Cell microenvironment stabilizer for capturing hydrogen ions, and preparation method and application thereof

Abstract

This invention provides a cell microenvironment stabilizer that captures hydrogen ions, its preparation method, and its application, belonging to the field of biomedical technology. The preparation method of this cell microenvironment stabilizer includes the following steps: (1) mineralizing CaCl2 with TGF-β1, catalase, and polyethylene glycol-polyglutamic acid block copolymer to obtain mineralized nanoparticles; (2) preparing hydrogel microspheres under freezing conditions using a microfluidic device, and then photocrosslinking the frozen hydrogel microspheres; (3) mixing the hydrogel microspheres with the mineralized nanoparticles to prepare the cell microenvironment stabilizer. This invention prepares a mineralized hydrogel microsphere, which, as a cell microenvironment stabilizer, can be used to block the NLRP3 cascade in chronic inflammation, capturing excess hydrogen ions through the mineralization layer to neutralize the acidic microenvironment. This micro / nano bioreactor based on hydrogel microspheres can effectively interfere with NLRP3 in the microenvironment of degenerated tissues from inside and outside the cells, inhibiting chronic inflammation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a cell microenvironment stabilizer that captures hydrogen ions, its preparation method, and its application. Background Technology

[0002] Inflammation is a key factor in tissue regeneration. Moderate acute inflammation can accelerate tissue regeneration, while chronic inflammation can cause irreversible tissue damage. The Nod-like receptor protein 3 (NLRP3) inflammasome pathway is an important pathway for the formation of chronic inflammation, and its abnormal activation has been shown to be a crucial molecular mechanism in many inflammatory diseases, metabolic diseases, degenerative diseases, and age-related diseases.

[0003] In chronic inflammation, the NLRP3 cascade is activated by endogenous danger signals and damage-associated molecular patterns (DAMPs), including inflammatory cytokines, acidity, and reactive oxygen species (ROS). Activated NLRP3 inflammasomes recruit connexins ASC and Caspase-1 precursors to assemble into inflammasome polymers. The assembled inflammasomes produce activated cleaved Caspase-1, which further splices inactive IL-1β precursors into active IL-1β. This NLRP3 / Caspase-1 / IL-1β cascade, known as the classic NLRP3 inflammatory cascade, participates in the formation of the local inflammatory microenvironment by releasing IL-1β, leading to long-term chronic inflammation and persistent tissue damage. Therefore, controlling the activity level of NLRP3 inflammasomes can effectively inhibit the progression of these diseases.

[0004] Currently, several small molecule inhibitors of NLRP3 (such as MCC950) have entered clinical trials for the purpose of suppressing systemic inflammation. [1] However, MCC950 exhibits strong hepatotoxicity when administered systemically, while local injection carries a high risk of infection due to repeated trauma and inflammation suppression. In the damaged microenvironment, NLRP3 activation is primarily caused by alterations in microenvironmental risk factors such as ATP, cholesterol, particles, or physicochemical factors. Therefore, effectively eliminating external microenvironmental factors while maintaining the long-term stability of the extracellular microenvironment holds promise for achieving sustained and effective suppression of inflammation while avoiding the local toxicity of small molecule inhibitors.

[0005] Based on this, people are eager to develop engineered biomaterials that can specifically inhibit the abnormal activation of NLRP3 inflammasomes and suppress chronic inflammation, in order to use them to promote the regeneration of degenerative tissues.

[0006] The root cause of aberrant NLRP3 activation is the abnormal environment resulting from extracellular matrix (ECM) damage. Considering the loss and inactivation of local drugs, even if NLRP3 is inhibited early by stabilizing the intracellular and extracellular environment, the environment will gradually deteriorate over time due to ECM destruction, thus losing the ability to inhibit the NLRP3 inflammasome. ECM renewal and repair can further maintain the stability of the intracellular and extracellular environment, thereby providing a favorable environment for cells to perform their functions. Therefore, initiating ECM renewal while inhibiting NLRP3 is key to ultimately eliminating chronic inflammation and completing tissue regeneration. This can be achieved through methods such as using cytokines or small molecule compounds. [2,3] Strategies to enhance ECM synthesis in nucleus pulposus cells (NPCs) were among the earliest attempts in this direction. However, in environments with low pH, high ROS, and high inflammation, cell renewal capacity is severely impaired, and cells become insensitive to exogenous drug stimulation. Furthermore, direct injection of drugs into the intervertebral disc presents problems such as rapid drug loss and leakage through the needle insertion site.

[0007] Some researchers [4-6] By constructing injectable hydrogel microspheres based on microfluidics technology to mimic the intervertebral disc remodeling (ECM) over a short period, a stable environment can be provided for endogenous or exogenous cells, effectively alleviating tissue degeneration through local drug delivery. However, pure hydrogel microspheres cannot inhibit inflammation, and their degradation rate is significantly accelerated in harsh microenvironments, failing to provide a long-term stable environment for cell remodeling. Therefore, cells loaded in microspheres are also easily affected by the inflammatory microenvironment after entering the intervertebral disc, making it difficult for them to exert their ECM remodeling capabilities.

[0008] Therefore, how to develop hydrogel microsphere biomaterials that can specifically inhibit the abnormal activation of NLRP3 inflammasomes and suppress chronic inflammation, and how to fully utilize the environmental stabilization ability of microspheres to give full play to the remodeling function of ECM, better promote tissue regeneration, reshape the benign cell-environment cycle, and thoroughly improve the environment for cell survival has become an urgent technical problem to be solved.

[0009] The cited references are as follows:

[0010] [1]H.Li,Y.Guan,B.Liang,P.Ding,X.Hou,W.Wei,Y.Ma,Therapeutic potential of MCC950,a specific inhibitor of NLRP3 inflammasome.Eur J Pharmacol 928,175091(2022).

[0011] [2] J.S. Kang, C. Liu, R. Derynck, New regulatory mechanisms of TGF-beta receptor function. Trends Cell Biol 19, 385 - 394 (2009).

[0012] [3] B. Costachescu, A.G. Niculescu, R.I. Teleanu, B.F. Iliescu, M. Radulescu, A.M. Grumezescu, M.G. Dabija, Recent Advances in Managing Spinal Intervertebral Discs Degeneration. Int J Mol Sci 23, (2022).

[0013] [4] H. Ruan, Y. Li, D. Zheng, L. Deng, G. Chen, X. Zhang, Y. Tang, W. Cui, Engineered extracellular vesicles for ischemic stroke treatment. The Innovation, 100394 (2023).

[0014] [5] Y. Lei, Y. Wang, J. Shen, Z. Cai, Y. Zeng, P. Zhao, J. Liao, C. Lian, N. Hu, X. Luo, W. Cui, W. Huang, Stem Cell-Recruiting Injectable Microgels for Repairing Osteoarthritis. Advanced Functional Materials 31, 2105084 (2021).

[0015] [6] X. Li, X. Li, J. Yang, J. Lin, Y. Zhu, X. Xu, W. Cui, Living and Injectable Porous Hydrogel Microsphere with Paracrine Activity for Cartilage Regeneration. Small, e2207211 (2023). Summary of the Invention

[0016] This invention aims to solve the aforementioned technical problems by providing a cell microenvironment stabilizer that captures hydrogen ions, its preparation method, and its applications. The technical objective of this invention is to address the shortcomings of existing technologies that use hydrogel microspheres to stabilize the cell microenvironment. These hydrogel microspheres cannot inhibit inflammation, degrade too rapidly in harsh microenvironments, fail to provide a long-term stable environment, and thus cannot effectively perform ECM remodeling. This invention provides a cell microenvironment stabilizer with acid regulation and ROS inhibition capabilities. It can effectively stabilize the extracellular microenvironment, block the activation of endogenous NLRP3, remodel a stable anti-inflammatory microenvironment, and enhance the cell's own ECM synthesis capacity. This allows for the formation of a long-term stable cellular environment, promoting the repair of intervertebral disc degeneration and effectively inhibiting the maintenance and development of chronic inflammation.

[0017] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0018] This invention first provides a method for preparing a cell microenvironment stabilizer that captures hydrogen ions, comprising the following steps:

[0019] (1) CaCl2 solution was mixed with HEPES buffer containing TGF-β1, catalase, polyethylene glycol-polyglutamic acid block copolymer and CaCO3, and a mineralization reaction was carried out under stirring to obtain mineralized nanoparticles.

[0020] (2) GelMA hydrogel microspheres and serum leucine aminopeptidase (LAP) were dissolved in PBS buffer as the aqueous phase, and paraffin oil and Span80 were thoroughly mixed as the oil phase. Hydrogel microspheres were prepared under freezing conditions using a microfluidic device. The frozen hydrogel microspheres were then photocrosslinked and freeze-dried.

[0021] (3) The hydrogel microspheres obtained in step (2) are mixed with the mineralized nanoparticles obtained in step (1) to prepare the cell microenvironment stabilizer.

[0022] The method provided by this invention constructs an injectable artificial active "cellular microenvironment stabilizer" through biomimetic mineralization and microfluidic technology, which is a mineralized hydrogel microsphere (GMNP). This invention first utilizes carboxyl groups and Ca... 2+Through a chelation reaction, a mineralized nanoparticle stabilizer (MNP) loaded with catalase (CAT) and TGF-β was successfully designed and integrated into porous GelMA microspheres to form a "cellular soil" (GMNP). This GMNP acts as a "cellular microenvironment stabilizer," blocking the NLRP3 cascade in chronic inflammation. The GMNP neutralizes the acidic microenvironment by capturing excess hydrogen ions through the mineralization layer. Furthermore, the released cytokine transforming growth factor-β helps promote extracellular matrix (ECM) remodeling, collectively eliminating exogenous NLRP3 activators. Under a stable extracellular environment, the catalase in the GMNP further blocks mitochondrial ROS production and inhibits endogenous NLRP3 activation. In in vitro experiments, GMNP inhibits the activation of the TXNIP / NLRP3 / IL-1β cascade and enhances ECM synthesis in nucleus pulposus cells. In vivo, GMNP, with microspheres at its core, constructs a sustainable and stable niche, inhibiting chronic inflammation and promoting the regeneration of degenerated intervertebral discs. Therefore, the present invention provides a cellular microenvironment stabilizer that captures hydrogen ions, which can effectively interfere with NLRP3 in the microenvironment of degenerated tissues both inside and outside the cell, thereby inhibiting chronic inflammation. This microsphere system can inhibit the activation of NLRP3 inflammasomes, reshape a stable anti-inflammatory microenvironment, and simultaneously enhance the cell's own ECM synthesis capacity, thus forming a long-term stable cellular environment and promoting the repair of degenerated intervertebral discs.

[0023] The MNP in this invention utilizes Ca in CAT. 2+ The mineralized nanoparticles (MNPs) were prepared by biomineralization through chelation of carboxyl groups with TGF-β. Furthermore, polyethylene glycol-modified polyglutamic acid (mPEG-P(Glu)) was synthesized to provide additional carboxyl groups and a PEG shell, making the MNPs more stable and biocompatible. The MNPs have a diameter of approximately 100 nm, possess acid-neutralizing capabilities, and retain the activity of the contained proteins.

[0024] Experiments have confirmed that the mineralized shell of the nanoparticles can neutralize lactic acid, improve the microenvironment, and protect cells. Then, the released CAT and TGF-β inhibit excessive mitochondrial ROS and remodeled ECM synthesis, respectively, exerting anti-inflammatory and regenerative effects. Ultimately, the GelMA microspheres provide a therapeutic core and replenish ECM. In summary, the cell microenvironment stabilizer system constructed in this invention provides a novel strategy for treating chronic inflammation by blocking the NLRP3 inflammasome cascade.

[0025] Furthermore, the mineralization reaction in step (1) is carried out under the condition of stirring at 4°C for 12 hours.

[0026] Furthermore, the weight ratio of TGF-β1, catalase and polyethylene glycol-polyglutamic acid block copolymer in step (1) is 1:150:5.

[0027] Furthermore, the freezing conditions in step (2) are -40°C, and the photocrosslinking conditions are crosslinking under 405nm light for 15 minutes.

[0028] Furthermore, the polyethylene glycol-polyglutamic acid block copolymer is synthesized via the Fuchs-Farthing reaction.

[0029] Furthermore, the CaCl2 is dissolved in Tris-HCl buffer solution with a concentration of 1 mM, a pH of 7.6, and a CaCl2 concentration of 100 mM.

[0030] Furthermore, the HEPES buffer in step (1) has a concentration of 50 mM, a pH of 7.1, contains 140 mM NaCl, and has a concentration of 10 mM Na2CO3.

[0031] Furthermore, in step (2), the flow rate of the oil phase in the microfluidic device is controlled to be 400 μl / min, and the flow rate of the water phase is controlled to be 40 μl / min.

[0032] A second objective of this invention is to provide a cell microenvironment stabilizer that captures hydrogen ions, prepared by the method described above.

[0033] A third objective of this invention is to provide the application of the aforementioned cell microenvironment stabilizer that captures hydrogen ions, which is to prepare the cell microenvironment stabilizer into a drug that inhibits chronic inflammation or promotes the regeneration of degenerated intervertebral discs.

[0034] The beneficial effects of this invention are as follows:

[0035] (1) The present invention prepared a cell microenvironment stabilizer (GMNP) capable of capturing hydrogen ions. Cell experiments confirmed that the GMNP can block the NLRP3 / Caspase-1 / IL-1β cascade, stabilize the intracellular and extracellular environment, and promote ECM synthesis. In vivo retention experiments showed that the GMNP has enhanced intervertebral disc retention capacity.

[0036] (2) The artificial “cell microenvironment stabilizer” provided by the present invention can effectively inhibit inflammation and significantly promote the regeneration of degenerated intervertebral discs. Attached Figure Description

[0037] Figure 1 Preparation scheme and therapeutic mechanism of cellular microenvironment stabilizer (GMNP); A) Construction of MNP; B) Preparation of GMNP by microfluidic device; C) How GMNP stabilizes the microenvironment in intervertebral disc degeneration by blocking the NLRP3 inflammasome cascade and enhancing ECM synthesis.

[0038] Figure 2 The 1H-NMR spectrum of mPEG-P(Glu) and the substitution rate of P(Glu) are shown.

[0039] Figure 3 Preparation and characterization of GMNPs; A) TEM image of MNPs, scale bar: 50 nm; B) DLS analysis of MNP size distribution; C) Evaluation of the acid neutralization capacity of MNPs; D) Evaluation of MNPs. CAT Antioxidant capacity under H2O2 and acidic conditions; E) Schematic diagram and MS micrograph of microfluidic device; F) MS / FITC-NP confocal image, scale bar: 50 μm; GH) SEM images of GM(G) and GMNP(H), scale bar: 50 μm; IJ) Surface elemental analysis of GM(I) and GMNP (J); K) Drug release spectra of MNP and GMNP under neutral or acidic pH conditions, data are expressed as mean ± SD, n = 3, ***p < 0.001.

[0040] Figure 4 To demonstrate the in vivo retention and in vitro cell protection capabilities of GMNP; A) Schematic diagram of the in vivo retention experiment; B) In vivo retention rate of Cy5.5-labeled BSA at different time points in different groups using IVIS imaging; C) Percentage and relative mean fluorescence intensity (MFI) of MNP and GMNP groups at different time points, with initial MFI set at 100% for each group; E) Percentage of final local fluorescence retention rate of each group on day 9; F) Confocal images of different formulations and NPCs under different conditions, scale bar: 50 μm; G1) Quantitative analysis of the number of adherent cells at different time points; G2) Quantitative analysis of the number of adherent cells of different microspheres at different time points (3 days, H3) 5 days, I4) 7 days, and J5), data are expressed as mean ± SD, n = 3, **p < 0.01, ***p < 0.001.

[0041] Figure 5 For GMNP CAT In vitro inhibition of NLRP3-mediated inflammation; A) GMNP CATA) Schematic diagram of NLRP3 inhibition mediated by NPCs; B) DCFH-DA staining, JC-1 staining and Mitotracker staining of NPCs, scale bar: 50 μm; C) Immunofluorescence staining of TXNIP and NLRP3 in NPCs treated with different formulations for 48 h, scale bar: 50 μm; D) Flow cytometry analysis of ROS intensity of NPCs; E) Quantitative analysis of JC-1 staining in NPCs; F) Quantitative analysis of Mitotracker staining in NPCs; G) WB bands of NLRP3 inflammasome-related proteins in NPCs; HJ) Quantitative analysis of protein expression, including TXNIP, NLRP3 and Cleaved Caspase-1; K) ELISA quantitative analysis of IL-1β protein, data are expressed as mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ns: no significant difference.

[0042] Figure 6 To verify GMNP using PCR and WB experiments CAT Changes in the NLRP3 inflammatory cascade during anti-inflammatory treatment.

[0043] Figure 7 For GMNP CAT The effect of inhibiting ROS (A); and the inhibition of NLRP3 activation level (B).

[0044] Figure 8 To verify the changes in mRNA levels in cells of each treatment group using PCR.

[0045] Figure 9 and Figure 10 To demonstrate the effect of each treatment group on the ECM synthesis ability of NPCs using immunofluorescence.

[0046] Figure 11 To promote in vivo intervertebral disc regeneration with GMNP; A) Schematic diagram of rat intervertebral disc degeneration construction and treatment, including control group, PBS (puncture), GMNP, GMNPTGF, GMNPCAT, and GM; B) X-ray and MRI images of rat intervertebral disc degeneration at different time points (including 4 weeks and 8 weeks); C) Changes in intervertebral disc height; D) Quantitative analysis of intervertebral disc gray values ​​at different time points in different groups, data are expressed as mean ± SD, n = 6, **p < 0.01, ***p < 0.001; E) Schematic diagram of material composition for each group; F) HE staining of rat intervertebral discs, scale bar: 500μm; G) Rapid green staining of rat intervertebral discs with erythromycin, scale bar: 500μm; #: statistically significant difference from the control group; *: statistically significant difference from the puncture group, data are expressed as mean ± SD, n = 3, **p < 0.01, ***p < 0.001.

[0047] Figure 12The effects of different treatment durations on in vivo intervertebral disc regeneration in each treatment group.

[0048] Figure 13 To demonstrate the role of GMNP in inhibiting intervertebral disc inflammation and promoting intervertebral disc regeneration in vivo; A) Immunofluorescence staining of NLRP3 in rat intervertebral discs, scale bar: 50 μm; B) Immunofluorescence staining of IL-1β in rat intervertebral discs, scale bar: 50 μm;

[0049] C) Quantitative analysis of NLRP3 fluorescence intensity; D) Analysis of IL-1β-positive area of ​​rat intervertebral discs; E) Immunofluorescence staining of type II collagen in rat intervertebral discs, scale bar: 50 μm; F) Immunofluorescence staining of intervertebral disc aggregates in rats, scale bar: 50 μm; G) Quantitative analysis of type II collagen fluorescence intensity in rat intervertebral discs; H) Quantitative analysis of intervertebral disc aggregates fluorescence intensity in rats, #: statistically significant difference from the control group; *: statistically significant difference from the puncture group. Data are expressed as mean ± SD, n = 3, *p < 0.05.

[0050] Figure 14 To assess the level of extracellular matrix remodeling in the intervertebral discs of each treatment group: A) Histological scores of different treatment groups; B) Proteoglycan content (%) of intervertebral discs in different treatment groups. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this invention.

[0052] The codes and their meanings involved in the embodiments are as follows:

[0053] FBS: Fetal bovine serum; DMEM: Culture medium; CAT: Catalase; TGF-β1: Transforming growth factor β1; MNP: Mineralized nanoparticles containing both CAT and TGF-β1; MNP CAT Mineralized nanoparticles containing only CAT; MNP TGF Mineralized nanoparticles containing only TGF-β1; MNP blank : Blank mineralized nanoparticles without CAT and TGF-β1 loading; GelMA: Methacrylic anhydride-modified gelatin hydrogel microspheres; GM: GelMA hydrogel microspheres without MNP loading; GMNP: GelMA hydrogel microspheres loaded with MNP; GMNP blank GelMA hydrogel microspheres loaded with blank mineralized nanoparticles.

[0054] Example 1

[0055] I. Experimental Content and Methods

[0056] (1) Materials

[0057] Fetal bovine serum (FBS), penicillin-streptomycin, DMEM, and type II collagenase were purchased from Gibco, USA; protein assay kits, dead / live staining kits, Dil, DiR, and Trizol were purchased from Thermo Fisher Scientific, USA; TGF-β was purchased from Peprotech, USA; CAT, paraffin, SPAM80, LAP, and phosphotungstic acid were purchased from Aladin, China; CCK-8, Hoechest 33258, RIPA, DCFH-DA, JC-1assay, and mitotracker were purchased from Beyotime, China; PrimeScript TM RT Master Mix and TB Premix Ex Taq TM II was purchased from Takara Corporation, Japan; Anti-TXNIP (ab188865), Anti-NLRP3 (ab263899), Anti-pro Caspase-1+p10+p12 (ab179515), Anti-MMP2 (ab92536), Anti-MMP13 (ab39012), and Anti-rabbit IgG H&L (Alexa) 488)(ab150077) and anti-rabbit IgG H&L (Alexa) 647)(ab150075) was purchased from Abcam, Inc., USA; β-Actin (8H10D10) mouse monoclonal antibody (3700S), anti-rabbit IgG HRP-linked Antibody (7074S) and anti-mouse IgG HRP-linked Antibody (7076S) were purchased from Cell Signaling Technology, Inc., USA; Collagen II Antibody (AF0135) and Aggrecan Antibody (DF7561) were purchased from Affinity Biosciences, Inc., USA.

[0058] (2) Preparation of MNP

[0059] First, polyethylene glycol-polyglutamic acid (PEG-P(Glu)) block copolymers were synthesized using the Fuchs-Farthing reaction (referencing the method in the literature: L. Bacakova, E. Filova, M. Parizek, T. Ruml, V. Svorcik, Modulation of cell adhesion, proliferation and differentiation on materials designed for body implants. Biotechnol Adv 29, 739-767 (2011).). The substitution rate of P(Glu) was determined by 1H-NMR (300 MHz; solvent: D2O).

[0060] Then, 1 ml of Tris-HCl buffer (1 mM, pH 7.6) containing 100 mM CaCl2 was mixed with 1 ml of HEPES buffer (50 mM, pH 7.1, NaCl 140 mM) containing 2 μg TGF-β1, 300 μg CAT (catalase), 10 mg mPEG-P (Glu) polymer, and 10 mM Na2CO3. The mixture was stirred at 4°C for 12 h, centrifuged (14800 rpm, 10 min) to remove free cytokines, and resuspended in physiological saline to obtain MNPs.

[0061] MNPs loaded with single-drug agents were synthesized using the same method described above. CAT and MNP TGF .

[0062] (3) Particle size analysis of MNP

[0063] The nanoparticle size of MNPs was determined using a Zetasizer (Malvern) microsample cell. First, the Zetasizer microsample cell was rinsed with distilled water. Then, 10 μl of MNPs was diluted to 1 ml with distilled water and added to the microsample cell for dynamic light scattering (DLS). The measurement was repeated three times.

[0064] (4) Preparation of GMNP hydrogel microspheres

[0065] GelMA hydrogel microspheres (denoted as GM) were prepared using a self-made coaxial microfluidic device. 250 mg of GelMA and 25 mg of LAP were dissolved in 5 ml of PBS buffer to form the aqueous phase, while 30 g of paraffin oil was thoroughly mixed with 1.2 g of Span 80 to form the oil phase. The aqueous and oil phases were connected to separate syringe pumps, with the oil phase flow rate set at 400 μl / min and the aqueous phase flow rate at 40 μl / min. The prepared hydrogel microspheres were collected in a 10 cm culture dish at -40℃. The frozen hydrogel microspheres were crosslinked under 405 nm light for 15 min, then washed three times with isopropanol and deionized water. The washed hydrogel microspheres were lyophilized and stored at room temperature.

[0066] GelMA hydrogel microspheres loaded with MNP were prepared and named GMNP. The lyophilized microspheres were incubated with PBS containing 1% MNP, and then the microspheres were lyophilized again. The lyophilized microspheres were stored in a freezer at -80°C for later use.

[0067] (5) Transmission electron microscopy analysis

[0068] Transmission electron microscopy (TEM) analysis: The sample was equilibrated at room temperature for 30 min, then 5 μl of sample was dropped onto a copper grid and allowed to stand for 5 min to remove excess liquid. The morphology of the MNPs was then observed using a 120 kV TEM, and the elemental distribution of the MNPs was analyzed using a Tecnai G2 TEM energy dispersive spectroscopy (EDS) system. After lyophilization, the sample was uniformly spread on a conductive adhesive for scanning electron microscopy (SEM) observation and energy dispersive spectroscopy analysis. After gold sputtering for 45 s, the sample was observed using a high-resolution field emission scanning electron microscope (FET).

[0069] (6) Isolation of rat nucleus pulposus cells

[0070] Four-week-old SD rats were selected as the source of NPCs (nucleus pulposus cells). After sacrificing the rats, nucleus pulposus tissue was removed and cut into 1mm × 1mm × 1mm pieces. The tissue was digested in high-glucose DMEM medium containing 0.1% type II collagenase at 37°C for 3 hours. The digested nucleus pulposus tissue was filtered through a 40μm cell strainer to remove undigested tissue fragments. After centrifugation at 1500 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in high-glucose DMEM medium containing 10% fetal bovine serum and 1% antibiotics. The cells were cultured in a 37°C incubator containing 5% CO2. Fresh medium was added every three days, and the cells were passaged at a 1:3 ratio when the cell density reached 80%. P3-P6 cells were used for subsequent experiments.

[0071] To simulate a degenerative acidic environment, in the acid-treated group, high-glucose DMEM medium containing 10% FBS and 1% antibiotics was adjusted to pH 6.2 with lactic acid and co-incubated with NPCs for 48 h. In the G-MNP group, 10% GMNP was added by volume to the acidified medium and co-incubated with NPCs for 48 h.

[0072] (7)G-MNP blank Biocompatibility evaluation of hydrogel microspheres

[0073] GM and GMNP were evaluated using the live-dead staining method and the CCK8 kit. blank To assess biocompatibility, 100 μl of lyophilized GelMA microspheres were dispersed in 1 ml of culture medium at a concentration of 1 × 10⁻⁶ microspheres per well. 4 NPCs were seeded into 24-well plates at a density of 30% and cultured. The cells were then treated with a culture medium containing hydrogel microspheres.

[0074] Cell staining: Add 20 μl of EthD-1 and 5 μl of calcein AM to 10 ml of sterile D-PBS, mix thoroughly, and incubate with cells at 37°C for 25 min. After washing three times with D-PBS, observe the stained cells under a fluorescence microscope. To evaluate CCK8, incubate cells with 10% CCK8 working solution at 37°C for 45 min. Measure the absorbance of the culture medium at 450 nm using a microplate reader.

[0075] (8) Evaluation of GMNP's effect on the extracellular microenvironment

[0076] The ability of GMNPs to improve the extracellular environment was assessed using cellular fluorescence staining. 1×10⁶ cells were collected. 7 NPCs were centrifuged at 1500 rpm and resuspended in high-glucose DMEM medium containing 0.1% Dil-red. The cells were incubated at 37°C for 30 min. Stained cells were collected, resuspended in 100 μl of medium, and slowly added to 100 μl of lyophilized GMNP microspheres. After 6 h, 1 ml of medium was added, and the cells were observed under a confocal microscope at specific time points.

[0077] (9) Cell fluorescence staining

[0078] NPCs were stained for ROS using serum-free DMEM medium containing 10 μM DCFH-DA (a reactive oxygen species fluorescent probe) and incubated at 37°C for 20 min. Cells were washed three times with serum-free cell culture medium to remove unbound DCFH-DA. Cells were then collected and observed under a microscope or analyzed by flow cytometry.

[0079] For JC-1 staining, according to the manufacturer's instructions, dilute an appropriate amount of JC-1 (200X) with ultrapure water and JC-1 staining buffer (5X) to prepare the JC-1 working solution. Incubate the JC-1 working solution with the treated NPCs at 37°C for 20 min. Wash the cells three times with JC-1 staining buffer (1X) and observe under a microscope or measure using a fluorescence microplate reader.

[0080] Mitotracker staining: The treated NPCs were treated with 200 nM Mitotracker in DMEM medium and incubated at 37°C for 20 min. After washing the cells three times with the medium, they were observed under a microscope.

[0081] Immunofluorescence staining: Treated NPCs were fixed with 4% paraformaldehyde for 30 min, followed by immunofluorescence blocking. Cells were incubated overnight at 4°C with the corresponding primary antibody (1:200). Cells were then treated with anti-rabbit green fluorescent secondary antibody (1:1000) or anti-rabbit red fluorescent secondary antibody (1:1000) for 1 hour, stained with Hoechst 33258 for 5 minutes, and observed under a confocal microscope. Between the above experimental steps, cells were washed three times with PBS buffer for 5 minutes each time.

[0082] (10) Real-time PCR

[0083] Total RNA was extracted from treated NPCs using Trizol and PrimeScript was used. TM RT MasterMix was used for reverse transcription into cDNA. TB was then used. Premix Ex Taq TM The II kit was used to detect the relative mRNA levels of BMAL and CLOCK. Primer sequences are shown in Table 1. Real-time quantitative PCR was performed using the ABI Step 1+ real-time PCR system, and the quantification of relevant genes was performed using a 24-hour PCR kit. -ΔΔCt The method is complete.

[0084] Table 1

[0085]

[0086]

[0087] (11) Protein expression analysis

[0088] To assess cellular protein expression, total protein was extracted by lysing NPCs using RIPA, and proteins of different molecular weights were separated using 8%, 10%, or 12% SDS-PAGE gels. Subsequently, protein bands were transferred to 0.45 μm PVDF membranes. QuickBlock was used for analysis. TM After blocking with blocking buffer, the PVDF membrane was incubated with primary antibody overnight. Then, the PVDF membrane was incubated with secondary antibody for 2 hours. Finally, the HRP-labeled protein bands were observed using an enhanced chemiluminescence detection system. Protein quantification was performed using ImageJ software.

[0089] (12) Local preservation ability

[0090] The local retention ability of the material was evaluated using 12-week-old SD rats and IVIS. First, Cy5.5-BSA-loaded MNPs were constructed by replacing CAT and TGF-β cytokines with Cy5.5-labeled BSA protein, following the method described above. After general anesthesia, the C5-6 and C7-8 nuclei of five SD rats were harvested, and 0.1 ml of the corresponding material (MNP or GMNP) was taken in 10 ml syringes. The retention of fluorescence signals in the rat tails was observed using IVIS on postoperative days 0, 3, 6, and 9.

[0091] (13) Establishment of an in vivo model of intervertebral disc degeneration

[0092] All animal husbandry protocols and experimental procedures were approved by the Institutional Animal Welfare and Use Committee (IACUC) of Beijing Viterhe Laboratory Animal Technology Co., Ltd. Ten-week-old male SD rats were ordered from Viterhe. After two weeks of rearing, 12 rats were used to establish an intervertebral disc degeneration model. After disinfection, an 18G needle was used to puncture the center of the C5-6, C6-7, C7-8, C8-9, and C9-10 intervertebral discs. The needle was rotated 360 degrees, held for 30 seconds, and then removed. The corresponding material (GMNP) was injected locally using a 1ml syringe. all GMNP TGF GMNP CAT 0.1 ml of GM was administered. All animal experiments were conducted under general anesthesia and in a sterile environment.

[0093] (14) In vivo imaging evaluation

[0094] X-ray images of the rat caudal vertebrae were obtained at 0, 4, and 8 weeks post-surgery. Intervertebral disc height was measured using ImageJ software. MRI imaging of the rat caudal vertebrae was also obtained at 0, 4, and 8 weeks post-surgery. The average grayscale value of the nucleus pulposus region during the T2W1 phase was used to assess the intervertebral disc water content. MRI image parameters were as follows: transient velocity: 150 mT / m / ms; field gradient: 30 mT / m; spin echo sequence T2W1 parameters were as follows: TR, TE = 2500 ms / 30 ms; scan matrix: 256 × 256; reconstruction matrix: 512 × 512; FOV (mm) = 100.00; Rfov (%) = 100.00; slice thickness = 1 mm; scan resolution = 0.1 mm.

[0095] (15) In vivo histological evaluation

[0096] Eight weeks post-surgery, rat caudal intervertebral disc specimens were collected. The isolated samples were fixed in 4% paraformaldehyde for 72 hours and decalcified with 10% EDTA for 4 weeks. The specimens were embedded in paraffin, cut into 3 μm sections, and stained for evaluation. HE staining was used to observe the intradiscal structure, safflower green staining was used to observe proteoglycan deposition, and immunohistochemistry and immunofluorescence were used to observe the expression of NLRP3, IL-1β, type II collagen, and Aggrecan within the intervertebral disc.

[0097] (16) Data Analysis

[0098] All data are expressed as mean ± SD. Data analysis was performed using GraphPadprism software 8.0. Statistical differences between data were analyzed using one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant, and P < 0.05 was considered not statistically significant (ns) (* represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001).

[0099] II. Results and Discussion

[0100] The principle and route of this invention are as follows: Figure 1 The corresponding experimental route verification and results are as follows.

[0101] (1) Preparation and characterization of MNPs

[0102] The MNP in this invention utilizes Ca in CAT. 2+ Biomineralization was performed using chelation of carboxyl groups with TGF-β. Furthermore, polyethylene glycol-modified polyglutamic acid (mPEG-P(Glu)) was synthesized to provide additional carboxyl groups and a PEG shell, making the mineralized nanoparticles more stable and biocompatible. mPEG-P(Glu) was synthesized via a Fuchs-Farthing reaction, and the substitution rate of P(Glu) was determined to be 40% by 1H-NMR. Figure 2 ).

[0103] Furthermore, the performance of the MNP was evaluated using TEM and DLS. For example... Figure 3 As shown in section AB, MNPs appear as opaque spherical particles on the TEM, with a size of approximately 100 nm, consistent with the average diameter measured by DLS.

[0104] Next, a lactic acid solution with a pH of 6.3 was prepared to simulate the acidic microenvironment of intervertebral disc degeneration, in order to verify the acid-neutralizing ability of MNP. After adding 0.3 mg / ml MNP, the pH of the lactic acid solution rose to 6.89. When the MNP concentration increased to 0.6 mg / ml, the pH of the solution further increased to 7.05, approaching the pH of a healthy intervertebral disc. Figure 3 (Part C). Finally, an acidic hydrogen peroxide (H₂O₂) solution was constructed to verify the activity of CAT in MNP. Figure 3 As can be seen from part D, CAT encapsulated in nanoparticles can be released under acidic stimulation and still retains its original catalytic ability, which can catalyze H2O2 into water and oxygen.

[0105] In summary, the MNP prepared by this invention has a diameter of approximately 100 nm, possesses acid neutralization capability, and retains the activity of the contained proteins.

[0106] (2) Preparation and characterization of GMNP

[0107] While MNPs can dynamically respond to tissue damage, they still require a physical core with high drug loading capacity and biocompatibility. This physical core must provide adhesion sites for cells while preventing excessive loss of MNPs due to local diffusion and intradiscal compression. To achieve minimally invasive treatment, the physical core we construct should also be injectable. This injectable core can deliver MNPs into the intervertebral disc without causing structural damage and provides sustained release capability.

[0108] Based on this, the present invention utilizes microfluidic technology to construct injectable GelMA microspheres, which are loaded with uniformly sized nanoparticles (GMNPs), and the microspheres are endowed with porosity through freeze-thaw technology. Figure 3 As shown in section E, hydrogel microspheres of approximately 200 μm can be mass-produced using a self-made coaxial microfluidic chip. The prepared microspheres were rapidly frozen in a -20°C paraffin oil solution, freezing the water within the microspheres into ice crystals. Through photo-induced crosslinking, the frozen microspheres could be solidified and maintain their porous morphology even after the ice crystals dissolved. Figure 3 The SEM results for the middle G section are shown. The porous hydrogel microspheres (GM) have a higher surface area, providing greater contact between the extracellular microenvironment and the MNPs. Furthermore, the rough surface further promotes cell adhesion, making the hydrogel microspheres an ideal physical core.

[0109] Next, the MNP was mixed into a GelMA solution to prepare a microfluidic GMNP with microenvironment responsiveness. Figure 3 As shown in section H, numerous nanoscale particles are visible on the surface of the microspheres, while the surface of pure microspheres is smooth. The elemental distribution on the microsphere surface was characterized using energy dispersive spectroscopy (EDS). The figure shows that without MNP, the microspheres are predominantly composed of C, N, and O, consistent with the composition of GelMA material. With the addition of MNP, the atomic proportion of oxygen increased from 19.47% to 35.86%, and the atomic proportion of calcium increased from 0.00% to 5.93%, both showing significant increases. Furthermore, the distribution of calcium is consistent with the distribution of nanoscale particles on the microsphere surface, indicating that the nanoscale particles observed by scanning electron microscopy are indeed MNP (…). Figure 3 (Part IJ). By loading FTIC-tagged proteins into MNPs, their distribution in GM can be observed under a confocal microscope. Figure 3 (Part F of the middle section).

[0110] Finally, the release pattern of GMNP under different pH conditions was verified. Figure 3 As shown in section K, MNP was significantly activated under acidic conditions, with the release of FTIC-labeled protein increasing significantly to 91.4% after 120 hours. Integrating MNP into the core of the hydrogel microspheres confined its action to the area surrounding the core, thus significantly reducing drug consumption; 72.8% of the drug was released after 120 hours. In contrast, under normal physiological conditions (pH 7.4), MNP was not triggered, with only 33.8% and 30.0% of the drug released in the MNP and GMNP groups, respectively. This demonstrates that by combining MNP with the hydrogel microsphere core, GMNP maintains a normal environment around the microspheres while significantly reducing nanoparticle consumption, ensuring long-term efficacy even under abnormal pathological conditions of the intervertebral disc and providing a sufficient time window for tissue regeneration.

[0111] (3) The ability of GMNP to be retained in the body

[0112] To further verify the preservation rate of MNP in the intervertebral disc in vivo, a rat caudal intervertebral disc preservation model was constructed. Figure 4 (Part A). Cy5.5-labeled bovine serum albumin was loaded into MNPs and GMNPs, respectively, and injected into two non-adjacent caudal intervertebral discs. Fluorescence signals were measured on days 0, 3, 6, and 9. From Figure 4 As shown in the middle BD section, due to the positive pressure of the intervertebral disc, pure MNPs seeped out from the needle puncture site after injection. Due to the adhesive ability of the hydrogel microspheres, most of the GMNPs with GM as the core remained within the intervertebral disc after injection, resulting in a higher and more significant immediate postoperative fluorescence signal in the GMNP group. The trends on days 3, 6, and 9 were similar to those on day 0, with the nanoparticle content in the GMNP group still significantly higher than that in the NPs group. Further data analysis showed that the loss rate in the GMNP group was also lower than that in the MNP group during the experiment. Figure 4 (Part E). These results confirm that GM, as a physical core, not only enhances the local preservation of MNPs during injection but also continuously reduces MNP loss in the harsh intervertebral disc environment. This allows GMNPs to exert long-term effects even with only a single injection.

[0113] (4) In vitro cell protection ability of GMNP

[0114] The in vitro cell protection capacity of GMNP nucleus pulposus cells is a functional basis for intervertebral disc regeneration. GMNP continuously maintains the normal environment around the hydrogel microspheres under degenerative conditions through MNP, thereby ensuring the normal function of surrounding cells and laying the foundation for further regeneration. To verify the physical core (GM) and blank GMNP (GMNP)... blankTo investigate the effect of cell adhesion on cell survival, we simulated normal physiological and pathological environments and observed cell adhesion using confocal microscopy.

[0115] Lactic acid was added to the normal culture medium, and the pH was maintained at 6.2 to simulate the local microenvironment during severe intervertebral disc degeneration. NPCs were first stained with Dil-red, and then compared with pure GM and GMNP. blank Co-cultured, and observed on days 3, 5, and 7. From Figure 4 As can be seen from the F section, under normal circumstances, pure GM and GMNP blank Both methods provide a mild and comfortable environment for the cells. In both groups, the cells were well stretched and able to expand. Figure 4 (Part F of the middle section). Interestingly, in GMNP... blank In the group, the cell adhesion level at each time point was higher than that in the GM group ( Figure 4 (In the GJ portion), this may be due to the rough surface caused by the distribution of MNPs on the GM surface. On the other hand, under acidic pathological conditions, the transgene itself lacks the ability to regulate the surrounding environment and cannot provide sufficient protection for the cell. Figure 4 As shown in section F, some cells initially adhere to the GM, but gradually die in the acidic environment, appearing as floating spheres around the GM and eventually disintegrating over time. Meanwhile, in GMNP... blank Within the GM, the acidic environment surrounding the MNP is effectively neutralized due to the protection of MNPs, and this mild environment can be maintained continuously. Therefore, under the continuous protection of NPs, the adherent cells maintain normal morphology and satisfactory proliferation rate. Figure 4 (FJ part). After 7 days, the proliferating cells even partially degraded the transgenic core, gradually forming natural cellular papillae.

[0116] The above results further demonstrate that GMNP can serve as a precursor to a "cell microenvironment stabilizer," providing protection against harsh pathological environments for cells while also creating anchor points for cell adhesion, thus laying the foundation for further modification of NPCs.

[0117] (5) In vitro observation of GMNP CAT Blocking effect on NLRP3 cascade

[0118] In vitro NLRP3 cascade blockade effect: Chronic inflammation remains the most prominent pathological feature of intervertebral disc degeneration. Due to the harsh acidic and hypoxic environment during intervertebral disc degeneration, the NLRP3 inflammasome in NPCs is activated, leading to active IL-1β secretion. The NLRP3 pathway is a conserved inflammatory mechanism whose main purpose is to defend against the invasion of foreign bacteria. Various abnormal environmental signals can activate NLRP3, hence it is also known as a danger signal sensor. Interestingly, both acidic environment and active IL-1β can activate the NLRP3 inflammasome by upregulating the production of mitochondrial ROS. Since the positive inflammatory feedback formed by IL-1β and NLRP3 is another reason for the gradual aggravation of intervertebral disc inflammation besides acidic stimulation, eliminating excess ROS can comprehensively stabilize the cellular microenvironment.

[0119] This invention loads CAT into MNP, which reduces excessive ROS involved in the NLRP3 inflammatory cycle while inhibiting the acidic environment, thereby inhibiting the positive inflammatory cycle caused by NLRP3 activation. Figure 5 (Part A). To simulate a pathological environment, cells were cultured in an acidic environment. The ASIC-3 expression results show that acid-induced damage was successfully constructed under acidic conditions. Figure 6 (Part A). Since excessive ROS production due to mitochondrial dysfunction is a key mediator of NLRP3 activation, we explored GMNP for the first time. CAT Effects on cellular ROS production. Intracellular ROS production can be observed using DCFH-DA staining and flow cytometry. From... Figure 5 As can be seen from sections B and D, the production of cellular ROS increases significantly under acidic conditions, while the addition of GMNP... blank Subsequently, due to the neutralizing effect of the acidic environment, the production of cellular ROS significantly decreased. However, due to the previously activated NLRP3 pathway, the presence of IL-1β could still stimulate ROS production. In GMNP CAT In this group, the release of CAT further decomposed hydrogen peroxide, an important second messenger of ROS, and the production of cellular ROS was suppressed to levels close to those in the normal group. Figure 7 Part A of the middle section.

[0120] In addition, we used JC-1 mitochondrial membrane potential assays and Mitotracker staining to assess changes in mitochondrial function. Figure 5 (Parts B, E, and F). Under acidic conditions, the green fluorescence of JC-1 staining was significantly enhanced, while the red fluorescence was weakened. The fluorescence intensity of Mitotracker was also significantly reduced, indicating impaired mitochondrial integrity and function. (GMNP) blank In the group, neutralization of the acidic environment partially restored mitochondrial function in the cells, while GMNP CATThe ROS depletion effect further improved mitochondrial function, bringing it to a level similar to that of the normal group. These results indicate that mitochondria in NPCs damaged in an acidic environment are protected by GMNP. CAT This dual intervention is a crucial step in blocking the activation of the NLRP3 cascade cycle.

[0121] GMNP was validated using immunofluorescence, PCR, and Western blotting. CAT Changes in the NLRP3 inflammatory cascade during anti-inflammatory treatment. These changes were determined by immunofluorescence assays using TXNIP. Figure 5 In section C, it can be observed that under normal conditions, TXNIP mainly accumulates in the cell nucleus, while acid stimulation causes TXNIP in most cells to migrate into the cytoplasm, thus significantly enhancing the fluorescence intensity of NLRP3. (Adding GMNP...) blank Subsequently, the migration of TXNIP caused by the acidic environment decreased, and NLRP3 was also inhibited, but IL-1β could still activate NLRP3 through ROS / TXNIP. Adding GMNP... CAT Subsequently, blocking ROS generation further suppressed the ROS / TXNIP / NLRP3 axis, and the activation level of NLRP3 was suppressed to be close to that of the normal group. Figure 5 Part C, Figure 7 Part B). The WB and PCR results for TXNIP and NLRP3 were consistent with the immunofluorescence trend, and the proportion of Cleaved-Caspase-1 activity after splicing in the MS / CAT-NP group was also significantly inhibited. Figure 5 The Chinese GJ part, Figure 6 ).

[0122] Subsequently, the level of IL-1β in the cell supernatant was measured using ELISA, such as... Figure 5 As shown in section K, the acidic group had the highest IL-1β concentration. MS / NP inhibited the release of IL-1β from cells, while MS / Cat-NP further reduced the IL-1β concentration, making it the lowest among the four groups. In summary, MS / CAT-NPs can inhibit the activation of the NLRP3 pathway by jointly inhibiting the acidic environment and extracellular ROS. Therefore, MS / CAT-NPs can effectively reverse the inflammatory state of NPCs, stop the deterioration of the intradiscal inflammatory environment, and create a microenvironment suitable for intervertebral disc regeneration.

[0123] (6) GMNP in vitro regeneration ECM

[0124] After improving the harsh environment within the intervertebral disc and preventing further deterioration of local inflammation, reshaping the microenvironment within the intervertebral disc using NPCs is the final step in regeneration. By guiding cells into a regenerative state, cells can enhance the synthesis of extracellular matrix, gradually replacing the damaged tissue outside the hydrogel microspheres, ultimately achieving complete tissue regeneration of the intervertebral disc.

[0125] To guide NPCs into a regenerative state, we further loaded the growth factor TGF-β into GMNP. CAT In this study, CAT and TGF-β co-loaded microspheres (GMNPs) with the ability to target cell transformation were constructed. TGF-β has been shown to enhance the extracellular matrix synthesis capacity of cells, but it is easily inactivated in acidic and inflammatory environments; therefore, GMNPs... CAT The lifespan of TGF-β can be significantly extended by altering the environment early on.

[0126] First, PCR was used to verify changes in mRNA levels in cells. For example... Figure 8 As shown, under acidic conditions, although Aggrecan RNA levels remained unchanged, Col II expression was significantly downregulated, while MMP2 and MMP13 expression was significantly increased, confirming that cellular behavior at this time was directed towards extracellular matrix degradation. GMNP CAT After pH adjustment, the expression of Aggrecan and Col II increased significantly, even exceeding that of cells under normal conditions. This may be due to GMNP. CAT The rough outer surface stimulates NPCs to synthesize more ECM proteins. Furthermore, the expression of MMP2 and MMP13 was significantly inhibited. After TGF-β supplementation, the expression of Aggrecan and Col II further increased, while the expression of MMP2 and MMP13 decreased. The protein expression results were consistent with PCR, and immunofluorescence also showed that the GMNP group of NPCs had the strongest ECM synthesis capacity among the four groups. Figure 9 These results demonstrate that, although GMNP CAT It can maintain normal ECM metabolism of cells, but the addition of TGF-β further enhances its synthetic capacity, thereby achieving targeted remodeling of residual cells and effectively accelerating the remodeling of the intervertebral disc environment.

[0127] (7) GMNP promotes radiographic improvement of intervertebral disc degeneration in vivo.

[0128] To further verify the ability of GMNP to promote tissue regeneration, an in vivo model of caudal intervertebral disc degeneration in rats was constructed, and the regeneration of the intervertebral disc was observed through imaging and pathological staining. To better evaluate the efficacy, PBS (puncture), GMNP, and GMNP were administered intraoperatively. TGF GMNP CAT Different groups, including GM, were injected into the intervertebral disc for in vivo evaluation. Figure 11 (Parts A and E). When the intervertebral disc degenerates, the mechanical properties of the disc decrease significantly due to the loss of water from the nucleus pulposus, leading to narrowing of the intervertebral disc space. Simultaneously, the signal intensity of the intervertebral disc decreases in the T2 phase of MRI (measuring water content). From... Figure 11 Parts B and D of the middle school and Figure 12 As shown in section A, postoperative disc degeneration had not yet begun, and there were no significant changes in the intervertebral spaces in each group; MRI findings were normal. Four weeks later, due to needle puncture damage to the intervertebral disc, the intervertebral space in the puncture group (Puncture) was significantly narrowed, and the T2 signal within the nucleus pulposus was also significantly reduced. This contrasts with the puncture group (Puncture) and GMNP. TGF Group, GMNP CAT Compared with the GM group, the GMNP group showed the strongest protective effect, with a significant increase in intervertebral space and higher T2 signal intensity. Figure 11 Parts B and D, Figure 12 (Part B). After 8 weeks, the intervertebral space in the puncture group further narrowed, and the signal intensity within the nucleus pulposus significantly decreased. The signal intensity in the GMNP group gradually increased, approaching that of the normal group. GMNP TGF GMNP CAT The trend for the GM group is similar to that of week 4. Figure 11 Parts B and D, Figure 12 Part C). Figure 11 As can be seen in section C, the intervertebral disc height in the GMNP group showed a rebound trend within 8 weeks, and the rebound rate was much higher than that in the puncture group and the GMNP group. CAT The study compared the GM group and the GM group, confirming that GMNP has a stronger regenerative capacity. Although at 4 weeks, GMNP... TGF The intervertebral disc height in this group was higher than that in GMNP. CAT The levels of TGF-β in the three groups were similar at 8 weeks compared to the GM group. This indicates that although early TGF-β release can play a certain role in tissue protection, in the later stages, the continuous aggravation of inflammation and the depletion of TGF-β exhaust its tissue regeneration potential, failing to prevent the progression of degenerative changes.

[0129] (8) The inhibitory effect of GMNP on nucleus pulposus inflammation in vivo

[0130] Next, the activation of the NLRP3 inflammatory cascade axis of the intervertebral disc was investigated using tissue staining. HE staining was used to observe the morphology of the nucleus pulposus, the integrity of the annulus fibrosus, and the size of the nucleus pulposus region. Figure 11 (Part F of the study). The histological scoring described in previous studies was used to assess intervertebral disc degeneration. At 4 and 8 weeks, the histological scores of all experimental groups were significantly higher than those of the control group, indicating intervertebral disc injury. However, at weeks 4 and 8, the scores of the GMNP group were significantly lower than those of the other three groups (GMNP...). TGF GMNP CAT The presence of GMNP (and GM) indicates that GMNP has a protective effect on the intervertebral disc structure when the intervertebral disc is damaged.

[0131] In addition, NLRP3 immunofluorescence and IL-1β immunohistochemistry were used to assess intradiscal inflammation. At 4 weeks, the Puncture group and GMNP group... TGFBoth the NLRP3 positive rate and the GM group had high rates, with the highest positive rate in the puncture group. In contrast, the GMNP group showed a higher positive rate. CAT In both the puncture group and the GMNP group, NLRP3 expression was significantly suppressed. After 8 weeks, the expression of NLRP3 was significantly suppressed in both the puncture group and the GMNP group. TGF The positivity rates of NLRP3 in the GM, GMNP, and GMNP groups were similar at 4 weeks, but those in the GMNP group were different. CAT The positive rate in the group increased significantly ( Figure 13 (Parts A and C). The trend of IL-1β detected by immunohistochemistry was similar to that at 4 weeks ( Figure 13 (Parts B and D). The results of these two inflammatory markers indicate that although mineralized nanoparticles can prevent the deterioration of the microenvironment, the positive inflammatory feedback brought about by NLRP3 activation still gradually transforms the intervertebral disc into a highly inflammatory environment. GMNP CAT It can suppress the progression of inflammation in the early stages of degenerative changes, but lacks the ability to remodel the extracellular matrix in the later stages, and GMNPs are depleted after 8 weeks. CAT It cannot stop the progression of chronic inflammation.

[0132] (9) GMNP enhances nucleus pulposus regeneration in vivo

[0133] The level of extracellular matrix remodeling within the intervertebral disc was further assessed using safflower-red-green staining and immunofluorescence. The content of intervertebral disc proteoglycans was evaluated using safflower-red-green staining. Figure 11 (Middle G section). At 4 and 8 weeks, the deposition of normal proteoglycans in the nucleus pulposus was most pronounced in the GMNP group, while the deposition in the puncture group and GMNP group was significantly lower. TGF Group, GMNP CAT In both the GM and GM groups, proteoglycans in the intervertebral discs were mainly deposited within the annulus fibrosus, and their content was significantly reduced. Figure 14 B).

[0134] In addition, the deposition of type II collagen in the intervertebral disc was studied using immunofluorescence. Figure 13 (Part E). In the GMNP group, at weeks 4 and 8, high levels of red fluorescence (type II collagen) were deposited around nucleus pulposus cells, similar to the normal group, indicating that type II collagen secreted by the cells was successfully deposited in the nucleus pulposus environment. (Needle aspiration group, GMNP) TGF Group, GMNP CAT The amount of type II collagen deposition in both the GM group and the GM group was significantly lower than that in the GMNP group, and the GMNP group... TGF A certain amount of type II collagen deposition was still visible in the group at 4 weeks, but it basically disappeared at 8 weeks. Figure 13 (Middle G part). Immunofluorescence staining results for another important extracellular matrix protein, Aggrecan, were similar to those for type II collagen ( Figure 13(F and H parts). These results indicate that GMNP's environmental regulation and cell behavior guidance capabilities are key steps in tissue regeneration, and the absence of certain components can delay or even hinder the tissue regeneration process.

Claims

1. A method for preparing a cell microenvironment stabilizer that captures hydrogen ions, characterized in that, Includes the following steps: (1) A Tris-HCl buffer solution containing CaCl2 was mixed with HEPES buffer solution containing TGF-β1, catalase, polyethylene glycol-polyglutamic acid block copolymer, and NaCO3. The mixture was stirred to carry out a mineralization reaction and obtain mineralized nanoparticles. (2) GelMA hydrogel microspheres and serum leucine aminopeptidase were prepared in a microfluidic device under freezing conditions to obtain hydrogel microspheres. The frozen hydrogel microspheres were then photocrosslinked and freeze-dried. (3) The freeze-dried hydrogel microspheres obtained in step (2) are mixed with the mineralized nanoparticles obtained in step (1) to prepare the cell microenvironment stabilizer.

2. The preparation method according to claim 1, characterized in that, The mineralization reaction in step (1) is carried out under the condition of stirring at 4°C for 12 hours.

3. The preparation method according to claim 1 or 2, characterized in that, The weight ratio of TGF-β1, catalase and polyethylene glycol-polyglutamic acid block copolymer in step (1) is 1:150:

5.

4. The preparation method according to claim 1 or 2, characterized in that, The freezing conditions in step (2) are -40°C, and the photocrosslinking conditions are crosslinking under 405nm light for 15min.

5. The preparation method according to claim 1 or 2, characterized in that, The polyethylene glycol-polyglutamic acid block copolymer is synthesized via the Fuchs-Farthing reaction.

6. The preparation method according to claim 1 or 2, characterized in that, The CaCl2 was dissolved in Tris-HCl buffer solution with a concentration of 1 mM, a pH of 7.6, and a CaCl2 concentration of 100 mM.

7. The preparation method according to claim 1 or 2, characterized in that, The HEPES buffer solution mentioned in step (1) has a concentration of 50 mM, a pH of 7.1, and contains 140 mM NaCl.

8. The preparation method according to claim 1 or 2, characterized in that, The concentration of Na2CO3 in step (1) is 10 mM.

9. The preparation method according to claim 1 or 2, characterized in that, Step (2) control the flow rate of the oil phase in the microfluidic device to 400 μl / min and the flow rate of the water phase to 40 μl / min.

10. A cell microenvironment stabilizer for capturing hydrogen ions prepared by the method according to any one of claims 1-9.

11. The application of the cellular microenvironment stabilizer for capturing hydrogen ions as described in claim 10, characterized in that, The goal is to prepare this cell microenvironment stabilizer into a drug that promotes the regeneration of degenerated intervertebral discs.

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