Bioactive microspheres for treating intervertebral disc degeneration, their preparation methods and applications

Abstract

This invention discloses bioactive microspheres for treating intervertebral disc degeneration, their preparation method, and applications, belonging to the field of biomedical technology. The bioactive microspheres, from the inside out, consist of an ultra-small Prussian blue nanozyme modified with mitochondrial-targeting peptides, a biomimetic scaffold layer, and a cell layer. The biomimetic scaffold layer is formed by cross-linking methacryloyl gelatin and methacryloyl hyaluronic acid. The cell layer is composed of nucleus pulposus progenitor cells adhered to the surface of the biomimetic scaffold layer. Through the synergistic effect of its three-layer structure, the bioactive microspheres of this invention, after implantation into the degenerated intervertebral disc, can simultaneously achieve protective delivery of transplanted cells, efficient scavenging of reactive oxygen species, restoration of mitochondrial homeostasis, and regeneration of the extracellular matrix, thereby synergistically promoting the structural and mechanical functional repair of the intervertebral disc. This product provides a novel integrated regenerative strategy for treating intervertebral disc degeneration.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to bioactive microspheres for treating intervertebral disc degeneration, their preparation methods, and applications. Background Technology

[0002] The core pathological process of intervertebral disc degeneration includes the progressive depletion of functional cells (nucleus pulposus cells and their stem cells) within the nucleus pulposus tissue, and a harsh microenvironment characterized by persistent reactive oxygen species (ROS) bursts. This oxidative stress microenvironment not only accelerates the aging and death of endogenous cells, but also severely hinders the survival and function of exogenous regenerative cells (such as transplanted stem cells), resulting in the limited efficacy of current single therapies based on cell transplantation or biomaterials.

[0003] Current technologies cannot sustain cell transplantation in the long term in the oxidative stress environment of degenerated tissues; simple antioxidant therapy lacks the ability to structurally repair degenerated tissues; and traditional biomaterial scaffolds mainly provide physical support and lack the biological function of actively regulating cell fate and microenvironment. Therefore, there is an urgent need to develop an integrated and intelligent treatment system that can combine regenerative cell replenishment, oxidative microenvironment remodeling, and bioactive support to synergistically overcome the above bottlenecks. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides bioactive microspheres for treating intervertebral disc degeneration, their preparation method, and applications. The purpose of this invention is to provide bioactive microspheres that combine cell delivery, targeted antioxidant activity, and matrix regeneration functions, which can be used in synergistic treatment of intervertebral disc degeneration or in medical devices. The bioactive microspheres of this invention can simultaneously achieve protective delivery of nucleus pulposus progenitor cells, precise remodeling of the oxidative stress microenvironment within the degenerated intervertebral disc, and active regeneration of the newly formed extracellular matrix.

[0005] This invention is achieved through the following technical solution:

[0006] The first objective of this invention is to provide a bioactive microsphere for treating intervertebral disc degeneration, wherein the bioactive microsphere comprises, from the inside out, an ultra-small Prussian blue nanozyme modified with mitochondrial-targeting peptides, a biomimetic scaffold layer, and a cell layer.

[0007] The mitochondrial-targeting peptide-modified ultrasmall Prussian blue nanozyme has mitochondrial targeting and reactive oxygen species scavenging capabilities, and can be slowly released from the scaffold layer in a physiological environment.

[0008] The biomimetic scaffold layer is formed by cross-linking methacrylamide gelatin and methacrylamide hyaluronic acid, providing a biomimetic microenvironment for three-dimensional colonization, proliferation, and secretion of extracellular matrix for the cell layer;

[0009] The cell layer is composed of nucleus pulposus progenitor cells that adhere to the surface of the biomimetic scaffold layer.

[0010] In one embodiment of the present invention, the mitochondrial targeting peptide is SS31 peptide; the SS31 peptide is a tetrapeptide (D-Arg-Dmt-Lys-Phe-NH2) with mitochondrial targeting function.

[0011] And / or, the mass ratio of the ultrasmall Prussian blue nanozyme to the mitochondrial targeting peptide is 1:150-1:100;

[0012] And / or, the particle size of the ultrasmall Prussian blue nanozyme is 3 nm-10 nm; preferably 5 nm;

[0013] And / or, the diameter of the bioactive microspheres is 100 μm-200 μm, preferably 200 μm.

[0014] In one embodiment of the present invention, the bioactive microspheres have a porous structure; the pore size of the porous structure is 20 μm-50 μm, preferably 25 μm.

[0015] The second objective of this invention is to provide a method for preparing the bioactive microspheres for treating intervertebral disc degeneration, comprising the following steps:

[0016] (1) The ultra-small Prussian blue nanozyme was carboxylated and activated, and then mitochondrial-targeting peptides were added and reacted overnight. After centrifugation and purification, ultra-small Prussian blue nanozyme modified with mitochondrial-targeting peptides was obtained.

[0017] (2) Dissolve methacrylated gelatin and methacrylated hyaluronic acid in a photoinitiator solution, add the ultrasmall Prussian blue nanozyme modified with mitochondrial-targeting peptide obtained in step (1), and disperse evenly to obtain a mixed prepolymer solution.

[0018] (3) Using the mixed prepolymer solution obtained in step (2) as the aqueous phase, it forms uniform droplets with the oil phase through a microfluidic device, cross-links and solidifies to form hydrogel microspheres of ultrasmall Prussian blue nanozymes modified with mitochondrial-targeting peptides.

[0019] (4) The nucleus pulposus progenitor cells are co-cultured with the hydrogel microspheres obtained in step (3) so that the nucleus pulposus progenitor cells adhere to the surface of the hydrogel microspheres, and bioactive microspheres for the treatment of intervertebral disc degeneration are obtained.

[0020] In one embodiment of the present invention, in step (1), the ultra-small Prussian blue nanozyme is prepared by the following method: polyvinylpyrrolidone and potassium ferricyanide are dissolved in a mixed solvent of HCl and alcohol to react and obtain ultra-small Prussian blue nanozyme; the reaction conditions are: 70℃-90℃ for 3 h-5 h.

[0021] In one embodiment of the present invention, in step (2), the final concentration of the methacrylamide gelatin is 100 mg / mL-150 mg / mL;

[0022] And / or, the final concentration of the methacrylamide hyaluronic acid is 10 mg / mL to 20 mg / mL;

[0023] And / or, the final concentration of the ultrasmall Prussian blue nanozyme is 0.1 mg / mL to 0.15 mg / mL.

[0024] In one embodiment of the present invention, in step (2), the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphite;

[0025] And / or, the photoinitiator concentration in the photoinitiator solution is 0.25%-0.5% by mass volume, preferably 0.25%. The solvent is deionized water or phosphate buffer.

[0026] In one embodiment of the present invention, in step (3), the oil phase is a mineral oil containing Span 80; the flow rate of the oil phase is 3 mL / min-5 mL / min;

[0027] And / or, the flow rate of the aqueous phase is 30 μL / min-40 μL / min.

[0028] In one embodiment of the present invention, in step (4), the density of the nucleus pulposus progenitor cells added is 1×10⁻⁶. 6 cells / mL - 5 × 10 6 The cells / mL; the culture conditions were: co-culture at 37℃ and 5% CO2 for 72 h.

[0029] A third objective of this invention is to provide the application of the aforementioned bioactive microspheres in the preparation of drugs or medical devices for treating intervertebral disc degeneration. The bioactive microspheres achieve therapeutic effects through the synergistic effect of their three-layer structure:

[0030] Ultra-small Prussian blue nanozymes remove local ROS and target damaged mitochondria, creating a "safe zone" for cell survival; the biomimetic scaffold layer provides a three-dimensional biomimetic microenvironment for cell colonization, proliferation, and secretion of extracellular matrix; with the support of the scaffold layer, the cell layer proliferates, differentiates, and secretes extracellular matrix, achieving tissue regeneration.

[0031] Cell protection and regeneration: Reduces TBHP-induced apoptosis in nucleus pulposus progenitor cells and promotes differentiation into nucleus pulposus cells.

[0032] Three-dimensional adhesion platform: microspheres load nucleus pulposus progenitor cells, promoting cell proliferation and extracellular matrix secretion.

[0033] Mechanical adaptability: The surface of the microspheres adheres strongly to the intervertebral disc tissue, preventing displacement; and promoting the recovery of mechanical function of degenerated intervertebral discs.

[0034] The bioactive microspheres prepared in this invention for treating intervertebral disc degeneration not only have good biocompatibility, mitochondrial-targeted antioxidant properties, and cell protection and regeneration characteristics, but can also effectively load nucleus pulposus progenitor cells to achieve histological and mechanical functional repair of degenerated nucleus pulposus tissue in rats.

[0035] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0036] (1) This invention provides bioactive microspheres for treating intervertebral disc degeneration, their preparation method and application. This invention proposes and constructs a three-in-one bioactive therapeutic system of "cell layer-bionic scaffold layer-functional nanolayer", which realizes the synergy of cell replenishment, microenvironment remodeling and structural support, and effectively solves the contradiction faced by single therapy where cells cannot survive or there are no repair cells.

[0037] (2) This invention precisely directs the antioxidant effect of ultra-small Prussian blue nanozymes to mitochondria through the modification of mitochondrial-targeting peptides, greatly improving the ROS scavenging efficiency and actively regulating mitochondrial autophagy to restore cellular energy homeostasis and protect cells from apoptosis and aging.

[0038] (3) The hydrogel microspheres of the present invention serve as a sustained-release carrier, enabling the continuous release of functional nanozymes and providing long-lasting antioxidant protection. Their porous biomimetic structure is conducive to long-term cell colonization and functional performance, and promotes the deposition of new extracellular matrix.

[0039] (4) The bioactive microspheres of this invention are specifically designed for intervertebral disc degeneration and can be applied through minimally invasive injection. Experiments have shown that these bioactive microspheres can significantly promote the repair and functional recovery of degenerated intervertebral discs at the imaging, histological and biomechanical levels, especially showing excellent regenerative potential in severe degeneration models, and have clear clinical application prospects and broad industrialization value. Attached Figure Description

[0040] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0041] Figure 1The images show the microstructure, FTIR, XPS, and XRD results of the bioactive hydrogel microspheres with mitochondrial-targeted antioxidant function of this invention; where A is a high-resolution transmission electron microscope image; B is an X-ray photoelectron spectroscopy (XPS) image; C is a Fourier transform infrared (FTIR) image; and D is an X-ray diffraction (XRD) image.

[0042] Figure 2 This is a graph showing the evaluation results of the ROS elimination efficiency of the bioactive hydrogel microspheres with mitochondrial-targeted antioxidant function of the present invention; where A is the •OH elimination capacity graph; B is the O2· - Elimination capacity diagram; C represents the elimination capacity diagram for H2O2; D represents the elimination capacity diagram for ¹O2;

[0043] Figure 3 This is a comparative diagram showing the effects of the bioactive hydrogel microspheres with mitochondrial-targeted antioxidant function of the present invention on eliminating ROS in cells and maintaining mitochondrial membrane potential. In the diagram, A shows intracellular ROS detection using the DCFH-DA probe; B shows mitochondrial membrane potential detection using the JC-1 probe; C shows cell viability / death staining; F shows the cell viability / death ratio; D shows cell apoptosis detection using TUNNEL; E shows the DCF fluorescence quantitative PCR statistical diagram; F shows the JC-1 monomer fluorescence statistical diagram; G shows the cell viability / death ratio statistical diagram; and H shows the TUNNEL positive staining statistical diagram.

[0044] Figure 4 This is a comparison of fluorescence staining of the bioactive hydrogel microspheres with mitochondrial-targeted antioxidant function of the present invention promoting mitochondrial autophagy in nucleus pulposus progenitor cells; where A is a co-staining image of autophagy protein LC3 and mitochondria; B is a statistical graph of co-localization of LC3 and MitoTracker.

[0045] Figure 5 Flow cytometry data of stemness indicators in nucleus pulposus progenitor cells isolated and cultured in vitro.

[0046] Figure 6 This is a comparative diagram showing the characterization of the proliferation and differentiation capacity of nucleus pulposus progenitor cells and the effect of bioactive hydrogel microspheres on cell proliferation and cytoskeleton spreading in this invention; wherein, A is a light micrograph at different co-culture time points; B is a cytoskeleton staining image at different co-culture time points; C is a cell liveness / death image at different co-culture time points; D is a statistical graph of cell proliferation rate; E is a statistical graph of the proportion of live cells.

[0047] Figure 7 The images show the imaging results of tissue repair in a rat model of intervertebral disc degeneration induced by nucleus pulposus removal using bioactive hydrogel microspheres with mitochondrial-targeted antioxidant function, as described in this invention; A is the imaging assessment image; B is the intervertebral disc height statistical image; C is the Priffimann degeneration grade statistical image.

[0048] Figure 8These are pathological staining images of tissue repair in a rat model of intervertebral disc degeneration induced by nucleus pulposus removal, using bioactive hydrogel microspheres with mitochondrial-targeted antioxidant function as described in this invention. A is an H&E staining image of the intervertebral disc tissue; B is an O+ and Fast Green staining image of the intervertebral disc tissue.

[0049] Figure 9 This image shows a pathological section staining analysis of tissue repair in a rat model of intervertebral disc degeneration induced by nucleus pulposus removal, using bioactive hydrogel microspheres with mitochondrial-targeting antioxidant function as described in this invention; where A is a statistical chart of histological scoring items; and B is a chart of histological scoring total score analysis.

[0050] Figure 10 The images show a comparison of the results of the bioactive hydrogel microspheres with mitochondrial-targeted antioxidant function promoting the repair of intervertebral disc mechanical function according to the present invention; where A is a real-world mechanical test image; B is a mechanical-displacement curve; and C is a statistical chart of compression stroke. Detailed Implementation

[0051] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0052] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0053] Example 1: Preparation and characterization of SS31 peptide-modified ultrasmall Prussian blue nanozyme (SS31-UPN)

[0054] (1) Synthesis of ultrasmall Prussian blue nanozyme: 1 g PVP and 0.2 g K3[Fe(CN)6] were dissolved in 40 mL of a mixed solvent containing 0.01 M HCl and 75% (v / v) ethanol. The reaction was stirred continuously in an 80°C water bath for 4 hours. After the reaction solution was cooled to room temperature, it was transferred to a 100 kDa molecular cutoff ultrafiltration tube, centrifuged at 1000 g for 30 minutes, and the filtrate was collected to obtain a purified ultrasmall Prussian blue nanozyme (UPN) aqueous dispersion.

[0055] (2) SS31 modification: Take the above-mentioned aqueous dispersion of ultrasmall Prussian blue nanozyme, add 0.1 M sodium citrate solution (pH 5.5), and stir at room temperature for 2 hours. Then add EDC and NHS to the final concentrations of 10 mM and 5 mM, respectively, and activate at room temperature for 3 hours. Dialyze the activated UPN solution for 2 hours to remove excess reagents. Then add SS31 peptide (molar ratio of UPN:SS31≈1:150-1:100), and stir at 4°C overnight. After the reaction is completed, purify again by centrifugation using a 100 kDa ultrafiltration tube to obtain SS31-modified ultrasmall Prussian blue nanozyme (SS31-UPN).

[0056] Comparative Example 1:

[0057] This comparative example provides an ultrasmall Prussian blue nanozyme, similar to Example 1, except that only the ultrasmall Prussian blue nanozyme (UPN) is synthesized without SS31 modification.

[0058] Nanozyme characterization (see Figure 1-2 ): By high-resolution transmission electron microscopy ( Figure 1 Observation A) shows that SS31-UPN consists of uniform particles with a diameter of approximately 5 nm; XPS analysis ( Figure 1 B) confirmed the existence of the Fe²⁺ / Fe³⁺ mixed valence state; FTIR spectroscopy ( Figure 1 C) shows that SS31-UPN also has the characteristic peak of UPN (2067 cm⁻¹). - ¹, -C≡N) and the characteristic peak of SS31 peptide (1655 cm⁻¹) - ¹, Amide I band); XRD pattern ( Figure 1 The D-values ​​in the image show that the diffraction peaks of SS31-UPN perfectly correspond to the (200), (220), and (400) crystal planes of standard Prussian blue (Fe4[Fe(CN)6]3, JCPDS # 73-0687), demonstrating its good crystallinity. Electron spin resonance (ESR) analysis ( Figure 2 (A in section A and B in section B) indicates that the SS31-UPN nanozyme can effectively scavenge hydroxyl radicals (•OH) and superoxide anions (O2•⁻). Furthermore, experiments on the scavenging of hydrogen peroxide (H2O2) and ¹O2 radicals (…) Figure 2 (C in 2 and D in 2) confirm that SS31-UPN has a broad-spectrum ROS scavenging ability.

[0059] Example 2: Preparation of hydrogel microspheres and characterization of restored nucleus pulposus cell function

[0060] Preparation of the mixed prepolymer solution: GelMA (10% w / v) and HAMA (2% w / v) were dissolved in deionized water containing 0.25% (w / v) LAP photoinitiator. SS31-UPN (final concentration 1 mg / ml) prepared in Example 1 was ultrasonically dispersed in the above solution.

[0061] Microfluidic preparation: Using a microfluidic device (internal phase: the above-mentioned mixed prepolymer solution, flow rate 30 μL / min; external phase: mineral oil containing 5% Span 80, flow rate 3 mL / min), crosslinking and curing were performed at the outlet under 365 nm UV irradiation to generate SS31-UPN-loaded GelMA / HAMA hydrogel microspheres (SS31-UPN@GH). The microspheres were washed three times with PBS before use. Light microscopy and SEM showed that the microsphere diameter was approximately 200 μm and the internal pore size was approximately 25 μm.

[0062] Comparative Example 2:

[0063] This comparative example provides a microsphere similar to Example 2, except that only blank microspheres (GH) are prepared using GelMA / HAMA, and the SS31-UPN prepared in Example 1 is not dispersed in the microspheres. Otherwise, it is consistent with Example 2.

[0064] Comparative Example 3:

[0065] This comparative example provides a microsphere similar to Example 2, except that UPN@GH microspheres are prepared by replacing SS31-UPN with unmodified UPN. Otherwise, the microspheres are the same as in Example 2.

[0066] See also: Promoting the survival and functional characterization of nucleus pulposus progenitor cells Figure 3-4 A cell model of oxidative stress was established by stimulating nucleus pulposus progenitor cells with tert-butyl hydroperoxide (TBHP, 50 μM, 4 hours). After cell modeling, cells were co-cultured with various microsphere systems (including Comparative Example 2 and Comparative Example 3) at 37°C and 5% CO2 for 72 hours. Intracellular ROS and mitochondrial membrane potential levels were detected using the DCFH-DA probe and the JC-1 probe. Cell viability and apoptosis levels were assessed using cell liveness staining and TUNEL staining. Results showed that, compared to Comparative Example 2 and Comparative Example 3, UPN-SS31@GH maximally reduced intracellular ROS (…). Figure 3 (A and E in the text) and the level of restoration of mitochondrial membrane potential ( Figure 3 The B and F components showed a strong ability to restore oxidative stress-induced cell function; cell viability and TUNEL staining results showed that UPN-SS31@GH could significantly reduce apoptosis and promote cell survival. Figure 3(C and D in the text); Immunofluorescence staining results showed that UPN-SS31@GH significantly promoted the co-localization of autophagy protein LC3 with MitoTracker ( Figure 4 (A and B in the figure) suggests that UPN-SS31@GH can activate mitochondrial autophagy in nucleus pulposus progenitor cells, thereby promoting the functional recovery of nucleus pulposus progenitor cells.

[0067] Example 3: Preparation and characterization of bioactive microspheres:

[0068] Extraction and identification of nucleus pulposus progenitor cells: Nucleus pulposus progenitor cells (NPPCs) were isolated from the intervertebral discs of SD rats using enzymatic digestion and differential adhesion methods, and stemness indicators were detected by flow cytometry.

[0069] Preparation of bioactive microspheres: P3 generation nucleus pulposus progenitor cells were used at a concentration of 1×10⁻⁶. 6 Cells / mL were co-cultured with SS31-UPN@GH microspheres at 37°C and 5% CO2 for 72 hours. Calcein AM / PI staining and cytoskeleton staining were performed at different co-culture time points to detect the adhesion and growth status of nucleus pulposus progenitor cells on the surface of the microspheres.

[0070] Characterization of nucleus pulposus progenitor cells and bioactive microspheres is shown in [reference needed]. Figure 5-6 Flow cytometry was used to detect stemness markers in in vitro cultured nucleus pulposus progenitor cells. The positive rates for CD29, CD44, and CD90 were 90.4%, 92.6%, and 98.6%, respectively, while the negative rates for CD45 and CD34 were 0.05% and 0.25%, respectively. Figure 5 This confirmed that the cultured nucleus pulposus progenitor cells had good stemness; calcein AM / propidium iodide staining and cytoskeleton staining were performed on days 1, 3, 5, and 7 of co-culture, and the results indicated that the nucleus pulposus progenitor cells could adhere and grow on different microsphere surfaces. Figure 6 In comparison to Comparative Examples 2 and 3, cells adhering to and growing on the surface of UPN-SS31@GH microspheres (A) exhibited a higher cell proliferation rate. Figure 6 (B and D in the middle) and more extensive actin fiber extension ( Figure 6 (C in the middle).

[0071] Example 4: Therapeutic effect of bioactive microspheres on intervertebral disc degeneration in animals

[0072] Establishment of an animal model of intervertebral disc degeneration: After surgically exposing the annulus fibrosus of the target intervertebral disc in the coccyx, the nucleus pulposus was partially removed using a miniature curette to establish a model of severe structural defects. After modeling, the discs were randomly assigned to groups.

[0073] NPPCs@SS31-UPN@GH group: Bioactive hydrogel microspheres (NPPCs@SS31-UPN@GH) were injected into the intervertebral disc immediately after nucleus pulposus removal and modeling.

[0074] SS31-UPN@GH group: Immediately after nucleus pulposus removal and modeling, SS31-UPN@GH microspheres without adherent cells were injected into the intervertebral disc.

[0075] NPPCs group: Immediately after nucleus pulposus removal and modeling, a suspension of simple nucleus pulposus progenitor cells was injected into the intervertebral disc.

[0076] PBS group (model control group): Immediately after nucleolysis modeling, an equal volume of PBS was injected into the intervertebral disc.

[0077] Sham surgery group: Only surgical exposure (cutting the skin and exposing the annulus fibrosus) was performed, but the nucleus pulposus was not removed, and the wound was then sutured.

[0078] See test results Figure 7-10 :

[0079] Histological assessment:

[0080] In radiographic evaluation, the NPPCs@SS31-UPN@GH group showed the largest intervertebral disc height on X-ray. Figure 7 In cases A and B), MRI T2-weighted images show the highest signal intensity in the nucleus pulposus and the lowest grade of radiographic degeneration (Pfirrmann classification). Figure 7 The C group was significantly superior to all other treatment groups (SS31-UPN@GH group, NPPCs group, PBS group); in histological evaluation, intervertebral disc histological staining indicated the best repair effect on the nucleus pulposus and annulus fibrosus. Figure 8 In the A and B groups, the tissue morphology and cell viability scores were closest to those of the sham surgery group. Figure 9 (A and B in the text).

[0081] The SS31-UPN@GH group and the NPPCs group showed no statistically significant difference in imaging and histological scores compared to the PBS group, demonstrating that simply supplementing cells or microspheres cannot achieve effective repair.

[0082] PBS group (model control group): lowest imaging and histological scores, severely decreased intervertebral disc height and nucleus pulposus MRI signal, severe loss of intervertebral disc nucleus pulposus tissue, and disordered annulus fibrosus structure, confirming successful modeling.

[0083] The sham surgery group had intact intervertebral disc structure, full nucleus pulposus tissue, and clear annulus fibrosus layers. The scores were not significantly different from those of healthy intervertebral discs, proving that simple surgical trauma does not lead to significant intervertebral disc degeneration.

[0084] Biomechanical assessment: The biomechanical displacement curve of the NPPCs@SS31-UPN@GH group was closest to that of the sham surgery group, while the other treatment groups (SS31-UPN@GH group and NPPCs group) showed no significant improvement compared to the PBS group. Figure 10 (B and C in the middle).

[0085] The above embodiments fully demonstrate that the bioactive microsphere preparation method provided by the present invention is feasible, the product structure is well-defined, and through the synergistic effect of the three-layer structure, it can effectively remove ROS, protect transplanted cells, and promote the structural repair and functional recovery of intervertebral disc tissue in in vitro and in vivo models, showing outstanding effects and application potential in the treatment of intervertebral disc degeneration.

[0086] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A bioactive microsphere for treating intervertebral disc degeneration, characterized in that, The bioactive microspheres consist of, from the inside out, an ultra-small Prussian blue nanozyme modified with mitochondrial-targeting peptides, a biomimetic scaffold layer, and a cell layer. The biomimetic scaffold layer is formed by cross-linking methacrylamide gelatin and methacrylamide hyaluronic acid; The cell layer is composed of nucleus pulposus progenitor cells that adhere to the surface of the biomimetic scaffold layer; The mitochondrial targeting peptide is SS31 peptide; The method for preparing the bioactive microspheres for treating intervertebral disc degeneration includes the following steps: (1) The ultra-small Prussian blue nanozyme was carboxylated and activated, and then mitochondrial-targeting peptides were added and reacted overnight. After centrifugation and purification, ultra-small Prussian blue nanozyme modified with mitochondrial-targeting peptides was obtained. (2) Dissolve methacrylated gelatin and methacrylated hyaluronic acid in a photoinitiator solution, add the ultrasmall Prussian blue nanozyme modified with mitochondrial-targeting peptide obtained in step (1), and disperse evenly to obtain a mixed prepolymer solution. (3) Using the mixed prepolymer solution obtained in step (2) as the aqueous phase, it forms uniform droplets with the oil phase through a microfluidic device, cross-links and solidifies to form hydrogel microspheres of ultrasmall Prussian blue nanozymes modified with mitochondrial-targeting peptides. (4) The nucleus pulposus progenitor cells are co-cultured with the hydrogel microspheres obtained in step (3) so that the nucleus pulposus progenitor cells adhere to the surface of the hydrogel microspheres, and bioactive microspheres for the treatment of intervertebral disc degeneration are obtained.

2. The bioactive microspheres according to claim 1, characterized in that, The mass ratio of the ultrasmall Prussian blue nanozyme to the mitochondrial-targeting peptide is 1:150-1:

100. And / or, the particle size of the ultrasmall Prussian blue nanozyme is 3 nm-10 nm; And / or, the diameter of the bioactive microspheres is 100 μm-200 μm.

3. The bioactive microspheres according to claim 1, characterized in that, The bioactive microspheres have a porous structure; the pore size of the porous structure is 20 μm-50 μm.

4. The bioactive microspheres according to claim 1, characterized in that, In step (1), the ultra-small Prussian blue nanozyme is prepared by the following method: polyvinylpyrrolidone and potassium ferricyanide are dissolved in a mixed solvent of HCl and alcohol to react and obtain the ultra-small Prussian blue nanozyme.

5. The bioactive microspheres according to claim 1, characterized in that, In step (2), the final concentration of the methacrylamide gelatin is 100 mg / mL-150 mg / mL; And / or, the final concentration of the methacrylamide hyaluronic acid is 10 mg / mL to 20 mg / mL; And / or, the final concentration of the ultrasmall Prussian blue nanozyme is 0.1 mg / mL to 0.15 mg / mL.

6. The bioactive microspheres according to claim 1, characterized in that, In step (2), the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphite; And / or, the mass-volume percentage concentration of the photoinitiator in the photoinitiator solution is 0.25%-0.5%.

7. The bioactive microspheres according to claim 1, characterized in that, In step (3), the oil phase is mineral oil containing Span 80; the flow rate of the oil phase is 3 mL / min-5 mL / min; And / or, the flow rate of the aqueous phase is 30 μL / min-40 μL / min.

8. The bioactive microspheres according to claim 1, characterized in that, In step (4), the density of the nucleus pulposus progenitor cells added is 1×10⁻⁶. 6 cells / mL - 5 × 10 6 cells / mL.

9. The use of the bioactive microspheres according to any one of claims 1-8 in the preparation of drugs or medical devices for treating intervertebral disc degeneration.

Images