Chordoma cell-derived small extracellular vesicles loaded with cp05 functionalized pva / tspba hydrogel and applications thereof

Abstract

The present application relates to the technical field of medical materials, and provides a notochord cell-derived small extracellular vesicle loaded CP05 functionalized PVA / TSPBA hydrogel and application thereof, which comprises the following steps: mixing a notochord cell-derived small extracellular vesicle suspension and a CP05 functionalized PVA / TSPBA hydrogel precursor solution to obtain a notochord cell-derived small extracellular vesicle loaded CP05 functionalized PVA / TSPBA hydrogel. The present application takes the notochord cell-derived small extracellular vesicle as a core active ingredient, can improve the degenerative phenotype of degenerative nucleus pulposus cells, reduce decomposition metabolism and inflammation-related indexes, promote the expression of extracellular matrix-related proteins, and improve the nucleus pulposus cell homeostasis. By loading the small extracellular vesicle in the CP05 functionalized PVA / TSPBA hydrogel, the anchoring, embedding and retention capacity of the small extracellular vesicle in the intervertebral disc can be further improved, the sustained release can be realized, and the biological activity can be maintained.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, and in particular to a PVA / TSPBA hydrogel functionalized with CP05 loaded with notochord cell-derived small extracellular vesicles and its applications. Background Technology

[0002] Notochord cells play an important role in maintaining intervertebral disc homeostasis, promoting nucleus pulposus cell survival, and regulating extracellular matrix metabolism. However, direct transplantation of notochord cells has problems such as limited cell sources, the degenerated intervertebral disc microenvironment being unfavorable for cell survival, and the difficulty in clinical translation.

[0003] Small extracellular vesicles, as cell-free delivery carriers of active ingredients, can carry active components such as proteins and RNA and regulate recipient cell function, exhibiting good biocompatibility and tissue repair potential. Notochord cell-derived small extracellular vesicles can improve the degenerative phenotype of degenerated nucleus pulposus cells, reduce the expression of catabolism and inflammation-related molecules, promote the expression of extracellular matrix-related proteins, and improve nucleus pulposus cell homeostasis.

[0004] However, simply injecting small extracellular vesicles into the intervertebral disc still has limitations such as short local retention time, easy diffusion or leakage, and insufficient duration of action, which restricts its repair effect. Existing PVA / TSPBA hydrogels can be used as local delivery carriers to achieve sustained release of small extracellular vesicles and are suitable for local injection into the intervertebral disc, but there is still room for further optimization in terms of local anchoring and utilization efficiency of small extracellular vesicles.

[0005] Therefore, it is necessary to develop a composite system that combines the biological repair function of small extracellular vesicles with the local delivery advantages of functionalized hydrogels to improve the retention, sustained release and repair effects of small extracellular vesicles on the intervertebral disc. Summary of the Invention

[0006] The purpose of this invention is to provide a CP05-functionalized PVA / TSPBA hydrogel loaded with notochord cell-derived small extracellular vesicles and its application, to address the problems of insufficient effective regeneration and repair methods for intervertebral disc degeneration and the insufficient retention and short duration of action during the local delivery of small extracellular vesicles. The composite material uses notochord cell-derived small extracellular vesicles as the core active ingredient, achieving intervertebral disc degeneration repair by improving the homeostasis of degenerated nucleus pulposus cells, inhibiting matrix degradation, and promoting matrix synthesis. The CP05-functionalized PVA / TSPBA hydrogel serves as a local delivery carrier, enhancing the anchoring, embedding, retention, and sustained-release capabilities of the small extracellular vesicles in the intervertebral disc, thereby further strengthening its repair effect.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing a PVA / TSPBA hydrogel functionalized with CPO5 loaded with small extracellular vesicles derived from notochord cells, comprising the following steps: (1) After initial cell culture, change the culture medium, continue culturing, and collect the supernatant; (2) After centrifuging the supernatant, filter it to obtain a clear supernatant; (3) The clarified supernatant was separated and purified, and then resuspended to obtain a suspension of small extracellular vesicles derived from notochord cells; (4) Mix PVA with deionized water to obtain a PVA solution; (5) Mix TSPBA with deionized water or buffer solution to obtain TSPBA solution; (6) Mix PVA solution and TSPBA solution to form PVA / TSPBA mixed solution, add CP05 peptide, and mix to obtain CP05 functionalized PVA / TSPBA hydrogel precursor solution; (7) Mix the suspension of small extracellular vesicles derived from notochord cells with the precursor solution to obtain a PVA / TSPBA hydrogel with CP05 loaded from small extracellular vesicles derived from notochord cells.

[0008] Preferably, the cells in step (1) are notochord cells, notochord-like cells, or nucleus pulposus-derived cells with a notochord phenotype; the initial culture is carried out using α-MEM medium containing 8-12% fetal bovine serum and 0.8-1.2% penicillin-streptomycin; the initial culture conditions are 35-39℃ and 4-6% CO2 until the cell confluence is 80%-90%; the replaced medium is α-MEM medium containing 8-12% exosome-free fetal bovine serum, and the continued culture conditions are 35-39℃ and 4-6% CO2 for 44-52 h.

[0009] Preferably, in step (2), the centrifugation is performed by first centrifuging at 280~320×g for 8~12 min, then centrifuging at 1800~2200×g for 18~22 min, and then centrifuging at 9000~11000×g for 25~35 min, with the centrifugation temperature being 2~6℃; and a 0.22 μm filter membrane is used for filtration.

[0010] Preferably, the separation and purification in step (3) is carried out by one or more of the following methods: ultracentrifugation, ultrafiltration concentration, size exclusion chromatography or reagent precipitation. The ultracentrifugation method is as follows: the filtered clear supernatant is ultracentrifuged at 2~6℃ and 90000~110000×g for 70~90 min, the supernatant is discarded, the precipitate is washed with phosphate buffer, and then ultracentrifuged again at 2~6℃ and 90000~110000×g for 70~90 min, and the precipitate is collected. The ultrafiltration concentration method is as follows: the clarified supernatant is concentrated using an ultrafiltration device with a molecular weight cutoff of 100 kDa, and then washed and replaced with phosphate buffer solution to collect the concentrate. The size exclusion chromatography method is as follows: a size exclusion chromatography column is pre-equilibrated with phosphate buffer, the clear supernatant or concentrate is loaded onto the column, eluted with phosphate buffer, and the eluted fraction rich in small extracellular vesicles is collected. The method of the reagent precipitation method is as follows: add the vesicle precipitation reagent to the clear supernatant, mix well, incubate at 2~6℃ for 8~16 h, and then collect the precipitate by centrifugation; The resuspension was performed using phosphate buffer, and the total protein concentration of the small extracellular vesicles in the notochord cell-derived small extracellular vesicle suspension was 0.05~5 mg / mL.

[0011] Preferably, the PVA concentration in the PVA solution in step (4) is 2% to 15% by mass or volume.

[0012] Preferably, the TSPBA concentration in the TSPBA solution in step (5) is 1% to 15% by mass or volume.

[0013] Preferably, the ratio of CP05 peptide, PVA solution and TSPBA solution used in step (6) is 100 μg: 0.9~1.1 mL: 0.9~1.1 mL by mass-volume ratio.

[0014] Preferably, during the mixing process in step (7), the suspension of small extracellular vesicles derived from notochord cells is added to the precursor solution and gently blown or stirred at low speed for 50-70 s at 22-27°C. Then, the mixture is allowed to stand or incubate at 22-27°C for 25-35 min, so that the small extracellular vesicles derived from notochord cells are retained in the PVA / TSPBA hydrogel network through the binding of CP05 peptide, thus obtaining a CP05-functionalized PVA / TSPBA hydrogel containing small extracellular vesicles derived from notochord cells.

[0015] The present invention also provides a PVA / TSPBA hydrogel with CP05-loaded small extracellular vesicles prepared by the above preparation method.

[0016] The present invention also provides the application of the notochord cell-derived small extracellular vesicle-loaded CP05-functionalized PVA / TSPBA hydrogel in the preparation of a drug for treating intervertebral disc degeneration.

[0017] Beneficial effects: This invention uses notochord cell-derived microvesicles as the core active ingredient, which can improve the degenerative phenotype of degenerated nucleus pulposus cells, reduce catabolism and inflammation-related indicators, promote the expression of extracellular matrix-related proteins, and improve nucleus pulposus cell homeostasis. By loading the microvesicles into CP05-functionalized PVA / TSPBA hydrogel, the anchoring, embedding, and retention capabilities of the microvesicles in the intervertebral disc can be further improved, achieving sustained release and maintaining their biological activity. Compared with free microvesicles, the microvesicle-loaded functionalized hydrogel composite system can better improve local delivery efficiency, which is more conducive to maintaining intervertebral disc height, reducing radiographic degeneration, and improving histological structure. Furthermore, the functionalized PVA / TSPBA hydrogel exhibits responsive release characteristics in the oxidative stress microenvironment of degenerated intervertebral discs, thereby further enhancing the local retention, sustained-release delivery, and intervertebral disc degeneration repair effects of the microvesicles.

[0018] The notochord cell-derived small extracellular vesicle loaded with CP05-functionalized PVA / TSPBA hydrogel includes a CP05-functionalized PVA / TSPBA hydrogel matrix and notochord cell-derived small extracellular vesicles loaded in the hydrogel matrix. The CP05 peptide is used to enhance the binding effect between the notochord cell-derived small extracellular vesicles and the PVA / TSPBA hydrogel system, and improve the loading stability, local retention capacity and sustained release capacity of the notochord cell-derived small extracellular vesicles in the hydrogel system. The PVA / TSPBA hydrogel is used to form an injectable three-dimensional network structure locally in the intervertebral disc to reduce diffusion, leakage or rapid clearance of notochord cell-derived small extracellular vesicles after direct injection. Attached Figure Description

[0019] Figure 1 The figures shown are characterization results of small extracellular vesicles derived from notochord cells in this embodiment of the invention; where A is the flow cytometry detection result of notochord-like cell-related markers, B is the transmission electron microscopy image of small extracellular vesicles derived from notochord cells, C is the particle size distribution detection result of small extracellular vesicles, D is the detection result of small extracellular vesicle marker proteins CD9, CD63, CD81 and negative marker protein Calnexin, and E is the result of fluorescently labeled small extracellular vesicles being taken up by nucleus pulposus cells.

[0020] Figure 2Figures showing the characterization and biocompatibility testing results of PVA / TSPBA hydrogel materials in this embodiment of the invention; wherein, A is an appearance image of PVA solution, TSPBA solution and PVA / TSPBA hydrogel, B is an image of the detection results of nucleus pulposus cell activity at different treatment times, and C is an image of the live / dead staining results of nucleus pulposus cells after treatment in the NC-exo group and the Gel-exo group.

[0021] Figure 3 Figure 1 shows the release performance and post-release bioactivity detection results of PVA / TSPBA hydrogel loaded with small extracellular vesicles derived from notochord cells in this embodiment of the invention; wherein, A is the in vitro cumulative release curve of small extracellular vesicles, B is the protein detection result of the effect of released small extracellular vesicles on the expression of COL2A1 and MMP13, and C is... Figure 3 The results of quantitative analysis of B in the figure.

[0022] Figure 4 This is a graph showing the detection results of the effects of notochord cell-derived small extracellular vesicles on the expression of degeneration-related proteins, inflammation-related proteins, and extracellular matrix-related proteins in degenerated nucleus pulposus cells in an embodiment of the present invention; wherein, A is the protein detection results of ADAMTS5, IL-6, P53, and MMP9, and B is... Figure 4 The graph shows the quantitative analysis results of A, C shows the protein detection results of ADAMTS5, COL2A1, and MMP9, and D shows... Figure 4 The results of quantitative analysis of C in the figure.

[0023] Figure 5 The images shown are immunofluorescence detection results of small extracellular vesicles derived from notochord cells on degenerated nucleus pulposus cells in an embodiment of the present invention; wherein, A is the immunofluorescence detection result of ADAMTS5, B is the immunofluorescence detection result of MMP9, C is the immunofluorescence detection result of MMP13, D is the immunofluorescence detection result of COL2A1, and E is the DCFH-DA fluorescence detection result of reactive oxygen species level.

[0024] Figure 6 As described in the embodiments of the present invention Figure 5 Quantitative analysis graphs of relevant immunofluorescence and reactive oxygen species detection results; where A is the quantitative analysis result graph of the fluorescence intensity of ADAMTS5, MMP9, MMP13 and COL2A1, and B is the quantitative analysis result graph of the fluorescence intensity of DCFH-DA.

[0025] Figure 7The diagram shows the detection results of the activation of the Hedgehog signaling pathway by small extracellular vesicles derived from notochord cells in an embodiment of the present invention. Among them, A is a heatmap of the differential proteome analysis between small extracellular vesicles derived from notochord cells and small extracellular vesicles derived from nucleus pulposus cells; B is a volcano diagram of the differential proteome analysis between small extracellular vesicles derived from notochord cells and small extracellular vesicles derived from nucleus pulposus cells; C is a heatmap of the changes in the expression of genes related to the Hedgehog signaling pathway after Shh-related treatment; and D is a diagram showing the detection results of the expression of proteins related to the Hedgehog signaling pathway and degeneration-related proteins after Shh-related treatment.

[0026] Figure 8 The images shown are immunofluorescence detection results of proteins related to the Hedgehog signaling pathway activated by small extracellular vesicles derived from notochord cells in this embodiment of the invention; wherein, A is the immunofluorescence detection result of Smo, B is the immunofluorescence detection result of Gli1, C is the immunofluorescence detection result of PTCH1, and D is the immunofluorescence detection result of SUFU.

[0027] Figure 9 As described in the embodiments of the present invention Figure 7 and Figure 8 Quantitative analysis of Hedgehog signaling pathway protein expression and immunofluorescence detection results; where A is the quantitative analysis result of Smo, Gli1, PTCH1, MMP9, MMP13 and SUFU protein expression, and B is the quantitative analysis result of Smo, Gli1, PTCH1 and SUFU immunofluorescence intensity.

[0028] Figure 10 This is a diagram illustrating the verification results of the mechanism by which circDHX8 regulates the Hedgehog signaling pathway and SUFU expression in this embodiment of the invention; wherein, A is a schematic diagram of the origin and structure of circDHX8, B is a sequencing result of the circDHX8 backsplicing site, C is a verification result of the circDHX8 circular structure, D is a detection result of the expression of Hedgehog signaling pathway-related proteins and degeneration-related proteins after SUFU overexpression or silencing, and E is... Figure 10 The quantitative analysis results of D are shown in Figure F, which shows the detection results of Hedgehog signaling pathway-related proteins and SUFU expression after circDHX8 overexpression or silencing, and Figure G shows the results of quantitative analysis of D. Figure 10 The results of quantitative analysis of F in the middle are shown in the figure.

[0029] Figure 11 This is a diagram showing the verification results of the circDHX8 / LNX1 / SUFU axis regulating SUFU protein stability and ubiquitination degradation in the embodiments of the present invention. In this diagram, A represents the detection results of Hedgehog signaling pathway-related proteins and SUFU expression after circDHX8 overexpression or silencing, and B represents... Figure 11Figure A shows the quantitative analysis results; Figure C shows the SUFU protein stability detection results after treatment with small extracellular vesicles derived from notochord cells; Figure D shows the SUFU ubiquitination detection results after treatment with small extracellular vesicles derived from notochord cells; Figure E shows the detection results of the effect of different candidate E3 ubiquitin ligases on SUFU ubiquitination; Figure F shows the detection results of the binding relationship between circDHX8 and candidate E3 ubiquitin ligases; Figure G shows the detection results of circDHX8 promoting the interaction between LNX1 and SUFU; and Figure H shows the detection results of LNX1-mediated SUFU ubiquitination degradation.

[0030] Figure 12 The images show the imaging results of the in vivo repair effect, where A is the X-ray detection result, B is the magnetic resonance imaging result, C is the micro-CT detection result, D is the intervertebral disc height index analysis result, and E is the Pfirrmann grading analysis result.

[0031] Figure 13 The images show the histological repair effect of hydrogel loaded with small extracellular vesicles derived from notochord cells on intervertebral disc degeneration in an embodiment of the present invention; wherein, A is the result of hematoxylin-eosin staining, B is the result of safranin O / fast green staining, and C is the result of intervertebral disc histological scoring. Detailed Implementation

[0032] This invention provides a method for preparing a PVA / TSPBA hydrogel functionalized with CPO5 loaded with small extracellular vesicles derived from notochord cells, comprising the following steps: (1) After initial cell culture, change the culture medium, continue culturing, and collect the supernatant; (2) After centrifuging the supernatant, filter it to obtain a clear supernatant; (3) The clarified supernatant was separated and purified, and then resuspended to obtain a suspension of small extracellular vesicles derived from notochord cells; (4) Mix PVA with deionized water to obtain a PVA solution; (5) Mix TSPBA with deionized water or buffer solution to obtain TSPBA solution; (6) Mix PVA solution and TSPBA solution to form PVA / TSPBA mixed solution, add CP05 peptide, and mix to obtain CP05 functionalized PVA / TSPBA hydrogel precursor solution; (7) Mix the suspension of small extracellular vesicles derived from notochord cells with the precursor solution to obtain a PVA / TSPBA hydrogel with CP05 loaded from small extracellular vesicles derived from notochord cells.

[0033] In this invention, the cells mentioned in step (1) are notochord cells, notochord-like cells, or nucleus pulposus-derived cells with a notochord phenotype; the initial culture is preferably carried out using α-MEM medium containing 8-12% fetal bovine serum and 0.8-1.2% penicillin-streptomycin, and more preferably α-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin; the initial culture conditions are preferably cultured at 35-39°C and 4-6% CO2 until the cell confluence is 80%-90%, and more preferably cultured at 37°C and 5% CO2 until the cell confluence is 80%-90%; the replaced medium is α-MEM medium containing 8-12% exosome-free fetal bovine serum, and more preferably 10%; the continued culture conditions are preferably cultured at 35-39°C and 4-6% CO2 for 44-52 h, and more preferably cultured at 37°C and 5% CO2 for 48 h.

[0034] In this invention, the centrifugation in step (2) is preferably performed by first centrifuging at 280~320×g for 8~12 min, then centrifuging at 1800~2200×g for 18~22 min, followed by centrifugation at 9000~11000×g for 25~35 min, and more preferably by first centrifuging at 300×g for 10 min, then centrifuging at 2000×g for 20 min, followed by centrifugation at 10000×g for 30 min; the centrifugation temperature is preferably 2~6℃, and more preferably 4℃; the filtration is preferably performed using a 0.22 μm filter membrane.

[0035] In this invention, the separation and purification in step (3) is carried out by one or more of the following methods: ultracentrifugation, ultrafiltration concentration, size exclusion chromatography or reagent precipitation. The preferred method of ultracentrifugation is as follows: the filtered clear supernatant is ultracentrifuged at 2-6℃ and 90,000-110,000×g for 70-90 min, the supernatant is discarded, the precipitate is washed with phosphate buffer, and then ultracentrifuged again at 2-6℃ and 90,000-110,000×g for 70-90 min, and the precipitate is collected. More preferably, the filtered clear supernatant is ultracentrifuged at 4℃ and 100,000×g for 80 min, the supernatant is discarded, the precipitate is washed with phosphate buffer, and then ultracentrifuged again at 4℃ and 100,000×g for 80 min, and the precipitate is collected. The ultrafiltration concentration method is as follows: the clarified supernatant is concentrated using an ultrafiltration device with a molecular weight cutoff of 100 kDa, and then washed and replaced with phosphate buffer solution to collect the concentrate. The size exclusion chromatography method is as follows: a size exclusion chromatography column is pre-equilibrated with phosphate buffer, the clear supernatant or concentrate is loaded onto the column, eluted with phosphate buffer, and the eluted fraction rich in small extracellular vesicles is collected. The method of reagent precipitation is as follows: add the vesicle precipitation reagent to the clear supernatant, mix well, incubate at 2-6℃ for 8-16 h, and then collect the precipitate by centrifugation, preferably incubating at 4℃ for 12 h. The resuspension is performed using phosphate buffer, and the total protein concentration of the small extracellular vesicles in the notochord cell-derived small extracellular vesicle suspension is preferably 0.05~5 mg / mL, more preferably 0.1~2 mg / mL.

[0036] In this invention, the mass-volume concentration of PVA in the PVA solution in step (4) is preferably 2% to 15%, more preferably 4% to 8%, and even more preferably 6%.

[0037] In this invention, the TSPBA mass-volume concentration in the TSPBA solution in step (5) is 1% to 15%, more preferably 4% to 8%, and even more preferably 6%.

[0038] In this invention, the ratio of the amount of CP05 peptide, PVA solution and TSPBA solution in step (6) is preferably 100 μg: 0.9~1.1 mL: 0.9~1.1 mL by mass-volume ratio, and more preferably 100 μg: 1 mL: 1 mL.

[0039] In this invention, during the mixing process described in step (7), it is preferable to add the suspension of small extracellular vesicles derived from notochord cells to the precursor solution, gently blow or stir at low speed for 50-70 s at 22-27°C, and then let stand or incubate at 22-27°C for 25-35 min, so that the small extracellular vesicles derived from notochord cells are retained in the PVA / TSPBA hydrogel network through the binding and mediation of CP05 peptide, thereby obtaining a CP05-functionalized PVA / TSPBA hydrogel of small extracellular vesicles derived from notochord cells. More preferably, the mixture is gently blown or stirred at low speed for 60 s at 25°C, and then let stand or incubate at 25°C for 30 min.

[0040] The present invention also provides a PVA / TSPBA hydrogel with CP05-loaded small extracellular vesicles prepared by the above preparation method.

[0041] The present invention also provides the application of the notochord cell-derived small extracellular vesicle-loaded CP05-functionalized PVA / TSPBA hydrogel in the preparation of a drug for treating intervertebral disc degeneration.

[0042] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1: Preparation of PVA / TSPBA hydrogels functionalized with CP05 loaded from small extracellular vesicles derived from notochord cells

[0044] (1) Preparation of small extracellular vesicles derived from notochord cells

[0045] Notochord cells, notochord-like cells, or nucleus pulposus-derived cells with a notochord phenotype were cultured. Once the cells reached a suitable degree of confluence, the culture medium was replaced with serum containing de-vesicle serum, and the cell culture supernatant was collected. The supernatant was then subjected to low-speed centrifugation to remove intact cells, followed by further centrifugation to remove cell debris and larger particulate impurities. The supernatant was then filtered through a 0.22 μm filter to obtain a clear supernatant. Subsequently, the clear supernatant was purified using one or more of the following methods: ultracentrifugation, ultrafiltration concentration, size exclusion chromatography, or reagent precipitation. The precipitate or eluted fraction was collected and resuspended in phosphate buffer to obtain small extracellular vesicles derived from notochord cells.

[0046] The obtained notochordine-derived small extracellular vesicles were characterized. Transmission electron microscopy was used to observe their morphology, nanoparticle tracking analysis was used to detect their particle size distribution and concentration, and Western blotting was used to detect small extracellular vesicle marker proteins such as CD9, CD63, and CD81. Calnexin was used as a negative control. The results showed that the obtained vesicles exhibited a typical membranous vesicle-like structure, with a particle size distribution consistent with the characteristics of small extracellular vesicles, and expressed marker proteins such as CD9, CD63, and CD81, indicating that notochordine-derived small extracellular vesicles were successfully obtained.

[0047] Furthermore, the notochord cell-derived small extracellular vesicles can be labeled with fluorescent dyes such as PKH26, DiI, or DiR, and then co-incubated with degenerated nucleus pulposus cells. Observations using fluorescence microscopy or laser confocal microscopy show that the notochord cell-derived small extracellular vesicles can be taken up by nucleus pulposus cells, indicating their ability to deliver active ingredients to nucleus pulposus cells.

[0048] (2) Preparation of CP05 functionalized PVA / TSPBA hydrogel precursor solution

[0049] Preparation of PVA and TSPBA solutions: Add PVA to deionized water and stir under heating until completely dissolved to obtain a PVA solution with a mass-volume concentration of 6%; add TSPBA to deionized water or buffer solution and stir until completely dissolved to obtain a TSPBA solution with a mass-volume concentration of 6%.

[0050] The CP05 peptide was added to a PVA solution, a TSPBA solution, or a PVA / TSPBA mixture and thoroughly mixed to introduce the CP05 peptide into the PVA / TSPBA hydrogel system, resulting in a CP05-functionalized PVA / TSPBA hydrogel precursor solution. The CP05 peptide can bind to related proteins on the surface of small extracellular vesicles, thus serving as a binding medium between notochord-derived small extracellular vesicles and the PVA / TSPBA hydrogel, thereby improving the anchoring, embedding, and retention capabilities of small extracellular vesicles in the hydrogel system.

[0051] (3) Preparation of PVA / TSPBA hydrogels with CP05 loaded on small extracellular vesicles

[0052] The notochordinal cell-derived small extracellular vesicles obtained in step (1) are added to the CP05-functionalized PVA / TSPBA hydrogel precursor solution obtained in step (2), and gently mixed to avoid vigorous shaking that could damage the structure of the small extracellular vesicles. After mixing, PVA and TSPBA form a three-dimensional hydrogel network, while the notochordinal cell-derived small extracellular vesicles are anchored, embedded, or retained in the hydrogel network through the binding of the CP05 peptide, thus obtaining a notochordinal cell-derived small extracellular vesicle-loaded CP05-functionalized PVA / TSPBA hydrogel.

[0053] The resulting composite hydrogel comprises a CP05-functionalized PVA / TSPBA hydrogel matrix and notochord cell-derived small extracellular vesicles loaded within the hydrogel matrix. The PVA / TSPBA hydrogel matrix is ​​used to form an injectable three-dimensional network structure, and the CP05 peptide enhances the binding effect between the notochord cell-derived small extracellular vesicles and the hydrogel system. The notochord cell-derived small extracellular vesicles, as the core active ingredient, improve the homeostasis of degenerated nucleus pulposus cells, inhibit matrix degradation, and promote extracellular matrix synthesis.

[0054] Example 2 Characterization and release performance verification of PVA / TSPBA hydrogels loaded with CP05 from notochord cell-derived small extracellular vesicles

[0055] The appearance, structure and release performance of the CP05-functionalized PVA / TSPBA hydrogel loaded with CP05 from the small extracellular vesicles of notochord cells prepared in Example 1 were tested.

[0056] (1) Observe the appearance of PVA solution, TSPBA solution and CP05 functionalized PVA / TSPBA hydrogel. The results show that the PVA solution and TSPBA solution can form a uniform hydrogel system after mixing, indicating that the PVA / TSPBA system can form a gel structure suitable for local injection and local filling.

[0057] (2) Fourier transform infrared spectroscopy was used to detect PVA, TSPBA and CP05-functionalized PVA / TSPBA hydrogels to verify the introduction and interaction of each component in the hydrogel system. The results showed that the CP05-functionalized PVA / TSPBA hydrogel had characteristic absorption peaks related to PVA, TSPBA and CP05, indicating that the CP05 peptide was introduced into the PVA / TSPBA hydrogel system and the resulting hydrogel had the expected composite structure.

[0058] (3) Verification of the release performance of small extracellular vesicles. Small extracellular vesicles derived from notochord cells loaded with CP05-functionalized PVA / TSPBA hydrogels were placed in phosphate buffer and incubated at 37°C. The release solution was collected at predetermined time points, and an equal volume of fresh buffer was added. The content of small extracellular vesicles in the release solution was determined using the BCA protein quantification method, nanoparticle tracking analysis, or extracellular vesicle marker protein detection method. The cumulative release amount was calculated, and a release curve was plotted.

[0059] The results showed that, compared with free notochord cell-derived small extracellular vesicles, the CP05-loaded PVA / TSPBA hydrogel of the notochord cell-derived small extracellular vesicles enabled the gradual release of small extracellular vesicles, reducing the occurrence of rapid diffusion or release in a short period of time, indicating that the composite hydrogel system has the ability to continuously release small extracellular vesicles.

[0060] (4) A control group consisting of PVA / TSPBA hydrogels without CP05 peptide was set up. Compared with the hydrogel group without CP05 peptide, the CP05-functionalized PVA / TSPBA hydrogel group showed stronger retention of small extracellular vesicles and a more stable release process, indicating that CP05 peptide helps to enhance the binding between small extracellular vesicles and the hydrogel system, and improves the loading stability and retention capacity of small extracellular vesicles in the hydrogel.

[0061] Example 3: In vitro efficacy verification of CP05-functionalized PVA / TSPBA hydrogel loaded with CP05 from notochord cell-derived small extracellular vesicles

[0062] Degenerated nucleus pulposus cells were cultured in vitro, and three groups were established: a blank hydrogel group, a group of free notochord cell-derived small extracellular vesicles, and a group of notochord cell-derived small extracellular vesicles loaded with CP05-functionalized PVA / TSPBA hydrogel. After treatment, cell viability, extracellular matrix metabolism, inflammatory response, matrix degradation, and oxidative stress levels of nucleus pulposus cells were measured.

[0063] Cell viability and proliferation were assessed using the CCK-8 assay. Results showed that the CP05-functionalized PVA / TSPBA hydrogel and its composite system loaded with small extracellular vesicles exhibited no significant cytotoxicity to nucleus pulposus cells and demonstrated good cell compatibility.

[0064] Real-time quantitative PCR was used to detect extracellular matrix synthesis-related indicators and matrix degradation-related indicators. Extracellular matrix synthesis-related indicators included type II collagen and Aggrecan; matrix degradation-related indicators included MMP9, MMP13, and ADAMTS5; and inflammation or degeneration-related indicators included IL-6 and TP53.

[0065] The results showed that, compared with the model control group, small extracellular vesicles derived from free notochord cells could reduce the expression of matrix degradation, inflammation and degeneration-related indicators in degenerated nucleus pulposus cells and promote the expression of extracellular matrix-related proteins. Compared with the small extracellular vesicles derived from free notochord cells, the small extracellular vesicles derived from notochord cells loaded with CP05-functionalized PVA / TSPBA hydrogel could more stably improve the phenotype of degenerated nucleus pulposus cells, reduce catabolism-related indicators and promote extracellular matrix synthesis by continuously releasing small extracellular vesicles.

[0066] The above results demonstrate that the CP05-loaded PVA / TSPBA hydrogel for small extracellular vesicles derived from notochord cells prepared in this invention can maintain the biological activity of small extracellular vesicles and improve the homeostasis of degenerated nucleus pulposus cells through continuous delivery.

[0067] Example 4: In vivo efficacy verification of CP05-functionalized PVA / TSPBA hydrogel loaded with notochord cell-derived small extracellular vesicles

[0068] An animal model of intervertebral disc degeneration was established to verify the in vivo repair effect of the composite hydrogel system of this invention. Experimental animals were anesthetized, and the caudal intervertebral disc was exposed. Disc degeneration was induced by puncture. After modeling, the animals were randomly divided into a normal control group, a degeneration model group, a blank hydrogel group, a group of free notochord cell-derived small extracellular vesicles, and a group of notochord cell-derived small extracellular vesicles loaded with CP05-functionalized PVA / TSPBA hydrogel.

[0069] Local injections were performed on the intervertebral discs in different treatment groups. The normal control group did not undergo degeneration modeling treatment; the degeneration model group was injected with phosphate buffer; the blank hydrogel group was injected with CP05-functionalized PVA / TSPBA hydrogel without small extracellular vesicles; the free notochord cell-derived small extracellular vesicle group was injected with free small extracellular vesicles; and the notochord cell-derived small extracellular vesicle-loaded CP05-functionalized PVA / TSPBA hydrogel group was injected with the composite hydrogel prepared in Example 1.

[0070] Following treatment, imaging and histological evaluations were performed at predetermined time points. Imaging evaluations included X-ray examination, magnetic resonance imaging (MRI), and micro-CT. The intervertebral disc height index was calculated using X-ray examination; changes in T2 signal intensity of the intervertebral disc were observed and Pfirrmann classification was performed using MRI; and changes in the intervertebral disc and adjacent structures were observed using micro-CT.

[0071] The results showed that the intervertebral disc height decreased, the magnetic resonance signal weakened, and the Pfirrmann grade increased in the degeneration model group, indicating significant intervertebral disc degeneration. The free notochord cell-derived small extracellular vesicle group could alleviate the above-mentioned degeneration manifestations to some extent. The notochord cell-derived small extracellular vesicle group loaded with CP05-functionalized PVA / TSPBA hydrogel group was able to maintain intervertebral disc height better, reduce the degree of radiographic degeneration, and improve intervertebral disc structure than the free small extracellular vesicle group.

[0072] Subsequently, intervertebral disc tissue was taken, fixed, decalcified, embedded, and sectioned, and then stained with hematoxylin-eosin and safranin O / fast green. Histological observation results showed that the nucleus pulposus tissue structure in the degeneration model group was significantly damaged, the annulus fibrosus was disordered, and the boundary between the nucleus pulposus and the annulus fibrosus was unclear. The free notochord cell-derived small extracellular vesicle group could partially improve the intervertebral disc tissue structure. In the group of notochord cell-derived small extracellular vesicles loaded with CP05-functionalized PVA / TSPBA hydrogel, the nucleus pulposus structure was better preserved, the annulus fibrosus was relatively intact, the boundary between the nucleus pulposus and the annulus fibrosus was clearer, and the degree of histological degeneration was reduced.

[0073] The above results demonstrate that the CP05-loaded PVA / TSPBA hydrogel of notochord cell-derived small extracellular vesicles provided by this invention can improve the retention and continuous release capacity of notochord cell-derived small extracellular vesicles in the intervertebral disc, thereby enhancing their effects on improving nucleus pulposus cell homeostasis, inhibiting matrix degradation, promoting extracellular matrix synthesis, and repairing intervertebral disc degeneration.

[0074] As can be seen from the above embodiments, the present invention provides a CP05-functionalized PVA / TSPBA hydrogel loaded with notochord cell-derived small extracellular vesicles and its application. The present invention uses notochord cell-derived small extracellular vesicles as the core active ingredient, which can improve the degenerative phenotype of degenerated nucleus pulposus cells, reduce catabolism and inflammation-related indicators, promote the expression of extracellular matrix-related proteins, and improve nucleus pulposus cell homeostasis. By loading the small extracellular vesicles into the CP05-functionalized PVA / TSPBA hydrogel, the anchoring, embedding, and retention capabilities of the small extracellular vesicles in the intervertebral disc can be further improved, achieving continuous release and maintaining their biological activity.

[0075] Example 5: Functional verification of the regulation of the Hedgehog signaling pathway by small extracellular vesicles derived from notochord cells.

[0076] To verify the mechanism by which notochord cell-derived small extracellular vesicles improve the homeostasis of degenerated nucleus pulposus cells, this embodiment verifies the function of notochord cell-derived small extracellular vesicles in regulating the Hedgehog signaling pathway.

[0077] (1) Proteomic analysis was performed on small extracellular vesicles derived from notochord cells and small extracellular vesicles derived from nucleus pulposus cells. The results showed that there were differences in the protein composition of small extracellular vesicles derived from notochord cells and small extracellular vesicles derived from nucleus pulposus cells, with Shh being enriched in small extracellular vesicles derived from notochord cells. Further treatment of degenerated nucleus pulposus cells with small extracellular vesicles derived from notochord cells, Shh overexpression, Shh silencing, and recombinant Shh protein were performed, and the expression changes of Hedgehog signaling pathway-related proteins and degeneration-related proteins were detected. The results showed that treatment with small extracellular vesicles derived from notochord cells, Shh overexpression, or recombinant Shh protein treatment could increase the expression of Smo, Gli1, and PTCH1, and decrease the expression of MMP9 and MMP13; while after Shh silencing, the activation of the above Hedgehog signaling pathway was weakened. Immunofluorescence detection results also showed that after treatment with small extracellular vesicles derived from notochord cells, the expression of Smo, Gli1, and PTCH1 in degenerated nucleus pulposus cells was enhanced. The above results indicate that small extracellular vesicles derived from notochord cells can activate the Hedgehog signaling pathway in degenerated nucleus pulposus cells through their enriched Shh components and inhibit the expression of degeneration-related catabolic markers.

[0078] (2) Analysis of circRNAs in small extracellular vesicles derived from notochord cells and small extracellular vesicles derived from nucleus pulposus cells. The results showed that there were differences in the circRNA expression profiles between the two groups. After screening and verification of candidate circRNAs enriched in small extracellular vesicles derived from notochord cells, it was found that circDHX8 had a more significant regulatory effect on molecules related to the Hedgehog signaling pathway. Further verification of the circular structure of circDHX8 was performed by Sanger sequencing and amplification using divergent and convergent primers. The results showed that circDHX8 has a backsplicing site and can be amplified from a cDNA template, indicating that circDHX8 is a circular RNA.

[0079] (3) The effects of circDHX8 on the Hedgehog signaling pathway and SUFU were examined. The results showed that overexpression of circDHX8 increased the expression of Smo, Gli1, and PTCH1, while decreasing SUFU expression; silencing of circDHX8 decreased the expression of Smo, Gli1, and PTCH1, while increasing SUFU expression. Simultaneously, SUFU overexpression decreased the expression of Smo, Gli1, and PTCH1, and increased the expression of MMP9 and MMP13; SUFU silencing increased the expression of Smo, Gli1, and PTCH1, and decreased the expression of MMP9 and MMP13. These results indicate that SUFU has an inhibitory effect on the Hedgehog signaling pathway, while circDHX8 in small extracellular vesicles derived from notochord cells can promote Hedgehog signaling pathway activation by reducing SUFU protein levels.

[0080] (4) The stability and ubiquitination level of SUFU protein were detected. The results showed that the rate of SUFU protein degradation accelerated after treatment with small extracellular vesicles derived from notochord cells; in the presence of MG132, treatment with small extracellular vesicles derived from notochord cells increased the ubiquitination level of SUFU. The above results indicate that small extracellular vesicles derived from notochord cells can promote the ubiquitination and degradation of SUFU, thereby weakening the inhibitory effect of SUFU on the Hedgehog signaling pathway.

[0081] (5) RNA pull-down combined with mass spectrometry was used to screen for proteins associated with circDHX8, and candidate E3 ubiquitin ligases were analyzed to obtain candidate molecules such as NEDD4, RNF4, LNX1, MDM2, and FBXW7. SUFU ubiquitination detection of these candidate molecules revealed that LNX1 had the most significant promoting effect on SUFU ubiquitination. RNA immunoprecipitation results showed that circDHX8 was enriched in the LNX1 immunoprecipitation complex, suggesting an association between circDHX8 and LNX1. Further investigation of the effect of LNX1 on SUFU protein levels showed that LNX1 silencing increased SUFU protein levels, while LNX1 overexpression decreased SUFU protein levels.

[0082] (6) The relationship between LNX1 and SUFU was detected by immunoprecipitation assay. The results showed that circDHX8 could enhance the interaction between LNX1 and SUFU. The ubiquitination assay showed that LNX1 overexpression could enhance SUFU ubiquitination, while LNX1 silencing could reduce SUFU ubiquitination, indicating that LNX1 is involved in mediating the ubiquitination and degradation of SUFU.

[0083] (7) Verification of combined intervention in degenerated nucleus pulposus cells. The results showed that treatment with notochord cell-derived small extracellular vesicles, Shh stimulation, and circDHX8 overexpression all increased the expression of Smo, Gli1, and PTCH1; SUFU overexpression weakened the activation of the above-mentioned Hedgehog signaling pathway, while SUFU silencing showed the opposite effect. The above results indicate that notochord cell-derived small extracellular vesicles can jointly promote the activation of the Hedgehog signaling pathway through Shh-mediated upstream activation and the circDHX8 / LNX1 / SUFU axis-mediated SUFU inhibition relief, thereby improving the homeostasis of degenerated nucleus pulposus cells and reducing the level of degeneration-related catabolism.

[0084] As can be seen from the above embodiments, the present invention provides a PVA / TSPBA hydrogel with CP05 loaded from small extracellular vesicles derived from notochord cells and its application, including the following steps: (1) after preliminary culture of cells, the culture medium is replaced, and after further culture, the supernatant is collected; (2) the supernatant is centrifuged and filtered to obtain a clear supernatant; (3) the clear supernatant is separated and purified, and resuspended to obtain a suspension of small extracellular vesicles derived from notochord cells; (4) PVA is mixed with deionized water to obtain a PVA solution; (5) TSPBA is mixed with deionized water or buffer to obtain a TSPBA solution; (6) after mixing the PVA solution and TSPBA solution to form a PVA / TSPBA mixed solution, CP05 peptide is added and mixed thoroughly to obtain a CP05-functionalized PVA / TSPBA hydrogel precursor solution; (7) the suspension of small extracellular vesicles derived from notochord cells and the hydrogel precursor solution are mixed to obtain a PVA / TSPBA hydrogel with CP05 loaded from small extracellular vesicles derived from notochord cells. This invention uses small extracellular vesicles derived from notochord cells as the core active ingredient, which can improve the degenerative phenotype of degenerated nucleus pulposus cells, reduce catabolism and inflammation-related indicators, promote the expression of extracellular matrix-related proteins, and improve nucleus pulposus cell homeostasis. By loading the small extracellular vesicles into CP05-functionalized PVA / TSPBA hydrogel, the anchoring, embedding, and retention capabilities of the small extracellular vesicles in the intervertebral disc can be further improved, achieving continuous release and maintaining their biological activity.

[0085] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a PVA / TSPBA hydrogel functionalized with CP05 loaded with small extracellular vesicles derived from notochord cells, characterized in that, Includes the following steps: (1) After initial cell culture, change the culture medium, continue culturing, and collect the supernatant; (2) After centrifuging the supernatant, filter it to obtain a clear supernatant; (3) The clarified supernatant was separated and purified, and then resuspended to obtain a suspension of small extracellular vesicles derived from notochord cells; (4) Mix PVA with deionized water to obtain a PVA solution; (5) Mix TSPBA with deionized water or buffer solution to obtain TSPBA solution; (6) Mix PVA solution and TSPBA solution to form PVA / TSPBA mixed solution, add CP05 peptide, and mix to obtain CP05 functionalized PVA / TSPBA hydrogel precursor solution; (7) Mix the suspension of small extracellular vesicles derived from notochord cells with the precursor solution to obtain a PVA / TSPBA hydrogel with CP05 loaded from small extracellular vesicles derived from notochord cells.

2. The preparation method according to claim 1, characterized in that, The cells mentioned in step (1) are notochord cells, notochord-like cells, or nucleus pulposus-derived cells with a notochord phenotype; the initial culture is carried out using α-MEM medium containing 8-12% fetal bovine serum and 0.8-1.2% penicillin-streptomycin; the initial culture conditions are 35-39℃ and 4-6% CO2 until the cell confluence reaches 80%-90%; the replaced medium is α-MEM medium containing 8-12% exosome-free fetal bovine serum, and the continued culture conditions are 35-39℃ and 4-6% CO2 for 44-52 h.

3. The preparation method according to claim 1, characterized in that, In step (2), the centrifugation is first performed at 280~320×g for 8~12 min, then at 1800~2200×g for 18~22 min, and then at 9000~11000×g for 25~35 min. The centrifugation temperature is 2~6℃. A 0.22 μm filter membrane is used for filtration.

4. The preparation method according to claim 1, characterized in that, The separation and purification described in step (3) employs one or more of the following methods: ultracentrifugation, ultrafiltration concentration, size exclusion chromatography, or reagent precipitation. The ultracentrifugation method is as follows: the filtered clear supernatant is ultracentrifuged at 2~6℃ and 90000~110000×g for 70~90 min, the supernatant is discarded, the precipitate is washed with phosphate buffer, and then ultracentrifuged again at 2~6℃ and 90000~110000×g for 70~90 min, and the precipitate is collected. The ultrafiltration concentration method is as follows: the clarified supernatant is concentrated using an ultrafiltration device with a molecular weight cutoff of 100 kDa, and then washed and replaced with phosphate buffer solution to collect the concentrate. The size exclusion chromatography method is as follows: a size exclusion chromatography column is pre-equilibrated with phosphate buffer, the clear supernatant or concentrate is loaded onto the column, eluted with phosphate buffer, and the eluted fraction rich in small extracellular vesicles is collected. The method of the reagent precipitation method is as follows: add the vesicle precipitation reagent to the clear supernatant, mix well, incubate at 2~6℃ for 8~16 h, and then collect the precipitate by centrifugation; The resuspension was performed using phosphate buffer, and the total protein concentration of the small extracellular vesicles in the notochord cell-derived small extracellular vesicle suspension was 0.05~5 mg / mL.

5. The preparation method according to claim 1, characterized in that, The PVA concentration in the PVA solution in step (4) is 2% to 15% by mass volume.

6. The preparation method according to claim 1, characterized in that, The TSPBA concentration in the TSPBA solution described in step (5) is 1% to 15% by mass or volume.

7. The preparation method according to claim 1, characterized in that, The ratio of CP05 peptide, PVA solution and TSPBA solution used in step (6) is 100 μg: 0.9~1.1 mL: 0.9~1.1 mL by mass-volume ratio.

8. The preparation method according to claim 1, characterized in that, During the mixing process described in step (7), the suspension of small extracellular vesicles derived from notochord cells is added to the precursor solution and gently blown or stirred at low speed for 50-70 seconds at 22-27°C. Then, the mixture is allowed to stand or incubate at 22-27°C for 25-35 minutes, so that the small extracellular vesicles derived from notochord cells are retained in the PVA / TSPBA hydrogel network through the binding of CP05 peptide, thus obtaining a CP05-functionalized PVA / TSPBA hydrogel containing small extracellular vesicles derived from notochord cells.

9. The notochord cell-derived small extracellular vesicle-loaded CP05-functionalized PVA / TSPBA hydrogel prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the notochord cell-derived small extracellular vesicle-loaded CP05-functionalized PVA / TSPBA hydrogel according to claim 9 in the preparation of a medicament for treating intervertebral disc degeneration.

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