Composite hydrogel for targeting cartilage endplate as well as preparation method and application of composite hydrogel

Abstract

The invention mainly relates to composite hydrogel of a targeted cartilage end plate as well as a preparation method and application of the composite hydrogel, and belongs to the technical field of biological medicines. The composite hydrogel of the targeting cartilage end plate comprises a cartilage targeting peptide modified drug-loaded exosome, calcium carbonate, chitosan and water, and the cartilage targeting peptide modified drug-loaded exosome comprises an exosome, a cartilage targeting peptide and salvianolic acid A. The composite hydrogel of the targeting cartilage end plate is prepared by the following steps: encapsulating salvianolic acid A by exosome derived from human mesenchymal stem cells, modifying by cartilage targeting peptide to obtain drug-loaded exosome modified by the cartilage targeting peptide, and mixing the drug-loaded exosome with calcium carbonate, chitosan and water. The compound can be used for accurately targeting cartilage endplate cells (CEPCs), remarkably relieving the inflammation of the CEPCs, changing the secretion phenotype of the CEPCs, delaying the senescence of nucleus pulposus cells (NPCs), relieving the inflammation of the NPCs and effectively treating intervertebral disc diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a composite hydrogel targeting cartilage endplates and a preparation method and application thereof. Background Art

[0002] Intervertebral disc disease (IVDD) is a widespread and debilitating disease and the leading cause of lower back pain. This degenerative disease severely affects the quality of life of patients and imposes a huge economic burden on society. Despite various surgical and conservative treatment strategies, spinal degenerative diseases including IVDD remain incurable. Surgical interventions such as total disc replacement and anterior cervical discectomy and fusion have the potential to exacerbate intervertebral disc (IVD) stress and increase the risk of adjacent segment disease. Although conservative treatments can effectively relieve pain associated with IVDD, they cannot prevent the progression of degeneration. Therefore, the development of novel therapeutic agents targeting the pathological process of IVDD is urgent.

[0003] The pathophysiology of IVDD is a complex process that mainly involves oxidative stress, cellular senescence, imbalance in extracellular matrix (ECM) metabolism, autophagy dysfunction, and advanced glycation end products (AGEs). The pathogenesis of IVDD is intricate and involves multiple cells and their surrounding microenvironment, including annulus fibrosus cells (AFCs), cartilage endplate cells (CEPCs), nucleus pulposus cells (NPCs), and infiltrating immune cells. The complex interactions between these cells make it more difficult to understand the pathophysiology of IVDD and hinder the development of effective treatments. Therefore, in order to overcome the current limitations of IVDD treatment and repair, it is imperative to develop multi-target drug therapies. However, most current IVDD treatment studies focus on NPCs. Since there is no vascular tissue in the IVD, the permeability of the cartilage endplate (CEP) is the nutrient metabolism of NPCs. As the main pathway for nutrient delivery, CEP degeneration leads to endplate calcification, which hinders this supply pathway, resulting in nutrient depletion and accumulation of metabolites in the IVD, which deteriorates the IVD microenvironment, impairs endogenous nucleus pulposus (NP) repair, and exacerbates IVDD progression. In addition, the acidic microenvironment also poses a huge challenge to the treatment of IVDD. Currently, there is no strategy for CEPCs that can reduce inflammation while solving the problems of ROS damage and cell senescence to better treat IVDD. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a composite hydrogel targeting cartilage endplate and a preparation method thereof, which can target CEP and better treat intervertebral disc diseases.

[0005] The present invention provides a composite hydrogel targeting cartilage endplates, comprising cartilage targeting peptide-modified drug-loaded exosomes, calcium carbonate, chitosan and water, wherein the dosage ratio of the cartilage targeting peptide-modified drug-loaded exosomes, calcium carbonate, chitosan and water is (90-110 μg): (9-11 mg): (90-110 mg): (3-5 mL); the cartilage targeting peptide-modified drug-loaded exosomes comprise cartilage targeting peptide, exosomes and salvianolic acid A.

[0006] Preferably, the exosomes are exosomes derived from human mesenchymal stem cells.

[0007] The present invention also provides a method for preparing the composite hydrogel, comprising the following steps:

[0008] Exosomes and salvianolic acid A were mixed, ultrasonically treated, and then incubated for the first time, followed by centrifugation to obtain drug-loaded exosome particles;

[0009] The drug-loaded exosome particles are mixed with the cartilage-targeting peptide in water, incubated for a second time, and centrifuged to obtain the cartilage-targeting peptide-modified drug-loaded exosomes;

[0010] The cartilage targeting peptide-modified drug-loaded exosomes, calcium carbonate, chitosan and water are mixed and gelled to obtain a composite hydrogel targeting the cartilage endplate.

[0011] Preferably, the mass ratio of the exosomes to salvianolic acid A is 3 to 7:1.

[0012] Preferably, the ultrasonic treatment is: ultrasonic amplitude 15-25%, on 30s / off 30s cycle for 2-4min, repeated 5-7 times, each time with an interval of 1-3min.

[0013] Preferably, the first incubation is at 35-39° C. for 0.8-1.2 h.

[0014] Preferably, the mixing ratio of the drug-loaded exosome particles and the cartilage targeting peptide is (0.8×10 12 ~1.2×10 12 )Particles / mL: (8~12)μM.

[0015] Preferably, the second incubation is performed at 3-5° C. for 8-14 hours.

[0016] Preferably, the gelation comprises shaking and standing.

[0017] The present invention also provides application of the composite gel or its preparation method in preparing medicine for treating intervertebral disc diseases.

[0018] Beneficial effects of the present invention:

[0019] The composite hydrogel (CAP-sEXOs@Gel) targeting cartilage endplates of the present invention includes exosomes (CAP-sEXOs) modified with cartilage targeting peptide (CAP) and CaCO3, chitosan and water. CAP-sEXOs contains salvianolic acid A (SAA). CAP-sEXOs@Gel is designed to release drug-loaded exosomes that specifically target CEPCs, thereby limiting the influx of iron ions, reducing oxidative stress, and inhibiting the mtDNA-cGMP-AMP synthase (cGAS)-interferon gene stimulator (STING) pathway. These biological effects jointly reduce CEPC inflammation, improve the secretion of regenerative cytokines, and alleviate the aging of NPC. The CaCO3 component in the hydrogel can neutralize H + , restore the activity of Akt protein kinase, inhibit the NLRP3-Caspase1 pathway, and thus reduce the inflammatory response of NPC. In vitro and in vivo experiments verified the long-term retention of CAP-sEXOs@Gel at the injection site in the intervertebral disc and its ability to target CEP. The IVDD rat model induced by CEP degeneration showed that the CAP-sEXOs@Gel of the present invention can effectively alleviate CEPC inflammation, inhibit NP inflammation, and promote IVD regeneration. In summary, the composite hydrogel targeted to the cartilage endplate provided by the present invention can play a therapeutic role in IVDD against a variety of pathogenic factors, providing a new idea for the treatment of IVDD. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The diagram shows the process of hiPSCs (human induced pluripotent stem cells) differentiating into iMSCs (human mesenchymal stem cells); the left picture is a light microscopy image of long-term cultured hiPSCs before differentiation, the middle picture is a mid-stage image of hiPSCs induced differentiation, and the right picture is a light microscopy image of iMSCs; scale bar = 100 μm.

[0021] Figure 2 These are graphs for detecting the tri-lineage differentiation ability of iMSCs; the left graph is an Oil Red O staining graph, the middle graph is an Alizarin Red staining graph, and the right graph is an Alcian Blue staining graph; scale bar = 100 μm.

[0022] Figure 3 Flow cytometric analysis of iMSCs surface markers; the first row is a positive marker graph, from left to right: CD105, CD90 and CD73; the second row is a negative marker graph, from left to right: CD14, CD45 and CD34.

[0023] Figure 4Characterization diagram of EXOs (iMSC-EXOs, exosomes); wherein, A is the TEM image of EXOs, scale bar = 100 nm; B is the western blot detection diagram of EXOs protein markers; C is the size distribution diagram of EXOs detected by DLS.

[0024] Figure 5 Preparation and characterization of CAP-sEXOs@Gel; A is a fluorescence microscopy image of CAP-sEXOs (cartilage-targeting peptide-modified drug-loaded exosomes) captured by Cy3-labeled protein A / G magnetic beads; B is a TEM image of CAP-sEXOs, scale = 100 nm; C is a particle size distribution of CAP-sEXOs measured by DLS; D is a stability graph of the particle size (orange) and zeta potential (brown) of CAP-sEXOs; E is a SAA release curve of CAP-sEXOs incubated with PBS or FBS (n = 3); F is a freeze-dried CAP-sEXOs@Ge l is the SEM image of CAP-sEXOs@Gel after re-dissolution; H is the exosome release curve under different pH; I is the miRNA expression spectrum heat map of CAP-sEXOs and BMSC-EXOs (human bone marrow mesenchymal stem cell exosomes); J is the dot plot of GO analysis enriched BP; K is the dot plot of graphene oxide analysis enriched MF; L is the dot plot of graphene oxide analysis enriched CC; M is the signal pathway dot plot of KEGG cluster analysis enriched endogenous miRNA carried by CAP-sEXOs; N is a pie chart showing the proportions of six miRNAs with cartilage protection function.

[0025] Figure 6Figure 2 is a diagram for detecting the ability of CAP-sEXOs@Gel to target CEPC in vitro and in vivo; A is a schematic diagram for the design of the in vitro targeting CEPC ability test; B is a fluorescence image (red) of DiR-labeled sEXOs (drug-loaded exosomes) and CAP-sEXOs after incubation with NPCs and CEPCs for 6 hours, scale bar = 100 μm; C is a quantitative analysis of DiR fluorescence intensity in the sEXOs group and the CAP-sEXOs group; D is a schematic diagram for the design of the in vivo targeting CEPC ability test; E is a fluorescence image of CAP-sEXOs and CAP-sEXOs@Gel injected into IVD at different time points and sEXOs@Gel (non-targeted drug-loaded exosome composite hydrogel) local distribution IVIS bioimaging; F is the quantitative evaluation of in vivo fluorescence intensity in each group; G is the IVIS image of the main organs (heart, liver, spleen, lung, and kidney) 24 hours after IVD injection; H is the fluorescence staining of IVD sections after injection of DiR-labeled CAP-sEXOs@Gel and sEXOs@Gel; I is the quantitative analysis of DiR fluorescence intensity of IVD sections; n=3, NS, no significant difference, *P<0.05, **P<0.01, ***P<0.001, #P<0.0001.

[0026] Figure 7 Figure 2 shows the detection results of CAP-sEXOs@Gel alleviating inflammation in CEPCs by inhibiting iron influx and mtDNA-cGAS-STING signaling pathway; A is a graphic description of the molecular mechanism, which shows that CAP-sEXOs@Gel alleviates inflammation in CEPCs; B is an immunofluorescence staining of HIF-2ɑ and TIR1, scale bar = 10μm; C is a quantitative analysis of immunofluorescence staining of HIF-2ɑ (top) and TIR1 (bottom); D is a graph showing the expression of Fe in CEPCs after 60 minutes of intervention with different gels 2+ Fluorescence image of the probe; E is Fe 2+ Fluorescence quantitative analysis graph; F is the quantitative analysis graph of intracellular ROS; G is the fluorescence staining graph of active mitochondrial probe; H is the quantitative analysis graph of mitochondrial fluorescence activity; I is the quantitative analysis graph of intracellular double-stranded DNA immunofluorescence staining; J is the intracellular double-stranded DNA immunofluorescence staining; K is the quantitative analysis graph of cGAMP in cytoplasmic lysate; L is the quantitative analysis graph of p-STING in cytoplasmic lysate; M is the quantitative analysis graph of p-IRF3 in nuclear lysate; n=5, NS, no significant difference, *P<0.05, **P<0.01, ***P<0.001, #P<0.0001.

[0027] Figure 8Figure 2 shows the detection results of CAP-sEXOs@Gel changing the secretory phenotype of CEPCs and indirectly alleviating NPC aging; A is a schematic diagram illustrating the experimental design of this part; B is a heat map showing the changes in cytokines in the conditioned medium of CEPCs after different interventions; C is a protein immunoblotting experiment result of NPCs aging-related markers (p16, p21 and p53) and stem cell-related markers (Nanog and OCT-4); D is a quantitative analysis of p16; E is a quantitative analysis of p21; F is a quantitative analysis of p53; G is a quantitative analysis of Nanog; H is a quantitative analysis of OCT-4; I is a quantitative analysis of Col2 mRNA expression; J is a quantitative analysis of Acan mRNA expression after treatment with different conditioned media; n=3, NS, no significant difference, *P<0.05, **P<0.01, ***P<0.001, #P<0.0001.

[0028] Fig. 9 Neutralization of H by CAP-sEXOs@Gel + Figure 3 shows the test results of reducing NPC inflammation; A is a schematic diagram illustrating the experimental design of this stage; B is the live / dead staining of NPCs after lactate and each group intervention, dead cells show PI red fluorescence, and live cells show calcium green fluorescence; C is the cell proliferation curve determined by CCK-8 method; D is the quantitative graph of the percentage of live cells by live / dead staining; E is the quantitative analysis graph of Akt kinase activity; F is the p21 / Col2 immunofluorescence staining (top) and p27 / Acan immunofluorescence staining (bottom); G is the fluorescence intensity measurement graph of p21 / Col2 (left) and p27 / Acan (right); H is the Nrf2 immunofluorescence staining; I is the molecular mechanism of CAP-sEXOs@Gel in reducing NPCs inflammation; J is the quantitative bar graph of NLRP3, Cleaved Caspase1 and IL-1β; *p<0.05; **p<0.01; ***p<0.001; #p<0.0001, NS, no significant difference.

[0029] Fig.10The figure shows the therapeutic effect of CAP-sEXOs@Gel on the CEP degenerative IVDD rat model; A is a schematic diagram of the experimental design of this part, B is a list of materials for different groups; C is an MRI image of the coccyx at 4 and 8 weeks after different treatments; D is a quantitative analysis of the grayscale value of IVD; E is a picture of Safranin O fast green staining (upper row), Alcian blue staining (middle row), and H&E staining (lower row) of rat IVD, scale bar: 500μm; F is the histological score of IVD in each group of rats; G is Immunofluorescence staining of TfR1, p-STING and SOX9 in CEP tissue, scale: 50μm; H is the quantitative analysis of p-STING fluorescence intensity in CEP tissue; I is the immunofluorescence staining of NLRP3, IL-1β and Acan in NP tissue, scale: 50μm; J is the quantitative analysis of NLRP3 fluorescence intensity in CEP tissue; n=5, *p<0.05; **p<0.01; ***p<0.001; #p<0.0001, NS, no significant difference. DETAILED DESCRIPTION

[0030] The present invention provides a composite hydrogel targeting cartilage endplates, comprising cartilage targeting peptide-modified drug-loaded exosomes, calcium carbonate, chitosan and water. The dosage ratio of the cartilage targeting peptide-modified drug-loaded exosomes, calcium carbonate, chitosan and water is (90-110 μg): (9-11 mg): (90-110 mg): (3-5 mL), preferably (95-105 μg): (9.5-10.5 mg): (95-105 mg): (3.5-4.5 mL); more preferably 100 μg: 10 mg: 100 mg: 4 mL.

[0031] The present invention does not specifically limit the sources of calcium carbonate, chitosan and water, and conventional commercial products in the art can be used. The cartilage targeting peptide-modified drug-loaded exosomes include cartilage targeting peptides, exosomes and salvianolic acid A (SAA). The exosomes (denoted as EXOs) are preferably exosomes derived from human mesenchymal stem cells. Exosomes can be prepared using commercially available exosomes or homemade methods. When the exosomes are homemade, they are preferably isolated from the conditioned medium of mesenchymal stem cells after inducing differentiation of human induced pluripotent stem cells into mesenchymal stem cells.

[0032] The present invention also provides a method for preparing the composite hydrogel targeting the cartilage endplate, comprising the following steps:

[0033] In the present invention, exosomes and salvianolic acid A are mixed, and the first incubation is performed after ultrasonic treatment, and centrifugation is performed to obtain drug-loaded exosome particles (referred to as sEXOs); the mass ratio of the exosomes to salvianolic acid A is preferably 3 to 7:1, more preferably 4 to 6:1, and further preferably 5:1. The ultrasonic treatment is preferably: ultrasonic amplitude 15 to 25%, on 30s / off 30s cycle for 2 to 4min, repeated 5 to 7 times, each interval 1 to 3min; in one embodiment, the ultrasonic amplitude can be selected to be 15%, 18%, 20%, 23% or 25%, on 30s / off 30s cycle for 2min, 2.5min, 3min, 3.5min or 4min, repeated 5 times, 6 times or 7 times, each interval 1min, 1.5min, 2min, 2.5min or 3min. After ultrasound, the first incubation is performed to promote the recovery of the exosome membrane; the first incubation is preferably incubated at 35-39°C for 0.8-1.2h. In one embodiment, 35°C, 36°C, 37°C, 38°C or 39°C can be selected for incubation for 0.8h, 0.9h, 1h, 1.1h or 1.2h. After incubation, centrifugation is performed to remove unencapsulated salvianolic acid A. The present invention does not specifically limit the centrifugation method, and conventional centrifugation methods in the art can be used. In one embodiment, centrifugation at 1200rpm for 10min can be selected; centrifugation is performed again to purify the product to obtain drug-loaded exosome particles. The present invention does not specifically limit the centrifugation method, and conventional centrifugation methods in the art can be used. In one embodiment, centrifugation at 120000g for 30-70min can be selected. In one embodiment, centrifugation for 30min, 50min or 70min can be selected.

[0034] In the present invention, the drug-loaded exosome particles obtained above are mixed with the cartilage targeting peptide in water, incubated for a second time, and centrifuged to obtain cartilage targeting peptide-modified drug-loaded exosomes (denoted as CAP-sEXOs); the mixing ratio of the drug-loaded exosome particles and the cartilage targeting peptide is preferably (0.8×10 12 ~1.2×10 12 ) particles / mL: (8-12) μM, more preferably (0.9×10 12 ~1.1×10 12 ) particles / mL: (9-11) μM, more preferably 1×10 12Particles / mL: 10 μM. The cartilage targeting peptide is preferably a cartilage targeting peptide commonly used in the art that can modify CAP to exosomes, and more preferably a DSPE-PEG-NHS-modified CAP, i.e., DSPE-PEG-NHS-CAP. The preparation method of the DSPE-PEG-NHS-CAP preferably comprises the following steps: taking the cartilage targeting peptide CAP, dissolving it in a 5mM TCEP solution, and adjusting the final concentration of the cartilage targeting peptide to 1 mg / mL. Take DSPE-PEG-NHS and dissolve it in a 5mM HEPES buffer, and also adjust the final concentration of DSPE-PEG-NHS to 1 mg / mL. Add the cartilage targeting peptide and DSPE-PEG-NHS to a beaker in a 1:1 ratio of the amount of substance, mix evenly at room temperature (any temperature between 20°C and 25°C, 20°C is used in the embodiment of the present invention), and stir overnight with a magnetic stirrer to obtain a crude product of the conjugate DSPE-PEG-NHS-CAP. The crude product solution is transferred to a dialysis bag and dialyzed for 24 hours. The final solution in the dialysis bag is freeze-dried to obtain powdered DSPE-PEG-NHS-CAP. The second incubation is preferably incubated at 3 to 5°C for 8 to 14 hours. In one embodiment, 3°C, 4°C or 5°C can be selected for incubation for 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours or 14 hours. After incubation, centrifugation is performed to remove unreacted cartilage targeting peptides. The present invention does not specifically limit the centrifugation method, and conventional centrifugation methods in the art can be used. In one embodiment, centrifugation at 1200rpm for 10 minutes can be selected; centrifugation is performed again to wash the product. The present invention does not specifically limit the centrifugation method, and conventional centrifugation methods in the art can be used. In one embodiment, centrifugation at 120000g can be selected for 70 minutes. After washing, CAP-sEXOs are obtained, and CAP-sEXOs are preferably resuspended in water for standby use.

[0035] In the present invention, the above-obtained cartilage targeting peptide-modified drug-loaded exosomes are mixed with calcium carbonate, chitosan and water for gelation to obtain a composite hydrogel targeting the cartilage endplate (denoted as CAP-sEXOs@Gel). The dosage ratio of the cartilage targeting peptide-modified drug-loaded exosomes, calcium carbonate, chitosan and water is (90-110 μg): (9-11 mg): (90-110 mg): (3-5 mL), preferably (95-105 μg): (9.5-10.5 mg): (95-105 mg): (3.5-4.5 mL); more preferably 100 μg: 10 mg: 100 mg: 4 mL. The gelation preferably includes shaking and standing. The present invention does not specifically limit the shaking method, and the conventional shaking method in the art can be used to mix the components evenly by shaking. The mixture is then allowed to stand for 25 to 35 minutes at preferably 20 to 25°C. In one embodiment, the mixture can be allowed to stand for 25 minutes, 28 minutes, 30 minutes, 32 minutes or 35 minutes at 20°C, 21°C, 22°C, 23°C, 24°C or 25°C to completely gel the mixture to obtain CAP-sEXOs@Gel.

[0036] In the present invention, there is no special limitation on the source of salvianolic acid A, and conventional commercial products in the art may be used.

[0037] The present invention also provides the use of the composite gel or its preparation method in the preparation of a drug for treating intervertebral disc disease. The composite gel is used to treat intervertebral disc disease, can target CEPCs to exert its effects, reduce inflammation of CEPCs, change the secretory phenotype of CEPCs, indirectly alleviate NPC aging, and can also inhibit NPC inflammation, effectively treating intervertebral disc disease.

[0038] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0039] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0040] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0041] In the following embodiments of the present invention, human iPSC (induced pluripotent stem cells) were purchased from the cell bank of the Chinese Academy of Sciences, SAA (salvianolic acid A) was purchased from the Wangjing Hospital of the China Academy of Chinese Medical Sciences; DSPE-PEG-NHS-CAP was prepared using the above-mentioned preparation method (commissioned by Shanghai Sixth People's Hospital for preparation), calcium carbonate was purchased from Shanghai Zhichuang Haotian Biotechnology Co., Ltd., and chitosan was purchased from Shanghai Zhichuang Haotian Biotechnology Co., Ltd.

[0042] Example 1

[0043] 1. Preparation of Exosomes (EXOs)

[0044] 1.1 Human iPSC cell line DYR0100 was used to induce iMSCs (human mesenchymal stem cells). A commercial iPSC-mesenchymal stem cell differentiation kit (ShowninBiotech) was used to differentiate iPSCs into iMSCs according to the kit instructions. During the differentiation process, iPSCs gradually lost their unique colony morphology. After differentiation, the cells became spindle-shaped, indicating that iPSCs had successfully differentiated into iMSCs. The differentiation process is as follows Figure 1 shown.

[0045] 1.2 The iMSCs obtained above were grown in serum-free mesenchymal stem cell medium for 48 hours. The conditioned medium was centrifuged twice, and non-adherent cells were extracted at 300g centrifugal force for 10 minutes; then cells and debris were extracted at 1030g centrifugal force for 15 minutes. Then, the supernatant was centrifuged at 10,400g for 60 minutes to extract larger vesicles from the supernatant. The residual supernatant was ultracentrifuged at 105,000g for 70 minutes, and the exosome pellet obtained was resuspended in PBS and ultracentrifuged again under the same conditions to remove contaminating proteins. All processes were performed at 4°C to obtain exosomes (EXOs).

[0046] 2. Preparation of CAP-sEXOs (cartilage-targeting peptide-modified drug-loaded exosomes)

[0047] 2.1 Encapsulate SAA into exosomes using ultrasound technology. Specifically: exosomes and SAA were mixed in a mass ratio of 5:1 and then ultrasonically treated. The ultrasonic parameters were an amplitude of 20%, 30s on / 30s off alternating cycle for 3min, repeated 6 times, with a 2min interval between each. The composition was then incubated at 37°C for 1h to promote the recovery of the exosome membrane. Unencapsulated SAA was removed by centrifugation at 1200rpm for 10min, and then the exosome particles loaded with SAA were further purified by centrifugation at 120,000g for 30min to obtain sEXOs (drug-loaded exosomes).

[0048] 2.2 sEXOs were modified with CAP (cartilage targeting peptide) and sEXOs (final concentration before incubation in ultrapure water was 10 12The sEXO particles were washed with DSPE-PEG-NHS-CAP (final concentration of 10 μM before incubation in ultrapure water) and incubated in ultrapure water at 4°C for 12 h. The unreacted DSPE-PEG-NHS-CAP was centrifuged at 1200 rpm for 10 min. After centrifugation at 120,000 g for 70 min, the CAP-modified sEXO particles were washed and resuspended in ultrapure water to obtain CAP-sEXOs.

[0049] 3. Preparation of composite hydrogel CAP-sEXOs@Gel targeting cartilage endplate

[0050] 100 mg chitosan, 10 mg CaCO3 and 100 μg CAP-sEXOs were placed in a centrifuge tube containing 4 mL ultrapure water. To fully gel, the mixture was shaken and placed at 20°C for 30 min to obtain the composite hydrogel CAP-sEXOs@Gel targeting the cartilage endplate. The sample was then stored at -80°C or freeze-dried for later use.

[0051] Example 2

[0052] 1. Preparation of Exosomes (EXOs)

[0053] Same as step 1 in Example 1.

[0054] 2. Preparation of CAP-sEXOs (cartilage-targeting peptide-modified drug-loaded exosomes)

[0055] 2.1 Encapsulate SAA into exosomes using ultrasound technology. Specifically: exosomes and SAA were mixed in a mass ratio of 3:1 and then ultrasonically treated. The ultrasonic parameters were an amplitude of 15%, 30s on / 30s off alternating cycle for 4min, repeated 5 times, with a 1min interval between each. The composition was then incubated at 39°C for 0.8h to promote the recovery of the exosome membrane. Unencapsulated SAA was removed by centrifugation at 1200rpm for 10min, and then the exosome particles loaded with SAA were further purified by centrifugation at 120,000g for 70min to obtain sEXOs (drug-loaded exosomes).

[0056] 2.2 sEXOs were modified with CAP (cartilage targeting peptide) and sEXOs (0.8×10 12 The sEXO particles (100 μg / mL) were incubated with DSPE-PEG-NHS-CAP (12 μM) in ultrapure water at 3°C ​​for 14 h. The unreacted DSPE-PEG-NHS-CAP was centrifuged at 1200 rpm for 10 min. After centrifugation at 120,000 g for 70 min, the CAP-modified sEXOs particles were washed and resuspended in ultrapure water to obtain CAP-sEXOs.

[0057] 3. Preparation of composite hydrogel CAP-sEXOs@Gel targeting cartilage endplate

[0058] 90 mg chitosan, 11 mg CaCO3 and 110 μg CAP-sEXOs were placed in a centrifuge tube containing 5 mL ultrapure water. To fully gel, the mixture was shaken and placed at 23°C for 25 min to obtain the composite hydrogel CAP-sEXOs@Gel targeting the cartilage endplate. The sample was then stored at -80°C or freeze-dried for later use.

[0059] Example 3

[0060] 1. Preparation of Exosomes (EXOs)

[0061] Same as step 1 in Example 1.

[0062] 2. Preparation of CAP-sEXOs (cartilage-targeting peptide-modified drug-loaded exosomes)

[0063] 2.1 Encapsulate SAA into exosomes using ultrasound technology. Specifically: exosomes and SAA were mixed in a mass ratio of 7:1 and then ultrasonically treated. The ultrasonic parameters were an amplitude of 25%, 30s on / 30s off alternating cycle for 2min, repeated 7 times, with a 3min interval between each. The composition was then incubated at 35°C for 1.2h to promote the recovery of the exosome membrane. Unencapsulated SAA was removed by centrifugation at 1200rpm for 10min, and then the exosome particles loaded with SAA were further purified by centrifugation at 120,000g for 70min to obtain sEXOs (drug-loaded exosomes).

[0064] 2.2 sEXOs were modified with CAP (cartilage targeting peptide) to 12 The sEXO particles (0.100 μg / mL) were incubated with DSPE-PEG-NHS-CAP (8 μM) in ultrapure water at 5°C for 8 h. The unreacted DSPE-PEG-NHS-CAP was centrifuged at 1200 rpm for 10 min. After centrifugation at 120,000 g for 70 min, the CAP-modified sEXOs particles were washed and resuspended in ultrapure water to obtain CAP-sEXOs.

[0065] 3. Preparation of composite hydrogel CAP-sEXOs@Gel targeting cartilage endplate

[0066] 110 mg chitosan, 9 mg CaCO3 and 90 μg CAP-sEXOs were placed in a centrifuge tube containing 3 mL ultrapure water. To fully gel, the mixture was shaken and placed at 25°C for 35 min to obtain the composite hydrogel CAP-sEXOs@Gel targeting the cartilage endplate. The sample was then stored at -80°C or freeze-dried for later use.

[0067] Comparative Example 1

[0068] Preparation of sEXOs@Gel (non-targeted drug-loaded exosome composite hydrogel)

[0069] 100 mg chitosan, 10 mg CaCO3 and 100 μg sEXOs obtained in step 2.1 of Example 1 were placed in a centrifuge tube containing 4 mL ultrapure water. To fully gel, the mixture was shaken and placed at 20°C for 30 min to obtain a composite hydrogel sEXOs@Gel targeting the cartilage endplate. The sample was then stored at -80°C or freeze-dried for later use.

[0070] Comparative Example 2

[0071] Preparation of Gel (chitosan gel containing CaCO3)

[0072] Place 100 mg chitosan and 10 mg CaCO3 in a centrifuge tube containing 4 mL ultrapure water. To fully gel, shake the mixture and place it at 20°C for 30 minutes. A chitosan gel containing CaCO3 is obtained. Then store the sample at -80°C or freeze-dry it for later use.

[0073] Comparative Example 3

[0074] CAP-EXOs@Gel (composite hydrogel targeting cartilage endplate without drug loading)

[0075] The EXOs prepared in step 1.1 of Example 1 were used to replace the sEXOs in step 2.2 of Example 1, and CAP-EXOs@Gel was prepared according to steps 2.2 and 3 of Example 1.

[0076] Comparative Example 4

[0077] Gel blank Preparation of blank gel without CaCO3

[0078] 100 mg of chitosan was placed in a centrifuge tube containing 4 mL of ultrapure water. To fully gelate the mixture, the mixture was shaken and placed at 20°C for 30 min. A chitosan gel without CaCO3 was obtained. blank The samples were then stored at -80°C or freeze-dried for later use.

[0079] Comparative Example 5

[0080] CAP-sEXOs@Gel blank Preparation of composite hydrogels targeting cartilage endplates without CaCO3

[0081] 100 mg chitosan and 100 μg CAP-sEXOs prepared in step 2 of Example 1 were placed in a centrifuge tube containing 4 mL ultrapure water. To fully gelate the mixture, the mixture was shaken and placed at 20°C for 30 min to obtain a composite hydrogel CAP-sEXOs@Gel that does not contain CaCO3 and targets the cartilage endplate. blank The samples were then stored at -80°C or freeze-dried for later use.

[0082] Test example

[0083] In the following test example:

[0084] Primary NPCs and CEPCs were isolated by the following method: 12-week-old Sprague-Dawley rats were adapted for feeding for 7 days. NPCs and CEPCs were isolated by surgery on the lumbar and thoracic vertebrae, and NP and CEP tissues were obtained and cut into 1 cubic millimeter pieces. The tissue pieces were digested in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 0.2 mg / mL type II collagenase at 37°C for three hours. After digestion, the resulting slurry was passed through a 40 μm cell strainer to remove undigested tissue components. The supernatant was discarded after centrifugation at 1500 rpm for 5 minutes. The precipitated cell pellet was resuspended in high-glucose DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. The cell suspension was then cultured at 37°C in a humidified atmosphere containing 5% carbon dioxide. On the third day, the culture medium was changed once. After the cell confluence reached 80%, they were cultured at a ratio of 1:3. P3-P5 cells were used in the following experimental procedures.

[0085] The ELISA detection method is: the levels of cGAMP, p-STING, p-IRF3, NLRP3, IL-1β and Caspase-1 in the cytoplasm are quantitatively detected by ELISA kit.

[0086] Statistical analysis methods: numerical data are presented as mean ± SD. All statistical analyses were performed using GraphPad Prism (version 9.0). Student's t-test was used for comparisons between two groups, and one-way analysis of variance was used for comparisons between three or more groups. Statistically significant P values ​​were defined as < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001, and #P < 0.0001 indicated statistical significance.

[0087] Test Example 1

[0088] 1. Identification of iMSCs

[0089] The iMSCs prepared in step 1 of Example 1 were identified.

[0090] Observe the cell morphology. After differentiation, the cells become spindle-shaped. Oil red O, alizarin red and alcian blue staining were used to detect the three-lineage differentiation of iMSCs. The results are as follows Figure 2 As shown in Figure 2, iMSCs have the ability to form adipocytes, osteoblasts and chondrocytes. Figure 3 As shown, Figure 3 The surface antigens CD105, CD90, and CD73 were positive, and CD14, CD45, and CD34 were negative. These characteristics indicated that iPSCs had been successfully differentiated into iMSCs because they met the characteristics of traditional mesenchymal stem cells.

[0091] 2. Characterization of EXOs

[0092] The EXOs obtained in step 1 of Example 1 were characterized. These EXOs were observed using a transmission electron microscope (TEM). Figure 4 A in the figure shows that they present characteristic vesicle structures. Western Blot was performed to detect them. Figure 4 B in the figure shows that iMSC-EXOs have high expression levels of CD9, CD63, and TSG101. Dynamic light scattering (DLS) was then performed to measure the expression of CD9, CD63, and TSG101 in iMSC-EXOs. Figure 4 C shows that the average diameter of iMSC-EXOs is about 130 nm.

[0093] The above results show that the present invention successfully obtained iMSCs by inducing iPSCs differentiation and successfully extracted EXOs.

[0094] 3. Characterization of CAP-sEXOs

[0095] To verify whether the CAP modification was successful, the CAP in Example 1 was replaced with CAP labeled with red fluorescence of Cy3, and the method of steps 1 and 2 of Example 1 was used to prepare Cy3-labeled CAP-sEXOs, and the Cy3-labeled CAP-sEXOs were captured with CD63 immunomagnetic beads and characterized. Figure 5 As shown in A, orange fluorescence was observed under a fluorescence microscope, confirming that CAP had been successfully inserted into the surface of exosomes; TEM images showed ( Figure 5 B), the modified exosomes maintain the typical vesicle structure. DLS ( Figure 5 C) shows that the diameter of CAP-sEXOs is slightly larger than that of unmodified exosomes, about 133 nm.

[0096] After one week of culture in phosphate-buffered saline (PBS), the particle size and possible changes of CAP-sEXOs were evaluated by DLS. Figure 5As shown in D, there was no significant change in particle size and zeta potential, indicating that CAP-sEXOs have good stability in physiological environment. Then, the drug release characteristics of CAP-sEXOs were tested in PBS and fetal bovine serum (FBS). Figure 5 As shown in Figure E, the release rates of SAA in FBS and PBS reached 8% and 10% after 2 h, respectively. The release rates tended to be stable over time, and reached 97% and 95% in FBS and PBS after 48 h, respectively.

[0097] 4. Characterization of CAP-sEXOs@Gel

[0098] The freeze-dried composite hydrogel material was observed under a scanning electron microscope (SEM). Figure 5 As shown in Figure F, it can be clearly seen that the exosomes were successfully loaded into the hydrogel. The freeze-dried CAP-sEXOs@Gel was redissolved in pure water solvent and then subjected to scanning electron microscopy. Figure 5 G shows that the exosomes are still clearly visible.

[0099] The exosome release rate of CAP-sEXOs@Gel was evaluated under different pH conditions. Figure 5 As shown in Figure 3, in a neutral physiological environment (pH 7.4), the release of exosomes from CAP-sEXOs@Gel was relatively slow. However, as the acidity increased, the release of exosomes was greatly accelerated. Although the exosome release from the composite hydrogel stabilized after 24 h under different acidity conditions, in a weakly acidic (pH 6.5) microenvironment similar to IVD, CAP-sEXOs@Gel showed rapid release of CAP-sEXOs within the first 24 h, followed by a sustained slow release for up to 168 h. This release characteristic is conducive to the rapid accumulation and maintenance of the effective therapeutic concentration (ETC).

[0100] The miRNA signature of CAP-sEXOs was investigated using second-generation high-throughput sequencing technology. miRNA sequencing: The manufacturer's instructions were followed to extract total RNA from exosomes using Trizol reagent. The samples were then sent for high-throughput sequencing (Cloud-seqBiotech, Shanghai, China). Before library construction, the purity and concentration of RNA were measured using a Nanodrop ND-100 spectrophotometer (ThermoFisher Scientific, Waltham, USA). To construct the miRNA sequencing library, approximately 150 bp PCR amplicons (equivalent to approximately 22 nt miRNA) were selected from each total RNA sample. The results showed that CAP-sEXOs showed a unique endogenous miRNA expression profile compared with exosomes derived from bone marrow mesenchymal stem cells (BMSCs), a commonly used cell source in regenerative medicine ( Figure 5 I in ). Then, Kyoto Encyclopedia of Genomes (KEGG) and Gene Ontology (GO) were used to perform enrichment analysis of these miRNAs to elucidate molecular pathways and biological processes. In terms of biological processes (BP), these miRNAs were mainly involved in the signaling pathways of cell surface receptors, the response to reactive oxygen species, the negative regulation of ossification, and the negative regulation of cell death ( Figure 5 J in ). Molecular function (MF) analysis showed that these differentially expressed miRNAs were mainly involved in the structural components of the extracellular matrix, the activity of nucleobase-containing compound kinases, the activity of DNA-binding transcription factors, and the binding of metal ions ( Figure 5 K in ). Cellular component (CC) analysis showed that the subcellular localization of these differentially expressed miRNAs was mainly associated with membrane-bound organelles, nucleoplasm, intracellular membrane-bound organelles, membrane-enclosed cavities, and intracellular organelle cavities ( Figure 5 KEGG analysis showed that differential miRNAs were mainly associated with signaling pathways, including TNF, NF-κB, and AMPK, which are closely related to inflammation, cell aging, energy metabolism, mitochondrial function, and oxidative stress ( Figure 5 Among the significantly differentially expressed and highly expressed miRNAs in the expression profile of CAP-sEXOs, miR-100-5p, miR-423-5p, miR-148a-3p, miR-99b-5p, miR-22-3p, and let-7a-5p accounted for 50.6% of the total miRNA content ( Figure 5 These miRNAs can delay cell senescence, inhibit inflammation, and promote cartilage regeneration. In conclusion, CAP-sEXOs are expected to improve the efficacy of IVDD.

[0101] Test Example 2

[0102] Capability of CAP-sEXOs@Gel to target CEPCs in vitro and in vivo

[0103] The following tests were conducted using the sEXOs, CAP-sEXOs, CAP-sEXOs@Gel prepared in Example 1 and the sEXOs@Gel prepared in Comparative Example 1:

[0104] 1. Follow Figure 6 The targeting effect of CAP-sEXOs on CEPCs was evaluated using an in vitro uptake assay as shown in the process A in Figure 1. sEXOs and CAP-sEXOs were labeled with DiR and then cultured with NPCs and CEPCs for 6 h, respectively. Figure 6 B in Figure 6 As shown in Figure C, the red fluorescence accumulation of CEPCs cultured with CAP-sEXOs was significantly higher than that of NPCs. In contrast, when cultured with unmodified sEXOs, there was no significant difference in the red fluorescence accumulation between the two groups. The results showed that CAP modification enhanced the uptake of exosomes by CEPCs, demonstrating that CAP-sEXOs have the property of targeting CEPCs in vitro.

[0105] 2. Exosomes in liquid dispersion systems have a limited residence time at the site of administration and are easily diffused into the circulatory system, resulting in undesirable nonspecific distribution. To evaluate the retention of exosomes in vivo, DiR-labeled CAP-sEXOs, CAP-sEXOs@Gel, and sEXOs@Gel were injected into rat intervertebral discs. In vivo fluorescence imaging was performed using IVIS (Bruker, Billerica, MA, USA) to evaluate the retention of exosomes in the intervertebral disc at 0, 1, 3, and 7 days. The schematic diagram of the experimental process is shown in the figure. Figure 6 The result is shown in D. Figure 6 As shown in Figure E, both groups of gels remained in the IVD for up to seven days after injection. Over time, the radiation signals of all three groups gradually weakened, but the decay rate was slower in the sEXOs@Gel and CAP-sEXOs@Gel groups. Notably, the fluorescent labeling of CAP-sEXO was undetectable after day 3. In addition, the CAP-sEXOs@Gel group showed a higher fluorescence signal than the sEXOs@Gel group. This suggests that both the gel medium and CAP modification help to prolong the retention time of local exosomes. The biophoton imaging optical density data were quantitatively analyzed, and the results are shown in Figure 5. Figure 6 As shown in F, the same conclusion is confirmed.

[0106] The main organs of the rats, heart, liver, spleen, lung and kidney, were dissected and separated 24 hours after injection, and the distribution of DiR fluorescence was detected by biophoton imaging using the IVIS system 24 hours after injection. The results showed that a large number of fluorescent signals appeared in the liver of the rats receiving the CAP-sEXOs group, while no obvious fluorescent signals were observed in the sEXOs@Gel group and the CAP-sEXOs@Gel group ( Figure 6 G). This observation further supports the role of the gel medium in achieving local exosome retention.

[0107] IVD tissue sections were used to observe the in vivo targeting effect of CAP-sEXOs@Gel on CEPCs. Fluorescence microscopy showed that the ability of CEPCs in IVD of the CAP-sEXOs@Gel group to absorb exosomes was enhanced ( Figure 6 Quantitative analysis showed that the CEPC absorption rate of CAP-sEXOs@Gel was significantly improved compared with sEXOs@Gel ( Figure 6 I in.

[0108] Taken together, these results clearly demonstrated the in vivo CEPC targeting ability of CAP-sEXOs@Gel.

[0109] Test Example 3

[0110] CAP-sEXOs@Gel alleviates the inflammatory response of CEPCs by inhibiting iron influx and mtDNA-cGAS-STING signaling pathway

[0111] The following tests were conducted using the CAP-sEXOs@Gel prepared in Example 1, the Gel prepared in Comparative Example 2, and the CAP-EXOs@Gel prepared in Comparative Example 3:

[0112] This experimental example verifies that CAP-sEXOs@Gel downregulates HIF-2α expression through both direct inhibition and indirect mechanisms involving oxidative stress relief. Specifically, CAP-sEXOs@Gel reduces intracellular iron ions, thereby alleviating lipid peroxidation damage caused by the Fenton reaction. This subsequently alleviates ROS- and iron-induced mitochondrial damage, prevents mtDNA from leaking into the cytoplasm, thereby avoiding activation of the cGAS-STING pathway, and ultimately alleviates CEPC inflammation ( Figure 7 A).

[0113] The specific experimental process is: the expression of HIF-2α and TfR1 proteins was detected by immunofluorescence staining, and the process of cell immunofluorescence (IF) staining was: after fixation in 4% paraformaldehyde for 20 minutes, the CEPC cells were permeabilized with 0.5% Triton X-100 for 20 minutes. To prevent nonspecific binding, a 5% bovine serum albumin solution was used before overnight incubation with primary antibodies against HIF-2α and TIR1. The samples were treated with matching secondary antibodies in the dark for 40 minutes and then rinsed three times with PBS. Immunofluorescence staining was observed using a laser scanning confocal microscope (CLSM), and the results were quantified using ImageJ software (National Institutes of Health, Bethesda, USA). The results showed that CAP-sEXOs@Gel significantly reduced their expression in CEPC, while chitosan gel containing CaCO3 alone had no such effect ( Figure 7 (B, C). Although the effect was not as good as CAP-sEXOs@Gel, CAP-EXOs@Gel also reduced the expression of HIF-2α and TfR1 proteins to a certain extent.

[0114] The Fe content of CEPCs after different gel interventions was evaluated. 2+ It was found that CAP-sEXOs@Gel and CAP-EXOs@Gel could significantly reduce the intracellular Fe 2+ ( Figure 7 D and E), among which CAP-sEXOs@Gel has a more obvious effect. A large number of studies have shown that Fe 3+ and Fe 2+ Can be mutually converted in cells, Fe 2+ The Fenton reaction mediated by Fe generates a large number of free radicals, leading to mitochondrial damage and mtDNA leakage. 2+ The concentration changes are consistent with Figure 7 F).

[0115] The active mitochondria in the cells were tracked using the mitochondrial probe MitoTracker. Figure 7 As shown in G and H, CAP-sEXOs@Gel retained the largest number of active mitochondria. In contrast, mtDNA leakage was minimal in CEPCs treated with CAP-sEXOs@Gel ( Figure 7 I, J in .

[0116] Enzyme-linked immunosorbent assay (ELISA) was used to investigate the activation of the cGAS-STING signaling pathway. The concentration of cytoplasmic cyclic GMP-AMP (cGAMP) reflects the enzymatic activity of cGAS ( Figure 7 K in ), while the level of cytoplasmic phosphorylated STING (p-STING) indicated that STING was activated ( Figure 7 The concentration of nuclear phosphorylated interferon regulatory factor 3 (p-IRF3) reflects its transcriptional activity during nuclear translocation ( Figure 7 The above results all indicate that the intervention of CAP-sEXOs@Gel effectively inhibited the activation of cGAS-STING signaling pathway.

[0117] Test Example 4

[0118] CAP-sEXOs@Gel can change the secretory phenotype of CEPCs and indirectly alleviate the aging of NPCs

[0119] The following tests were conducted using the CAP-sEXOs@Gel prepared in Example 1 and the CAP-EXOs@Gel prepared in Comparative Example 3:

[0120] Given that CAP-sEXOs@Gel can alleviate inflammation by reducing iron influx into CEPCs and inhibiting the cGAS-STING pathway, and considering the established effects of inflammation on the cytokine secretion phenotype and the paracrine regulatory effects of CEPCs on NPCs, we used CEPCs from healthy young mice as controls and examined the cytokine profiles in the conditioned medium of CEPCs treated with LPS and co-intervention with CAP-sEXOs@Gel, as in the following procedures: Figure 8 As shown in A in the figure. The results showed that the expression of ossification-inhibiting cytokines (such as SOST and DKK3) in CEPCs treated with LPS was decreased compared with the control group. At the same time, the expression of growth factors with cartilage protective functions, including FGF-2, FGF-18, FGF-23, TGF-β1, TGF-β2 and TGF-β3, and the antioxidant enzyme SOD2 were also downregulated. In contrast, proinflammatory cytokines (TNF-α, IFN-γ, IL-1β, IL6, IL-18) and ECM-degrading cytokines (MMP-2, MMP-3, MMP-13) were upregulated. In contrast, the intervention of CAP-sEXOs@Gel reduced the expression of these cytokines to levels similar to those of the control group ( Figure 8 B), indicating that the secretory phenotype of CEPCs is altered under inflammatory conditions.

[0121] Subsequently, NPCs were treated with conditioned medium from CEPCs treated with LPS and different gels and healthy CEPCs, and then protein immunoblot analysis and quantitative analysis of senescence (p16, p21, p53) and cell stemness (Nanog, OCT-4) markers were performed: cellular proteins were separated using a commercial protein extraction kit (Beyotime). After SDS-PAGE electrophoresis, equal amounts of proteins were deposited on a PVDF membrane with a diameter of 0.22 μm. The membrane was blocked with a 5% skim milk solution to prevent nonspecific binding. The p16, p21, p53, Nanog and OCT-4 specific primary antibodies provided by Abcam were incubated overnight at room temperature. Subsequently, the membrane was treated with secondary antibodies and horseradish peroxidase (HRP) at room temperature for one hour. After the incubation period, the protein bands on the membrane were detected using an improved chemiluminescence detection technique. The results are shown in Figure 2. Figure 8 As shown in CH, the results indicate that conditioned medium from healthy CEPCs inhibited senescence and maintained stemness in NPCs. In contrast, inflammatory CEPCs promoted senescence and impaired stemness. Intervention with CAP-sEXOs@Gel effectively reversed this adverse effect. qRT-PCR analysis of Col2 and Acan gene expression in NPCs: Cells were collected and treated with Trizol reagent to isolate total RNA according to the manufacturer's instructions, using a ThermoScientific NanoDrop-2000 spectrophotometer. Then, the miRNA samples were reverse transcribed using a stem-loop reverse transcriptase primer kit (Ribobio, Guangzhou, China). Three sets of real-time quantitative PCR were performed using the SYBR Prime Script kit (TakaraBio Inc, Shiga, Japan). Comparative Ct(2 -ΔΔCt ) technology was used to determine the relative expression levels of Acan and Col2. Figure 8 As shown in I and J in Figure 1, it also confirms that Figure 8 Therefore, under inflammatory conditions, CAP-sEXOs@Gel can change the secretory phenotype of CEPCs and indirectly alleviate the aging of NPCs.

[0122] Test Example 5

[0123] CAP-sEXOs@Gel neutralizes H + To reduce the inflammatory response of NPC

[0124] CAP-sEXOs@Gel prepared in Example 1, Gel prepared in Comparative Example 2, and Gel prepared in Comparative Example 4 were used. blank Conduct the following test:

[0125] In order to study the role of CaCO3 in CAP-sEXOs@Gel in neutralizing H+ In order to reduce the inflammatory response of NPC, we used lactic acid to simulate the acidic microenvironment in vitro. Fig. 9 As shown in A. To evaluate cell viability, live-death staining assay, CCK-8 assay and Akt kinase activity were performed. The CCK-8 assay was performed as follows: cells were placed in CTRL (PBS), Gel blank , Gel, or CAP-sEXOs@Gel for different time periods. Cell viability was then assessed using a CCK-8 kit (NHC, Suzhou, China) according to the manufacturer's instructions. Results: Live / dead staining showed that lactate treatment affected the control group (CTRL) and the blank gel without CaCO3 (Gel blank ) group, while the CaCO3 gel carrier (Gel) and CAP-sEXOs@Gel promoted the survival of NPCs ( Fig. 9 BE in ), which was confirmed by the subsequent cell proliferation curve ( Fig. 9 C).

[0126] ELISA assays for Akt kinase activity revealed lactate-induced Akt inhibition, which was reversed by Gel and CAP-sEXOs@Gel. Studies on NPC senescence and ECM markers showed that lactate increased the expression of p21 and p27, while reducing the expression of Acan and Col2, which was not conducive to matrix regeneration. In contrast, Gel and CAP-sEXOs@Gel inhibited the expression of p21 and p27 and restored the expression of Acan and Col2 ( Fig. 9 F, G). As Akt phosphorylation promotes Nrf2 nuclear entry and subsequent antioxidant protein transcription, we observed Nrf2 nuclear translocation after Gel and CAP-sEXOs@Gel treatment. Fig. 9 In addition, quantitative analysis of cytoplasmic NLRP3, cleaved Caspase1, and IL-1β showed that Gel and CAP-sEXOs@Gel treatments reduced the production of these pro-inflammatory proteins or cytokines ( Fig. 9 In conclusion, CAP-sEXOs@Gel can neutralize extracellular H + , inhibiting Akt signaling pathway and inhibiting NLRP3 pathway activation to alleviate inflammatory response ( Fig. 9 I in.

[0127] Test Example 6

[0128] Therapeutic effect of CAP-sEXOs@Gel on IVDD rat model induced by CEP degeneration

[0129] EXOs, CAP-sEXOs@Gel prepared in Example 1, sEXOs@Gel prepared in Comparative Example 1, CAP-EXOs@Gel prepared in Comparative Example 3, and CAP-sEXOs@Gel prepared in Comparative Example 5 were used. blank Conduct the following test:

[0130] All animal experiments were approved by the Animal Care and Use Committee of the China Academy of Chinese Medical Sciences (No. WJEC-KT-2021-014-P001). SD rats were randomly divided into: normal control group (Normal), lipopolysaccharide group (Conrtol), sEXOs@Gel group, CAP-sEXOs@Gel group blank group, CAP-EXOs@Gel group, and CAP-sEXOs@Gel group. Anesthesia was induced by intraperitoneal injection of pentobarbital (40 mg / kg body weight). To create an IVDD rat model caused by CEP degeneration, the Co5 / 6 spinal region was found by digital palpation. A 22G lumbar puncture needle was used for the bone drilling process. 20 μL of lipopolysaccharide (LPS, 10 μg / mL) was injected subcutaneously with a microliter syringe. To evaluate the effectiveness of the composite gel, 20 μL of EXOs (100 μg / mL) was injected into the rat vertebral bone marrow. In addition, at weeks 1 and 5, LPS-treated rats were injected with 50 μL of sEXOs@Gel, CAP-sEXOs@Gel blank , CAP-EXOs@Gel and CAP-sEXOs@Gel. The normal control group and the lipopolysaccharide group received an equal amount of saline. After the operation, the animals were returned to the enclosure and allowed to move freely. Fig.10 The specific composition of each group is shown in Fig.10 B in.

[0131] Magnetic resonance imaging and histological staining were used to evaluate the repair effect on CEP and NP tissues. Magnetic resonance imaging (MRI): Magnetic resonance imaging (BioSpec 70 / 20USR, Bruker, Germany) was used to examine the caudal vertebrae of rats to measure signal intensity and structural changes in sagittal T2-weighted images. Histological staining: The intervertebral disc tissue was fixed with 4% (w / v) paraformaldehyde and decalcified in 10% (w / v) formic acid solution for four weeks. After paraffin fixation, the samples were cut into 4.0 μm thick sections using a Thermo Scientific rotary microtome and then mounted on slides with the assistance of Wuhan Pinnuofei Biotechnology Co., Ltd. The sections were then stained using H&E, Safranin O-Fast Green (SO) and Alcian Blue Staining Solution Kit to evaluate the tissue repair effect.

[0132] The results showed that at 4 weeks, LPS injection caused the intervertebral space to narrow significantly and the T2 signal of NP to decrease ( Fig.10 C). Compared with the LPS group, CAP-sEXOs@Gel treatment restored the T2 signal to a large extent. At week 8, the T2 signal of NPs was almost close to that of the normal group ( Fig.10 C, D in Figure 3). Tissue repair effect evaluation showed that the LPS group showed disordered tissue structure, a large reduction in NPCs, and a reduction in ECM compared with the normal control group. The IVDD histological score in the LPS group was also significantly higher ( Fig.10 CEP denaturation was clearly observed by Alcian blue staining, which clearly marked CEP destruction and discontinuous cartilage staining. The LPS group showed severe CEP destruction, while all of these composite gels showed varying degrees of improvement. Due to the lack of CEP targeting, sEXOs@Gel did not show satisfactory effects on endplate cartilage repair compared with other gels containing CAP-sEXOs or CAP-EXOs ( Fig.10 This phenomenon verifies the importance of CEP-targeted modification for CEP repair, which is further emphasized by the increased expression of TfR1 / p-STING in CEP tissues ( Fig.10 (G, H in Figure ). CAP-sEXOs@Gel blank Group showed insufficient NP regeneration ( Fig.10 E). CAP-sEXOs@Gel blank In the NP tissue of the group, although the expression of ECM components (such as Acan) was very low, NLRP3 and IL-1β also remained at a high level close to that of the LPS group ( Fig.10 I, J in Figure 3). Compared with CAP-sEXOs@Gel, CAP-EXOs@Gel also showed poor endplate cartilage repair due to the lack of SAA loading. As expected, intervention with CAP-sEXOs@Gel decreased NLRP3 / IL-1β in NPs and reduced p-STING and TfR1 in CEPs, while increasing the expression of Acan and SOX9. Undoubtedly, CAP-sEXO@Gel mediated the most thorough repair compared with the other composite gel groups.

[0133] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composite hydrogel targeting cartilage endplate, characterized in that: The invention comprises cartilage targeting peptide-modified drug-loaded exosomes, calcium carbonate, chitosan and water, wherein the dosage ratio of the cartilage targeting peptide-modified drug-loaded exosomes, calcium carbonate, chitosan and water is (90-110 μg): (9-11 mg): (90-110 mg): (3-5 mL); the cartilage targeting peptide-modified drug-loaded exosomes comprises cartilage targeting peptide, exosomes and salvianolic acid A.

2. The composite hydrogel according to claim 1, characterized in that The exosomes are exosomes derived from human mesenchymal stem cells.

3. The method for preparing the composite hydrogel according to claim 1 or 2, characterized in that: The steps include: Exosomes and salvianolic acid A were mixed, ultrasonically treated, and then incubated for the first time, followed by centrifugation to obtain drug-loaded exosome particles; The drug-loaded exosome particles are mixed with the cartilage-targeting peptide in water, incubated for a second time, and centrifuged to obtain the cartilage-targeting peptide-modified drug-loaded exosomes; The cartilage targeting peptide-modified drug-loaded exosomes, calcium carbonate, chitosan and water are mixed and gelled to obtain a composite hydrogel targeting the cartilage endplate.

4. The preparation method according to claim 3, characterized in that: The mass ratio of the exosomes to salvianolic acid A is 3-7:

1.

5. The preparation method according to claim 3, characterized in that: The ultrasonic treatment is as follows: ultrasonic amplitude is 15-25%, on 30s / off 30s cycle is performed for 2-4min, repeated 5-7 times, each time interval is 1-3min.

6. The preparation method according to claim 3, characterized in that: The first incubation is at 35-39° C. for 0.8-1.2 h.

7. The preparation method according to claim 3, characterized in that: The mixing ratio of the drug-loaded exosome particles and the cartilage targeting peptide is (0.8×10 12 ~1.2×10 12 )Particles / mL: (8~12)μM.

8. The preparation method according to claim 3, characterized in that: The second incubation is performed at 3-5° C. for 8-14 hours.

9. The preparation method according to claim 3, characterized in that: The gelation includes shaking and standing.

10. Use of the composite gel according to any one of claims 1 to 2 or the preparation method according to any one of claims 3 to 9 in preparing a drug for treating intervertebral disc diseases.

Images