Hybrid exosome nanomotor based on pathological microenvironment driving and preparation method and application thereof

Abstract

This invention provides a hybrid exosome nanomotor driven by a pathological microenvironment, its preparation method, and its applications, belonging to the field of biomedical technology. The hybrid exosome nanomotor provided by this invention can utilize the unique oxidative microenvironment of the osteoarthritis lesion area to achieve self-driven movement, thereby actively and efficiently penetrating the dense extracellular matrix of cartilage tissue, significantly enhancing its enrichment and retention capacity in the lesion area, and promoting the specific uptake of therapeutic molecules by chondrocytes. This system not only achieves intelligent response and utilization to the disease microenvironment but also simultaneously alleviates local oxidative stress, ultimately achieving efficient, precise, and sustained therapeutic effects. This invention provides a novel, proactively targeted delivery strategy and treatment platform with promising clinical translation prospects for diseases such as osteoarthritis involving dense tissues or inflammatory microenvironments.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a hybrid exosome nanomotor driven by a pathological microenvironment, its preparation method, and its application. Background Technology

[0002] Osteoarthritis (OA) is a common degenerative joint disease characterized by progressive wear and tear of articular cartilage, abnormal chondrocyte metabolism, and joint inflammation. Because articular cartilage is an avascular, nerveless tissue with a dense extracellular matrix, traditional systemic drug delivery methods struggle to effectively reach the lesion site. Therefore, intra-articular injection has become a primary clinical strategy for direct drug delivery. However, intra-articular injection still faces significant challenges: injected drugs or nanocarriers are rapidly cleared by the synovium, resulting in a short residence time within the joint cavity; more importantly, they primarily rely on passive diffusion to penetrate the cartilage tissue, while the dense, negatively charged cartilage matrix forms a strong "molecular sieve" barrier, severely limiting the penetration of most macromolecules and nanoparticles, making it difficult for drugs to reach deep chondrocytes and limiting therapeutic efficacy.

[0003] Exosomes, as natural nanovesicles secreted by cells, are considered highly promising drug delivery carriers due to their excellent biocompatibility, low immunogenicity, and ability to cross biological barriers. In the treatment of osteoarthritis, researchers have attempted to load nucleic acid molecules with chondrogenic effects (such as microRNA-140) into exosomes for delivery via intra-articular injection. However, existing exosome delivery systems are essentially passive delivery systems; their distribution and penetration depend entirely on concentration gradients and random diffusion. They cannot actively overcome the physical barrier of the cartilage matrix, nor can they intelligently accumulate at the lesion in response to disease states.

[0004] Therefore, developing an exosome delivery system that can actively navigate, penetrate barriers, and specifically remain in the lesion area is key to improving the efficiency of drug treatment for osteoarthritis. Summary of the Invention

[0005] The purpose of this invention is to provide a hybrid exosome nanomotor driven by the pathological microenvironment, its preparation method and application, which solves the technical problems of existing exosome delivery systems in the treatment of osteoarthritis, such as the inability to effectively penetrate the dense cartilage matrix due to reliance on passive diffusion, insufficient targeting, and short residence time in the joint cavity, which makes it difficult to efficiently deliver therapeutic molecules to deep chondrocytes.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] The present invention provides an exosome nanomotor system, comprising a hybrid exosome carrier, self-driven nanoparticles loaded inside the hybrid exosome carrier, and a targeting molecule modified on the surface of the hybrid exosome carrier;

[0008] The self-driven nanoparticles are manganese dioxide nanoparticles.

[0009] The hybrid exosome carrier is formed by membrane fusion of exosomes from at least two different sources;

[0010] The target molecule is a target peptide that can specifically recognize cartilage tissue or cartilage cells.

[0011] Preferably, the hybrid exosome carrier also contains therapeutic molecules.

[0012] Preferably, the therapeutic molecule is a microRNA, and the microRNA is miR-140.

[0013] Preferably, the at least two different sources of exosomes include a first exosome derived from bone marrow mesenchymal stem cells and a second exosome derived from breast milk;

[0014] The targeting peptide is the cartilage-targeting peptide CAP.

[0015] Preferably, the manganese dioxide nanoparticles have a particle size of 50 nm to 100 nm;

[0016] The hybrid exosome carrier has an average particle size of 90 nm to 150 nm.

[0017] This invention also provides a method for preparing the above-mentioned exosome nanomotor system, comprising the following steps:

[0018] S1: Provides the first and second exosomes;

[0019] S2: Mix the first exosome with the second exosome and perform membrane fusion treatment to obtain a hybrid exosome carrier;

[0020] S3: Load manganese dioxide nanoparticles into the hybrid exosome carrier;

[0021] S4: The target molecule is modified onto the surface of the hybrid exosome carrier to obtain the exosome nanomotor system.

[0022] Preferably, in step S1, the first exosome is a bone marrow mesenchymal stem cell-derived exosome loaded with therapeutic microRNA, obtained by transfecting bone marrow mesenchymal stem cells with a lentiviral vector carrying the microRNA and collecting the culture supernatant for separation.

[0023] The lentiviral vector has an infection multiplicity of 50 to 100;

[0024] In step S1, the second exosome is a milk-derived exosome;

[0025] The membrane fusion process described in step S2 includes: first incubating the mixed exosome solution with shaking at 35°C to 39°C for 10 to 14 hours, and then extruding it through a filter membrane with a pore size of 300 nm to 500 nm 8 to 12 times;

[0026] Step S3 specifically includes: mixing the manganese dioxide nanoparticle suspension with the hybrid exosome carrier and treating it under ultrasound with a power of 80 W to 120 W for 4 to 6 minutes, so that the manganese dioxide nanoparticles are loaded inside the hybrid exosome carrier;

[0027] The mass ratio of the manganese dioxide nanoparticles to the hybrid exosome carrier is 0.5:1 to 1.5:1.

[0028] Step S4 specifically includes: coupling the target molecule to the lipid molecule via a linker molecule to form a modified lipid; mixing the modified lipid with a hybrid exosome carrier loaded with the manganese dioxide nanoparticles; and extruding the modified lipid into the membrane of the hybrid exosome carrier.

[0029] The linker molecule is DSPE-PEG2000-MAL, and the target molecule is coupled to it via a thiol-maleimide click chemistry reaction.

[0030] Preferably, the hybrid exosome carrier is prepared by a method comprising the following steps: extracting a first exosome and a second exosome; mixing the first exosome and the second exosome at a protein ratio of 0.5:1 to 1.5:1; incubating the mixture at 35°C to 39°C and at a rotation speed of 50 rpm to 70 rpm for 10 to 14 hours; continuously extruding the incubated mixture through a filter membrane with a pore size of 300 nm to 500 nm 8 to 12 times to induce membrane fusion; and purifying to obtain the hybrid exosome carrier.

[0031] The extraction method of bone marrow mesenchymal stem cell-derived exosomes loaded with therapeutic microRNA includes: collecting the culture supernatant of transfected cells, centrifuging at 300 g to 500 g for 8 to 12 minutes, centrifuging at 2000 g for 18 to 22 minutes, centrifuging at 10000 g for 28 to 32 minutes, filtering at 0.22 μm, and ultracentrifuging at 100000 g for 68 to 72 minutes.

[0032] The method for extracting milk exosomes includes: mixing skim milk with glacial acetic acid at a final concentration of 0.8% to 1.2%, centrifuging at 10,000 g to 14,000 g for 18 to 22 minutes to remove the precipitate, filtering the supernatant through 0.22 μm, and then ultracentrifuging at 170,000 g to 180,000 g for 68 to 72 minutes;

[0033] The manganese dioxide nanoparticles described in step S3 are prepared by a biomineralization method, which includes: mixing an 80 mM to 120 mM manganese salt solution with an equal volume of an 8 mg / mL to 12 mg / mL bovine serum albumin solution, adjusting the pH to 9.5 to 10.5, stirring at 35°C to 39°C for 1.5 to 2.5 hours, and then dialysis purification.

[0034] The mass ratio of the modified lipid to the hybrid exosome carrier is 5% to 15%.

[0035] The present invention also provides the application of the above-described exosome nanomotor system or the exosome nanomotor system prepared by the above-described preparation method in the preparation of a drug for treating osteoarthritis.

[0036] The present invention also provides the application of the above-described exosome nanomotor system or the exosome nanomotor system prepared by the above-described preparation method in the preparation of drugs for treating degenerative arthritis, tendon or ligament injury, articular cartilage defects, fibrotic diseases, ischemia-reperfusion injury or chronic inflammatory diseases.

[0037] The beneficial effects of this invention are:

[0038] The hybrid exosome nanomotor provided by this invention can autonomously drive its movement by utilizing the unique oxidative microenvironment of the osteoarthritis lesion area. This allows it to actively and efficiently penetrate the dense extracellular matrix of cartilage tissue, significantly enhancing its accumulation and retention capacity in the lesion area and promoting the specific uptake of therapeutic molecules by chondrocytes. This system not only achieves intelligent response and utilization of the disease microenvironment but also simultaneously alleviates local oxidative stress, ultimately achieving highly efficient, precise, and sustained therapeutic effects. This invention provides a novel, proactively targeted delivery strategy and treatment platform with promising clinical translation prospects for diseases such as osteoarthritis involving dense tissues or inflammatory microenvironments.

[0039] Specifically, its beneficial effects are achieved synergistically through the following technical features: First, by encapsulating manganese dioxide nanoparticles within hybrid exosomes, an endogenous hydrogen peroxide-driven "nanoengine" is constructed. This engine catalytically decomposes hydrogen peroxide in the pathological microenvironment into oxygen, generating continuous thrust and enabling the delivery system to acquire autonomous movement capabilities. Its average movement speed is increased by orders of magnitude in a hydrogen peroxide environment, and it can actively migrate towards the lesion core area with higher concentrations. Second, this self-driving force is effectively converted into strong tissue penetration. In an ex vivo cartilage model, the penetration depth of this nanomotor can reach approximately 140 micrometers, which is about four times that of ordinary exosomes that rely solely on passive diffusion, breaking through the physical barrier of the dense cartilage matrix. Furthermore, by covalently modifying the exosome membrane surface with chondrocyte-targeting peptides, specific recognition and anchoring of target cells are achieved. Quantitative analysis by flow cytometry shows that its uptake efficiency of chondrocytes is more than 2.5 times higher than that of unmodified exosomes. The cascade mechanism of "autonomous movement-deep penetration-targeted anchoring" significantly prolongs the intra-articular retention time of this system in osteoarthritis model animals, with approximately 30% signal retention still observed two weeks after injection, far exceeding traditional formulations. Finally, while efficiently delivering therapeutic miR-140, the system's catalytic process continuously consumes hydrogen peroxide at the lesion site, directly improving the harmful oxidative microenvironment. Experiments have demonstrated its ability to effectively reduce intracellular reactive oxygen species levels, restore mitochondrial membrane potential, inhibit apoptosis, and upregulate the expression of matrix synthesis markers such as collagen II, achieving synchronization and synergistic effects between "delivery" and "treatment." Attached Figure Description

[0040] Figure 1 A schematic diagram of the system structure of a hybrid exosome nanomotor;

[0041] Figure 2 A schematic diagram illustrating the overall workflow of the hybrid exosome nanomotor;

[0042] Figure 3 Figure 1 shows the basic characteristic identification results of bExos and mExos; where: (A) representative TEM images and particle size distribution analysis; (B) WB detection results of exosome marker proteins;

[0043] Figure 4 Figure 1 shows the morphological characteristics and catalytic performance characterization results of MnO2 NPs; where: (A) representative TEM images; (B) catalytic activity evaluation under H2O2 conditions;

[0044] Figure 5 For DSPE-PEG 2000 Figure 1H-NMR characterization results of CAP;

[0045] Figure 6 For DSPE-PEG2000 -FT-IR analysis results of CAP;

[0046] Figure 7 The figure shows the verification results of CAP modification on the mExos surface based on Zeta potential analysis.

[0047] Figure 8 The figure shows the optimized results of the feeding ratio of MnO2 NPs and mExos.

[0048] Figure 9 Figure showing the results of optimizing lentivirus transduction conditions and corresponding quantitative analysis of fluorescence intensity using confocal microscopy imaging;

[0049] Figure 10 Figure showing the results of optimized transduction conditions and corresponding quantitative analysis of fluorescence intensity using confocal microscopy imaging (*: p ≤ 0.05; ***: p ≤ 0.001).

[0050] Figure 11 Characterization results of hybrid exosomes constructed by dual-source exosome membrane fusion; where: (A) Membrane fusion efficiency between exosomes under different construction strategies was evaluated based on FRET technology; (B) Particle size distribution and semi-quantitative analysis of PDI of the corresponding samples;

[0051] Figure 12 The SDS-PAGE analysis results for the protein composition integrity of each exosome preparation are shown in the figure.

[0052] Figure 13 Representative TEM images and particle size distribution results for each exosome formulation;

[0053] Figure 14 The graph shows the 7-day monitoring results of the hydrodynamic diameter and PDI of various exosome preparations in simulated synovial fluid;

[0054] Figure 15 XPS analysis results of manganese chemical state in various exosome preparations;

[0055] Figure 16 The graph shows the trajectory analysis and semi-quantitative velocity assessment results of exosomal nanomotors in the presence and absence of H2O2. In the graph, A is the trajectory diagram of different exosomal formulations, and B is the velocity statistics of different exosomal formulations (ns: p>0.05; **: p≤0.01).

[0056] Figure 17 The graph shows the kinematic response behavior of various exosome preparations induced by H2O2 concentration gradient.

[0057] Figure 18Confocal microscopy images of DiI-labeled exosome preparations after co-incubation with cartilage tissue for 2, 4, and 6 days;

[0058] Figure 19 Quantitative analysis results of tissue penetration depth of various DiI-labeled exosome preparations after co-incubation with cartilage tissue for 2, 4, and 6 days (***: p ≤ 0.001);

[0059] Figure 20 Confocal micrographs of chondrocyte uptake of various DiI-labeled exosome preparations and corresponding semi-quantitative analysis results (***: p ≤ 0.001).

[0060] Figure 21 Figure 1 shows the results of flow cytometry quantitative analysis of chondrocyte uptake of various DiI-labeled exosome preparations (***: p ≤ 0.001).

[0061] Figure 22 The figure shows the results of in vivo fluorescence imaging and semi-quantitative analysis of fluorescence signal retention time after various exosome preparations were injected into the joint cavity of rat OA via intra-articular injection.

[0062] Figure 23 Figure 1 shows the results of in vivo fluorescence imaging and semi-quantitative analysis of fluorescence signal retention in OA and healthy joints after intra-articular injection of CAP-Mn@hyExos.

[0063] Figure 24 Image showing the live / dead staining results of OA chondrocytes treated with various exosome preparations;

[0064] Figure 25 The graph shows the blood compatibility evaluation results of various exosome preparations;

[0065] Figure 26 Image showing cytoskeleton staining results of OA chondrocytes treated with various exosome preparations;

[0066] Figure 27 CCK-8 assay results for OA chondrocytes treated with various exosome preparations (ns: p>0.05; *: p ≤0.05; ***: p ≤ 0.001).

[0067] Figure 28 Figure 1 shows the results of scratch assay and migration analysis of OA chondrocytes treated with various exosome preparations (ns: p>0.05; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001).

[0068] Figure 29Figure 1 shows the results of ROS level detection (DCFH-DA) and semi-quantitative analysis of chondrocytes after treatment with various exosome preparations (***: p ≤ 0.001).

[0069] Figure 30 The results of JC-1 staining and semi-quantitative analysis of mitochondrial membrane potential in chondrocytes after treatment with various exosome preparations are shown in the figure (**: p ≤ 0.01; ***: p ≤ 0.001).

[0070] Figure 31 Figure 1 shows the flow cytometry results and semi-quantitative analysis of chondrocyte apoptosis after treatment with various exosome preparations (**: p ≤ 0.01; ***: p ≤ 0.001).

[0071] Figure 32 Figures showing the results of live / dead staining and semi-quantitative analysis of chondrocytes after treatment with various exosome preparations (***: p ≤ 0.001).

[0072] Figure 33 Figure 1 shows the results of detecting antioxidant-related indicators in chondrocytes after treatment with various exosome preparations (*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001).

[0073] Figure 34 Immunofluorescence detection and semi-quantitative analysis results of OA chondrocytes treated with various exosome preparations (**: p ≤ 0.01; ***: p ≤ 0.001);

[0074] Figure 35 Figures showing the results of Western blot (WB) detection and semi-quantitative analysis of OA chondrocytes treated with various exosome preparations (*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001).

[0075] Figure 36 qPCR detection results of OA chondrocytes treated with various exosome preparations (*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001);

[0076] Figure 37 Histological staining and semi-quantitative analysis results of OA chondrocytes treated with various exosome preparations (*: p≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001). Detailed Implementation

[0077] This invention provides an exosome nanomotor system driven by a pathological microenvironment. This system integrates delivery, actuation, and targeting functions, aiming to overcome the challenge of drugs failing to penetrate dense cartilage tissue and precisely target diseased cells in existing technologies. Specifically, this invention provides an exosome nanomotor system comprising a hybrid exosome carrier, self-driven nanoparticles loaded within the hybrid exosome carrier, and targeting molecules modified on the surface of the hybrid exosome carrier. In this invention, the "hybrid exosome carrier" refers to a single vesicle entity formed by fusing two or more exosome membrane structures from different cell sources through physical or chemical methods. This carrier possesses the membrane components and functional characteristics of exosomes from various sources; for example, it can simultaneously exhibit good biocompatibility and high structural stability. The "self-driven nanoparticles" refer to nanoscale particles capable of converting chemical energy or other forms of energy into their own propulsion under specific external stimuli or in the presence of endogenous substances. The term "targeting molecule" refers to a molecule that can specifically recognize and bind to biomarkers (such as receptors, antigens, etc.) on the surface of specific tissues, cells, or organelles, such as peptides, antibodies, nucleic acid aptamers, or fragments thereof.

[0078] In a preferred embodiment, the self-driven nanoparticles are manganese dioxide nanoparticles. Manganese dioxide is a common transition metal oxide, and its nanoparticles exhibit good catalytic activity and biocompatibility. In this invention, the manganese dioxide nanoparticles are preferably prepared by conventional methods such as biomineralization, hydrothermal methods, coprecipitation, or sol-gel methods. The particle size of the manganese dioxide nanoparticles is preferably 50 nm to 100 nm, for example, 60 nm, 70 nm, 80 nm, or 90 nm; more preferably 60 nm to 80 nm; and most preferably, the particle size is about 70 nm. Such a size facilitates effective encapsulation by exosomes and ensures a sufficient catalytic reaction interface.

[0079] In a preferred embodiment, the hybrid exosome carrier is formed by membrane fusion of exosomes from at least two different sources. Exosomes are extracellular vesicles with a diameter of approximately 30-150 nm secreted by cells, naturally present in various body fluids, and carrying bioactive molecules such as proteins and nucleic acids. "Different sources" can refer to exosomes from different species (e.g., humans, mice, cattle), different tissues (e.g., bone marrow, fat, milk), or different cell types (e.g., stem cells, immune cells, tumor cells). More preferably, the at least two different sources of exosomes include a first exosome derived from bone marrow mesenchymal stem cells and a second exosome derived from milk. Exosomes derived from bone marrow mesenchymal stem cells are typically rich in bioactive molecules related to tissue repair; while milk-derived exosomes (e.g., bovine milk exosomes) have the advantages of wide availability, high yield, and relatively low cost. The average hydrodynamic diameter of the hybrid exosome carrier is preferably 90 nm to 150 nm, for example, 100 nm, 110 nm, 120 nm, or 130 nm; more preferably 100 nm to 130 nm.

[0080] In a preferred embodiment, the targeting molecule is a targeting peptide capable of specifically recognizing cartilage tissue or chondrocytes. Peptides are a class of biomolecules composed of amino acids linked by peptide bonds; their molecular weight is typically smaller than that of proteins, and they possess low immunogenicity. The targeting peptide can be a linear peptide or a cyclic peptide, and can be obtained through phage display technology, peptide library screening, or rational design based on known receptor ligands. More preferably, the targeting peptide is the cartilage-targeting peptide CAP. CAP peptide is a known polypeptide sequence capable of specifically binding to components on the extracellular matrix or surface of chondrocytes.

[0081] In a further preferred embodiment, the hybrid exosome carrier further loads a therapeutic molecule. A therapeutic molecule is a chemical substance or biomolecule that has preventative, alleviating, or therapeutic effects on diseases. Preferably, the therapeutic molecule is a nucleic acid drug, more preferably a microRNA. MicroRNAs are a class of non-coding single-stranded RNA molecules approximately 18-25 nucleotides in length that can regulate gene expression at the post-transcriptional level. Most preferably, the microRNA is miR-140. miR-140 has been extensively studied and confirmed to play an important role in cartilage homeostasis and osteoarthritis, and can target and regulate multiple genes related to cartilage catabolism and inflammation.

[0082] This invention also provides a method for preparing the above-mentioned exosome nanomotor system. The method has clear steps and high reproducibility, and includes the following steps:

[0083] S1: Provides the first and second exosomes;

[0084] S2: Mix the first exosome with the second exosome and perform membrane fusion treatment to obtain a hybrid exosome carrier;

[0085] S3: Load manganese dioxide nanoparticles into the hybrid exosome carrier;

[0086] S4: The target molecule is modified onto the surface of the hybrid exosome carrier to obtain the exosome nanomotor system.

[0087] In step S1, the first exosome is preferably a bone marrow mesenchymal stem cell-derived exosome loaded with a therapeutic microRNA (such as miR-140). Its acquisition typically involves cell culture, genetic engineering, and ultracentrifugation. Specifically, bone marrow mesenchymal stem cells are transfected with a lentiviral vector carrying the target microRNA sequence, causing the cells to stably overexpress the microRNA and secrete it into exosomes. Lentiviral vectors are commonly used gene delivery tools, and their multiple of infection (MOI) is a key parameter affecting transfection efficiency and cytotoxicity. In this method, the MOI of the lentiviral vector is preferably 50 to 100, for example 60, 70, 80, or 90; more preferably 65 to 85; and most preferably about 75. After collecting the transfected cell culture supernatant, a series of separation and purification steps are required to obtain high-purity exosomes. The preferred extraction method includes: sequentially centrifuging at 300 g to 500 g (e.g., 350 g, 400 g, 450 g) for 8 to 12 minutes (e.g., 9, 10, 11 minutes) to remove suspended cells; followed by centrifugation at 2000 g for 18 to 22 minutes (e.g., 19, 20, 21 minutes) to remove cell debris; then centrifugation at 10000 g for 28 to 32 minutes (e.g., 29, 30, 31 minutes) to remove large vesicles; filtering the supernatant through a 0.22 μm microporous membrane to remove any remaining particulate matter; and finally, ultracentrifugation at 100000 g for 68 to 72 minutes (e.g., 69, 70, 71 minutes) to precipitate exosomes. The speed and time of the centrifugation steps can be routinely adjusted according to the specific centrifuge model and sample volume.

[0088] In step S1, the second exosome is preferably a milk-derived exosome. The extraction method typically includes steps such as defatting, removal of major proteins, and ultracentrifugation. Specifically, skim milk can be mixed with an acid (such as acetic acid or citric acid) to precipitate casein. Preferably, glacial acetic acid is used, with a final concentration preferably from 0.8% to 1.2% (v / v), for example, 0.9%, 1.0%, or 1.1%; more preferably from 0.9% to 1.1%. After centrifuging the mixture at 10,000 g to 14,000 g (e.g., 11,000 g, 12,000 g, or 13,000 g) for 18 to 22 minutes (e.g., 19, 20, or 21 minutes), the precipitate is discarded, and the supernatant (whey) is collected. The supernatant was filtered through a 0.22 μm filter membrane and then subjected to ultracentrifugation at 170,000 g to 180,000 g (e.g., 172,000 g, 175,000 g, 178,000 g) for 68 to 72 minutes (e.g., 69, 70, 71 minutes) to collect the milk exosome precipitate.

[0089] The membrane fusion process in step S2 is crucial for forming the hybrid carrier. Its purpose is to fuse exosome membranes from different sources, forming a single vesicle with a hybrid membrane structure. This process can include incubation, freeze-thaw cycles, ultrasonic treatment, or extrusion. Preferably, the membrane fusion process includes: first, incubating the exosome solution mixed in a certain ratio (e.g., a protein-to-protein ratio of 0.5:1 to 1.5:1) with shaking at 35°C to 39°C (e.g., 36°C, 37°C, 38°C) for 10 to 14 hours (e.g., 11, 12, 13 hours); then, extruding the incubated mixture through a polycarbonate filter membrane with a pore size of 300 nm to 500 nm (e.g., 350 nm, 400 nm, 450 nm) 8 to 12 times (e.g., 9, 10, 11 times). Incubation utilizes the membrane's fluidity to promote spontaneous fusion, while extrusion forces membrane rupture and recombination through physical shear force; the combination of both improves fusion efficiency and product uniformity.

[0090] Step S3 involves loading the driving component into the carrier. The loading of the manganese dioxide nanoparticles can be achieved through co-incubation, electroporation, ultrasonic loading, or active loading techniques. Preferably, step S3 specifically includes: mixing a pre-prepared manganese dioxide nanoparticle suspension with the hybrid exosome carrier obtained in step S2, and treating it under ultrasound at a power of 80 W to 120 W (e.g., 90 W, 100 W, 110 W) in a pulsed mode (e.g., 1 second on, 1 second off) for 4 to 6 minutes (e.g., 4.5, 5, 5.5 minutes), utilizing the ultrasonic cavitation effect to promote the nanoparticles to enter the exosome. The mass ratio of the manganese dioxide nanoparticles to the hybrid exosome carrier is an important factor affecting the loading rate and carrier integrity, preferably 0.5:1 to 1.5:1 (e.g., 0.8:1, 1:1, 1.2:1); more preferably 0.8:1 to 1.2:1; and most preferably about 1:1. The manganese dioxide nanoparticles used in step S3 can be synthesized by a variety of chemical methods, preferably by biomineralization, which is a mild method that allows for easy control of particle size. The preferred biomineralization method includes: mixing an equal volume of a manganese salt solution (e.g., manganese chloride, manganese sulfate) with a concentration of 80 mM to 120 mM (e.g., 90 mM, 100 mM, 110 mM) and a bovine serum albumin solution with a concentration of 8 mg / mL to 12 mg / mL (e.g., 9, 10, 11 mg / mL); adjusting the pH of the mixture to 9.5 to 10.5 (e.g., 9.8, 10.0, 10.2) using an alkali (e.g., sodium hydroxide); and magnetically stirring at 35°C to 39°C (e.g., 36°C, 37°C, 38°C) for 1.5 to 2.5 hours (e.g., 1.8, 2.0, 2.2 hours) to complete the reaction; subsequently placing the reaction solution in a dialysis bag and dialyzing with deionized water for 24-72 hours to remove impurities, thereby obtaining a purified manganese dioxide nanoparticle suspension.

[0091] Step S4 involves functionalizing the carrier surface to endow it with targeting capabilities. Methods for immobilizing the targeting molecule include physical adsorption, covalent coupling, and lipid insertion. Preferably, step S4 specifically includes: first, coupling the targeting molecule to a lipid molecule via a linker molecule to form a modified lipid. The linker molecule is preferably DSPE-PEG2000-MAL, an amphiphilic molecule whose DSPE (distearate phosphatidylethanolamine) portion can insert into the lipid membrane, the PEG (polyethylene glycol) chain provides steric hindrance and flexibility, and the terminal MAL (maleimide) group can undergo a click chemistry reaction with thiol-containing molecules. The targeting molecule, through its terminal modified thiol group, undergoes a thiol-maleimide click chemistry reaction with the terminal maleimide group of the DSPE-PEG2000-MAL molecule under mild conditions (room temperature, pH 6.5-7.5), thereby forming a stable thioether bond. The formed modified lipids are then mixed with a hybrid exosome carrier loaded with manganese dioxide nanoparticles. The modified lipids are then inserted into the lipid bilayer of the hybrid exosome carrier via extrusion (e.g., several times through a 400 nm filter membrane) or incubation, thereby displaying the targeting peptide on the carrier surface. The mass ratio of the modified lipids to the hybrid exosome carrier is preferably 5% to 15% (e.g., 6%, 8%, 10%, 12%); more preferably 8% to 12%; and most preferably about 10%.

[0092] The present invention also provides the application of the above-described exosome nanomotor system, or the exosome nanomotor system prepared by any of the above preparation methods, in the preparation of drugs.

[0093] The cascaded hybrid exosome nanomotor system driven by the oxidative microenvironment of osteoarthritis provided by this invention integrates a self-driven module, a target recognition module, and a therapeutic molecule delivery module in a single delivery carrier, thereby achieving active delivery and precise treatment of osteoarthritis lesions. Figure 1 From a system structure perspective, the technical solution of this invention includes at least the following functional modules: a hybrid exosome carrier module; an oxidative microenvironment-responsive self-driven module; a cartilage-targeting recognition module; and a therapeutic molecule loading and release module. These modules are not simply parallel, but rather form a cascaded delivery system through structural coupling and functional synergy. Their working sequence and functional logic are: autonomous motion navigation → cartilage matrix penetration → cellular uptake and internalization → therapeutic molecule release for treatment.

[0094] 1. Specific technical implementation scheme of hybrid exosome carrier module

[0095] 1) Structural composition of hybrid exosomes

[0096] The delivery carrier in this invention is a hybrid exosome structure, which is composed of at least two exosomes from different sources via membrane fusion: the first exosome is derived from bone marrow mesenchymal stem cells and is used to load therapeutic nucleic acid molecules; the second exosome is derived from milk exosomes or other high-yield biological sources and is used to carry inorganic nanoparticles and targeted peptide modifications, and to enhance structural stability. The fused hybrid exosome retains its complete lipid bilayer membrane structure and inherits the membrane protein characteristics of the original exosome.

[0097] 2) Preparation method of hybrid exosomes (method-based technical solution)

[0098] The preparation of this hybrid exosome includes at least the following steps: extracting exosomes from different sources; mixing the two types of exosomes in a predetermined ratio; and inducing membrane fusion by physical incubation, extrusion, freeze-thaw cycles, or ultrasonic treatment.

[0099] The fused hybrid exosomes were purified and characterized. Through the above steps, a hybrid exosome framework structure with multifunctional integration capabilities was obtained.

[0100] 2. Technical Implementation Scheme of Oxidation Microenvironment Response Self-Driven Module

[0101] 1) Structure of the self-driven module

[0102] This invention introduces manganese dioxide nanoparticles (MnO2 NPs) as a self-driven core component inside hybrid exosomes. The MnO2 NPs are encapsulated within the exosome membrane lumen, and there is no covalent disruption between them and the exosome membrane, thereby maintaining the overall structural integrity of the exosome.

[0103] 2) Implementation mechanism of self-driving function (principle-based technical solution)

[0104] In osteoarthritis lesions, the concentration of hydrogen peroxide (H2O2) is significantly higher than in normal tissue. MnO2 NPs can undergo a catalytic reaction with H2O2 to generate oxygen and produce local thrust, enabling exosomes to move autonomously. This reaction process simultaneously achieves: endowing exosomes with autonomous movement ability; consuming excess local H2O2, and reducing oxidative stress levels.

[0105] 3. Technical Implementation Scheme of Cartilage Targeting Recognition Module

[0106] 1) Structural connection method of the target module

[0107] This invention uses chemical linkers to immobilize cartilage-targeting peptides on the surface of hybrid exosome membranes. The targeting peptides specifically recognize cartilage tissue or chondrocyte surface structures. The targeting peptides are linked to the exosome membrane via stable chemical bonds, preventing detachment in the in vivo environment.

[0108] 2) The effectiveness of the targeted function

[0109] The introduction of this targeting module can achieve the following technical effects: improve the adhesion ability of exosomes to the cartilage surface; prolong their retention time in cartilage tissue; and increase the probability of chondrocytes taking up exosomes.

[0110] 4. Technical Implementation Scheme for Therapeutic Molecular Loading and Release Module

[0111] 1) Loading method of therapeutic molecules

[0112] The therapeutic molecule described in this invention is preferably a therapeutic microRNA (e.g., miR-140). Stable loading is achieved by genetically regulating donor cells to enrich the target microRNA in their secreted exosomes.

[0113] 2) Release and mechanism of action of therapeutic molecules

[0114] After exosomes are taken up by chondrocytes, the microRNA inside them is released into the cytoplasm through exosome membrane rupture or fusion, and exerts a therapeutic effect by regulating related signaling pathways.

[0115] The in vivo workflow of the cascaded hybrid exosome nanomotor described in this invention includes the following sequential and interconnected steps, which are cascaded in spatial location and temporal order. Figure 2 ):

[0116] 1. Exosome nanomotors are injected into the joint cavity via intra-articular injection.

[0117] First, the constructed hybrid exosome nanomotor was introduced into the target joint cavity via intra-articular injection. After injection, the exosome nanomotor distributed within the joint cavity along with the synovial fluid, and made initial contact with the cartilage surface under the influence of joint movement and synovial fluid flow. In this step, the exosome nanomotor maintained its structural integrity and did not yet exhibit significant autonomous movement; its main function was to enable the delivery system to enter the local environment of the lesion and complete initial localization.

[0118] 2. Activated in an oxidizing microenvironment, it generates autonomous movement.

[0119] When exosomal nanomotors enter the lesion area of ​​osteoarthritis, the MnO2 NPs loaded inside the exosomes undergo a catalytic reaction with H2O2 in the environment, influenced by the local oxidative microenvironment. This reaction generates oxygen and creates local thrust, transforming the exosomal nanomotors from a passive diffusion state to an activated state with autonomous movement capabilities. Simultaneously, H2O2 is continuously consumed, thereby reducing the level of local oxidative stress. Through this step, the present invention achieves a self-driven activation mechanism triggered by disease pathological characteristics, avoiding the need for external energy input.

[0120] 3. Gradually penetrates the cartilage matrix during movement.

[0121] After self-driven activation, exosomal nanomotors continuously generate movement behavior in the oxidative microenvironment, migrating towards damaged cartilage areas with high H2O2 concentrations. During this continuous movement, the exosomal nanomotors constantly contact the cartilage surface and extracellular matrix structures, effectively overcoming the physical resistance of the cartilage matrix through kinetic energy input, thereby gradually migrating from the superficial to the deep layers of cartilage and achieving active penetration of the dense cartilage matrix. This step is significantly different from existing technologies that rely entirely on...

[0122] First, this invention provides its application in the preparation of medicaments for treating osteoarthritis. Osteoarthritis is a chronic joint disease characterized by degenerative changes in articular cartilage, subchondral bone sclerosis, and osteophyte formation at the joint margins. The nanomotor system of this invention, through local drug delivery methods such as intra-articular injection, can actively target and penetrate into the diseased cartilage tissue, release therapeutic molecules, and regulate the local microenvironment, thereby achieving the therapeutic goal.

[0123] Secondly, based on its core capabilities of actively penetrating dense tissues and targeting and regulating the microenvironment, this invention also offers its potential application in a wider range of diseases. Specifically, this invention provides the application of the above system in the preparation of drugs for treating the following diseases: degenerative joint diseases (such as rheumatoid arthritis); tendon or ligament injuries; articular cartilage defects; fibrotic diseases (such as pulmonary fibrosis, liver fibrosis); ischemia-reperfusion injury (such as tissue damage after myocardial infarction, stroke); and chronic inflammatory diseases (such as inflammatory bowel disease, chronic dermatitis, etc.). In these applications, the targeting molecules of the exosome nanomotor system can be replaced according to the target disease and tissue, for example, replaced with peptides targeting cardiomyocytes, neurons, or intestinal epithelial cells, while its self-driving module can utilize hydrogen peroxide or other endogenous substances overexpressed at the corresponding lesion site as driving fuel.

[0124] 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.

[0125] Example 1

[0126] 1. Extraction and identification of BMSCs and milk-derived exosomes

[0127] 1) Extraction of BMSCs-derived exosomes (bExos)

[0128] After BMSCs were cultured to approximately 80% confluence, the original culture medium was discarded and replaced with exosome-free serum medium for another 24 h to reduce exogenous vesicle contamination and promote exosome secretion. The culture supernatant was collected and exosomes were isolated using a classic differential ultracentrifugation procedure at 4°C. The specific steps were as follows: first, centrifugation at 300 g for 10 min removed suspended cells; then centrifugation at 2000 g for 20 min removed cell debris and residual nuclear fragments; finally, centrifugation at 10,000 g for 30 min removed large vesicles and apoptotic bodies. The processed supernatant was filtered through a 0.22 μm filter and then ultracentrifuged at 100,000 g for 70 min to collect the exosome pellet. The pellet was gently resuspended in pre-chilled PBS and centrifuged again at 100,000 g for 70 min to improve purity. Finally, the exosome pellet was resuspended in PBS, aliquoted, and stored at −80°C for later use. The concentration of the obtained exosomes was determined using a BCA protein quantification kit to facilitate dosage adjustment and comparison in subsequent experiments.

[0129] 2) Extraction of milk-derived exosomes (mExos)

[0130] Fresh skim milk was preheated in a 37°C water bath to homogenize it and reduce its viscosity for subsequent processing. To remove casein and major milk proteins, 1% glacial acetic acid was added and gently mixed to precipitate the casein. After centrifugation at 12,000 g for 20 min at 4°C, the precipitate was discarded, and the clear whey supernatant was filtered through a 0.22 μm microporous membrane to further remove particulate impurities and residual protein aggregates. The filtered whey was then subjected to ultracentrifugation (174,900 g, 70 min) to precipitate exosomes. The resulting precipitate was resuspended in pre-cooled PBS and centrifuged again at high speed to improve purity. Finally, it was resuspended in PBS, aliquoted, and stored at −80°C.

[0131] result:

[0132] TEM Figure 3 A showed that both types of exosomes exhibited typical spheroidal vesicle structures with clear edges and intact membrane contours, without obvious collapse or contamination, consistent with the morphological characteristics of exosomes. Further quantitative determination of the particle size distribution of exosomes was performed using NTA. The results showed that the particle size of bExos was 118.1 ± 11.4 nm, and the particle size of mExos was 99.8 ± 3.3 nm, with peak distributions within the classic exosome size range, indicating that the extracted exosomes had good size uniformity. Figure 3A). Subsequently, Western blotting was used to detect characteristic exosome marker proteins. The results showed that bExos highly expressed exosome marker proteins ALIX, TSG101, and CD81, while mExos highly expressed exosome marker proteins ALIX, TSG101, and CD63. Figure 3 B). In summary, the morphological, particle size distribution, and molecular marker analyses corroborate each other, indicating that this study successfully obtained exosomes with intact structures, high purity, and conforming to the International Society for Extracellular Vesicles (ISEV) recommended standards, providing a reliable carrier basis for subsequent functionalization modifications and hybridization construction experiments.

[0133] 2. Biomineralization preparation of MnO2 NPs

[0134] MnO2 NPs were prepared using a protein template-induced biomineralization strategy. 100 mM manganese chloride solution was mixed with an equal volume of 10 mg / mL bovine serum albumin (BSA), serving as both a stabilizer and a nucleation template. The pH was then adjusted to 10 using NaOH to promote the oxidation of Mn²⁺ and the deposition of MnO2. The mixture was magnetically stirred continuously at 37 °C for 2 h to complete the biomineralization process. After the reaction, the mixture was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed against deionized water for 48 h to thoroughly remove unreacted ions and residual small molecule impurities, ultimately obtaining a stable suspension of dispersed MnO2 NPs.

[0135] result:

[0136] TEM results showed that the prepared MnO2 NPs were well dispersed, exhibiting a regular spherical structure with a relatively concentrated particle size distribution and an average particle size of approximately 70 nm. Figure 4 A). This particle size not only facilitates effective encapsulation by exosomes in subsequent processes, but also avoids potential damage to the integrity of the exosome membrane structure due to excessively large particle size while ensuring drug loading efficiency. Based on this, the catalytic performance of MnO2 NPs was further evaluated in the presence of H2O2. The results show that MnO2 NPs can efficiently catalyze the decomposition of H2O2 in a short time and continuously generate a large amount of gas ( Figure 4 (B) reflects its significant peroxidase-like activity and good gas generation capacity. This catalytic behavior is particularly crucial in inflammatory microenvironments, suggesting that MnO2 NPs can use H2O2 enriched in the lesion as "fuel" to achieve energy conversion, thereby providing a continuous power source for the system.

[0137] 3. Synthesis, characterization, and exosome surface modification of chondroitin-targeting peptide CAP

[0138] 1) Synthesis of chondroitin-targeting peptide CAP

[0139] To prepare modified lipid molecules that can self-assemble with lipid nanostructures and possess active targeting capabilities, a thiol-maleimide click chemistry reaction was used to couple CAP peptides to DSPE-PEG. 2000 -MAL. First, accurately weigh a certain amount of DSPE-PEG. 2000 -MAL was dissolved in an appropriate amount of anhydrous dimethyl sulfoxide (DMSO) to prepare a clear solution for later use. Separately, the CAP peptide was dissolved in an equimolar amount of DMSO, ensuring complete dissolution. Under magnetic stirring, the CAP peptide solution was slowly added dropwise to DSPE-PEG. 2000 In a 1MAL solution, the two components were brought into full contact to promote the specific addition reaction between maleimide and thiol groups. To improve reaction efficiency and suppress side reactions, a small amount of triethylamine was added as a basic catalyst, and the reaction was continued at 25°C for 24 h with stirring to ensure the amidation / coupling reaction proceeded fully. After the reaction was complete, the reaction mixture was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water for 48 h, with the dialysate changed every 4-6 h to thoroughly remove unreacted free CAP peptides and low-molecular-weight impurities. After dialysis, the sample was freeze-dried to obtain white or off-white DSPE-PEG. 2000 -CAP lyophilized powder, sealed and stored at -20℃ for later use.

[0140] 2) Evaluation of CAP grafting modification on exosome surface

[0141] To endow exosomes with active targeting capabilities, the chondroitin-targeting peptide CAP was subjected to DSPE-PEG. 2000 The carrier was integrated onto the surface of the mExos membrane. The specific procedure was as follows: a 1 mg / mL mExos sample was thoroughly mixed with 10% (w / w) DSPE-PEG2000-CAP, and then extruded 11 times consecutively through a 400 nm pore size polycarbonate membrane. A co-incubation group was set up as a control. During the extrusion process, lipid molecules rearranged under shear force and embedded themselves into the exosome lipid bilayer, achieving stable grafting of CAP molecules and obtaining CAP-modified exosomes (CAP-mExos). CAP grafting typically causes a slight increase in the surface potential of exosomes, and its modification effect was further verified by zeta potential (Zeta potential) measurement.

[0142] result:

[0143] ¹H-NMR spectral analysis results are as follows Figure 5As shown, while retaining the typical proton signal of the PEG backbone, DSPE-PEG2000-CAP exhibits a new characteristic peak in the low-field region, corresponding to the proton signal of the amino acid side chain in the CAP peptide, indicating that CAP has been successfully introduced into the molecular structure of DSPE-PEG2000 via covalent bonding.

[0144] FT-IR analysis further confirmed the successful grafting of CAP. Compared with DSPE-PEG2000-MAL, DSPE-PEG2000-CAP, while retaining the characteristic absorption peaks of the PEG skeleton, added amide bond-related C=O stretching vibration peaks and NH bending vibration peaks. Figure 6 This indicates that CAP is stably grafted via covalent bonding without disrupting the overall structure of the PEG molecule, providing a chemical basis for its subsequent stable anchoring on the exosome membrane.

[0145] DSPE-PEG 2000 -CAP was inserted into the surface of the mExos membrane to obtain CAP-modified exosomes (CAP-mExos). Zeta potential detection results showed that, compared with the mixed incubation group, the surface potential of exosomes in the extrusion group was approximately 2 mV higher than that in the unmodified group (Table 1 and ). Figure 7 Given that the CAP molecule itself carries a weak positive charge, this change suggests from a physicochemical perspective that CAP-modified lipids have been successfully anchored to the exosome membrane surface.

[0146] Table 1. Results of physicochemical properties of products from each group

[0147] Particle size (nm) polydispersion coefficient Potential (mV, 0.1×PBS) mExos 133.57±1.47 0.16±0.02 -11.17±0.45 DSPE-PEG2000-CAP+mExos (mixed) 123.97±1.56 0.13±0.01 -11.87±0.74 DSPE-PEG2000-CAP+mExos (Extrusion) 132.50±1.48 0.15±0.01 -9.84±0.80

[0148] 4. Loading and optimization of MnO2 NPs in exosomes

[0149] To load MnO2 NPs into exosomes, ultrasonic treatment was performed at different MnO2 NP to mExos feed ratios (100 W; 5 min; pulse mode: 1 s / 1 s). The optimal feed ratio was explored by comparing the particle size distribution and PDI of the products.

[0150] result:

[0151] When MnO2 NPs and mExos are fed in a 1:1 particle number ratio, the resulting exosome formulation exhibits the most concentrated particle size distribution and the lowest PDI. Figure 8 (See Table 2). These results indicate that under these conditions, a good balance is achieved between the loading efficiency of MnO2 NPs and the structural stability of exosomes, providing a reasonable parameter basis for the subsequent construction of a nanomotor system with uniform structure and stable performance.

[0152] Table 2

[0153] Grouping (particle number ratio) Particle size (nm) polydispersion coefficient Potential (mV, 0.1×PBS) mExos 118.47±0.54 0.15±0.02 -17.57±1.31 <![CDATA[MnO2NPs]]> 72.59±0.66 0.25±0.01 -17.78±0.90 <![CDATA[mExos:MnO2NPs=1:4]]> 80.16±1.70 0.31±0.03 —— <![CDATA[mExos:MnO2NPs=1:2]]> 92.36±1.28 0.28±0.03 —— <![CDATA[mExos:MnO2NPs=1:1]]> 106.50±2.58 0.24±0.02 —— <![CDATA[mExos:MnO2NPs=2:1]]> 113.00±2.55 0.28±0.03 ——

[0154] 5. Construction and identification of miR-140 overexpression exosomes

[0155] Stable high miR-140-expressing BMSCs were constructed using a lentivirus-mediated gene overexpression strategy. Following the manufacturer's standard operating procedure, BMSCs in the logarithmic growth phase were infected with a lentivirus vector (Lentivirus-miR140) carrying the miR-140 sequence. To achieve optimal transfection efficiency, infection conditions were optimized by setting different multiplicity of infection (MOI) gradients. Post-infection, the expression of green fluorescent protein in the viral vector was observed using an inverted fluorescence microscope to assess cell infection efficiency and determine optimal transfection parameters. After determining the optimal infection conditions, successfully transfected BMSCs were expanded and cultured. When cell confluence reached approximately 80%, the medium was replaced with exosome-free serum and cultured for another 24 h, and the culture supernatant was collected. Subsequently, the culture supernatant was processed using the aforementioned differential ultracentrifugation method to isolate and extract exosomes, ultimately obtaining exosomes derived from miR-140-overexpressing BMSCs (140@bExos). The obtained exosomes were stored at -80 °C and protein quantification was performed using the BCA method for dose standardization in subsequent experiments.

[0156] To verify that lentivirus-miR140 effectively overexpressed miR-140 in BMSCs, the expression level of miR-140 in transfected BMSCs was first detected by quantitative polymerase chain reaction (qPCR). Subsequently, 140@bExos was co-incubated with chondrocytes for 24 h. After co-incubation, chondrocytes were collected and total RNA was extracted. Changes in endogenous miR-140 expression were analyzed by qPCR to evaluate the efficiency and biological feasibility of exosome-mediated miRNA transcellular delivery.

[0157] result:

[0158] When the MOI was 75 and the transfection enhancer Hitrans A was used in combination, the fluorescence signal distribution in BMSCs was uniform and stable, and the fluorescence intensity was significantly enhanced under the lower MOI condition, but no significant difference was observed compared with the MOI group of 100. Figure 9 Taking into account transduction efficiency, potential cytotoxicity, and experimental economy, an MOI of 75 combined with Hitrans A was ultimately selected as the optimal transfection condition for subsequent experiments.

[0159] qPCR results showed that compared with untreated BMSCs and the control group transfected with Lentivirus-miR140-NC, the expression level of miR-140 in BMSCs transfected with Lentivirus-miR140 was significantly increased, indicating that the miR-140 overexpression model was successfully constructed. Further co-culturing the isolated 140@bExos cells with chondrocytes also significantly upregulated the expression level of miR-140 in the chondrocytes. Figure 10 The results suggest that 140@bExos can serve as an effective carrier to mediate the delivery of miR-140 to target cells, providing a prerequisite for it to perform its subsequent biological functions.

[0160] 6. Preparation of membranes by fusing bExos and mExos

[0161] To obtain hybrid exosomes with dual-source characteristics and improved biological functions, this study used binary fluorescent dyes to label exosome membranes and combined incubation and extrusion methods to achieve exosome membrane fusion. First, bExos (5 μg / mL) was mixed with the lipid-soluble red fluorescent probe DiI (0.5 mg / mL), and mExos (5 μg / mL) was mixed with the lipid-soluble green fluorescent probe DiO (1 mg / mL). The mixtures were incubated at room temperature in the dark for 40 min to allow the fluorescent dyes to fully insert into the exosome lipid bilayer membrane. After incubation, unbound free dyes were removed by ultrafiltration centrifugation (5000 rpm, 5 min), and the membranes were washed twice with PBS to reduce background fluorescence interference. Subsequently, DiI and DiO labeled bExos and mExos were mixed at a protein mass ratio of 1:1, and membrane fusion was performed using the following two methods: one was the incubation method, in which the mixed system was placed at a constant temperature of 37 ℃ and slowly oscillated at 60 rpm for 12 h, utilizing the fluidity of the exosome membrane and the self-assembly characteristics of lipids to promote membrane fusion; the other was the extrusion method, in which the mixed exosome solution was continuously extruded 11 times through a polycarbonate membrane with a pore size of 400 nm, causing the exosomes to undergo membrane lysis and recombination under shear force, thereby achieving efficient membrane fusion.

[0162] The degree of membrane fusion of hybrid exosomes was evaluated using the fluorescence resonance energy transfer (FRET) effect. DiO was used as the energy donor, and the fluorescence emission signal of the DiI acceptor was recorded at its specific excitation wavelength. As exosome membrane fusion occurred, DiI and DiO gradually approached and coexisted within the same nanovesicle membrane structure, thereby enhancing the FRET signal intensity to reflect the membrane fusion efficiency. Furthermore, dynamic light scattering (DLS) was used to detect the particle size distribution and polydispersity index (PDI) of exosome samples before and after fusion to further evaluate the impact of membrane fusion on the physical properties of exosomes.

[0163] result:

[0164] like Figure 11 As shown, the FRET signal in the sample significantly increased after simple incubation, indicating effective energy transfer between the donor and acceptor fluorescent dyes, suggesting that the membrane structures of mExos and bExos achieved close spatial proximity and substantial fusion. However, the hybrid exosomes obtained under this condition had a larger particle size and a higher PDI value, suggesting that some degree of particle aggregation or size inhomogeneity occurred during membrane fusion. In contrast, simple extrusion significantly reduced the average particle size and improved dispersibility, but no significant FRET signal enhancement was observed, suggesting that this treatment method is more likely to lead to physical rearrangement of the exosome membrane and is insufficient to achieve sufficient fusion of the dual-source membrane. Based on the above comparative results, this study adopted an optimized strategy of "incubation first, then extrusion" to prepare hybrid exosomes. This method ensures sufficient fusion of mExos and bExos membrane components while effectively controlling particle size and dispersibility through the extrusion process, providing a stable and reliable carrier basis for subsequent functionalization modification and biological performance research.

[0165] In summary, through stepwise construction and systematic characterization, we successfully integrated the hybrid exosome carrier module, the oxidative microenvironment response self-driven module, the cartilage target recognition module, and the therapeutic molecule loading and release module, thus constructing a structurally stable and functionally defined hybrid exosome nanomotor system. This lays a solid foundation for its subsequent physicochemical performance evaluation and biological function verification.

[0166] Example 2

[0167] 1. Analysis of the integrity of exosomal nanomotor protein composition

[0168] To assess whether multiple engineering procedures, including CAP modification, MnO2 NP loading, and dual-source exosome membrane fusion, affected the intrinsic protein composition of exosomes, SDS-PAGE was used to analyze the overall protein profiles of each exosome formulation. Results are as follows: Figure 12 As shown, each treatment group exhibited a clear and stable protein band distribution pattern, and the overall protein spectra among different samples were highly similar. No obvious protein band loss, abnormal migration, or degradation was observed. These results indicate that a series of chemical modifications and physical treatments did not disrupt the inherent protein composition and structural integrity of exosomes, providing an important structural basis for the stable performance of their biological functions.

[0169] 2. Microstructure and particle size distribution characteristics of exosome nanomotors

[0170] To visually assess the structural and morphological characteristics of exosomal nanomotors, TEM was used to observe various exosomal formulations. For example... Figure 13 As shown, all samples exhibited typical spherical vesicle structures with clear edges and intact membrane contours. No obvious collapse, rupture, or severe aggregation was observed, indicating that engineering operations such as CAP modification, MnO2 NPs loading, and membrane fusion did not damage the basic vesicle structure of exosomes.

[0171] Further quantitative determination of exosome particle size distribution was performed using NTA. The results showed that the particle size of exosomes in each group was mainly concentrated in the 100-140 nm range, which is within the classic exosome size range. Figure 13 Compared with unmodified exosomes, the hydrated particle size increased slightly after CAP modification and the introduction of MnO2 NPs, but the overall distribution remained concentrated, without obvious multi-peak distribution or abnormal increase, indicating that the engineering modification did not have an adverse effect on the dispersibility and uniformity of exosomes.

[0172] 3. Stability characteristics of exosomal nanomotors

[0173] To evaluate the structural stability of exosomal nanomotors in physiologically relevant environments, various formulations were incubated continuously for 7 days in vitro under simulated synovial fluid conditions, and their mean hydrodynamic diameter and platelet density (PDI) were monitored periodically using DLS. Results are as follows: Figure 14 As shown, the particle size and PDI value of each exosome formulation remained relatively stable throughout the entire incubation period, without significant fluctuations or abnormal increases. These results indicate that the constructed exosome nanomotors possess good physical stability in the simulated joint cavity microenvironment and can maintain their structural integrity and dispersion state over a long period.

[0174] 4. MnO2 NPs loading validation and drug loading efficiency analysis

[0175] To verify whether MnO2 NPs were successfully loaded into the exosome system, XPS was first used to analyze the elemental composition and chemical state of each formulation. For example... Figure 15 As shown, compared with the mExos, bExos, and CAP-mExos groups, CAP-Mn@mExos and CAP-Mn@hyExos both exhibit significant Mn 2p groups at binding energies of approximately 640 eV and 655 eV. 3 / 2 With Mn 2p 1 / 2 The characteristic peaks indicate that manganese was successfully introduced into the exosome system in the form of MnO2 NPs, thus confirming the effective loading of MnO2 NPs at the elemental level.

[0176] Based on this, ICP-OES was further used to quantitatively determine the manganese content in various exosome formulations to systematically evaluate the loading efficiency of MnO2 NPs (Table 3 below). The results showed that the mass fraction of manganese in free MnO2 NPs was 4.62%, with an encapsulation efficiency of approximately 72%. After exosome loading, the mass fractions of manganese in CAP-Mn@mExos and CAP-Mn@hyExos were 3.42% and 3.45%, respectively, with corresponding encapsulation efficiencies significantly increased to 87.8% and 88.4%. These results indicate that MnO2 NPs are more effectively encapsulated and retained in exosome systems compared to their free state.

[0177] Table 3. ICP-OES quantitative analysis of manganese content and encapsulation efficiency in various formulations.

[0178] <![CDATA[MnO2NPs]]> CAP-Mn@mExos CAP-Mn@hyExos Mass fraction of Mn element 4.62% 3.42% 3.45% Encapsulation rate 72.0% 87.8% 88.4%

[0179] In summary, through systematic characterization of exosome marker protein expression, overall protein composition, morphology, particle size distribution, stability, and MnO2 NP loading, this study fully demonstrates that the constructed exosome nanomotor maintains good structural integrity and physicochemical stability after multi-level engineering modification. Furthermore, this system exhibits high MnO2 NP drug loading efficiency and stable vesicle characteristics, providing solid experimental evidence for its long-term effects in the complex joint cavity microenvironment and subsequent in vivo functional studies.

[0180] Example 3

[0181] 1. Self-driving behavior and cartilage matrix penetration ability of exosome nanomotors

[0182] 1.1 Self-driven motion behavior of exosome nanomotors in H2O2 environment

[0183] Given the prevalent elevated H2O2 levels in the OA cartilage microenvironment, evaluating the motility of exosomal nanomotors under H2O2 conditions is crucial for verifying their functional feasibility. Using NTA technology, the motility trajectories and kinetic parameters of various exosomal formulations were recorded with and without H2O2. Results showed that in the control environment without H2O2, mExos, bExos, CAP-mExos, and exosomal formulations containing MnO2 NPs all exhibited typical random Brownian motion, with short trajectories, poor directionality, and low average displacement and velocity. In contrast, after the addition of H2O2, the CAP-Mn@mExos and CAP-Mn@hyExos groups containing only MnO2 NPs showed significantly prolonged and continuous trajectories, with significantly enhanced directionality and significantly higher average velocity than the exosomal group without MnO2 NPs. Figure 16 The above results indicate that MnO2 NPs can continuously catalyze reactions and generate driving forces in the presence of H2O2, thereby effectively endowing exosomes with stable self-driven motility, an effect not observed in exosomes without MnO2 NPs.

[0184] 1.2 Kinematic response of exosomal nanomotors to H2O2 concentration gradient

[0185] Based on the established self-driving capability of exosomal nanomotors, a Matrigel-based H2O2 concentration gradient model was further constructed to evaluate their kinematic response characteristics to oxidative stress signals. The migration behavior of different exosomal formulations under gradient conditions was studied. Figure 17 As shown in the figure. The results indicate that, in the presence of an H2O2 concentration gradient, the penetration depth of CAP-Mn@mExos and CAP-Mn@hyExos groups in Matrigel is significantly increased, and their migration direction exhibits a certain directionality. In contrast, the migration distances of the mExos, bExos, and CAP-mExos groups are shorter, and their distribution shows no obvious directional bias. These results demonstrate that the introduction of MnO2 NPs not only endows exosomes with active motility but also enables them to sense and respond to H2O2 concentration gradients, thereby enhancing their migration and potential lesion accumulation capabilities in an oxidative stress microenvironment.

[0186] 1.3 Exosomal nanomotors' matrix penetration ability in an in vitro cartilage tissue model

[0187] After confirming the self-driving and chemotactic capabilities of exosome nanomotors, an in vitro cartilage tissue model was further used to systematically evaluate their penetration performance in the dense chondrocyte extracellular matrix. Different DiI-labeled exosome preparations were co-cultured with cartilage sections, and confocal microscopy images were acquired at different time points to track their spatial distribution in the cartilage tissue. The results showed that in the early stages of culture, the fluorescence signals of each exosome group were mainly distributed in the cartilage surface. With prolonged culture time, the fluorescence signals of the mExos, bExos, and CAP-mExos groups remained confined to the superficial region, with limited diffusion into the deeper cartilage tissue. In contrast, the fluorescence signals of the CAP-Mn@mExos and CAP-Mn@hyExos groups gradually migrated into the cartilage interior over time, and significant fluorescence distribution was still observed in the deep cartilage region after 6 days of culture. Figure 18 ).

[0188] Further quantitative analysis was performed on the penetration depth of exosomes in cartilage tissue. The results showed that, compared with the exosome group without MnO2NPs, the average penetration depth of CAP-Mn@mExos and CAP-Mn@hyExos was significantly increased at all time points; at 6 days of culture, the maximum penetration depth reached approximately 140 μm, about four times that of the mExos, bExos, and CAP-mExos groups. Figure 19 ).

[0189] In summary, through systematic analysis of motor behavior, chemotactic experiments, and evaluation of cartilage tissue penetration, this study fully demonstrates that MnO2 NPs-driven exosome nanomotors exhibit significantly enhanced self-driving ability, H2O2 concentration gradient chemotactic properties, and excellent deep penetration ability into the cartilage matrix in an oxidative stress microenvironment. These functional advantages lay an important experimental foundation for their efficient and precise intra-articular delivery and deep treatment within osteoarthritis lesions.

[0190] 2. Chondrocyte-targeted uptake characteristics of exosome nanomotors

[0191] 2.1 Observation of cellular uptake of exosome nanomotors using confocal microscopy

[0192] To systematically evaluate the effect of CAP modification on the cell-targeting ability of exosomal nanomotors, different DiI-labeled exosomal formulations were co-incubated with chondrocytes. Under uniform imaging parameters, the distribution characteristics and relative intensity of intracellular fluorescence signals were observed using confocal laser scanning microscopy. The results showed that compared with the unmodified mExos and bExos groups, the CAP-modified exosomal formulations (CAP-mExos, CAP-Mn@mExos, and CAP-Mn@hyExos) exhibited significantly enhanced red fluorescence signals in chondrocytes, with the fluorescence mainly distributed in the cytoplasm rather than the extracellular background. Figure 20 These results suggest that CAP modification can effectively promote the specific recognition, adhesion, and endocytosis of exosomes with chondrocytes, thereby significantly improving their cellular uptake levels. Further comparison within the CAP-modified groups revealed that the intracellular fluorescence signal intensity in the CAP-Mn@mExos and CAP-Mn@hyExos groups was significantly higher than that in the CAP-mExos group. This indicates that, based on target peptide-mediated cell recognition, the self-driven properties conferred by MnO2 NPs may further promote efficient uptake by chondrocytes by enhancing the dynamic contact frequency and local enrichment effect of exosomes on the cell surface.

[0193] 2.2 Flow cytometry quantitative analysis of the uptake efficiency of exosome nanomotors

[0194] To further quantitatively validate the qualitative results obtained by confocal microscopy, flow cytometry was used to detect the fluorescence signal intensity of different exosome preparations in chondrocytes, and their uptake efficiency was statistically analyzed. The results are as follows: Figure 21 As shown, compared with the unmodified mExos and bExos groups, the mean intracellular fluorescence intensity of all CAP-modified groups was significantly increased, indicating that CAP modification effectively enhanced the cellular uptake capacity of exosomes overall. Among them, the mean fluorescence intensity of the CAP-Mn@mExos and CAP-Mn@hyExos groups was the highest, approximately 2.5 times that of the mExos and bExos groups, and the difference was statistically significant. This quantitative result is highly consistent with the confocal microscopy observation results, further confirming the key role of CAP modification in enhancing the targeted uptake of exosomes by chondrocytes, and also indicating that the self-driven nanomotor properties can synergistically promote the cellular endocytosis of exosomes.

[0195] 3. Retention behavior of exosome nanomotors in the joint cavity of OA

[0196] 3.1 Comparison of retention characteristics of different exosome preparations in the knee joint cavity of osteoarthritis (OA)

[0197] To systematically evaluate the retention capacity of different exosome formulations in the knee joint cavity of osteoarthritis (OA), fluorescently labeled mExos, bExos, CAP-mExos, CAP-Mn@mExos, and CAP-Mn@hyExos were injected into the knee joint cavity of OA rats. Under uniform imaging parameters, in vivo fluorescence imaging (IVIS) was used to continuously and dynamically monitor changes in fluorescence signals in the joint region. Results are as follows: Figure 22As shown, unmodified mExos and bExos exhibited significant fluorescence signal decay shortly after injection, with a substantial decrease in fluorescence intensity in the joint region within one week. This suggests that they primarily rely on passive diffusion within the joint cavity and are rapidly cleared by synovial fluid circulation and lymphatic drainage. In contrast, the fluorescence signal decay rate of the CAP-mExos group was significantly slower, and its retention time in the joint cavity was significantly longer than that of the mExos and bExos groups. This indicates that modification with the cartilage-targeting peptide CAP can enhance the specific interaction between exosomes and articular cartilage tissue, thereby promoting their adhesion and accumulation in local tissues. Further comparison among CAP-modified formulations revealed that the CAP-Mn@mExos and CAP-Mn@hyExos groups exhibited the most persistent fluorescence signal in the OA knee joint cavity. Even two weeks after injection, approximately 30% of the initial fluorescence intensity could still be detected in the joint region, significantly higher than other groups. These results indicate that, based on CAP-mediated targeting, the self-driven properties conferred by MnO2 NPs can further delay the clearance process of exosomes in the joint cavity, thereby significantly enhancing their local retention capacity.

[0198] 3.2 The Influence of the OA Pathological Microenvironment on the Joint Retention Behavior of Exosomal Nanomotors

[0199] To further investigate the role of the pathological microenvironment of osteoarthritis (OA) in joint retention of exosome nanomotors, CAP-Mn@hyExos was injected into the knee joint cavity of normal rats, and the retention behavior was compared with that in an OA rat model. The results showed that in the normal knee joint cavity, the fluorescence signal clearance rate of CAP-Mn@hyExos was significantly accelerated, and its joint retention time was significantly shorter than that in the OA joint model. Figure 23 These results suggest that the prolonged retention of CAP-Mn@hyExos in OA joints is not solely dependent on its structural or modification properties, but is closely related to the unique pathological microenvironment of OA joints. In particular, under inflammatory conditions, changes such as elevated H2O2 levels in the joint cavity and loosening of the cartilage matrix structure may collectively provide favorable conditions for the self-driven movement, tissue penetration, and local accumulation of exosome nanomotors, thereby significantly enhancing their retention capacity within OA joints.

[0200] In summary, the self-driving ability of exosomes endowed with MnO2 NPs significantly enhanced their cartilage penetration and deep retention in OA joints, while CAP modification further promoted the targeted enrichment of exosomes on cartilage tissue and chondrocytes. The synergistic effect of these two factors significantly slowed the clearance rate of exosomes within the joint cavity, thereby effectively prolonging the retention time of this hybrid exosome nanomotor in OA joints, laying an important functional foundation for its continuous, precise, and efficient intra-articular therapy.

[0201] 4. Evaluation of in vitro biocompatibility and cell activity of exosomal nanomotors

[0202] 4.1 Evaluation of biosafety using live / dead staining and hemolysis assays

[0203] By fusing 140@bExos with CAP-Mn@mExos, the final exosomal nanomotor CAP-Mn / 140@hyExos was successfully constructed. First, live / dead staining was used to evaluate the viability of chondrocytes treated with different exosomal formulations. The results showed that cells in all treatment groups exhibited predominantly green live-cell fluorescence, with almost no red dead-cell signal observed, and no significant differences were observed between the groups. Figure 24 The results indicate that the constructed exosome nanomotors do not exhibit significant cytotoxicity to chondrocytes.

[0204] Further evaluation of its blood compatibility was conducted using a hemolysis test. The results showed that, compared to the negative control group, no significant hemolysis was observed in erythrocytes treated with any of the exosome preparations, and the hemolysis rate remained within the permissible range of biomaterial safety standards. Figure 25 The above results demonstrate that this exosomal nanomotor system exhibits good biocompatibility in a blood environment, meeting the basic safety requirements for subsequent in vivo drug delivery and biomedical applications.

[0205] 4.2 Effects of cytoskeleton staining analysis on OA chondrocyte morphology

[0206] To further evaluate the regulatory role of exosomal nanomotors on pathological morphological changes in OA chondrocytes through cytoskeleton staining, such as... Figure 26 As shown, normal chondrocytes exhibit a regular polygonal morphology with a uniformly distributed cytoskeleton; however, OA chondrocytes induced by IL-1β are significantly elongated and exhibit an irregular spindle shape, indicating impaired cellular structural homeostasis. After exosome intervention, the abnormal morphology of OA chondrocytes in both the CAP-Mn@hyExos and 140@bExos treatment groups was improved to some extent, with cell outlines becoming more regular. Among them, CAP-Mn / 140@hyExos showed the most significant effect on restoring cell morphology, suggesting that the synergistic effect of nanomotor structure and miR-140 functional delivery helps alleviate cellular structural abnormalities under OA conditions.

[0207] 4.3 CCK-8 assay to assess the effect on OA chondrocyte proliferation

[0208] The effects of different exosome preparations on the proliferation capacity of OA chondrocytes were detected using the CCK-8 assay. Results are as follows: Figure 27As shown, compared with the IL-1β-induced OA model group, treatments with CAP-Mn@hyExos, 140@bExos, and CAP-Mn / 140@hyExos significantly improved cell viability and proliferation. Among them, the CAP-Mn / 140@hyExos group showed the most significant promoting effect on cell proliferation, indicating that it can effectively improve the state of chondrocyte proliferation restriction under OA conditions while maintaining good biocompatibility.

[0209] 4.4 Scratch assay to assess the regulatory effect on OA chondrocyte migration ability

[0210] Further scratch assays were used to analyze the effects of different exosome preparations on chondrocyte migration ability. For example... Figure 28 As shown, compared with the OA control group, all exosome treatments promoted the gradual closure of the scratch area to varying degrees, suggesting that exosomes as a whole have certain cell migration-promoting potential. However, the promoting effects varied among different formulations. CAP-Mn@hyExos showed relatively limited cell migration promotion, the 140@bExos group exhibited a moderate promotion effect, while the CAP-Mn / 140@hyExos group showed the most significant scratch closure rate at all time points. These results indicate that the synergistic effect between the functional regulation of miR-140 and the structural characteristics of nanomotors can significantly enhance the migration ability of chondrocytes, thereby facilitating the tissue repair process.

[0211] 5. Regulatory role of exosomal nanomotors in chondrocyte oxidative stress

[0212] During the occurrence and progression of osteoarthritis (OA), chondrocytes are exposed to a high-level ROS microenvironment for extended periods, leading to mitochondrial dysfunction, increased apoptosis, and exacerbated extracellular matrix degradation. Exogenous H2O2 treatment can stably induce oxidative stress in chondrocytes in vitro, and its pathological characteristics are highly consistent with those of OA. Therefore, this study established an H2O2-induced in vitro oxidative stress model to systematically evaluate the protective effect of CAP-Mn / 140@hyExos against oxidative damage in chondrocytes.

[0213] 5.1 Exosomal nanomotors reduce intracellular ROS levels in chondrocytes

[0214] First, ROS levels in chondrocytes treated with different exosome preparations were detected using a ROS fluorescent probe to evaluate their antioxidant capacity. The results are as follows: Figure 29As shown, compared with the normal group, H2O2 treatment significantly increased the intracellular ROS level in chondrocytes, indicating that the oxidative stress model was successfully established. 140@bExos treatment could reduce ROS accumulation to some extent, but the inhibitory effect was limited. In contrast, both CAP-Mn@hyExos and CAP-Mn / 140@hyExos significantly reduced intracellular ROS fluorescence intensity, with the CAP-Mn / 140@hyExos group showing the most significant ROS scavenging effect. These results indicate that after introducing MnO2 NPs, exosomal nanomotors can effectively respond to the oxidative stress microenvironment, rapidly scavenging excess ROS, thereby significantly alleviating intracellular oxidative stress.

[0215] 5.2 Exosomal nanomotors restore mitochondrial membrane potential

[0216] Given that mitochondria are one of the most critical subcellular targets of oxidative stress damage, JC-1 staining was further used to assess changes in mitochondrial membrane potential. Figure 30 As shown, the red / green fluorescence ratio of JC-1 cells in chondrocytes significantly decreased after H2O2 induction, indicating mitochondrial membrane potential depolarization and impaired function. After treatment with CAP-Mn@hyExos and CAP-Mn / 140@hyExos, the red fluorescence ratio significantly recovered, and the mitochondrial membrane potential significantly increased and approached normal levels. The CAP-Mn / 140@hyExos group showed the most significant improvement, indicating its stronger protective effect in maintaining mitochondrial functional homeostasis.

[0217] 5.3 Exosomal nanomotors inhibit oxidative stress-induced apoptosis

[0218] Annexin V / PI double staining was used to quantitatively analyze chondrocyte apoptosis. The results showed that H2O2 treatment significantly increased the proportion of early and late apoptosis in chondrocytes; after intervention with 140@bExos, the apoptosis level decreased, but was still significantly higher than the normal control group. In contrast, both CAP-Mn@hyExos and CAP-Mn / 140@hyExos significantly reduced the apoptosis rate, with CAP-Mn / 140@hyExos showing the most significant inhibitory effect. Figure 31 The above results suggest that exosomal nanomotors can effectively block the oxidative stress-induced apoptosis cascade by synergistically clearing oxidative stress damage and exerting the endogenous protective effect of miR-140, thereby significantly enhancing the survival ability of chondrocytes.

[0219] 5.4 Exosomal nanomotors improve chondrocyte survival

[0220] To visually assess the overall survival status of chondrocytes under different treatment conditions, live / dead cell staining was further used for observation. Figure 32 As shown, the H2O2 treatment group exhibited a significant increase in red dead cell signaling, indicating severe cell damage; while intervention with CAP-Mn@hyExos and CAP-Mn / 140@hyExos significantly increased the proportion of green live cells, indicating a marked improvement in cell viability. The CAP-Mn / 140@hyExos group demonstrated the best protective effect. These results are highly consistent with apoptosis analysis, further validating the protective effect of exosomal nanomotors against oxidative stress damage.

[0221] 5.5 Exosomal nanomotors enhance the cellular endogenous antioxidant defense system

[0222] Further analysis of intracellular antioxidant markers was conducted to assess their regulatory role in oxidative stress. Results showed that, compared to the H2O2 model group, CAP-Mn@hyExos and CAP-Mn / 140@hyExos treatments significantly increased CAT and SOD activities while significantly decreasing MDA content. Figure 33 The above results indicate that exosomal nanomotors can not only directly scavenge excess ROS, but also further enhance the endogenous antioxidant defense system of chondrocytes, thereby synergistically alleviating cell damage caused by oxidative stress.

[0223] In summary, CAP-Mn@hyExos and CAP-Mn / 140@hyExos exert their antioxidant protective effects synergistically through multiple levels and mechanisms, including efficiently scavenging excess ROS, maintaining mitochondrial functional stability, inhibiting oxidative stress-induced apoptosis, and enhancing the activity of endogenous antioxidant enzymes. Among them, CAP-Mn / 140@hyExos exhibits a more comprehensive and stable antioxidant synergistic effect, providing solid experimental evidence for its application as a novel exosomal nanomotor in the treatment of osteoarthritis (OA).

[0224] 6. Exosome nanomotors promote OA chondrocyte phenotype restoration and matrix homeostasis reconstruction

[0225] 6.1 Immunofluorescence analysis of the effect of exosomal nanomotors on the phenotype of OA chondrocytes

[0226] To visually evaluate the regulatory effects of different exosome preparations on the phenotype of osteoarthritis (OA) chondrocytes, immunofluorescence staining was used to detect the expression of COL2A1, a marker of chondrocyte matrix synthesis, and MMP13 and iNOS, matrix degradation and inflammation-related factors. Figure 34As shown, compared with the normal control group, the COL2A1 fluorescence signal was significantly weakened in the OA model group, while the MMP13 and iNOS fluorescence signals were significantly enhanced, exhibiting typical abnormal OA phenotype characteristics. After treatment with CAP-Mn@hyExos, COL2A1 expression was somewhat restored, and the expression of MMP13 and iNOS decreased to some extent, but the overall improvement was limited. In contrast, after intervention with 140@bExos and CAP-Mn / 140@hyExos, the COL2A1 fluorescence signal was significantly enhanced, and the expression of MMP13 and iNOS was significantly reduced. Among them, the phenotypic improvement effect of the CAP-Mn / 140@hyExos group was the most significant, suggesting that it has a stronger regulatory ability in restoring the normal phenotype of OA chondrocytes.

[0227] 6.2 Protein Level Analysis: Regulation of Cartilage Matrix and Inflammatory Factors by Exosomal Nanomotors

[0228] To further validate the immunofluorescence results at the protein expression level, Western blotting was used to detect the expression of COL2A1, MMP13, and iNOS in chondrocytes from different treatment groups. Figure 35 As shown, COL2A1 protein expression was significantly downregulated in the OA model group, while MMP13 and iNOS were significantly increased. CAP-Mn@hyExos treatment partially restored COL2A1 expression and inhibited the abnormal increase in MMP13 and iNOS to some extent; while 140@bExos and CAP-Mn / 140@hyExos treatments significantly upregulated COL2A1 protein expression, while significantly reducing MMP13 and iNOS expression. Among these, the CAP-Mn / 140@hyExos group showed the most significant regulatory effect at the protein level, which was highly consistent with the immunofluorescence results.

[0229] 6.3 Transcriptional analysis of the effects of exosomal nanomotors on cartilage metabolism-related genes

[0230] The mRNA expression levels of COL2A1, MMP13, and iNOS in chondrocytes from different treatment groups were further analyzed using qPCR. The results are as follows: Figure 36 As shown, compared with the normal control group, COL2A1 mRNA expression was significantly downregulated in the OA model group, while MMP13 and iNOS mRNA levels were significantly increased. After treatment with CAP-Mn@hyExos, the abnormal transcriptional levels of the above genes were improved to some extent; while treatment with 140@bExos and CAP-Mn / 140@hyExos significantly upregulated COL2A1 transcriptional levels and effectively inhibited the expression of MMP13 and iNOS. Among them, the CAP-Mn / 140@hyExos group showed the most significant regulatory effect at the transcriptional level, further confirming its advantage in regulating cartilage metabolic homeostasis.

[0231] 6.4 Histological staining evaluation of the role of exosomal nanomotors in extracellular matrix synthesis

[0232] To evaluate the effects of different exosome preparations on the extracellular matrix synthesis capacity of chondrocytes at the histological level, Safranin O and Alcian blue staining were used to detect changes in proteoglycans and collagen components in the extracellular matrix. Figure 37 As shown, both staining intensities were significantly weakened in the OA model group, indicating a significant decrease in the extracellular matrix synthesis capacity of chondrocytes. Treatment with CAP-Mn@hyExos slightly enhanced staining intensity; while intervention with 140@bExos and CAP-Mn / 140@hyExos significantly restored staining intensity. The CAP-Mn / 140@hyExos group exhibited the most significant enhancement of matrix staining, suggesting that it can effectively promote the synthesis and deposition of extracellular matrix in chondrocytes.

[0233] In summary, multiple in vitro experiments consistently demonstrate that the exosomal nanomotor CAP-Mn / 140@hyExos can significantly improve the abnormal phenotype of OA chondrocytes. This system significantly upregulates COL2A1 at both the transcriptional and protein levels, while effectively inhibiting the abnormal expression of MMP13 and iNOS, and significantly enhancing extracellular matrix synthesis. Compared to single-function exosomes, CAP-Mn / 140@hyExos exhibits a more prominent comprehensive repair advantage in restoring cartilage metabolic homeostasis and alleviating the inflammatory microenvironment.

[0234] As demonstrated by the above embodiments, this invention provides a cascade-response hybrid exosome nanomotor integrating self-driving, targeted recognition, and therapeutic functions. This system exhibits significant autonomous movement capabilities, chemotaxis towards oxidative stress regions, and excellent penetration performance into dense cartilage matrix in simulated and real osteoarthritis pathological environments. Simultaneously, it can efficiently target and be taken up by chondrocytes, achieving long-term retention within the joint cavity, effectively improving the oxidative stress state, abnormal phenotype, and metabolic imbalance of chondrocytes, significantly promoting extracellular matrix synthesis, and demonstrating outstanding comprehensive repair effects.

[0235] 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. An exosome nanomotor system, characterized in that, It includes a hybrid exosome carrier, self-driven nanoparticles loaded inside the hybrid exosome carrier, and targeting molecules modified on the surface of the hybrid exosome carrier; The self-driven nanoparticles are manganese dioxide nanoparticles. The hybrid exosome carrier is formed by membrane fusion of exosomes from at least two different sources; The target molecule is a target peptide that can specifically recognize cartilage tissue or cartilage cells. The at least two different sources of exosomes include a first exosome derived from bone marrow mesenchymal stem cells and a second exosome derived from breast milk; The targeting peptide is the cartilage-targeting peptide CAP.

2. The exosome nanomotor system of claim 1, wherein, The hybrid exosome carrier also carries therapeutic molecules.

3. The exosome nanomotor system of claim 2, wherein, The therapeutic molecule is a microRNA, specifically miR-140.

4. The exosome nanomotor system of claim 1, wherein, The manganese dioxide nanoparticles have a particle size of 50 nm to 100 nm. The hybrid exosome carrier has an average particle size of 90 nm to 150 nm.

5. The method of producing the exosome nanomotor system according to any one of claims 1 to 4, characterized by, Includes the following steps: S1: Provides the first and second exosomes; S2: Mix the first exosome with the second exosome and perform membrane fusion treatment to obtain a hybrid exosome carrier; S3: Load manganese dioxide nanoparticles into the hybrid exosome carrier; S4: The target molecule is modified onto the surface of the hybrid exosome carrier to obtain the exosome nanomotor system.

6. The preparation method according to claim 5, characterized in that, In step S1, the first exosome is a bone marrow mesenchymal stem cell-derived exosome loaded with therapeutic microRNA, which is obtained by transfecting bone marrow mesenchymal stem cells with a lentiviral vector carrying the microRNA and collecting the culture supernatant for separation. The lentiviral vector has an infection multiplicity of 50 to 100; In step S1, the second exosome is a milk-derived exosome; The membrane fusion process described in step S2 includes: first incubating the mixed exosome solution with shaking at 35°C to 39°C for 10 to 14 hours, and then extruding it through a filter membrane with a pore size of 300 nm to 500 nm 8 to 12 times; Step S3 specifically includes: mixing the manganese dioxide nanoparticle suspension with the hybrid exosome carrier, and treating it under ultrasound with a power of 80W to 120W for 4 to 6 minutes, so that the manganese dioxide nanoparticles are loaded inside the hybrid exosome carrier; The mass ratio of the manganese dioxide nanoparticles to the hybrid exosome carrier is 0.5:1 to 1.5:

1. Step S4 specifically includes: coupling the target molecule to the lipid molecule via a linker molecule to form a modified lipid; mixing the modified lipid with a hybrid exosome carrier loaded with the manganese dioxide nanoparticles; and extruding the modified lipid into the membrane of the hybrid exosome carrier. The linker molecule is DSPE-PEG2000-MAL, and the target molecule is coupled to it via a thiol-maleimide click chemistry reaction.

7. The preparation method according to claim 6, characterized in that, The hybrid exosome carrier is prepared by a method comprising the following steps: extracting a first exosome and a second exosome; mixing the first exosome and the second exosome at a protein ratio of 0.5:1 to 1.5:1; incubating the mixture at 35°C to 39°C with shaking at 50 rpm to 70 rpm for 10 to 14 hours; continuously extruding the incubated mixture through a filter membrane with a pore size of 300 nm to 500 nm 8 to 12 times to induce membrane fusion; and purifying to obtain the hybrid exosome carrier. The extraction method of bone marrow mesenchymal stem cell-derived exosomes loaded with therapeutic microRNA includes: collecting the culture supernatant of transfected cells, centrifuging at 300 g to 500 g for 8 to 12 minutes, centrifuging at 2000 g for 18 to 22 minutes, centrifuging at 10000 g for 28 to 32 minutes, filtering at 0.22 μm, and ultracentrifuging at 100000 g for 68 to 72 minutes. The extraction method of the milk-derived exosomes includes: mixing skim milk with glacial acetic acid at a final concentration of 0.8% to 1.2%, centrifuging at 10,000 g to 14,000 g for 18 to 22 minutes to remove the precipitate, filtering the supernatant through 0.22 μm, and then ultracentrifuging at 170,000 g to 180,000 g for 68 to 72 minutes; The manganese dioxide nanoparticles described in step S3 are prepared by a biomineralization method, which includes: mixing an 80 mM to 120 mM manganese salt solution with an equal volume of an 8 mg / mL to 12 mg / mL bovine serum albumin solution, adjusting the pH to 9.5 to 10.5, stirring at 35°C to 39°C for 1.5 to 2.5 hours, and then dialysis purification. The mass ratio of the modified lipid to the hybrid exosome carrier is 5% to 15%.

8. The use of the exosome nanomotor system according to any one of claims 1 to 4 or the exosome nanomotor system prepared by the preparation method according to any one of claims 5 to 7 in the preparation of a medicament for treating osteoarthritis.

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