was added to the culture medium of each well. After 24 hours, the expression of green fluorescence was observed under an inverted microscope. The transfection efficiency was detected by qPCR. The results are shown in Figure 2. Figure 2 (c) shown. Figure 3 The results showed that when the N / P ratio was 1:1, GPNPs were able to form a stable complex with the plasmid, indicating that it had a high binding capacity. Transfection experiments were conducted using complexes formed by (containing the green fluorescent protein Zs Green1 expression gene fragment) and GPNPs at different N / P ratios to investigate its transfection efficiency. The results showed that as the N / P ratio increased, the number of ZsGreen1-positive cells gradually increased, indicating that the transfection efficiency increased ( Figure 2 (b) Figure 4 ).

[0058] Example 4

[0059] Preparation of SIRT1@GPNPs: SIRT1 protein and GPNPs were mixed at a mass ratio of 2:1, vortexed and placed at room temperature for 30 min.

[0060] The particle size of SIRT1@GPNPs was measured using Zetasizer Nano-ZS. Figure 2 (d) shown.

[0061] Example 5

[0062] The difference from Example 4 is that the mass ratio of SIRT1 protein to GPNPs was 2:1 and the SIRT1 protein was co-treated with different groups, namely PBS group, PBS / LPS group, and GPNPs / PBS group. + Group, SIRT1 / LPS only + group and SIRT1@GP NPs / LPS + Reactive oxygen species staining of co-cultured nucleus pulposus cells.

[0063] Fluorescence imaging of different groups was observed using a fluorescence microscope.

[0064] The results showed that SIRT1 protein could inhibit LPS-induced oxidative stress only in the presence of GPNPs. Figure 2 (f) shown.

[0065] Comparative Example 1

[0066] Control group: treated with DMEM / F12 cell culture medium and PBS only.

[0067] Comparative Example 2

[0068] GPNPs group: treated with DMEM / F12 cell culture medium plus 1 μg / ml LPS

[0069] Comparative Example 3

[0070] BSA-FITC group: cells were treated with DMEM / F12 cell culture medium supplemented with 1 μg / ml LPS and 10 μg / ml SIRT1 protein.

[0071] Example 6

[0072] Preparation of BSA-FITC@GPNPs: Using BSA-FITC as a model protein, GPNPs and BSA-FITC (mass ratio of 2:1) were added to the cell culture medium.

[0073] Evaluation of GPNPs protein delivery efficiency: After treating Example 6 and Comparative Examples 1 to 3 for 6 h, the cells were fixed with 4% paraformaldehyde. Flavonoid staining was performed at 37°C in the dark for 1 h, and then nuclear staining was performed with DAPI staining solution. The fluorescence distribution was then observed under an inverted microscope. BSA-FITC was effectively delivered to NPCs and localized in the cytoplasm, as shown in Figure 2. Figure 2 (e) shown.

[0074] Example 7

[0075] Preparation of SIRT1 / SOX9@GPNPs: First, GPNPs and SOX9 expression plasmid were assembled at an N / P ratio of 8:1 to form SOX9@GPNPs. Then, SIRT1 was assembled on GPNPs at a mass ratio of SIRT1 protein to GPNPs of 2:1 to form SIRT1 / SOX9@GPNPs.

[0076] In order to characterize the ability of GPNPs to co-deliver SIRT1 protein and SOX9 expression plasmid, the particle size changes of GPNPs, SOX9@GPNPs prepared in Example 2, and SIRT1 / SOX9@GPNPs prepared in Example 7 were monitored by Zetasizer Nano-ZS within 36 h, and the morphology of SIRT1 / SOX9@GPNPs was characterized by TEM (JEOL JEM 2100F). Figure 2 g, i). To evaluate intracellular delivery, BSA-FITC and a plasmid expressing DsRed fluorescent protein were delivered into cells. The intracellular fluorescence distribution was then observed 6, 12, 24, and 48 h after delivery ( Figure 2 h).

[0077] The SOX9@GPNPs prepared in Example 2, the SIRT1@GPNPs prepared in Example 4, and the SIRT1 / SOX9@GPNPs prepared in Example 7 were stably combined to form nanoparticles with uniform particle size and were able to exert biological functions. In addition, SIRT1 and SOX9 appeared in a sequential order in the cells, as shown in FIG. Figure 2 shown.

[0078] Example 8

[0079] (1) Dissolve 1.5 g of hyaluronic acid (HA) in 150 ml of deionized water. After thorough stirring until dissolved, add 802 mg of sodium periodate, and stir the mixture in the dark for 3 h. Finally, dialyze the product against deionized water for 48 h, freeze-dry, and store at 4°C to obtain OHA.

[0080] (2) OHA (1%) and GelMA (5%) were dissolved in 1 ml of LAP to form a G-HA prepolymer solution. SIRT1@GPNPs prepared in Example 2, SOX9@GPNPs prepared in Example 4, and SIRT1 / SOX9@GPNPs prepared in Example 7 were then dissolved in the prepolymer solution at a volume ratio of 1:20. The prepolymers were then cross-linked by ultraviolet irradiation for 30 seconds to obtain SIRT1@GPNPs@G-HA, SOX9@GPNPs@G-HA, and SIRT1 / SOX9@GPNPs@G-HA. The hydrogels were then sterilized or freeze-dried for further experiments.

[0081] The above hydrogel samples were prepared by freeze-drying method and then analyzed using scanning electron microscopy (SEM) (Quanta 250, FEI, Hillsboro, OR, USA). The addition of SIRT1 / SOX9@GPNPs did not change the porous structure of the hydrogel, as shown in Figure 2. Figure 5 shown.

[0082] Example 9

[0083] In vivo release of GPNPs-Cy3 was monitored using an IVIS imaging system:

[0084] Preparation of GPNPs-cy3: To modify GPNPs with Cy3, first prepare MES buffer (EDC:NHS=1:1). Cy3 and GPNPs are then dissolved in the MES buffer, vortexed, and incubated overnight at room temperature to allow for complete reaction. After the reaction is complete, the mixture is centrifuged at 5000 rpm for 10 minutes, the supernatant discarded, and the precipitated GPNPs are washed with ethanol or DMF to remove unreacted Cy3. This washing step is repeated two to three times. The washed GPNPs are then placed in a dialysis bag and dialyzed against purified water for 24 hours to further remove unbound dye and other impurities, yielding purified GPNPs-cy3.

[0085] Six-week-old Sprague Dawley (SD) rats were randomly divided into two groups, one group received SIRT1 / SOX9@GPNPs-cy3@G-HA injection prepared in Example 8, and the other group received SIRT1 / SOX9@GPNPs-cy3 intervertebral disc injection prepared in Example 7. The fluorescence intensity on days 0, 7, and 14 after injection was observed using an IVIS system. The fluorescence intensity of the SIRT1 / SOX9@GPNPs-cy3 group decreased significantly on day 7, while the SIRT1 / SOX9@GPNPs-cy3@G-HA group still maintained clear fluorescence intensity on day 14. These results indicate that the composite hydrogel SIRT1 / SOX9@GPNPs@G-HA with sustained-release functional nanoplatform has higher retention efficiency in the body, such as Figure 6 shown.

[0086] Example 10

[0087] (1) Prepare four hydrogels prepared in step (2) of Example 8, namely SIRT1@GPNPs@G-HA, SOX9@GP NPs@G-HA, SIRT1 / SOX9@GPNPs@G-HA, and G-HA without any nanoparticles, and configure SIRT1 / SOX9@GPNPs@G-HA hydrogels containing 2 times and 3 times the SIRT1 / SOX9@GPNPs concentration (2X and 3X).

[0088] (2) Nucleus pulposus cells (NPCs) were extracted from the caudal vertebrae of male Sprague-Dawley rats (6 weeks old). Under sterile conditions, the nucleus pulposus tissue was removed from the intervertebral disc and incubated at 37°C for 4 h in a solution containing 0.25% type II collagenase (Shanghai, Yuanye). The digested mixture was then collected, centrifuged to precipitate the cells, and the supernatant was discarded. The cell pellet was resuspended in DMEM / F12 medium supplemented with 10% FBS. After the cells attached, the medium was changed every two days, and the cell morphology was checked regularly.

[0089] (3) In vitro cytotoxicity was assessed using the CCK8 assay. Approximately 6,000 cells were seeded in a 96-well plate and cultured overnight to allow attachment. The cells were grouped according to the above culture system, with three replicates per group. The next day, NPCs were treated with the different formulations for 24 h and then incubated with CCK8 solution for an additional 2 h. The absorbance at a wavelength of 450 nm was measured using a spectrophotometer (Inbitrogen, USA).

[0090] (4) Total RNA was extracted using TRIzol (Invitrogen, California, USA), and RNA concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA). 1 μg of total RNA was reverse transcribed into cDNA and amplified according to the manufacturer's instructions. Primer sequences were provided by Sangon Biotech (Shanghai, China). The expression of all genes, including FOXO3, Gp x, Sod2, and Cat, was normalized to GAPDH.

[0091] (5) Intracellular ROS levels were assessed using a ROS detection kit (Beyotime, China) according to the manufacturer's protocol. After treatment with different groups, NPCs were exposed to DCFH-DA (10 μM) and incubated for 20 minutes. NPC cells were then thoroughly washed three times with serum-free medium to remove excess DCFH-DA. Cells were then stained with Hoechst 33342 live cell stain (Beyotime, China) at 37°C for 10 minutes. ROS levels were subsequently analyzed using fluorescence microscopy.

[0092] (6) Mitochondrial membrane potential was detected using the mitochondrial membrane potential sensitive indicator JC-1 dye (Beyotime, China) according to the instructions provided by the manufacturer. The functional mechanism of JC-1 depends on its aggregation state in response to membrane potential: it forms red fluorescent J-aggregates at high potentials and exists as green fluorescent monomers at low potentials. Before staining, nasopharyngeal carcinoma cells were rinsed with PBS and then treated with preheated JC-1 dye and nuclear stain Hoechst 33342 at 37°C for 30 minutes. After staining, the cells were rinsed three times with PBS to remove unbound dye. Finally, the stained cells were observed under an inverted microscope to evaluate the intensity and distribution of red and green fluorescence, and the fluorescence intensity was analyzed using ImageJ software.

[0093] (7) To measure mitochondrial reactive oxygen species (mtROS), we used MitoSOX Red, a fluorescent probe specifically designed to detect superoxide in mitochondria (MCE, USA). For staining, a 10 μM MitoSOX Red working solution was first prepared. After discarding the culture medium, freshly prepared MitoSOX Red working solution was added to ensure that all cells were covered. The cells were then incubated in a dark environment at 37°C and 5% CO2 for 30 minutes and then stained with Hoechst 33342 under the same conditions. After the incubation period, the cells were rinsed three times with PBS to remove unbound dye. Finally, the cells were observed under an inverted microscope and analyzed using ImageJ software.

[0094] (8) After stimulation, nasopharyngeal carcinoma cells were collected and thoroughly centrifuged. They were then further fixed: washed three times with 0.1 M phosphate buffer (pH 7.2); refixed with 1% osmium tetroxide; and washed three times with 0.1 M phosphate buffer (pH 7.2). Afterwards, the samples were dehydrated in a series of graded alcohols, infiltrated, and embedded in resin. The resin blocks were then cut into ultrathin sections (50 nm) for staining. Finally, the sections were observed under a transmission electron microscope.

[0095] (9) Proteins were extracted from nasopharyngeal carcinoma cells under different experimental conditions using RIPA buffer (Thermo Fisher, USA) containing protease inhibitors. Protein concentration was determined using a BCA protein assay kit (Beyotime, China). Proteins were denatured at a temperature above 95°C and then loaded onto a 10% SDS-PAGE gel in equal volumes for electrophoresis. The proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membrane was blocked with Western blot blocking buffer (Beyotime, China) for 2 hours and then incubated with diluted primary antibodies against Col II, ACAN, NLRP3, Pro-Caspase-1, Caspase-1, GSDMD, GSDMD-NT, ASC, and GAPDH (1:1000) at 4°C overnight. The next day, the primary antibody was removed and the membrane was washed three times with TBST. The membrane was then incubated with diluted secondary antibodies (1:1000) for 2 hours. After washing three times with TBST, the membrane was scanned using a gel imaging system and the band intensity was analyzed using ImageJ software to assess protein expression levels. The antibody list is shown in Table 2 below.

[0096] Table 2 Antibody table

[0097] Antibody Vendor Catalog number COLⅡ Abcam, UK ab34712 ACAN Abcam, UK ab36861 KRT19 Abcam, UK ab76539 NLRP3 Abcam, UK ab263889 ASC Abcam, UK ab309497 Caspase-1 Proteintech,USA 22915-1-AP GSDMD Proteintech,USA 20770-1-AP GAPDH Proteintech,USA 60004-1-Ig MMP13 Abconal,China A11148

[0098] (10) Approximately 10,000 NPCs were seeded in a 24-well plate and allowed to attach overnight. The next day, different stimuli were applied.

[0099] Nasopharyngeal carcinoma cells were fixed with 4% paraformaldehyde at room temperature and blocked with Triton X-100-containing immunostaining blocking solution (Biotech, China) for 1 hour. Cells were then incubated with diluted primary antibodies (1:200, including COL II, KRT19, and NLRP3) at 4°C overnight. Finally, the cells were observed under an inverted microscope, and fluorescence intensity was semi-quantitatively analyzed using ImageJ software.

[0100] (11) Thirty adult male rats weighing approximately 300 g and aged between 8 and 10 weeks were randomly divided into four experimental groups: Sham group, Defect group, G-HA group, and SIRT1 / SOX9@GPNPs@G-HA group. Specific procedures: The Sham group was the control group and underwent surgical operation; the Defect group was the acupuncture-untreated group, in which a 21G needle was inserted into the intervertebral spaces between Co7 and Co8 and Co9 and Co10 for 2 cm, rotated 180 degrees, and retained for 30 seconds; the G-HA group was injected with 10 μl of G-HA hydrogel after acupuncture; the SIRT1 / SOX9@GPNPs@G-HA group was injected with 10 μl of SIRT1 / SOX9@GPNPs@G-HA hydrogel after acupuncture.

[0101] Before surgery, the workbench and rat coccyx were disinfected. To minimize interference with adjacent degenerated segments, a 20G needle was used to puncture the NP tissue in the center of the coccyx at segments 7-8 (Co7-8) and 9-10 (Co9-10) to a depth of 5 mm, rotated 360 degrees, and held for 30 seconds. PBS and hydrogels of different compositions were then injected into the puncture site. After surgery, the coccyx was disinfected again, and the rats were transferred to a constant temperature and ventilated environment for recovery.

[0102] At 4 and 8 weeks after surgery, rats underwent radiographic imaging under anesthesia and in the supine position. The disc height index (DHI%) was normalized using X-ray images using Image J software. T2-weighted images of the intervertebral disc were acquired using a 1.5T MRI scanner (Magnetom Essenza, Siemens Medical Solution, Erlangen, Germany) and analyzed using Image J software to assess the white signal intensity. Coccyx samples from SD rats were collected at 4 and 8 weeks and fixed in formalin solution for 24 hours. NP tissue was isolated and embedded in paraffin and cut into 5 μm thick sections. The sections were examined by H&E and Safranin O / Fast Green (SO / FG) staining to observe the disc structure and matrix component distribution. Finally, the degenerated discs were evaluated and graded according to the histological grading scheme described previously.

[0103] The nanoparticle composite hydrogel has excellent biocompatibility, which is manifested in its non-cytotoxicity determined by CCK8 method, and no significant difference between live-dead staining and phalloidin staining and the control group. Figure 7 Subsequently, ROS, MitoSOX, JC-1 staining and RT-qPCR were used to detect the recovery of the antioxidant function of the nucleus pulposus cells by the hydrogel, and the promotion of the recovery of the mitochondrial structure of the nucleus pulposus cells, as shown in Figure 2. Figure 8 As shown. Furthermore, the hydrogel demonstrated its repair function on the extracellular matrix of nucleus pulposus. Figure 9 Finally, the repair effect of the hydrogel on the intervertebral disc height, water content and structure was verified in the rat tail degeneration animal model, as shown in Figure 2. Figure 11 shown.

[0104] Example 11

[0105] (1) Preparation Step (2) of Example 8 was used to prepare four hydrogels: SIRT1@GPNPs@G-HA, SOX9@GPNPs@G-HA, SIRT1 / SOX9@GPNPs@G-HA, and G-HA without any nanoparticles.

[0106] (2) After disinfection, place 300 μl of hydrogel in each well of a 24-well plate. 4 Nasopharyngeal carcinoma cells were seeded on the hydrogel surface at a density of 10 cells / mL and cultured overnight. Cell viability was assessed on days 1, 3, and 5 using CCK-8 solution and live / dead staining. Cell morphology was observed using phalloidin staining, and cell proliferation was assessed using EDU (Beyotime, China) staining.

[0107] (3) Resuspend NPCs at a concentration of 2×10 6 cells / mL hydrogel solution and solidified. After thorough mixing, 50 μl of the culture system was dispensed into each well of a 24-well plate. The culture medium was changed every two days, and the cultures were fixed with 4% paraformaldehyde after seven days. The fixed samples were then dehydrated with a gradient sucrose solution, embedded in optimal cutting temperature (OCT) compound, and cryosectioned into 10 μm thick sections. The cryosections were treated with sodium citrate buffer at 85°C for 10 minutes for antigen retrieval, followed by peroxide treatment for 10 minutes. The sections were then incubated with diluted primary antibodies (1:200, NLRP3, COLII, KRT19) at 4°C overnight. The next day, after three rinses with PBST, fluorescent secondary antibodies were used for labeling. Finally, the sections were observed under an inverted microscope, and the number of positively stained cells was quantified using Image J software.

[0108] By implanting nucleus pulposus cells on the surface and inside of the nanoparticle composite hydrogel, we achieved two-dimensional and three-dimensional culture of the cells, and verified by EDU staining and immunofluorescence staining respectively that the hydrogel still had a therapeutic effect on nucleus pulposus cells. Activated caspase-1 helped promote the cleavage of GSDMD into its N-terminal fragment (GSDMD-NT), thereby inducing cell membrane perforation and triggering cell apoptosis

[16] . Subsequently, we verified by Western blot that after LPS induction, NOD-like receptor signaling pathway-related proteins were upregulated, and the intervention of SIRT1 and SIRT1 / SOX9@GPNPs partially reversed the negative effects of LPS. Slice immunofluorescence further confirmed the above experimental results ( Figure 10 The above experimental results indicate that the repair mechanism of SIRT1 / SO X9@GPNPs may involve the inhibition of NOD-like receptor signaling pathway.

[0109] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, and all of these should be included in the scope of the present invention.

Claims

1. A nanoparticle composite hydrogel for treating intervertebral disc degeneration, characterized by: The nanoparticle composite hydrogel includes a SOX9 expression plasmid, a SIRT1 protein, a fifth-generation dendrimer modified with phenylboronic acid, methacrylated gelatin, and hyaluronic acid modified with amino and aldehyde groups; Among them, SOX9 expression plasmid and SIRT1 protein are used as cargo; phenylboronic acid-modified fifth-generation dendrimers are used as carriers.

2. The nanoparticle composite hydrogel according to claim 1, wherein: The nitrogen-phosphorus ratio of the SOX9 expression plasmid to the fifth-generation dendrimer modified with phenylboronic acid is 1 to 16:

1.

3. The nanoparticle composite hydrogel according to claim 1, wherein: The mass ratio of the SIRT1 protein to the fifth-generation dendrimer modified with phenylboronic acid is 2 to 8:

1.

4. A method for preparing a nanoparticle composite hydrogel for treating intervertebral disc degeneration, characterized by: include, The SOX9 expression plasmid and SIRT1 were mixed with the fifth-generation dendrimer modified with phenylboronic acid to obtain multifunctional nanoparticles loaded with dual bioactive factors; The multifunctional nanoparticles loaded with dual bioactive factors are cross-linked with methacrylated gelatin and hyaluronic acid modified with amino and aldehyde groups through physical, chemical and photoinitiated cross-linking to obtain the nanoparticle composite hydrogel for treating intervertebral disc degeneration.

5. The preparation method according to claim 4, wherein: The preparation method of the fifth-generation dendrimer modified with phenylboronic acid comprises: The fifth-generation dendrimer is added to anhydrous methanol and mixed evenly to obtain a fifth-generation dendrimer solution; Phenylboronic acid was added to the fifth-generation dendrimer solution, heated and stirred, and then cooled to room temperature. The obtained product was dialyzed in anhydrous methanol and deionized water, respectively, and then ultrafiltered and concentrated to obtain the fifth-generation dendrimer modified with phenylboronic acid.

6. The preparation method according to claim 5, wherein: The molar ratio of the phenylboronic acid to the fifth-generation dendrimer solution is 2 to 10:

1.

7. The preparation method according to claim 4, wherein: The mass ratio of the multifunctional nanoparticles loaded with dual biologically active factors to the methacrylated gelatin is 1:12-48.

8. The preparation method according to claim 4, wherein: The preparation method of the amino- and aldehyde-modified hyaluronic acid comprises the following steps: dissolving the hyaluronic acid in deionized water, stirring thoroughly until dissolved, adding sodium periodate, stirring in the dark, dialyzing the product with deionized water, and freeze-drying.

9. The preparation method according to claim 8, wherein: The mass ratio of the hyaluronic acid to sodium periodate is 2-4:

1.

10. The preparation method according to claim 9, wherein: The ratio of the multifunctional nanoparticles loaded with dual biologically active factors to the aldehyde-modified hyaluronic acid is 1:18-54.

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