A nanoparticle and a vesicular delivery system comprising the nanoparticle, and a method of preparing and use thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses a kind of nanoparticle and vesicle delivery system comprising nanoparticle and its preparation method and application.The nanoparticle includes core and shell layer coated on the surface of core;Core includes nanoenzyme with antioxidant activity, and shell layer includes polypeptide.The vesicle delivery system includes extracellular vesicle and the nanoparticle encapsulated inside the extracellular vesicle. Optionally, nanoparticle can also include polysaccharide outer shell coated on the surface of shell layer.The nanoparticle and vesicle delivery system provided by the application can efficiently remove active oxygen, restore mitochondrial function, reverse nucleus cell stress-induced aging, and have significant anti-inflammatory, antioxidant, anti-aging and repair intervertebral disc structure effects verified by in vitro and in vivo experiments.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a nanoparticle and a vesicle delivery system containing the nanoparticle, as well as its preparation method and application. Background Technology

[0002] Intervertebral disc degeneration (IDD) is a major cause of lower back pain, placing a heavy burden on social and individual health. Its pathological features are mainly manifested as dysfunction of nucleus pulposus (NP) cells and excessive accumulation of reactive oxygen species (ROS). ROS can induce mitochondrial dysfunction, exacerbate cellular oxidative stress, and lead to NP cell senescence, thereby disrupting the metabolic homeostasis within the intervertebral disc and accelerating the degeneration process. Therefore, effectively clearing ROS, restoring mitochondrial function, and combating cellular senescence are key to the treatment of IDD.

[0003] Currently, treatments for intervertebral disc degeneration (IDD) include conservative therapy and surgery, but these methods cannot reverse or slow the degenerative process and have limitations in effectiveness and are highly invasive. Treatment strategies based on biomaterials are also being explored. For example, some nanozymes possess antioxidant enzyme activity that can scavenge reactive oxygen species (ROS), but they are easily eliminated in vivo, have poor targeting, and are difficult to maintain an effective concentration in the intervertebral disc when used alone. While some mitochondrial-targeting peptides can regulate cellular energy metabolism and stress responses, they have poor stability, are easily degraded, and have low delivery efficiency when used alone.

[0004] Existing research largely focuses on optimizing single functional components. For multi-component synergistic delivery systems, problems such as interference between components leading to reduced activity or unstable carrier structures causing premature leakage are often encountered. Existing single-component or traditional delivery systems suffer from poor targeting, easy clearance, insufficient stability, and difficulty in simultaneously exerting multiple biological activities, which limits their practical application in the treatment of IDD. Summary of the Invention

[0005] To address the problems of poor targeting, easy clearance, insufficient stability, and difficulty in simultaneously exerting multiple biological activities in the treatment of intervertebral disc degeneration using single-component or traditional delivery systems, this invention provides a nanoparticle and a vesicle delivery system containing nanoparticles, as well as their preparation method and application.

[0006] The inventors provide a nanoparticle comprising a cerium-tannic acid nanozyme core with antioxidant activity and a mitochondrial-derived peptide MOTS-c shell; optionally, the nanoparticle further comprises a hyaluronic acid shell coating the surface of the peptide shell.

[0007] The present invention also provides a vesicle delivery system, comprising extracellular vesicles and the aforementioned nanoparticles encapsulated within the extracellular vesicles; preferably, the extracellular vesicles are derived from nucleus pulposus cells.

[0008] The vesicle delivery system can achieve synergistic therapeutic effects of anti-oxidation and mitochondrial function regulation. Combined with the natural biocompatibility of vesicles, it effectively overcomes the shortcomings of limited efficacy of single components and insufficient uptake by target cells, providing a highly efficient and stable new regenerative treatment option for intervertebral disc degeneration.

[0009] In summary, the technical solution provided by this invention is as follows: In a first aspect, the present invention provides a nanoparticle comprising a core and a shell coating the surface of the core; the core comprising a cerium-tannic acid nanozyme with antioxidant activity; and the shell comprising a polypeptide.

[0010] In conjunction with the first aspect of the present invention, in some embodiments, the nanoparticles further include an outer shell layer covering the surface of the shell layer.

[0011] Furthermore, the outer shell layer comprises natural polysaccharides or derivatives thereof.

[0012] Furthermore, the polysaccharide includes hyaluronic acid, chitosan, or sodium alginate.

[0013] In conjunction with the first aspect of the present invention, in some embodiments, the nanozyme includes a metal-based nanozyme, a carbon-based nanozyme, or a single-atom nanozyme.

[0014] Furthermore, the metal-based nanozyme is a cerium-based nanozyme.

[0015] In conjunction with the first aspect of the present invention, in some embodiments, the polypeptide is the mitochondrial-derived peptide MOTS-c.

[0016] In a second aspect, the present invention provides a vesicle delivery system comprising extracellular vesicles and the aforementioned nanoparticles encapsulated within the extracellular vesicles.

[0017] In conjunction with a second aspect of the invention, in some embodiments, the extracellular vesicles are derived from nucleus pulposus cells.

[0018] Thirdly, the present invention provides a pharmaceutical composition comprising the above-described nanoparticles or the above-described vesicle delivery system, and a pharmaceutically acceptable carrier.

[0019] Fourthly, the present invention provides the use of the above-described nanoparticles, vesicle delivery systems, or pharmaceutical compositions in the preparation of medicaments for treating intervertebral disc degeneration.

[0020] Fifthly, the present invention provides a method for preparing the above-mentioned vesicle delivery system, comprising the following steps: Provides nanozymes with antioxidant activity; By coating peptides onto the surface of nanozymes to form a peptide shell, nanoparticles are obtained. Obtain extracellular vesicles; The nanoparticles are encapsulated inside the extracellular vesicles to obtain the vesicle delivery system.

[0021] In conjunction with the fifth aspect of the invention, some embodiments further include coating the surface of the nanoparticles with a polysaccharide shell layer.

[0022] Compared with the prior art, the present invention has at least the following beneficial effects: The vesicle delivery system provided by this invention combines cerium-tannic acid nanozymes with antioxidant functions with mitochondrial-derived peptide MOTS-c, which has the functions of regulating energy metabolism and anti-aging, to achieve synergistic treatment with multiple targets and mechanisms. It can not only directly eliminate reactive oxygen species, but also fundamentally reverse stress-induced aging of nucleus pulposus cells and reshape a healthy energy metabolism pattern by activating the integrated stress response (ISR) and key energy metabolism signaling pathways, which is significantly superior to single-component treatment. Moreover, the vesicle delivery system uses extracellular vesicles derived from nucleus pulposus cells as the encapsulation shell, which endows the system with extremely high biocompatibility and homologous targeting, significantly improving the delivery efficiency of active ingredients and effectively solving the problem of low delivery efficiency of existing single-functional molecules or traditional delivery systems. Validated by in vitro cell models and in vivo rat models, this vesicle delivery system has significant anti-inflammatory, antioxidant, anti-aging and intervertebral disc structure restoration effects, providing a new treatment strategy with clinical translational potential for intervertebral disc degeneration and similar oxidative stress-related degenerative diseases. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 TEM image and energy spectrum of Ce-TA.

[0025] Figure 2 Characterization of nanoparticles. A: Hydrated particle size distribution of nanoparticles; B: Potential (Zeta potential) of nanoparticles.

[0026] Figure 3 Efficiency of peptide encapsulation.

[0027] Figure 4 TEM image of Ce-MOTS@EV.

[0028] Figure 5 Hydrated particle size distribution of Ce-MOTS@EV.

[0029] Figure 6 Efficiency of vesicle-loaded nanoparticles.

[0030] Figure 7 Cellular uptake results. Where A: Representative image of cellular uptake; B: Quantitative analysis of cellular uptake.

[0031] Figure 8 ROS flow cytometry plot of Ce-MOTS@EV and its components after TBHP treatment of nucleus pulposus cells.

[0032] Figure 9 JC-1 fluorescence image after Ce-MOTS@EV and its components were treated with TBHP in nucleus pulposus cells.

[0033] Figure 10 JC-1 flow cytometry plots of Ce-MOTS@EV and its components after treatment with TBHP in nucleus pulposus cells. In the figure, A is a representative image, and B is the quantitative analysis of the corresponding JC-1 flow cytometry plot.

[0034] Figure 11 ATP content in nucleus pulposus cells treated with Ce-MOTS@EV and its components after treatment with TBHP.

[0035] Figure 12 GSH content in nucleus pulposus cells treated with Ce-MOTS@EV and its components after treatment with TBHP.

[0036] Figure 13 Transcriptome sequencing analysis results after Ce-MOTS@EV treatment of TBHP-treated nucleus pulposus cells. A: Heatmap of genes related to inflammatory factors; B: Heatmap of genes related to cell cycle regulation.

[0037] Figure 14 Western blot images and quantitative analysis of stress-related molecular proteins integrated by Ce-MOTS@EV after treatment with TBHP in nucleus pulposus cells. In the image, A represents a representative Western blot image; B represents the corresponding quantitative analysis.

[0038] Figure 15Western blot images and quantitative analysis of cellular senescence-related molecules in nucleus pulposus cells treated with Ce-MOTS@EV followed by TBHP treatment. In the image, A represents a representative Western blot image; B represents the corresponding quantitative analysis.

[0039] Figure 16 The images show changes in intervertebral disc height index and magnetic resonance imaging (MRI) degeneration grading in rats treated with Ce-MOTS@EV. A represents changes in intervertebral disc height index; B represents changes in MRI degeneration grading. Figure 17 Changes in the degree of intervertebral disc degeneration in rats treated with Ce-MOTS@EV.

[0040] Figure 18 Ce The synthesis of extracellular matrix (NP) in rats after intervertebral disc degeneration treated with MOTS@EV. A: Representative immunohistochemical images of extracellular matrix (type II collagen, proteoglycan); B: Quantitative analysis of type II collagen optical density in immunohistochemistry; C: Quantitative analysis of proteoglycan optical density in immunohistochemistry. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0042] the term: Ce-TA: refers to the core particles of cerium-based nanozymes formed by the coordination of cerium ions and tannic acid (TA), also known as cerium-tannic acid nanoparticles or Ce-TA nanoparticles, which have antioxidant activity.

[0043] MOTS-c: refers to the mitochondrial-derived peptide MOTS-c, which is used to coat the Ce-TA surface to form a polypeptide shell, also known as MOTS-c peptide.

[0044] Ce-TA / MOTS: refers to the core formed by MOTS-c peptides assembling on the Ce-TA surface through electrostatic adsorption. Shell-structured complexes (i.e., nanoparticles coated with polypeptide shells), sometimes referred to as Ce-MOTS in this invention.

[0045] Ce-MOTS@HA: refers to nanoparticles sequentially coated with MOTS-c peptide and hyaluronic acid shell, namely Ce-TA / MOTS@HA, also known as cerium nanozyme. MOTS-c Hyaluronic acid composite nanoparticles.

[0046] EVs: Extracellular vesicles, derived from nucleus pulposus cells, are used as delivery carriers and are also known as EVs.

[0047] Ce-MOTS@EV: refers to the vesicle delivery system obtained by encapsulating Ce-MOTS@HA nanoparticles inside EVs, which is the final product of this invention.

[0048] As described in the background section, existing treatments for intervertebral disc degeneration suffer from limited efficacy of single-component therapies and low delivery efficiency of active ingredients. To address these issues, in one specific embodiment, this invention synthesizes a cerium-tannic acid nanozyme core through coordination interactions, and uses electrostatic adsorption to assemble MOTS-c peptides onto the core surface to form a shell, constructing core-shell structured nanoparticles. Furthermore, a hyaluronic acid shell can be coated onto the surface of the nanoparticles, and they can be encapsulated into extracellular vesicles derived from nucleus pulposus cells through ultrasonic co-incubation, constructing a vesicle delivery system.

[0049] This system utilizes the dual-component effects of cerium-tannic acid nanoenzymes scavenging reactive oxygen species and MOTS-c regulating mitochondrial energy metabolism to achieve synergistic therapeutic effects and significantly reverse stress-induced senescence of nucleus pulposus cells. Validated in a rat intervertebral disc degeneration animal model, the delivery system of this invention exhibits excellent anti-inflammatory, antioxidant, and anti-aging effects, effectively restoring intervertebral disc structure and improving function. It overcomes the limitations of single-therapy approaches and provides a highly efficient regenerative treatment strategy for intervertebral disc degeneration.

[0050] Based on this, the present invention provides nanoparticles and a vesicle delivery system containing nanoparticles, as well as their preparation method and application.

[0051] In a first aspect, the present invention provides a nanoparticle comprising a core and a shell coating the surface of the core; the core comprising a nanoenzyme with antioxidant activity; and the shell comprising a polypeptide.

[0052] In conjunction with the first aspect of the present invention, in some embodiments, the nanoparticles further include an outer shell layer covering the surface of the shell layer.

[0053] Furthermore, the outer shell layer comprises natural polysaccharides or derivatives thereof.

[0054] Furthermore, the polysaccharide includes hyaluronic acid, chitosan, or sodium alginate.

[0055] Preferably, the polysaccharide is hyaluronic acid.

[0056] Natural polysaccharides or their derivatives possess good biocompatibility and modifiability, making them suitable for use as outer shell materials. Hyaluronic acid, used in Example 1, is a typical example of a natural polysaccharide. Those skilled in the art will understand that other natural polysaccharides or their derivatives, such as chitosan and sodium alginate, can also be used to construct outer shells, and their selection can be adjusted according to specific application requirements.

[0057] This invention reveals that the absolute value of the Zeta potential on the surface of nanoparticles without a polysaccharide shell is relatively low, indicating that the system may be in a relatively unstable state under physiological conditions and is prone to aggregation. By coating the surface of the nanoparticles with a negatively charged hyaluronic acid polysaccharide shell, the principle of charge repulsion can provide additional electrostatic repulsion, thereby significantly increasing the overall absolute value of the Zeta potential of the nanoparticle system. This effectively maintains the dispersion stability of the system during in vitro storage and in vivo treatment, prevents particle aggregation, and optimizes pharmacokinetic behavior.

[0058] It should be noted that the polysaccharide shell layer is a preferred embodiment of the present invention, designed to further improve the physical stability and in vivo circulation performance of the nanoparticles, and is not a necessary technical feature for achieving the core technical effects of the present invention. The core technical effects of the present invention mainly rely on the synergistic effect of nanozymes with antioxidant activity and bioactive peptides, as well as the natural targeted delivery capability of extracellular vesicles. Even in embodiments that do not include the polysaccharide shell layer (i.e., only extracellular vesicles and internally encapsulated nanoparticles), the vesicle delivery system can still achieve basic antioxidant, anti-inflammatory, and cell-protective functions based on the synergistic mechanism of nanozymes and peptides. Those skilled in the art can selectively add or omit the polysaccharide shell layer according to specific formulation stability requirements and application scenarios.

[0059] In conjunction with the first aspect of the present invention, in some embodiments, the nanozyme includes a metal-based nanozyme, a carbon-based nanozyme, or a single-atom nanozyme.

[0060] Preferably, the metal-based nanozyme is a cerium-based nanozyme.

[0061] Nanozymes are a class of nanomaterials with enzyme-like activity, which can be used for antioxidant therapy such as scavenging reactive oxygen species. This invention uses cerium-based nanozymes, a type of metal-based nanozyme, as an example for detailed verification (Example 1, step 3.1). These nanozymes are synthesized through coordination interactions and have a uniform hydrated particle size distribution (approximately 55 nm). Figure 2 Carbon-based nanozymes (such as carbon dots and graphene quantum dots) and single-atom nanozymes are also known nanomaterials with antioxidant activity in the art, and those skilled in the art can select appropriate nanozyme types based on the concept of this invention.

[0062] In conjunction with the first aspect of the present invention, in some embodiments, the polypeptide is a MOTS-c peptide.

[0063] MOTS-c peptide is a mitochondrial-derived peptide that regulates cellular energy metabolism and stress responses. As shown in step 3.2 of Example 1, MOTS-c peptide is assembled onto the surface of Ce-TA nanoparticles via electrostatic adsorption, achieving an encapsulation efficiency of up to 95%. Figure 3 In Example 2, MOTS-c peptide alone increased the survival rate of TBHP-damaged nucleus pulposus cells to 0.715 ± 0.034 (Table 1) and restored some ATP levels (relative value 0.645 ± 0.024, Table 2), demonstrating its function in protecting cellular energy metabolism. More importantly, when MOTS-c peptide and cerium nanozyme were co-encapsulated in extracellular vesicles derived from nucleus pulposus cells to form the Ce-MOTS@EV system, a strong synergistic effect was observed in ATP restoration (Q = 1.17) and GSH enhancement (Q = 1.23) (Tables 2 and 3), verifying the superiority of the combination.

[0064] In a second aspect, the present invention provides a vesicle delivery system comprising extracellular vesicles and the aforementioned nanoparticles encapsulated within the extracellular vesicles.

[0065] In conjunction with a second aspect of the invention, in some embodiments, the extracellular vesicles are derived from nucleus pulposus cells.

[0066] Utilizing extracellular vesicles derived from nucleus pulposus cells as an encapsulation shell endows the system with extremely high biocompatibility and homologous targeting, significantly improving the delivery efficiency of active ingredients. Figure 7 ).

[0067] Thirdly, the present invention provides a pharmaceutical composition comprising the above-described nanoparticles or the above-described vesicle delivery system, and a pharmaceutically acceptable carrier.

[0068] Fourthly, the present invention provides the use of the above-described nanoparticles, vesicle delivery systems, or pharmaceutical compositions in the preparation of medicaments for treating intervertebral disc degeneration.

[0069] In vitro experiments have shown that this vesicle delivery system (Ce) MOTS@EV increased the survival rate of oxidative stress-damaged nucleus pulposus cells to 0.943 and showed synergistic therapeutic effects compared to cerium-tannin nanozymes or MOTS-c peptides alone in terms of ATP restoration (Q=1.17) and GSH enhancement (Q=1.23). An in vivo rat intervertebral disc degeneration model confirmed that the nanoparticles (Ce-MOTS) and the vesicle delivery system (Ce...)... MOTS@EV can effectively maintain the intervertebral disc height index, improve MRI signal, reduce histological degeneration score, and promote the expression of type II collagen and proteoglycans, showing definite anti-inflammatory, antioxidant, anti-aging and structural repair effects, and therefore has clear pharmaceutical applications.

[0070] Fifthly, the present invention provides a method for the above-described vesicle delivery system, comprising the following steps: Provides nanozymes with antioxidant activity; By coating peptides onto the surface of nanozymes to form a peptide shell, nanoparticles are obtained. Obtain extracellular vesicles; The nanoparticles are encapsulated within the extracellular vesicle cavity to obtain the vesicle delivery system.

[0071] In conjunction with the fifth aspect of the invention, some embodiments further include coating the surface of the nanoparticles with a polysaccharide shell layer.

[0072] This invention involves mixing equimolar concentrations (24 mmol / L) of tannic acid and cerium nitrate under alkaline conditions (pH 8.0), stirring at room temperature in the dark for 6 hours, followed by dialysis and freeze-drying to obtain Ce-TA nanoparticles (hydrated particle size approximately 55 nm, Zeta potential -30 ± 2.1 mV). Subsequently, MOTS-c peptides are assembled onto the Ce-TA surface via electrostatic adsorption to form a Ce-TA / MOTS complex, increasing the hydrated particle size to 60 nm–70 nm, with an average of approximately 65 nm, a potential reversal of +10 ± 1.5 mV, and an encapsulation efficiency of up to 95% (…). Figure 3 This proves that the polypeptide shell was successfully coated.

[0073] The Ce-TA / MOTS complex was mixed with a hyaluronic acid solution (0.1%, w / v), stirred at 4°C in the dark for 24 hours, centrifuged at 5000g to remove excess hyaluronic acid solution, and then the precipitate was freeze-dried to obtain Ce. TA / MOTS@HA nanoparticles (abbreviated as Ce) MOTS@HA refers to nanoparticles containing a polysaccharide outer shell.

[0074] Finally, Ce MOTS@HA nanoparticles were encapsulated with extracellular vesicles (EVs) derived from nucleus pulposus cells via ultrasonic co-incubation to obtain Ce-MOTS@EVs. Transmission electron microscopy (TEM) was used to analyze the encapsulation process. Figure 4 The vesicle structure appears intact, with high-electron-density nanoparticles visible inside; dynamic light scattering (…) Figure 5 The measured hydrated particle size ranged from 200 nm to 400 nm, and the hydrated particle size curve showed a symmetrical single-peak shape, with the main peak concentrated around 300 nm; the loading efficiency reached 90%. Figure 6 The above data fully validates the feasibility and efficiency of this preparation method.

[0075] Intervertebral disc degeneration (IDD) exhibits a highly conserved pathological mechanism in both humans and rats, manifesting as oxidative stress in nucleus pulposus cells, mitochondrial dysfunction, cellular senescence, and extracellular matrix degradation. The rat caudal IDD model used in this invention is an internationally recognized standard animal model for evaluating the in vivo efficacy of IDD therapeutics, and its results are widely accepted for predicting human treatment outcomes. Furthermore, the molecular pathways (such as ROS clearance and related signaling pathways) and cellular processes (such as mitochondrial energy metabolism and cellular senescence) targeted by the delivery system of this invention share high homology between humans and rats. Therefore, based on the positive efficacy data obtained in the rat model, those skilled in the art can reasonably expect that this vesicle delivery system also has the potential to treat IDD in humans.

[0076] Unless otherwise specified, the experimental procedures described in the following examples are all conventional techniques in the art, including but not limited to cell culture, mRNA and total protein extraction, molecular biological detection (such as Western blot and immunohistochemistry), cell biological techniques (such as flow cytometry), H&E staining, Masson's trichrome staining, animal modeling, data collection, bioinformatics analysis, and statistical methods. All experimental reagents used were commercially available and met general quality standards in the art. The implementation methods of the above-mentioned conventional techniques are common knowledge to those skilled in the art, and specific operations can be referred to relevant authoritative literature. Experimental and control groups were set up according to the grouping methods commonly used in the art.

[0077] The primary nucleus pulposus (NP) cells used in the following examples were obtained from Wuhan Pronosei Biotechnology Co., Ltd., and their cell identity was confirmed by STR typing. The mycoplasma test results were negative. Male Sprague-Dawley rats were purchased from Shulaibao (Wuhan) Biotechnology Co., Ltd. All animal experiments have been approved by the Ethics Committee of the Animal Center of Tongji Medical College, Huazhong University of Science and Technology (Approval No.: IACUC〔2025〕4985).

[0078] In this invention, all data collection and processing in the statistical analysis section were performed using a blinded method, and the quantitative data were expressed as mean ± standard deviation. All data were analyzed using GraphPad Prism software. In the attached figures, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, and ns indicates no statistical difference (P ≥ 0.05).

[0079] The technical solution provided by the present invention will be described in detail below with reference to the embodiments.

[0080] Example 1: Ce Preparation and characterization of MOTS@EV 1. Purpose This embodiment aims to construct nanoparticles with core-shell structure (Ce-TA / MOTS) and vesicle delivery system (Ce-MOTS@EV), and systematically characterize their morphology, hydration size, potential, composition and encapsulation efficiency through physicochemical means to verify the feasibility of the preparation process and the structural integrity of the system.

[0081] 2. Experimental materials and instruments Biological material: primary rat nucleus pulposus cells.

[0082] Main reagents: tannic acid, cerium nitrate, sodium hydroxide, MOTS-c peptide, hyaluronic acid, 2% (w / v) phosphotungstic acid, potassium bromide, BCA kit (Beyotime, Shanghai).

[0083] Main instrument: Transmission electron microscope (JEOL JEM) F200 (Japan), DLS dynamic light scattering instrument (Malvern Zetasizer Nano ZS90, UK), Fourier transform infrared spectrometer (Nexus 670, Thermo Nicolet, USA), X-ray photoelectron spectrometer (Thermo ESCALAB 250XI, USA), spectrophotometer (BioTek Epoch, USA), high-speed centrifuge.

[0084] Data analysis: Avantage software.

[0085] 3. Experimental Methods 3.1 Synthesis of cerium-tannic acid nanoparticles (Ce-TA): Equimolar concentration (24 mmol / L) of tannic acid and cerium nitrate were mixed under alkaline conditions (pH 8.0), stirred at room temperature in the dark for 6 hours, and then dialyzed and freeze-dried to obtain Ce-TA nanoparticles.

[0086] 3.2 Load MOTS C-peptide (Ce-TA / MOTS): 10 mg MOTS C-peptide was dissolved in 50 mL of Tris-HCl solution at pH 8.0, and 25 mg of Ce was slowly added while stirring continuously. TA nanoparticles. The suspension was stirred at 4 °C in the dark for 24 hours to induce MOTS. c was fully adsorbed. Subsequently, centrifuged at 5000g for 1 hour, discarded the supernatant, resuspended the precipitate in buffer solution, and freeze-dried to obtain Ce. TA / MOTS composite, i.e. the aforementioned nanoparticles.

[0087] 3.3 Encapsulating hyaluronic acid shell (Ce TA / MOTS@HA): The Ce-TA / MOTS complex was mixed with a hyaluronic acid solution (0.1%, w / v), stirred at 4°C in the dark for 24 hours, centrifuged at 5000g to remove excess hyaluronic acid solution, and then the precipitate was freeze-dried to obtain Ce. TA / MOTS@HA nanoparticles (abbreviated as Ce) MOTS@HA refers to nanoparticles containing a polysaccharide outer shell.

[0088] 3.4 Extracellular vesicle extraction and encapsulation (Ce-MOTS@EV): EVs were extracted from NP cell culture supernatant by differential centrifugation; the concentration of EVs was determined using a BCA kit and adjusted to 50 μg / mL with PBS.

[0089] 5 mg Ce MOTS@HA nanoparticles were thoroughly dispersed in 10 mL of EVs solution (50 μg / mL). The nanoparticles were then... The EV mixture was sonicated (20% amplitude; 10 cycles, 5 seconds of sonication per cycle with a 10-second interval), and then incubated at 4 °C for 36 hours before centrifugation to remove uncoated nanoparticles, yielding the final product Ce-MOTS@EV.

[0090] 3.5 Characterization and Detection: 3.5.1 Morphology and Structure Ce-MOTS@EV samples or purified EVs were dropped onto a carbon-copper grid. EV samples were negatively stained with 2% (w / v) phosphotungstic acid for 1 minute, and the stain was removed. After drying at room temperature, the morphology was observed using transmission electron microscopy (TEM). Selected areas were subjected to energy dispersive spectroscopy (EDS) to determine the elemental composition and relative abundance.

[0091] The freeze-dried sample was mixed with potassium bromide (KBr), ground evenly, and prepared into a thin film. Infrared spectroscopy was performed using a Fourier transform infrared spectrometer, with a scanning range of 4000–400 cm⁻¹. -1 4 cm resolution -1 The background baseline was subtracted by pressing pure KBr pellets to obtain the transmission infrared spectrum of the sample.

[0092] The chemical valence state and relative surface content of cerium (Ce) in nanoparticles were analyzed using X-ray photoelectron spectroscopy. Data processing was performed using Avantage software, based on Ce... 3+ With Ce 4+The peak position and peak area of ​​characteristic peaks and satellite peaks are used to quantitatively determine the composition ratio of cerium in different valence states.

[0093] 3.5.2 Zeta potential analysis The zeta potential of nanoparticles was simultaneously measured using a dynamic light scattering instrument to evaluate the surface charge properties and colloidal dispersion stability of the samples. The samples were dispersed in a 1 mmol / L KCl solution (pH 7.4), and the test temperature was set at 25 °C. Each group of samples was measured in triplicate.

[0094] 3.5.3 Encapsulation efficiency determination a. MOTS c. Encapsulation efficiency determination: 10 mg MOTS Dissolve c in 50 mL of pH 8.0 solution, then add 25 mg of Ce. TA nanoparticles were stirred at 4 °C in the dark for 2, 4, 6, 8, 12, 24, and 48 hours, respectively. After the reaction was completed, the mixture was centrifuged at 5000 g for 1 hour, and the supernatant was collected. The residual MOTS in the supernatant was determined using a BCA kit. c content, based on which MOTS are calculated C-package efficiency.

[0095] b. Loading efficiency of Ce-MOTS@HA nanoparticles Five mg of Ce-MOTS@HA nanoparticles were dispersed in 10 mL of a 50 μg / mL extracellular vesicle (EV) solution. After sonication, the mixture was incubated at 4 °C for 1, 2, 4, 8, 12, 18, 36, and 48 hours, respectively. The mixture was centrifuged at 5000 g for 1 hour, and the concentration of free particles in the supernatant was measured at 600 nm using a spectrophotometer to calculate the loading efficiency of Ce-MOTS@HA nanoparticles.

[0096] 4. Results 4.1 Morphology and Structure: Transmission electron microscopy (TEM) images showed that the synthesized Ce-TA nanoparticles had uniform hydrated particle size and good monodispersity, with no obvious aggregation. Energy dispersive spectroscopy (EDS) elemental analysis detected obvious cerium (Ce), oxygen (O), and carbon (C) signals in the particle region. The Ce signal was uniformly distributed within the particles without significant segregation, confirming the successful synthesis of the cerium-based nanozyme core and the purity and uniform elemental distribution of the material. Figure 1 ).

[0097] like Figure 4As shown, natural EVs exhibit typical cup-shaped or circular vesicle structures with clear membrane boundaries, uniform internal electron density, and diameters of approximately 100–200 nm (scale bar = 100 nm). The final product, Ce-MOTS@EV, retains the complete vesicle structure of EVs, and distinct high-electron-density nanoparticles (marked with red arrows) can be observed inside the vesicles, confirming that the nanoparticles have been successfully loaded into the EVs without disrupting the basic vesicle morphology.

[0098] 4.2 Hydrated Particle Size and Distribution: DLS analysis showed that the Ce-TA nanoparticles had a narrow hydration size distribution, with an average diameter of approximately 55 nm; after loading with MOTS-c peptide, the hydration size of Ce-TA / MOTS increased slightly to approximately 65 nm, indicating successful peptide coupling; after coating with hyaluronic acid (HA), Ce... The hydrated particle size of MOTS@HA further increased to approximately 110 nm, confirming the formation of a core-shell structure. Figure 2 A). The final hydrated particle size of the Ce-MOTS@EV system is approximately 300 nm ( Figure 5 The hydration particle size distribution width was slightly increased compared to natural EVs. Both samples maintained a unimodal distribution with no obvious aggregation, indicating that the encapsulation process did not disrupt the colloidal stability of the EVs. Combined with TEM results, this change in hydration particle size further confirms the successful construction of the Ce-MOTS@EV nanocarrier, and its hydration particle size characteristics meet the requirements for in vivo delivery.

[0099] 4.3 Surface Potential: Zeta potential analysis ( Figure 2 (B) The results show that Ce-TA has a Zeta potential of -30 ± 2.1 mV and a negatively charged surface. After loading with MOTS-c peptide, the Zeta potential of Ce-TA / MOTS reverses to +10 ± 1.5 mV, indicating that the cationic peptide significantly neutralizes the negative charge on the material surface. Further encapsulation with HA reduces the Zeta potential of Ce-TA / MOTS@HA to -45 ± 2.8 mV, significantly increasing the negative charge density, demonstrating the regulatory effect of HA on the surface charge. The high absolute Zeta potential (>30 mV) indicates that Ce-TA and Ce-TA / MOTS@HA have good colloidal dispersion stability in aqueous solution, while the positive potential characteristic of Ce-TA / MOTS is beneficial for enhancing interaction with negatively charged cell membranes.

[0100] 4.4 Loading efficiency: Encapsulation efficiency measurements showed that the encapsulation efficiency of MOTS-c peptide on Ce-TA nanoparticles reached as high as 95%. Figure 3 Meanwhile, the loading efficiency of Ce-MOTS@HA nanoparticles by EVs can reach 90%. Figure 6 The two key data points above demonstrate that this preparation method can achieve efficient loading of functional components (MOTS-c peptides and nanocomposites).

[0101] 5. Conclusion This embodiment successfully established a reproducible fabrication process for constructing an extracellular vesicle-encapsulated cerium nanozyme / MOTS-c two-component vesicle delivery system (Ce-MOTS@EV). The system's structure was confirmed by TEM and other methods, and its size and surface properties met design expectations. Most importantly, the system achieved near-complete encapsulation and loading of functional components, laying a solid material foundation for subsequent efficient targeted delivery and synergistic biological effects.

[0102] Example 2: Ce In vitro biological function validation of MOTS@EV 1. Purpose This embodiment aims to verify Ce in vitro. The MOTS@EV vesicle delivery system protects nucleus pulposus (NP) cells from oxidative stress damage, and this study focuses on evaluating the synergistic therapeutic effects between its core components (cerium nanozyme and MOTS-c peptide). By comparing the effects of single components and the complete system in scavenging reactive oxygen species, restoring mitochondrial function, enhancing cell viability, and improving cellular energy metabolism, the synergistic advantage of "1+1>2" is clarified.

[0103] 2. Experimental Materials Cells: Rat primary nucleus pulposus cells.

[0104] Biomaterials: Ce prepared in Example 1 The MOTS@EV system, individual Ce-TA nanoparticles, individual MOTS-c peptide, and natural nucleus pulposus cell-derived EVs.

[0105] Main reagents: tert-butyl hydroperoxide (TBHP, used to construct oxidative stress models), DCFH-DA reactive oxygen species assay kit (MCE, Shanghai), ATP content assay kit (Wuhan Saiweier Biotechnology Co., Ltd., G4309), JC-1 mitochondrial membrane potential assay kit (MCE, Shanghai), glutathione (GSH) content assay kit (Wuhan Saiweier Biotechnology Co., Ltd., G4305), Calcein-AM / PI live / dead cell double staining kit (Beijing Lanjieke Technology Co., Ltd., BL130S), BeyoExo™ exosome labeling and tracing kit (PKH67, Beyotime, Shanghai).

[0106] Main instruments: Flow cytometer (BD Biosciences, USA), chemiluminescent microplate reader (Thermo Fisher Scientific, USA), fluorescence microscope.

[0107] 3. Experimental Methods 3.1 Cell uptake experiment Ce-MOTS@EV was labeled with PKH67, and Ce-TA / MOTS@HA was labeled with Cy2. Free dyes were removed by centrifugation at 20,000 g for 1 hour. The labeled Ce-TA / MOTS@HA or Ce-MOTS@EV was added to NP cells cultured to 50% confluence and incubated at 37°C for 24 hours. Cells were washed three times with PBS, fixed with 4% paraformaldehyde for 10 minutes, stained with phalloidin for 30 minutes, and stained with DAPI for 5 minutes. Cell uptake of Ce-TA / MOTS@HA and Ce-MOTS@EV was observed under a fluorescence microscope. Simultaneously, cells were collected and mean fluorescence intensity (MFI) was measured using flow cytometry. Each group had at least three replicates.

[0108] 3.2 Cell Model Establishment and Grouping: Rat nucleus pulposus cells were seeded into 6-well plates and cultured for 24 hours. Except for the control group, the other groups were treated with 200 μMTBHP for 2 hours to construct an oxidative stress model.

[0109] Group intervention: Cells were randomly divided into five groups, each treated with a corresponding drug or vector. The specific grouping details are as follows: Control group: Rat nucleus pulposus cells were not subjected to oxidative stress damage, and an equal volume of PBS was added during treatment; TBHP group (model group): Rat nucleus pulposus cells were treated with 200 μM TBHP for 2 hours to establish an oxidative stress model; TBHP + Evs group (EVs treatment group): After TBHP-induced damage, EVs solution with a final concentration of 50 ug / mL derived from nucleus pulposus cells was added for treatment; TBHP + MOTS-c group (MOTS-c peptide monotherapy group): After TBHP-induced damage, MOTS-c peptide was added for treatment at a final concentration of 100 μM. TBHP + Ce-TA group (Ce-TA monotherapy group): After TBHP-induced damage, Ce-TA nanoparticles were added for treatment at a final concentration of 0.5 mg / m³. TBHP + Ce-MOTS@EV group (two-component vesicle delivery system treatment group): After TBHP-induced damage, Ce-MOTS@EV at a final concentration of 50 ug / mL was added for treatment.

[0110] 3.3 Functional Indicator Testing: 3.3.1 Cell viability assay: Cell viability was analyzed using a Calcein-AM / PI live / dead cell double staining kit: The kit contained Calcein... The staining solutions of AM (for live cells, green) and propidium iodide (for dead cells, red) were incubated at 37°C in the dark for 30 minutes. The cells in each group were observed and photographed under a fluorescence microscope.

[0111] 3.3.2 Detection of intracellular reactive oxygen species (ROS): The DCFH-DA probe was used to quantitatively detect the ROS levels in each group of cells using flow cytometry.

[0112] 3.3.3 Detection of mitochondrial membrane potential: NP cells from each group were collected and resuspended in preheated JC-1 staining buffer, and JC-1 working solution with a final concentration of 10 μM was added. The cells were incubated at 37°C in the dark for 30 minutes, washed twice with pre-cooled buffer, and observed by fluorescence microscopy or detected by flow cytometry (488 nm laser, FITC and PE channels). The mitochondrial membrane potential level was analyzed using FlowJo X software.

[0113] 3.3.4 Cellular ATP content: After collecting NP cells from each group, resuspend them in lysis buffer, homogenize for 5 minutes, boil for 2 minutes, cool under running water, centrifuge at 4 ℃ (10000 g) for 15 minutes, and collect the supernatant for later use on ice. Prepare gradient ATP standard solutions to plot a standard curve. Mix enzyme reagent and buffer at a ratio of 1:50 to prepare working solution. Mix the sample and standard with the working solution and shake for 15 seconds. Use a Thermo Fisher chemiluminescent microplate reader to detect the luminescence value and calculate the ATP content.

[0114] 3.3.5 Glutathione (GSH) content: Collect NP cells from each group, resuspend homogenate in PBS, centrifuge at 10000g for 15 minutes at 4℃, mix the supernatant with an equal volume of protein removal reagent, vortex, and centrifuge again under the same conditions. Collect the supernatant for testing. Prepare GSH gradient standard solutions to plot a standard curve. Mix the probe with pure water at a ratio of 1:19 to prepare a working solution, add the sample, mix and vortex for 1 minute, incubate at room temperature for 5 minutes, and measure the absorbance at 412 nm using an ELISA reader.

[0115] 3.4 Evaluation of Synergistic Effect The combined protective effect of the Ce-MOTS@EV vesicle delivery system was evaluated using the Jin Zhengjun Q-value method. Since this experiment aimed to assess the protective effect of the drug on cells (i.e., increased survival rate and restoration of ATP / GSH levels), survival rate or relative concentration was directly used as the effect value (E) and substituted into the formula: Q = Ea+b / (E a + E b - E a × E b ); where E a+b : Effect value of the Ce-MOTS@EV vesicle delivery system treatment group, i.e., survival rate / relative content of the Ce-MOTS@EV vesicle delivery system treatment group; E a and E b The effect values ​​are for each individual component, namely the survival rate / relative content in the MOTS-c peptide monotherapy group and the Ce-TA monotherapy group.

[0116] The criteria for judgment are as follows: Q<0.85 indicates antagonism (the combined protective effect is lower than expected), 0.85≤Q≤1.15 indicates summation (the combined effect is comparable to the expected effect), and Q>1.15 indicates synergy (the combined effect is better than expected).

[0117] Table 1 Ce Cell viability of MOTS@EV and its components after TBHP treatment of nucleus pulposus cells

[0118] Table 2 Ce Relative values ​​of ATP content in TBHP-treated nucleus pulposus cells after treatment with MOTS@EV and its components

[0119] Table 3 Ce Relative values ​​of GSH content in TBHP-treated nucleus pulposus cells after treatment with MOTS@EV and its components

[0120] 4. Results 4.1 Cellular uptake efficiency like Figure 7 As shown in Figure A, under confocal microscopy, the intracellular fluorescence signal of the Ce-MOTS@EV group was significantly stronger than that of the free nanoparticle group (Ce-TA / MOTS@HA), and the fluorescence was mainly distributed in the cytoplasm, proving that the nanoparticles had successfully entered the cell interior; the cytoskeleton (red) and cell nucleus (blue) morphology of both groups remained intact, indicating that the two nanomaterials had no obvious cytotoxicity at the experimental concentration; flow cytometry quantitative analysis ( Figure 7 B) shows that the mean fluorescence intensity (MFI) of the Ce-MOTS@EV group was 2.47 times that of the free nanoparticle group (p<0.01), indicating that vesicle encapsulation significantly improved the efficiency of the delivery system into NP cells.

[0121] 4.2 Cell viability Cell survival was significantly reduced in the TBHP model group. Treatment with MOTS-c peptide or Ce-TA nanozyme alone partially improved survival (0.715 ± 0.034 and 0.723 ± 0.042, respectively), but the effects were limited. In contrast, the Ce-MOTS@EV treatment group showed a survival rate as high as 0.943 ± 0.046, almost completely reversing TBHP damage, with a Q value (synergistic coefficient) of 1.02, demonstrating an additive protective effect (Table 1).

[0122] 4.3 ROS removal capability Flow cytometry ( Figure 8 ) and JC-1 testing ( Figure 9-10 The results showed that Ce-MOTS@EV could most effectively remove excess ROS induced by TBHP and significantly restore the reduced mitochondrial membrane potential, with a protective effect significantly superior to any single component.

[0123] 4.4 Synergistically reverse metabolic disorders and enhance cellular energy: Figure 11 The results showed that Ce-MOTS@EV most effectively restored cellular energy levels, exhibiting the best effect and being closest to the control group. After normalization, its relative ATP content was 1.000 ± 0.013, significantly higher than the MOTS-c group (0.645 ± 0.024) and the Ce-TA group (0.583 ± 0.019), with a synergistic Q value of 1.17, demonstrating that the synergistic effect of the two components greatly promoted energy metabolism (Table 2).

[0124] Ce-MOTS@EV also demonstrated the strongest ability to enhance GSH, a key intracellular antioxidant, far exceeding that of single-component formulations. Figure 12 After normalization using the Ce-MOTS@EV group as a baseline, the relative GSH content in the Ce-MOTS@EV group was 1.000 ± 0.022, significantly higher than that in the MOTS-c group (0.560 ± 0.016) and the Ce-TA group (0.572 ± 0.028). The Q value calculated according to the formula was 1.23, indicating that the Ce-MOTS@EV vesicle delivery system has a strong synergistic effect in enhancing the cellular antioxidant defense system (Table 3).

[0125] 5. Conclusion This embodiment demonstrates through in vitro cell experiments that the Ce-MOTS@EV vesicle delivery system can efficiently enter nucleus pulposus cells, significantly clear ROS, and restore mitochondrial function. Key data show that, compared to cerium-tannic acid nanozymes or MOTS-c peptides used alone, this system exhibits synergistic therapeutic effects in ATP restoration (Q = 1.17) and GSH enhancement (Q = 1.23), and also shows an additive effect in cell survival protection (Q = 1.02). Its therapeutic effect is significantly superior to that of single components, verifying the effectiveness and superiority of this vesicle delivery system in combating oxidative stress and aging in nucleus pulposus cells.

[0126] Example 3: Ce MOTS@EV Mechanism of Action 1. Purpose This embodiment aims to explore the molecular mechanism of the two-component vesicle delivery system (Ce-MOTS@EV) in treating intervertebral disc degeneration, focusing on verifying its activation of key signaling pathways and its regulatory mechanism on cellular senescence-related proteins, and elucidating the specific pathway by which cerium nanozymes and MOTS-c peptide synergistically reverse stress-induced senescence of nucleus pulposus cells.

[0127] 2. Experimental Materials Cell grouping and treatment: Same as in Example 2.

[0128] Main reagents and instruments: Transcriptome sequencing: RNA extraction kit (ThermoFisher, 15596026CN), high-throughput sequencing platform and related bioinformatics analysis software Illumina.

[0129] Western blot: protein extraction lysis buffer, SDS-PAGE gel, PVDF membrane, chemiluminescence imaging system, and other antibodies were purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0130] 3. Experimental Methods 3.1 Transcriptome Sequencing and Bioinformatics Analysis Total RNA was extracted from NP cells using TRIzol, and its purity, concentration, and integrity were assessed. RNA sequencing libraries were constructed using the extracted RNA. After quality control, the libraries were sequenced on a high-throughput sequencing platform (Illumina), generating raw image data, which was then converted into raw reads. The raw data underwent quality control filtering to remove low-quality sequences, yielding clean reads. The clean reads were aligned to a reference genome for quantification. Differentially expressed genes were identified using FDR < 0.05 and |log2FC| ≥ 1 as screening criteria. Based on these differentially expressed genes, GO functional annotation, KEGG pathway enrichment analysis, and gene set enrichment analysis (GSEA) were performed. GO and KEGG analyses were performed using the DAVID functional annotation bioinformatics tool. GSEA was performed using GSEA 4.3.2 software with default parameters.

[0131] 3.2 Western blot verification of protein expression levels Cellular protein samples were collected from each group to detect the expression levels of proteins in the following key signaling pathways: integrated stress response (ISR)-related proteins and cellular senescence-related marker proteins (such as p16 and p21). The specific experimental method was as follows: 20 μg of protein from each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane. The membrane was blocked in blocking buffer for 30 minutes and then incubated overnight with diluted primary antibody. Subsequently, the membrane was washed three times with TBST buffer and then incubated for one hour with HRP-labeled secondary antibody against rabbit or mouse. After washing three more times with TBST, the membrane was developed on a Bio-Rad system using ECL chemiluminescence reagent. The band density of the three independent experiments was quantified using ImageJ 1.5 software.

[0132] 4. Results 4.1 Transcriptome sequencing: Considering cost and data analysis complexity, differentially expressed genes were analyzed only in the control group (PBS) and the Ce-MOTS@EV treatment group. Transcriptome results ( Figure 13 The results showed that Ce-MOTS@EV treatment significantly upregulated genes related to antioxidant and glutathione metabolism, while downregulating pathways related to inflammation, cell cycle, and aging.

[0133] 4.2 Protein level validation: Western blot analysis Figure 14-15The results showed that, compared with the control group, TBHP treatment significantly downregulated the protein expression levels of p-EIF2α and ATF4, while significantly upregulating the expression of aging markers P21 and P16 (p<0.001), indicating the successful construction of a model of oxidative stress-induced cell damage and aging. Extracellular vesicle treatment alone did not show statistically significant improvement in the above indicators, suggesting that extracellular vesicles alone have no significant protective effect. In contrast, Ce-MOTS@EV treatment significantly reversed the protein expression changes induced by TBHP: on the one hand, it significantly upregulated the expression of p-EIF2α and ATF4, restoring the activation of the endoplasmic reticulum stress pathway; on the other hand, it significantly downregulated the expression of P21 and P16, inhibiting the cellular senescence phenotype (p<0.001), and both effects were significantly better than the EV group.

[0134] 5. Conclusion This embodiment demonstrates at the molecular level that Ce-MOTS@EV effectively alleviates TBHP-induced cell damage by synergistically regulating mitochondrial homeostasis, endoplasmic reticulum stress adaptation, and senescence inhibition pathways, providing a basis for Ce... MOTS@EV provides a solid mechanistic basis for the treatment of intervertebral disc degeneration.

[0135] Example 4: Verification of Ce In vivo efficacy of MOTS@EV in a rat model of intervertebral disc degeneration 1. Purpose This embodiment aims to evaluate the in vivo structural repair capability of the two-component vesicle delivery system (Ce-MOTS@EV) by establishing a rat caudal intervertebral disc degeneration model, and to verify its actual efficacy in treating intervertebral disc degeneration through three core indicators: intervertebral disc height, magnetic resonance signal, and histopathological score.

[0136] 2. Experimental Materials Experimental animals: 30 male Sprague-Dawley rats, each 8 weeks old and weighing approximately 200g, of SPF grade.

[0137] Model inducer: tumor necrosis factor-α (TNF-α, 100 ng / mL).

[0138] Therapeutic formulations: PBS, EVs derived from nucleus pulposus cells (50 μg / mL), Ce-TA / MOTS complex prepared in Example 1 (0.5 mg / mL, i.e., the nanoparticles described in this invention), and Ce-MOTS@EV (50 μg / mL).

[0139] Main reagents: Hematoxylin-eosin (H&E) staining solution, Safranin O-Fix Green staining solution, Masson's trichrome staining solution, anti-COL2A1 antibody (Abcam, ab307674), anti-Aggrecan antibody (Abcam, ab315487).

[0140] Main instruments: X-ray imaging system, small animal magnetic resonance imaging (MRI) instrument, paraffin slicer.

[0141] 3. Experimental Methods 3.1 Model Building and Grouping All animal experiments were approved by the Institutional Animal Care and Use Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. Male Sprague-Dawley rats were selected, and the intervertebral discs of the 8th and 9th caudal vertebrae were accurately located by palpation and imaging. In the sham-operated group, only a 33G puncture needle was inserted into the intervertebral disc and an equal volume of PBS was injected, without any other treatment. In the IDD model group, 2 μL of TNF-α at a concentration of 100 ng / mL was slowly injected into the nucleus pulposus with a 33G puncture needle. Intervention was carried out twice a week for two consecutive weeks to establish an intervertebral disc degeneration model.

[0142] After modeling, Sprague-Dawley rats were randomly divided into 5 groups (n = 6), and the intervention details are as follows: Sham surgery group: A 33G needle was inserted into the Co8 / 9 intervertebral disc of the coccyx, and an equal volume and frequency of PBS were injected.

[0143] IDD model group: 2 μL of TNF-α was injected into the nucleus pulposus after puncture, twice a week for 2 weeks to establish the IDD model.

[0144] EVs treatment group: After modeling, 2 μL of nucleus pulposus cell-derived EVs (50 μg / mL) were injected into the nucleus once a week for 4 weeks.

[0145] Ce-MOTS treatment group: After modeling, 2 μL Ce-MOTS (0.5 mg / mL) was injected into the nucleus once a week for 4 weeks.

[0146] Ce-MOTS@EV treatment group: After modeling, 2 μL of Ce-MOTS@EV (50 μg / mL) was injected into the nucleus once a week for 4 weeks.

[0147] 3.2 In vivo efficacy assessment: 3.2.1 Imaging assessment: X-ray examination: X-ray images of the rat caudal spine were taken at 4 and 8 weeks after treatment to measure the intervertebral disc height. The degree of intervertebral disc structural collapse and height loss caused by intervertebral disc degeneration was quantitatively assessed by the change in the %DHI value, according to the formula: Intervertebral Disc Height Index (%DHI) = Intervertebral Disc Height / (Height of Adjacent Superior Vertebral Body + Height of Adjacent Inferior Vertebral Body) × 100%.

[0148] Magnetic resonance imaging (MRI) examination: T2-weighted magnetic resonance imaging was used for scanning and detection. The changes in intervertebral disc water content were assessed based on the Pfirrmann grading system, and the severity of intervertebral disc degeneration was determined accordingly.

[0149] 3.2.2 Histological and Molecular Assessment: Rats were sacrificed 6 weeks after the last treatment to obtain intervertebral disc tissue.

[0150] Histological staining: Sections were stained with H&E (to observe the overall structure), Safranin O-Fix Green (to assess proteoglycan content), and Masson stain (to observe collagen fibers), and the tissue degeneration grade was scored.

[0151] Immunohistochemistry: Detect the expression levels of type II collagen (COL2A1) and aggrecan (ACAN) in nucleus pulposus tissue to quantify the synthesis of extracellular matrix.

[0152] 4. Results 4.1 Imaging improvements: like Figure 16 As shown in Figure A, the percentage of intervertebral disc height index in the IDD model group decreased significantly over time, indicating disc collapse. The Ce-MOTS@EV treatment group significantly maintained intervertebral disc height, and its percentage of intervertebral disc height index was significantly higher than that of the model group, EVs treatment group, and Ce-MOTS treatment group, approaching the level of the sham surgery group.

[0153] like Figure 16 As shown in Figure B, the intervertebral discs in the IDD model group exhibited decreased and uneven signal intensity on T2-weighted images, indicating dehydration and structural disorder, with an increased degeneration grade. In the Ce-MOTS@EV treatment group, the T2 signal intensity of the intervertebral discs was well maintained, and the degeneration grade was significantly lower than that in the model group, demonstrating its ability to effectively delay intervertebral disc dehydration and structural damage.

[0154] 4.2 Histology and Matrix Synthesis Restoration: like Figure 17 As shown, based on the general pathological scoring criteria for intervertebral disc tissue, the histological score of the intervertebral disc degeneration model group was significantly higher than that of the sham surgery group, indicating that the pathological injury model of intervertebral disc degeneration was successfully constructed. Compared with the IDD group, the histological score of the EVs intervention group was not significantly reduced, and the improvement effect was weak; while the histological score of the Ce-MOTS@EV intervention group was significantly reduced (p<0.001), and the effect was significantly better than that of the Ce-MOTS intervention group, confirming that Ce-MOTS@EV can significantly alleviate the structural damage of the intervertebral disc nucleus pulposus tissue, inhibit the loss of extracellular matrix, and has the best anti-degeneration effect.

[0155] Immunohistochemical results further showed that the expression levels of type II collagen and proteoglycans in the intervertebral disc tissue of the Ce-MOTS@EV treatment group were significantly higher than those in the model group and other treatment groups, indicating that this system can effectively promote the synthesis of functional extracellular matrix by nucleus pulposus cells and reverse the degenerative process. Figure 18 ).

[0156] 5. Conclusion This embodiment demonstrates, using a rat IDD model, that the Ce-MOTS@EV two-component vesicle delivery system can significantly restore intervertebral disc height, improve MRI signal intensity, and repair histopathological structures in vivo. It not only effectively delays disc height loss and water loss on a macroscopic imaging basis, but also protects the nucleus pulposus structure and promotes the synthesis and retention of extracellular matrix (type II collagen, proteoglycans) on a microscopic histological basis. These results prove that the nanoparticles and their delivery system of this invention still possess significant therapeutic activity in the complex in vivo environment, providing strong in vivo experimental evidence for the regenerative treatment of intervertebral disc degeneration.

[0157] In summary, Ce-MOTS (nanoparticles) or Ce-MOTS@EV (vesicle delivery system) offer a novel treatment strategy with clear clinical translational potential for reversing or delaying intervertebral disc degeneration.

[0158] In the description of this specification, the references to terms such as "one embodiment / mode," "some embodiments / modes," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment / mode or example is included in at least one embodiment / mode or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment / mode or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments / modes or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments / modes or examples described in this specification, as well as the features of different embodiments / modes or examples.

[0159] It should be noted that in this invention, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise expressly specified.

[0160] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A vesicle delivery system, characterized in that, Includes extracellular vesicles and nanoparticles encapsulated within the extracellular vesicles; The nanoparticles include a core, a shell covering the surface of the core, and an outer shell covering the surface of the shell. The core includes a cerium-tannic acid nanozyme with antioxidant activity; The shell includes the mitochondrial-derived peptide MOTS-c; The outer shell layer includes hyaluronic acid; The extracellular vesicles are derived from nucleus pulposus cells.

2. The vesicle delivery system according to claim 1, characterized in that, The hydrated particle size of the vesicle delivery system is 200 nm to 400 nm.

3. A pharmaceutical composition, characterized in that, Includes the vesicle delivery system as described in claim 1 or 2, and a pharmaceutically acceptable carrier.

4. Use of the vesicle delivery system of claim 1 or 2 or the pharmaceutical composition of claim 3 in the preparation of a medicament for treating intervertebral disc degeneration.

5. A method for preparing the vesicle delivery system according to claim 1 or 2, characterized in that, Includes the following steps: Provides cerium-tannic acid nanozymes with antioxidant activity; The mitochondrial-derived peptide MOTS-c was coated onto the surface of the nanozyme to form a polypeptide shell, thus obtaining nanoparticles. Coating the surface of nanoparticles with a hyaluronic acid shell layer; Obtain extracellular vesicles derived from nucleus pulposus cells; The nanoparticles are encapsulated inside the extracellular vesicles to obtain the vesicle delivery system.

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